US20070003610A1 - Application of lipid vehicles and use for drug delivery - Google Patents

Application of lipid vehicles and use for drug delivery Download PDF

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Publication number
US20070003610A1
US20070003610A1 US11/438,912 US43891206A US2007003610A1 US 20070003610 A1 US20070003610 A1 US 20070003610A1 US 43891206 A US43891206 A US 43891206A US 2007003610 A1 US2007003610 A1 US 2007003610A1
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bladder
kcl
liposomes
lipid
saline
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US11/438,912
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Michael Chancellor
Matthew Fraser
Yao-Chi Chuang
William de Groat
Leaf Huang
Naoki Yoshimura
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University of Pittsburgh
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University of Pittsburgh
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Priority to US11/438,912 priority Critical patent/US20070003610A1/en
Priority to US11/546,025 priority patent/US8110217B2/en
Priority to PCT/US2006/039614 priority patent/WO2007044748A2/en
Priority to JP2008535629A priority patent/JP5815915B2/en
Priority to EP06825726A priority patent/EP1933813A4/en
Publication of US20070003610A1 publication Critical patent/US20070003610A1/en
Priority to US12/651,075 priority patent/US20100104631A1/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY OF PITTSBURGH - OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION
Priority to US13/350,326 priority patent/US20120128762A1/en
Priority to US13/569,968 priority patent/US20120301540A1/en
Priority to US14/376,590 priority patent/US20150037402A1/en
Priority to JP2013171944A priority patent/JP2013234202A/en
Priority to US15/629,087 priority patent/US20170290773A1/en
Assigned to NATIONAL INSTITUTES OF HEALTH (DEITR) reassignment NATIONAL INSTITUTES OF HEALTH (DEITR) CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY OF PITTSBURGH
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/127Liposomes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0034Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents

Definitions

  • the present invention relates to compositions and methods for the Instillation of lipid vehicles (e.g., micelles, microemulsions, macroemulsions, and liposomes) to treat various disorders, including bladder inflammation and dysfunction.
  • lipid vehicles e.g., micelles, microemulsions, macroemulsions, and liposomes
  • the vehicles of the present invention are useful for prolonged delivery of drugs such as antibiotics and anticancer agents to the bladder, genitourinary tract, gastrointestinal system, pulmonary system, and other organs or body systems.
  • the present invention relates to liposome-based delivery of resiniferatoxin, capsaicin, tinyatoxin, and other vanilloid compounds for the treatment of bladder pain, inflammation, incontinence, and voiding dysfunction.
  • liposome-based delivery of toxins such as botulinum toxin
  • toxins such as botulinum toxin
  • the vehicles of the present invention are used alone or in conjunction with antibodies, e.g., uroplakin antibodies, that improve duration of liposome attachment, and provide a long-term intravesical delivery platform for drug, peptide, polypeptide, or nucleic acid delivery.
  • the present invention relates to liposomes coated with antibodies against nerve growth factor (NGF) receptor and containing NGF antisense nucleic acids, which are useful as treatments for neurogenic bladder dysfunction.
  • NGF nerve growth factor
  • Neuropathic pain is thought to occur because of a sensitization in the peripheral and central nervous systems after an initial injury to the peripheral system (see N. Attal, 2000, Clin. J. Pain 16(3 Suppl):S118-30). Direct injury to the peripheral nerves as well as many systemic diseases including AIDS/HIV, Herpes Zoster, syphilis, diabetes, and various autoimmune diseases, can induce this disorder. Such pain is also associated with conditions of the bladder, including interstitial cystitis. Neuropathic pain is typically experienced as burning, shooting, and unrelenting in its intensity, and can sometimes be more debilitating that the initial injury or the disease process which induced it. Unfortunately, the few remedies that have been reported to alleviate this condition are effective in only a small percentage of patients.
  • Interstitial cystitis is characterized by bladder pain, irritative voiding symptoms, and sterile urine (see R. Doggweiler-Wiygul et al., 2000, Curr. Rev. Pain 4(2):137-41).
  • the bladder wall shows inflammatory infiltration with mucosal ulceration and scarring that causes smooth muscle contraction, diminished urinary capacity, hematuria, and frequent, painful urination.
  • solutes such as potassium
  • therapies that restore the mucosal lining or surface GAG layer e.g., administration of heparine, hyaluronic acid, or pentosanpolysulfate, can reduce the leakage of irritant and result in palliation of IC symptoms (see, e.g., C. L. Parsons et al., 1994, Br. J. Urol. 73:504; J. I. Bade et al., 1997, Br. J. Urol. 79:168; J. C. Nickel et al., 1998, J. Urol. 160:612).
  • Capsaicin is a homovanillic acid derivative (8-methyl-N-vanillyl-6-nonenamid). It is the active component of the red pepper of the genus Capsicum, and has been used in humans for topical treatment of cluster headache, herpes zoster, and vasomotor rhinitis (see P. Holzer, 1994, Pharmacol. Rev. 43:143; Sicuteri et al., 1988, Med. Sci. Res. 16-1079; Watson et al., 1988, Pain 33:333; Marabini et al., 1988, Regul. Pept. 22:1).
  • capsaicin In vitro capsaicin modulates cellular growth, collagenase synthesis, and prostaglandin secretion from rheumatoid arthritis synoviocytes (see Matucci-Cerinic et al., 1990, Ann. Rheum. Dis. 49:598). Capsaicin has also been shown to be immunomodulatory as indicated by its ability to modulate lymphocyte proliferation, antibody production, and neutrophil chemotaxis (see Nilsson et al., 1988, J. Immunopharmac. 10:747; Nilsson et al., 1991, J. Immunopharmac. 13:21; and Eglezos et al., 1990, J. Neuroimmunol. 26:131).
  • capsaicin induces mitochondrial swelling, inhibits NADH oxidase, induces apoptosis of transformed cells, stimulates adenylate cyclase, activates protein kinase C, inhibits superoxide anion generation and alters the redox state of the cell.
  • capsaicin The various effects of capsaicin are mediated through a specific cellular receptor referred to as a vanilloid receptor.
  • This receptor is shared by resiniferatoxin, an alkaloid derived from plants of the genus Euphorbia.
  • Resiniferatoxin is a structural homologue of capsaicin, and has been shown to mimic many of the actions of capsaicin.
  • Resiniferatoxin is also structurally similar to phorbol esters (phorbol myristate acetate), which interact with distinct binding sites and activate protein kinase C (see Szallasi, et al., 1989, Neurosci. 30:515; and Szallasi and Blumberg, 1989, Neurosci. 30:515).
  • capsaicin Unlike resiniferatoxin, capsaicin has no homology to phorbol myristate acetate. However, capsaicin can activate protein kinase C, suggesting that such activation is not due entirely to the phorbol ester-like moiety on resiniferatoxin.
  • Capsaicin has been used as an experimental tool because of its selective action on the small diameter afferent nerve fibers, or C fibers, which mediate pain. From studies in animals, capsaicin appears to trigger C fiber membrane depolarization by opening cation selective channels for calcium and sodium.
  • capsaicin mediated effects include: (I) activation of nociceptors in peripheral tissues; (ii) eventual desensitization of peripheral nociceptors to one or more stimulus modalities; (iii) cellular degeneration of sensitive unmyelinated C fiber afferents; (iv) activation of neuronal proteases; (v) blockage of axonal transport; and (vi) the decrease of the absolute number of C fibers without affecting the number of myelinated fibers.
  • transdermal therapeutic formulation comprising capsaicin, a non-steroidal anti-inflammatant, and pamadorm (a diuretic agent) where the composition is said to contain from about 0.001-5% by weight capsaicin and to be useful in treating the pain and discomfort associated with menstrual cramps, bloating, and/or muscular pain such as muscular back pain.
  • Several studies have assessed intravesical capsaicin as a treatment for urge incontinence in patients with spinal detrusor hyperreflexia or bladder hypersensitivity disorders (see F. Cruz, 1998, Int. Urogynecol. J. Pelvic Floor Dysfunct. 9:214-220).
  • capsaicin application itself frequently causes burning pain and hyperalgesia apart from the neuropathic pain being treated, patient compliance has been poor and the drop out rates during clinical trials have exceeded fifty percent.
  • the spontaneous burning pain and heat hyperalgesia are believed to be due to intense activation and temporary sensitization of the peripheral nociceptors at the site of capsaicin application (primary hyperalgesia).
  • Mechanical hyperalgesia evident in areas surrounding the site of topical application appears to originate from central sensitization of dorsal horn neurons involved in pain transmission (secondary hyperalgesia). Because of these side effects, the maximal capsaicin concentration used in previous human studies has usually been limited to 0.075%.
  • Dystonias are neurological movement disorders characterized by involuntary muscle contractions that force certain parts of the body into abnormal, sometime painful, movements or postures (see S. B. Bressman, 2000, Clin. Neuropharmacol. 23(5):239-51). Dystonia disorders cause uncontrolled movement and prolonged muscle contraction, which can result in spasms, twisting body motions, tremor, or abnormal posture. These movements may involve the entire body, or only an isolated area, such as the arms and legs, trunk, neck, eyelids, face, bladder sphincter, or vocal cords.
  • Dystonias result from environmental or disease-related damage to the basal ganglia, birth injury, (particularly due to lack of oxygen), certain infections, reactions to certain drugs, heavy-metal or carbon monoxide poisoning, trauma, or stroke can cause dystonic symptoms. Dystonias can also be symptoms of other diseases, some of which may be hereditary.
  • Urinary detrusor-sphincter dyssynergia (UDSD; also called detrusor-external sphincter dyssynergia and urethral dyssynergia) is a specific type of neurological movement disorder (see H. Madersbacher, 1990, Paraplegia 28(4):217-29; J. T. Andersen et al., 1976, J. Urol. 116(4):493-5).
  • UDSD is characterized by involuntary urinary sphincter spasms occurring simultaneously with bladder contractions. The lack of coordination between detrusor contraction and urethral relaxation causes urinary obstruction (i.e., partial or complete block of urination).
  • UDSD results from lesions of the corticospinal tract, which are caused by spinal chord injury, multiple sclerosis, or related conditions.
  • hyperactive neurogenic bladder Another neurological movement disorder is hyperactive (also called contracted; spastic) neurogenic bladder (see M. H. Beers and R. Berkow (eds), 1999, The Merck Manual of Diagnosis and Therapy, Section 17:Genitourinary Disorders, Chapter 216: Myoneurogenic Disorders).
  • hyperactive bladder the bladder contracts more frequently than normal, due to instability and inappropriate contraction of detrusor muscles (see, e.g., C. F. Jabs et al., 2001, Int. Urogynecol. J. Pelvic Floor Dysfunct. 12(1):58-68; S. K. Swami and P. Abrams, 1996, Urol. Clin. North Am. 23(3):417-25).
  • Hyperactive bladders can empty spontaneously and result in urinary incontinence (urge incontinence). In addition, the uncoordinated contraction between the bladder and bladder outlet (vesical neck or external urinary sphincter) can result in vesicoureteral reflux with concomitant renal damage. Hyperactive bladder is usually due to brain or suprasacral spinal cord damage. The most common cause is spinal cord injury from transverse myelitis or traumatic cord transection.
  • Hyperactive bladder can also be caused by conditions such as anxiety, aging, infections (e.g., syphilis), diabetes mellitus, brain and spinal cord tumors, stroke, ruptured intervertebral disk, and demyelinating and degenerative diseases (e.g., multiple sclerosis and amyotrophic lateral sclerosis).
  • infections e.g., syphilis
  • diabetes mellitus e.g., diabetes mellitus
  • brain and spinal cord tumors e.g., stroke, ruptured intervertebral disk
  • demyelinating and degenerative diseases e.g., multiple sclerosis and amyotrophic lateral sclerosis.
  • Botulinum toxins are zinc endopeptidases produced by the anaerobic bacterium Clostridium botulinum. Previously known as a cause of a serious and often fatal paralysis acquired through ingestion of contaminated food, botulinum neurotoxins are presently used in both therapeutic and cosmetic applications (see N. Mahant et al., 2000, J. Clin. Neurosci. 7(5):389-94; A. Carruthers and J. Carruthers, 2001, Semin. Cutan. Med. Surg. 20(2):71-84). In particular, these toxins are used in the treatment of conditions involving involuntary muscle spasms, frown lines, and facial wrinkles.
  • A-G botulinum toxins
  • the serotypes differ in their cellular targets, potency, and duration of action, but all exert their paralytic effect by inhibiting acetylcholine release at the neuromuscular junction (see M. F. Brin, 1997, Muscle Nerve 20(suppl 6):S146-S168).
  • Each serotype acts by cleaving one or more proteins involved in vesicle transport and membrane fusion.
  • botulinum toxin A is internalized by endocytosis at the axon terminal, where it is fully activated by disulfide reduction reactions, and it targets SNAP-25 (see M. F.
  • Botulinum toxin A causes reversible denervation atrophy that is typically terminated by axon sprouting within 2-6 months (see M. F. Brin, 1997, Muscle Nerve 20(suppl 6):S146-S168).
  • a major drawback of current botulinum toxin therapies is the development of antitoxin antibodies in patients.
  • Antitoxin antibodies result in resistance to botulinum toxin, and the reduction or elimination of its therapeutic effect. It has been estimated that the prevalence of neutralizing antibodies among patients receiving chronic treatment at the higher doses for torticollis or spasticity is probably at least 3% (see M. F. Brin, 1997, Muscle Nerve 20(suppl 6):S146-S168).
  • Patients with botulinum toxin A resistance may benefit from injections with other serotypes, including botulinum toxin B, C, and F.
  • differences in the duration of the effects of the other serotypes can be significant, and cause dramatic reductions in treatment efficacy (see M. F. Brin, 1997, Muscle Nerve. 20(suppl 6):S146-S168).
  • Liposomes are self-assembling structures comprising concentric amphipathic lipid (e.g., phospholipid) bilayers separated by aqueous compartments (see, e.g., K. Reimer et al., 1997, Dermatology 195(suppl. 2):93; M. Schafer-Korting et al., 1989, Dermatology 21:1271).
  • the amphipathic lipid molecules comprise a polar headgroup region covalently linked to one or two non-polar acyl chains. The energetically unfavorable contact between the hydrophobic acyl chains and the aqueous solution surrounding the lipid molecules causes the polar headgroups and acyl chains to rearrange.
  • the polar headgroups become oriented toward the aqueous solution, while the acyl chains orient towards the interior part of the bilayer.
  • the lipid bilayer structure thereby comprises two opposing monolayers, wherein the acyl chains are shielded from contact with the surrounding medium.
  • Liposomes are excellent vehicles for drug delivery and gene therapy (K. Reimer et al., 1997, Dermatology 195(suppl. 2):93; T. Tsuruta et al., 1997, J. Urol. 157:1652; F. Szoka, 2000, Mol. Therapy 1:S2; G. Gregoriadis, 1976, New Eng. J. Med. 295:704).
  • Previous studies have demonstrated that submucosal injection of liposomal doxorubicin into bladder wall provides an effective and safe treatment for bladder cancer with pelvic lymph node metastasis (I. Tsuruta et al., 1997, J. Urol. 157:1652).
  • an active ingredient such as a drug
  • an active ingredient is encapsulated or entrapped in the liposome and then administered to the patient to be treated.
  • the active ingredient is lipophilic, it may be associated with the lipid bilayer.
  • Active ingredients encapsulated by liposomes reduce toxicity, increase efficacy, or both.
  • liposomes are thought to interact with cells by stable absorption, endocytosis, lipid transfer, and fusion (R. B. Egerdie et al., 1989, J. Urol. 142:390). In this way, liposomes comprise molecular films, which fuse with cells and provide optimal conditions for wound healing (K.
  • liposomes have low antigenicity and can be used to encapsulate and deliver components that cause undesirable immune responses in patients (see A. Natsume et al., 2000, Jpn. J. Cancer Res. 91:363-367).
  • the present invention encompasses improved treatments for pain (e.g., neuropathic pain), pain-intensive disorders (e.g., IC), muscle contraction disorders (e.g., IC, hyperactive bladder, and UDSD), and related conditions by providing compositions and methods for the intravesical administration of lipid vehicles.
  • Liposomes provide non-toxic vehicles for the delivery of lipophilic therapeutic agents that have irritative side effects (e.g., vanilloids such as capsaicin) or undesirable antigenicity (e.g., botulinum toxin).
  • the disclosed lipid vehicles can be used simultaneously deliver and ameliorate irritation caused by irritating therapeutic agents.
  • the vehicles can also be used to reduce or prevent antibody-mediated resistance to antigenic therapeutic agents.
  • the disclosed lipid vehicles can be utilized as an intravesical drug delivery platform for antibiotic and anticancer agents in the bladder and other luminal organ systems, e.g., the distal colon and vagina.
  • compositions comprising lipid vehicles (e.g., micelles, microemulsions, macroemulsions, and liposomes) for use as intravesical instillation vehicles for cells or tissues.
  • lipid vehicles e.g., micelles, microemulsions, macroemulsions, and liposomes
  • Such vehicles may further include antibodies, for example, uroplakin or NGF receptor antibodies. These antibodies may be conjugated to the surface of the liposome, and act to target the liposome to specific cell: types and/or receptors.
  • the vehicles may include compositions, including capsaicin, resiniferatoxin, tinyatoxin, and other vanilloids, which can be delivered to the cells.
  • the vehicles may also include compositions comprising bioactive agents (e.g., antisense nucleic acids or peptides), drugs (e.g., pain therapeutics, anticancer treatments, or antibiotics), toxins (e.g., botulinum toxin), or other agents.
  • bioactive agents e.g., antisense nucleic acids or peptides
  • drugs e.g., pain therapeutics, anticancer treatments, or antibiotics
  • toxins e.g., botulinum toxin
  • the present invention further encompasses methods of treating various disorders, e.g., defects or diseases of the genitourinary tract, gastrointestinal tract, pulmonary system, and other body systems, using the disclosed lipid vehicles.
  • the disclosed vehicles can be administered via intravesical instillation to treat interstitial cystitis (IC), urinary detrusor-sphincter dyssynergia (UDSD), spastic neurogenic bladder, hyperactive bladder, or other conditions of the genitourinary system.
  • IC interstitial cystitis
  • UDSD urinary detrusor-sphincter dyssynergia
  • spastic neurogenic bladder e.g., spastic neurogenic bladder
  • hyperactive bladder e.g., hyperactive bladder
  • the disclosed vehicles can also be administered intravesically to treat systemic infections and cancers, utilizing the unique interaction of the disclosed vehicles as a novel route for prolonged delivery of such therapies.
  • the invention also encompasses methods of treating pain (e.g., neuropathic pain) associated with cancers and/or disorders of the bladder, genitourinary tract, gastrointestinal tract, pulmonary system, and other body systems, using the disclosed lipid vehicles.
  • the disclosed vehicles can be administered via intravesical instillation to treat pain associated with IC, or other conditions of the bladder, such as bladder infections and bladder cancer.
  • these vehicles may comprise vanilloids, e.g., capsaicin, resiniferatoxin, or tinyatoxin, and may further comprise surface antibodies, e.g., uroplakin or NGF receptor antibodies, to target pain relief to the affected sites.
  • the disclosed vehicles can be administered via intravesical instillation to treat muscle contractions caused by IC, UDSD, spastic neurogenic bladder, or related conditions.
  • the vehicles may be empty or may carry toxins, e.g., botulinum toxins, to deliver relief from muscle contractions at the affected sites.
  • FIG. 1 shows the experimental design for the studies described in Examples 1-2 (below).
  • FIGS. 2A-2F show CMG tracing results. Treatments included saline (control), protamine sulfate in potassium chloride (PS/KCl), and liposomes in KCl (LP/KCl) or KCl alone. PS/KCl elicited bladder hyperactivity. LP/KCl partly reversed the irritative effect of LP/KCl, and this reversal was maintained after switching to KCl.
  • FIGS. 2B, 2D , and 2 F show saline infusion (control), PS/KCl infusion, and KCl infusion, respectively, in the control animal.
  • FIGS. 2A, 2C , and 2 E show saline infusion (control), PS/KCl infusion, and liposomal infusion in the presence of maintenance KCl, respectively.
  • FIGS. 3A-3F show CMG tracing results. Treatments included saline (control), acetic acid (M), and liposomes (LP) or saline. AA elicited bladder hyperactivity. LP partly reversed the irritative effect of AA, and this reversal was maintained after switching to saline.
  • FIGS. 3B, 3D , and 3 F show saline infusion (control), AA infusion and saline infusion, respectively, in the control animal.
  • FIGS. 3A, 3C , and 3 E show saline infusion (control), AA infusion, and liposomal infusion in the presence of maintenance M, respectively.
  • FIGS. 4A-4D show CMG tracing results. Treatments included saline (control) and various concentrations of protamine sulfate (PS). High concentrations of PS induced bladder hyperactivity (decreased ICl), whereas low concentrations of PS produced no effect.
  • FIG. 4A shows a control CMG measured before PS treatment.
  • FIG. 4B shows a CMG measured during treatment with low concentrations of PS.
  • FIG. 4C shows a control CMG measured before PS treatment.
  • FIG. 4D shows a CMG measured during treatment with high concentrations of PS.
  • FIGS. 5A-5F show CMG tracing results.
  • Treatments included saline (control) and various concentrations of potassium chloride (KCl) following one hour of PS (10 mg/ml). High concentrations of KCl induced bladder hyperactivity (decrease ICl), whereas low concentrations of KCl had no effect.
  • FIG. 5A shows a control CMG measured before KCl treatment.
  • FIG. 5B shows a CMG measured during treatment with 100 mM KCl.
  • FIG. 5C shows a control CMG measured before KCl treatment.
  • FIG. 5D shows a CMG measured during treatment with 300 mM KCl.
  • FIG. 5E shows a control CMG measured before KCl treatment.
  • FIG. 5F shows a CMG measured during treatment with 500 mM KCl.
  • FIGS. 6A-6B show CMG tracing results. Treatments included saline (control) and KCl (500 mM) infusion following PS (10 mg/ml) infusion in micturition reflex suppressed animals. KCl stimulated the detrusor muscle and decreased bladder compliance.
  • FIG. 6A shows a control CMG measured before KCl treatment.
  • FIG. 6B shows a CMG measured during KCl treatment.
  • FIG. 7 shows the efficacy of liposomal delivery of capsaicin utilizing bladder contraction frequency as a bioassay of the irritative effects of the vanilloid.
  • Column 1 saline
  • Column 2 liposomes
  • Column 3 liposomes plus capsaicin.
  • Inclusion of capsaicin into the liposomal preparation allowed for effective capsaicin delivery.
  • the addition of saline or liposomes produced no change in bladder contraction frequency.
  • the combination of liposome and capsaicin produced a significant increase in bladder contraction frequency.
  • the present invention relates to the administration of lipid vehicles to provide long-lasting drug delivery to diseased or dysfunctional cells, tissues, or body systems.
  • the invention relates to treatments for urinary system components, e.g., kidneys, ureters, bladders, sphincter muscles, and urethras.
  • urinary system components e.g., kidneys, ureters, bladders, sphincter muscles, and urethras.
  • bladder irritation and irritation-induced bladder dysfunction are included in accordance with the present invention.
  • nonionic liposomes are formulated to act as a drug with prolonged efficacy for topical bladder instillation, and bladder-protective effects. The efficacy and protective effects of such formulations are unexpected and surprising results.
  • the disclosed liposome vehicles can be used to simultaneously deliver and ameliorate irritation caused by irritating therapeutic agents, e.g., resiniferatoxin or other vanilloid agents.
  • the disclosed methods of intravesical administration of liposomes provide novel treatments for IC patients. Such methods can also be employed for the treatment of other disorders of the urinary system, bladder, genitourinary tract, gastrointestinal tract, pulmonary system, and other body organs and systems, including cancers, infections, and spasticity.
  • the lipid vehicles of the present invention encompass micelles, microemulsions, macroemulsions, liposomes, and similar carriers.
  • the term micelles refers to colloidal aggregates of amphipathic (surfactant) molecules that are formed at a well-defined concentration known as the critical micelle concentration. Micelles are oriented with the nonpolar portions at the interior and the polar portions at the exterior surface, exposed to water. The typical number of aggregated molecules in a micelle (aggregation number) is 50 to 100.
  • microemulsions are essentially swollen micelles, although not all micellar solution can be swollen to form microemulsion. Microemulsions are thermodynamically stable, are formed spontaneously, and contain particles that are extremely small.
  • Droplet diameters in microemulsions typically range from 10-100 nm.
  • macroemulsions refers to droplets with diameters greater than 100 nm.
  • liposomes are closed lipid vesicles comprising lipid bilayers that encircle aqueous interiors. Liposomes typically have diameters of 25 nm to 1 ⁇ m (see, e.g., D. O. Shah (ed), 1998, Micelles, Microemulsions, and Monolayers: Science and Technology, Marcel Dekker; A. S. Janoff (ed), 1998, Liposomes: Rational Design, Marcel Dekker).
  • Lipid vehicles of the invention may carry a bioactive agent (e.g., a nucleic acid, polypeptide, peptide, or antibody molecule) or drug (e.g., one or more pepper extract compounds such as capsaicin, resiniferatoxin, tinyatoxin and other vanilloids, as well as antibiotics, anti-inflammatory agents, and antispasmodics).
  • a bioactive agent e.g., a nucleic acid, polypeptide, peptide, or antibody molecule
  • drug e.g., one or more pepper extract compounds such as capsaicin, resiniferatoxin, tinyatoxin and other vanilloids, as well as antibiotics, anti-inflammatory agents, and antispasmodics.
  • nucleic acid and polynucleotide are synonymous, and refer to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribon
  • protein and polypeptide are synonymous as used herein, and refer to polymers comprising amino acid residues linked by peptide bonds.
  • Peptides are defined as fragments or portions of polypeptides, preferably fragments or portions having at least one functional activity (e.g., binding, antigenic, or catalytic activity) as the complete polypeptide sequence (see, e.g., by H. Lodish et al., 1999, Molecular Cell Biology, W. H. Freedman and Sons, N.Y.; L. Stryer, 2001, Biochemistry, W. H. Freedman and Sons, N.Y.; B. Lewin, 1999, Genes VII, Oxford University Press).
  • the vehicle is a liposome formulation
  • the drug is an organic or inorganic small molecule.
  • the principal lipid of the vehicle is, preferably, phosphatidylcholine, but can include various natural (e.g., tissue derived L- ⁇ -phosphatidyl: egg yolk, heart, brain, liver, soybean) and/or synthetic (e.g., saturated and unsaturated 1,2-diacyl-SN-glycero-3-phosphocholines, 1-acyl-2-acyl-SN-glycero-3-phosphocholines, 1,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the same.
  • Such lipids can be used alone, or in combination with a helper lipid.
  • Preferred helper lipids are non-ionic or uncharged at physiological pH.
  • Particularly preferred non-ionic lipids include, but are not limited to, cholesterol and DOPE (1,2-dioleolylglyceryl phosphatidylethanolamine), with cholesterol being most preferred.
  • DOPE 1,2-dioleolylglyceryl phosphatidylethanolamine
  • the molar ratio of a phospholipid to helper lipid can range from about 3:1 to about 1:1, more preferably from about 1.5:1 to about 1:1, and most preferably, the molar ratio is about 1:1.
  • a liposome used for the preparation of a vehicle of the invention is, in simplest form, composed of two lipid layers.
  • the lipid layer may be a monolayer, or may be multilamellar and include multiple layers.
  • Constituents of the liposome may include, for example, phosphatidylcholine, cholesterol, phosphatidylethanolamine, etc.
  • Phosphatidic acid which imparts an electric charge, may also be added.
  • Exemplary amounts of these constituents used for the production of the liposome include, for instance, 0.3 to 1 mol, preferably 0.4 to 0.6 mol of cholesterol; 0.01 to 0.2 mol, preferably 0.02 to 0.1 mol of phosphatidylethanolamine; 0.0-0.4 mol, preferably 0-0.15 mol of phosphatidic acid per 1 mol of phosphatidylcholine.
  • Liposomes of the present invention can be constructed by well-known techniques (see, e.g., G. Gregoriadis (ed.), 1993, Liposome Technology Vols. 1-3, CRC Press, Boca Raton, Fla.). Lipids are typically dissolved in chloroform and spread in a thin film over the surface of a tube or flask by rotary evaporation. If liposomes comprised of a mixture of lipids are desired, the individual components are mixed in the original chloroform solution. After the organic solvent has been eliminated, a phase consisting of water optionally containing buffer and/or electrolyte is added and the vessel agitated to suspend the lipid.
  • the suspension is then subjected to ultrasound, either in an ultrasonic bath or with a probe sonicator, until the particles are reduced in size and the suspension is of the desired clarity.
  • ultrasound either in an ultrasonic bath or with a probe sonicator
  • the aqueous phase is typically distilled water and the suspension is sonicated until nearly clear, which requires several minutes depending upon conditions, kind, and quality of the sonicator.
  • lipid concentrations are 1 mg/ml of aqueous phase, but could be higher or lower by about a factor of ten.
  • Liposomes according to the invention optionally have one or more amphiphiles.
  • the exact composition of the liposomes will depend on the particular circumstances for which they are to be used. Those of ordinary skill in the art will find it a routine matter to determine a suitable composition.
  • the liposomes of the present invention comprise at least one compound of the present invention.
  • the liposomes of the present invention consist essentially of a single type of phospholipid.
  • the liposomes comprise mixtures of phospholipids.
  • the liposomes of the present invention comprise one or more phospholipids in a mixture with one or more natural or synthetic lipids, e.g., cholesterol or DOPE.
  • Liposomes can be produced in accordance with established methods. For example, a mixture of the above-mentioned lipids, from which the solvents have been removed, can be emulsified by the use of a homogenizer, lyophilized, and melted to obtain multilamellar liposomes. Alternatively, unilamellar liposomes can be produced by the reverse phase evaporation method (Szoka and Papahadjopoulos, 1978, Proc. Natl. Acad. Sci. USA 75:4194-4198). Unilamellar vesicles can also be prepared by sonication or extrusion. Sonication is generally performed with a bath-type sonifier, such as a Branson tip sonifier (G.
  • a bath-type sonifier such as a Branson tip sonifier (G.
  • Extrusion may be carried out by biomembrane extruders, such as the Lipex Biomembrane Extruder (Northern Lipids Inc, Vancouver, British Columbia, Canada). Defined pore size in the extrusion filters may generate unilamellar liposomal vesicles of specific sizes.
  • the liposomes can also be formed by extrusion through an asymmetric ceramic filter, such as a Ceraflow Microfilter (commercially available from the Norton Company, Worcester, Mass.).
  • the liposomes that have not been sized during formation may be sized by extrusion to achieve a desired size range and relatively narrow distribution of liposome sizes.
  • a size range of about 0.2-0.4 microns will allow the liposome suspension to be sterilized by filtration through a conventional filter (e.g., a 0.22 micron filter).
  • the filter sterilization method can be carried out on a high throughput basis.
  • the size of the liposomal vesicles may be determined by quasi-elastic light scattering (QELS) (see Bloomfield, 1981, Ann. Rev. Biophys. Bioeng. 10:421-450). Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.
  • QELS quasi-elastic light scattering
  • Liposomes can be extruded through a small-pore polycarbonate membrane or an asymmetric ceramic membrane to yield a well-defined size distribution. Typically, a suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved. The liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size.
  • liposomes For use in the present invention, liposomes have a size of about 0.05 microns to about 0.5 microns. More preferred are liposomes having a size of about 0.05 to about 0.2 microns.
  • Various conditions can be used to trigger the liposome to release its payload or active agent, including pH, ionic strength, controlled release and antibody attachment.
  • Research related to pH-sensitive liposomes has focused principally on anionic liposomes comprised largely of phosphatidylethanolamine (PE) bilayers (see, Huang et al., 1989, Biochemistry 28:9508-9514; Duzgunes et al., 1990, “pH-Sensitive Liposomes” Membrane Fusion J. Wilschut and D. Hoekstra (eds.), Marcel-Decker Inc., New York, N.Y. pp. 713-730; Yatvin et al., 1980, Science, 210, 1253-1255).
  • PE phosphatidylethanolamine
  • pH-sensitive cationic liposomes have been developed to mediate transfer of DNA into cells. For instance, researchers have described a series of amphiphiles with headgroups containing imidazole, methylimidazole, or aminopyridine moieties (see, Budker et al., 1996, Nature Biotech. 14:760-764). Also described are lipid molecules within liposome assemblies that are capable of structural reorganization upon a change in pH (see, e.g., U.S. Pat. No. 6,200,599 to Nantz et al.).
  • the vehicles of the present invention are useful for both in vitro and in vivo applications.
  • the vehicles of the present invention will find use for nearly any in vitro or in vivo application requiring delivery of bioactive agents (e.g., nucleic acids, peptides, polypeptides, or antibodies) and/or drugs (e.g., pain therapeutics, anticancer treatments, or antibiotics) into cells.
  • bioactive agents e.g., nucleic acids, peptides, polypeptides, or antibodies
  • drugs e.g., pain therapeutics, anticancer treatments, or antibiotics
  • nucleic acids of all types may be associated with the lipid vehicles of the present invention.
  • nucleic acids may be single- or double-stranded molecules, i.e., DNA, RNA, or DNA-DNA, DNA-RNA or RNA-RNA hybrids, or protein nucleic acids (PNAs) formed by conjugating bases to an amino acid backbone.
  • Nucleic acids may also be oligonucleotides such as antisense oligonucleotides, chimeric DNA-RNA polymers, and ribozymes, as well as modified versions of these nucleic acids wherein the modification may be in the base, the sugar moiety, the phosphate linkage, or in any combination thereof.
  • the nucleic acids may comprise an essential gene or fragment thereof, in which the target cell or cells is deficient in some manner. This can occur where the gene is lacking or where the gene is mutated resulting in under- or over- expression.
  • the nucleic acids can also comprise antisense oligonucleotides. Such antisense oligonucleotides may be constructed to inhibit expression of a target gene.
  • DNA containing all or part of the coding sequence for a polypeptide, or a complementary sequence thereof is incorporated into a vector and inserted into a lipid vehicle for gene therapy applications.
  • Gene therapy can be defined as the transfer of DNA for therapeutic purposes. Improvement in gene transfer methods has allowed for development of gene therapy protocols for the treatment of diverse types of diseases. Gene therapy has also taken advantage of recent advances in the identification of new therapeutic genes, improvement in both viral and non-viral gene delivery systems, better understanding of gene regulation, and improvement in cell isolation and transplantation. Gene therapy can be carried out according to generally accepted methods as described by, for example, Friedman, 1991, Therapy for Genetic Diseases, Friedman, Ed., Oxford University Press, pages 105-121.
  • Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector may be used.
  • Methods for introducing DNA into cells are known in the art, and the choice of method is within the competence of one skilled in the art (Robbins (ed), 1997, Gene Therapy Protocols, Human Press, N.J.).
  • Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and non-viral transfer methods.
  • viruses have been used as gene transfer vectors, including polyoma, i.e., SV40 (Madzak et al., 1992, J. Gen. Virol., 73:1533-1536), adenovirus (Berkner, 1992, Curr. Top. Microbiol. Immunol. 158:39-6; Berkner et al., 1988, Bio Techniques, 6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Natl. Acad. Sci.
  • polyoma i.e., SV40 (Madzak et al., 1992, J. Gen. Virol., 73:1533-1536), adenovirus (Berkner, 1992, Curr. Top. Microbiol. Immunol. 158:39-6; Berkner et al., 1988, Bio Techniques, 6:616
  • Non-viral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Graham et al., 1973, Virology, 52:456-467; Pellicer et al., 1980, Science, 209:1414-1422), mechanical techniques, for example microinjection (Anderson et al., 1980, Proc. Natl. Acad. Sci. USA, 77:5399-5403; Gordon et al., 1980, Proc. Natl. Acad. Sci.
  • plasmid DNA is complexed with a polylysine-conjugated antibody specific to the adenovirus hexon protein, and the resulting complex is bound to an adenovirus vector.
  • the trimolecular complex is then used to infect cells.
  • the adenovirus vector permits efficient binding, internalization, and degradation of the endosome before the coupled DNA is damaged.
  • liposome/DNA is used to mediate direct in vivo gene transfer. While in standard liposome preparations the gene transfer process is non-specific, localized in vivo uptake and expression have been reported in tumor deposits, for example, following direct in situ administration (Nabel, 1992, Hum. Gene Ther., 3:399-410).
  • Suitable gene transfer vectors possess a promoter sequence, preferably a promoter that is cell-specific and placed upstream of the sequence to be expressed.
  • the vectors may also contain, optionally, one or more expressible marker genes for expression as an indication of successful transfection and expression of the nucleic acid sequences contained in the vector.
  • vectors can be optimized to minimize undesired immunogenicity and maximize long-term expression of the desired gene product(s) (see Nabe, 1999, Proc. Natl. Acad. Sci. USA 96:324-326).
  • vectors can be chosen based on cell-type that is targeted for treatment.
  • vector constructs for transfection or infection of the host cells include replication-defective viral vectors, DNA virus or RNA virus (retrovirus) vectors, such as adenovirus, herpes simplex virus and adeno-associated viral vectors.
  • Adeno-associated virus vectors are single stranded and allow the efficient delivery of multiple copies of nucleic acid to the cell's nucleus.
  • the vectors will normally be substantially free of any prokaryotic DNA and may comprise a number of different functional nucleic acid sequences.
  • An example of such functional sequences may be a DNA region comprising transcriptional and translational initiation and termination regulatory sequences, including promoters (e.g., strong promoters, inducible promoters, and the like) and enhancers which are active in the host cells. Also included as part of the functional sequences is an open reading frame (polynucleotide sequence) encoding a protein of interest. Flanking sequences may also be included for site-directed integration. In some situations, the 5′-flanking sequence will allow homologous recombination, thus changing the nature of the transcriptional initiation region, so as to provide for inducible or non-inducible transcription to increase or decrease the level of transcription, as an example.
  • promoters e.g., strong promoters, inducible promoters, and the like
  • enhancers which are active in the host cells.
  • an open reading frame polynucleotide sequence
  • Flanking sequences may also be included for site-directed integration. In some situations, the 5
  • an encoded and expressed polypeptide may be intracellular, i.e., retained in the cytoplasm, nucleus, or in an organelle, or may be secreted by the cell.
  • the natural signal sequence present in a polypeptide may be retained.
  • a signal sequence may be provided so that, upon secretion and processing at the processing site, the desired protein will have the natural sequence.
  • Specific examples of coding sequences of interest for use in accordance with the present invention include the polypeptide-coding sequences disclosed herein.
  • a marker may be present for selection of cells containing the vector construct.
  • the marker may be an inducible or non-inducible gene and will generally allow for positive selection under induction, or without induction, respectively.
  • Examples of marker genes include neomycin, dihydrofolate reductase, glutamine synthetase, and the like.
  • the vector employed will generally also include an origin of replication and other genes that are necessary for replication in the host cells, as routinely employed by those having skill in the art.
  • the replication system comprising the origin of replication and any proteins associated with replication encoded by a particular virus may be included as part of the construct.
  • the replication system must be selected so that the genes encoding products necessary for replication do not ultimately transform the cells.
  • Such replication systems are represented by replication-defective adenovirus (see G. Acsadi et al., 1994, Hum. Mol. Genet. 3:579-584) and by Epstein-Barr virus.
  • replication defective vectors particularly, retroviral vectors that are replication defective, are BAG, (see Price et al., 1987, Proc. Natl. Acad. Sci. USA, 84:156; Sanes et al., 1986, EMBO J., 5:3133).
  • the final gene construct may contain one or more genes of interest, for example, a gene encoding a bioactive metabolic molecule.
  • cDNA, synthetically produced DNA or chromosomal DNA may be employed utilizing methods and protocols known and practiced by those having skill in the art.
  • a vector containing an antisense sequence or encoding a polypeptide is directly injected into the recipient cells (in vivo gene therapy).
  • cells from the intended recipients are explanted, genetically modified to contain the antisense or encode the polypeptide, and reimplanted into the donor (ex vivo gene therapy).
  • An ex vivo approach provides the advantage of efficient viral gene transfer, which is superior to in vivo gene transfer approaches.
  • the host cells are first transfected with engineered vectors containing at least one nucleic acid sequence, suspended in a physiologically acceptable carrier, excipient, or diluent such as saline or phosphate buffered saline, and the like; and then administered to the host.
  • a physiologically acceptable carrier such as saline or phosphate buffered saline, and the like.
  • the desired protein and/or RNA is expressed by the injected cells.
  • the introduced gene products are thereby utilized to treat or ameliorate a disorder that is related to altered expression or function of a gene.
  • an antisense nucleic acid sequence is carried by a lipid vehicle of the invention.
  • An antisense sequence can be wholly or partially complementary to a target nucleic acid, and can be DNA, or its RNA counterpart (i.e., wherein T residues of the DNA are U residues in the RNA counterpart).
  • Antisense nucleic acids can be produced by standard techniques (see, for example, Shewmaker et al., U.S. Pat. No. 5,107,065).
  • An antisense nucleic acid may comprise a sequence complementary to a portion of a protein coding sequence. A portion, for example a sequence of 16 nucleotides, may be sufficient to inhibit expression of the protein. Or, an antisense nucleic acid or oligonucleotide, complementary to 5′ or 3′ untranslated regions, or overlapping the translation initiation codons (5′ untranslated and translated regions), of target genes, or genes encoding a functional equivalent can also be effective. Accordingly, antisense nucleic acids or oligonucleotides can be used to inhibit the expression of the gene encoded by the sense strand or the mRNA transcribed from the sense strand.
  • antisense nucleic acids and oligonucleotides can be constructed to bind to duplex nucleic acids either in the genes or the DNA:RNA complexes of transcription, to form stable triple helix-containing or triplex nucleic acids to inhibit transcription and/or expression of a gene (Frank-Kamenetskii, M. D. and Mirkin, S. M., 1995, Ann. Rev. Biochem. 64:65-95).
  • Such oligonucleotides of the invention can be constructed using the base-pairing rules of triple helix formation and the nucleotide sequences of the target genes.
  • At least one of the phosphodiester bonds of an antisense oligonucleotide has been substituted with a structure that functions to enhance the ability of the compositions to penetrate into the region of cells where the RNA whose activity is to be modulated is located. It is preferred that such substitutions comprise phosphorothioate bonds, methyl phosphonate bonds, or short chain alkyl or cycloalkyl structures.
  • the phosphodiester bonds are substituted with structures which are, at once, substantially non-ionic and non-chiral, or with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in the practice of the invention.
  • Oligonucleotides may also include species that include at least some modified base forms. Thus, purines and pyrimidines other than those normally found in nature may be so employed. Similarly, modifications on the furanosyl portions of the nucleotide subunits may also be affected, as long as the essential tenets of this invention are adhered to. Examples of such modifications are 2′-O-alkyl- and 2′-halogen-substituted nucleotides.
  • modifications at the 2′ position of sugar moieties which are useful in the present invention include OH, SH, SCH 3 , F, OCH 3 , OCN, O(CH 2 ) n NH 2 and O(CH 2 ) n CH 3 , where n is from 1 to about 10.
  • Such oligonucleotides are functionally interchangeable with natural oligonucleotides or synthesized oligonucleotides, which have one or more differences from the natural structure. All such analogs are comprehended by this invention so long as they function effectively to hybridize with a nucleic acid to inhibit the function thereof.
  • the antisense oligonucleotides in accordance with this invention preferably comprise from about 3 to about 50 subunits. It is more preferred that such oligonucleotides and analogs comprise from about 8 to about 25 subunits and still more preferred to have from about 12 to about 20 subunits.
  • a subunit is a base and sugar combination suitably bound to adjacent subunits through phosphodiester or other bonds.
  • the antisense oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
  • Equipment for such synthesis is available from several vendors, including PE Applied Biosystems (Foster City, Calif.). Any other means for such synthesis may also be employed, however, the actual synthesis of the oligonucleotides is well within the abilities of the practitioner.
  • methods for preparing modified oligonucleotides such as phosphorothioates and alkylated derivatives.
  • oligonucleotides of this invention are designed to be hybridizable with target RNA (e.g., mRNA) or DNA.
  • target RNA e.g., mRNA
  • DNA oligonucleotide e.g., DNA oligonucleotide
  • an oligonucleotide that hybridizes to the translation initiation site of an mRNA molecule can be used to prevent translation of the mRNA.
  • oligonucleotides that bind to double-stranded DNA can be administered. Such oligonucleotides can form a triplex construct and inhibit the transcription of the DNA.
  • Triple helix pairing prevents the double helix from opening sufficiently to allow the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described (see, e.g., J. E. Gee et al., 1994, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.).
  • antisense oligonucleotides may be targeted to hybridize to the following regions: mRNA cap region; translation initiation site; translational termination site; transcription initiation site; transcription termination site; polyadenylation signal; 3′ untranslated region; 5′ untranslated region; 5′ coding region; mid coding region; and 3′ coding region.
  • the complementary oligonucleotide is designed to hybridize to the most unique 5′ sequence of a gene, including any of about 15-35 nucleotides spanning the 5′ coding sequence.
  • Appropriate oligonucleotides can be designed using OLIGO software (Molecular Biology Insights, Inc., Cascade, Colo.; http://www.oligo.net).
  • an antisense oligonucleotide can be synthesized, formulated as a pharmaceutical composition, and administered to a subject.
  • the synthesis and utilization of antisense and triplex oligonucleotides have been previously described (e.g., H. Simon et al., 1999, Antisense Nucleic Acid Drug Dev. 9:527-31; F. X. Barre et al., 2000, Proc. Natl. Acad. Sci. USA 97:3084-3088; R. Elez et al., 2000, Biochem. Biophys. Res. Commun. 269:352-6; E. R. Sauter et al., 2000, Clin. Cancer Res. 6:654-60).
  • expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population.
  • recombinant vectors which will express a nucleic acid sequence that is complementary to a target gene. These techniques are described both in Sambrook et al., 1989 and in Ausubel et al., 1992.
  • gene expression can be inhibited by transforming a cell or tissue with an expression vector that expresses high levels of untranslatable sense or antisense sequences. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and even longer if appropriate replication elements included in the vector system.
  • RNA levels can be assessed by Northern blot analysis (Sambrook et al., 1989; Ausubel et al., 1992; J. C. Alwine et al. 1977, Proc. Natl. Acad. Sci. USA 74:5350-5354; I. M. Bird, 1998, Methods Mol. Biol. 105:325-36), quantitative or semi-quantitative RT-PCR analysis (see, e.g., W. M. Freeman et al., 1999, Biotechniques 26:112-122; Ren et al., 1998, Mol. Brain Res.
  • polypeptide levels can be measured, e.g., by western blot analysis, indirect immunofluorescence, or immunoprecipitation techniques (see, e.g., J. M. Walker, 1998, Protein Protocols on CD-ROM, Humana Press, Totowa, N.J.).
  • the lipid vehicles of the present invention carry nucleotide sequences encoding cytotoxins (e.g., diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), and the pertussis adenylate cyclase (CYA)), antisense nucleic acids (e.g., NGF antisense), ribozymes, labeled nucleic acids, and nucleic acids encoding tumor suppressor genes such as p53, p 110Rb, and p72.
  • cytotoxins e.g., diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), and the pertussis adenylate cyclase (CYA)
  • antisense nucleic acids e.g., NGF antisense
  • ribozymes e.g., labeled nucle
  • NGF antisense nucleic acids have been described by, e.g., K. A. Chang et al., 1999, J. Mol. Neurosci. 12(1):69-74; C. Culmsee et al., 1999, Neurochem. Int. 35(1):47-57; F. Hallbook et al., 1997, Antisense Nucleic Acid Drug Dev. 7(2):89-100.
  • Such antisense nucleic acids can be used with the lipid vehicles of the invention for treating NGF-related diseases, including disorders of the brain (e.g., Alzheimer's) (see, e.g., K. A. Chang et al., 1999, J. Mol. Neurosci. 12(1):69-74; R.
  • Lipid vehicles of the present invention can be conjugated to antibodies, i.e., polyclonal and/or monoclonal antibodies, fragments thereof, or immunologic binding equivalents thereof.
  • the term antibody is used both to refer to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities.
  • Antibodies can include whole antibody molecules, hybrid antibodies, chimeric antibodies, and univalent antibodies. Also included are antibody fragments, including Fc, Fv, Fab 1 , and F(ab) 2 fragments of antibodies.
  • Antibodies may be obtained from commercial sources, e.g., Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.; Advanced Targeting Systems, San Diego, Calif.; Connex GmbH (Martinsried, Germany), Covance Research Products, Cumberland, Va.; Pierce Endogen, Rockford, Ill.; DiaSorin, Stillwater, Minn.; and DAKO Corporation, Carpinteria, Calif.
  • antibodies may be produced in an animal host (e.g., rabbit, goat, mouse, or other non-human mammal) by immunization with immunogenic components.
  • Antibodies may also be produced by in vitro immunization (sensitization) of immune cells.
  • the antibodies may also be produced in recombinant systems programmed with appropriate antibody-encoding DNA.
  • the antibodies may be constructed by biochemical reconstitution of purified heavy and light chains.
  • An isolated polypeptide or portion thereof can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation (see, e.g., E. Harlow and D. Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
  • a full-length polypeptide can be used or, alternatively, antigenic peptide portions can be used as immunogens.
  • An antigenic peptide typically comprises at least 5 contiguous amino acid residues, and encompasses an epitope of a polypeptide such that an antibody raised against the peptide forms a specific immune complex with the peptide.
  • the immunogenic polypeptides or peptides for use with the present invention may be isolated from cells or may be chemically synthesized.
  • An appropriate immunogenic preparation can contain, for example, a recombinantly produced polypeptide or a chemically synthesized polypeptide, or portions thereof.
  • the preparation can further include an adjuvant or similar immunostimulatory agent.
  • adjuvants are known and used by those skilled in the art.
  • suitable adjuvants include incomplete Freund's adjuvant, mineral gels such as alum, aluminum phosphate, aluminum hydroxide, aluminum silica, and surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • adjuvants include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3 hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/TWEEN®80 emulsion.
  • thr-MDP N-acetyl-muramyl-L-thre
  • a particularly useful adjuvant comprises 5% (wt/vol) squalene, 2.5% Pluronic L121 polymer and 0.2% polysorbate in phosphate buffered saline (Kwak et al., 1992, New Eng. J. Med. 327:1209-1215).
  • Preferred adjuvants include complete BCG, Detox, (RIBI, Immunochem Research Inc.), ISCOMS, and aluminum hydroxide adjuvant (Superphos, Biosector). The effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against the immunogenic peptide.
  • Polyclonal antibodies to polypeptides can be prepared as described above by immunizing a suitable subject with an immunogen.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide or peptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique (see Kohler and Milstein, 1975, Nature 256:495-497; Brown et al., 1981, J. Immunol. 127:539-46; Brown et al., 1980, J. Biol. Chem. 255:4980-83; Yeh et al., 1976, PNAS 76:2927-31; and Yeh et al., 1982, Int. J.
  • hybridomas The technology for producing hybridomas is well-known (see generally R. H. Kenneth, 1980, Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y.; E. A. Lerner, 1981, Yale J. Biol. Med., 54:387-402; M. L. Gefter et al., 1977, Somatic Cell Genet. 3:231-36).
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds to the polypeptides or peptides.
  • a monoclonal antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the corresponding polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No.
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies comprising both human and non-human portions
  • recombinant antibodies can be made using standard recombinant DNA techniques.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No.
  • Fv fragments of monoclonal antibodies may be produced in bacteria using single chain antibody technology (U.S. Pat. No. 4,946,778 and International Application No. WO 88/09344).
  • Fv fragments can be genetically engineered to contain glycosylation sites. These engineered Fv fragments can then be produced in mammalian cells, to result in a fragment containing carbohydrate moieties.
  • Fab or F(ab′) 2 fragments of monoclonal antibodies may be produced by enzymatic cleavage of whole IgG which is produced by a hybridoma or a transfected cell lines (e.g., a myeloma or a cell line such as Chinese Hamster Ovary (CHO) cells), using pepsin or papain digestion, respectively.
  • a hybridoma or a transfected cell lines e.g., a myeloma or a cell line such as Chinese Hamster Ovary (CHO) cells
  • pepsin or papain digestion respectively.
  • the antibodies or antibody fragments can be conjugated to liposomes using conventional techniques (see, e.g., M. J. Ostro (ed.) 1987, Liposomes: from Biophysics to Therapeutics, Marcel Dekker, New York, N.Y.).
  • One preferred method of preparing liposomes and conjugating immunoglobulins to their surface is described by Y. Ishimoto et al., 1984, J. Immunol Met. 75:351-360.
  • multilamillar liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, and phosphotidylethanolamine are prepared.
  • Purified fragments are then coupled to the phosphatidylethanolamine by the cross-linking agent N-hydroxysuccinimidyl 3-(2-pyridyidithio) propionate.
  • the coupling of the antibody or fragment to the liposome is demonstrated by the release of a pre-trapped marker, e.g., carboxyfluorescence, from the liposomes. This release occurs upon incubation with a secondary antibody against the conjugated antibody, fragment, or complement.
  • the antibodies or antibody fragments can also be coupled to a liposome or another carrier of the invention via carbohydrate moieties. Such coupling can be used provided that the carbohydrate moiety is not in the hypervariable region or at the antibody binding sites. In this way, conjugation via the cross-linking with the carbohydrate will not affect binding, and the binding sites will still be available to bind to cell surface antigens.
  • One preferred method for coupling antibodies or antibody fragments of the invention (other than Fv) to a polymer backbone or a liposome involves conjugation through the carbohydrate moieties in the constant regions. This maximizes the number of available antigen-binding sites.
  • Binding of a monoclonal antibody to the surface of a liposome may also be accomplished by the formation of cross-linkage between phosphatidylethanolamine and the antibody using glutaraldehyde.
  • a thiolated antibody can be allowed to react with a liposome comprising a lipid into which a maleimide group has been incorporated. Remaining maleimide groups on the surface of the liposome may be further reacted with a compound containing thiolated polyalkyleneglycol moiety.
  • Thiolation of an antibody or antibody fragment may be achieved through use of N-succinimidyl-3-(2-pyridyidithio)propionate (SPDP), which is usually used for thiolation of protein, iminothiolane, or mercaptoalkylimidate.
  • SPDP N-succinimidyl-3-(2-pyridyidithio)propionate
  • a dithiol group endogenous to an antibody may be reduced to form a thiol group.
  • the latter method is preferred for maintaining antibody function.
  • whole antibodies are treated with an enzyme such as pepsin to form F(ab) 2 fragments, which are then reduced with dithiothreitol (DTT) to form Fab fragments, which results in the production of one to three thiol groups.
  • DTT dithiothreitol
  • the conjugation of the thiolated antibody to the maleimide group-containing liposome may be accomplished by reacting the components in
  • the lipid vehicles of the present invention are conjugated to antibodies or antibody fragments directed to NGF receptor or uroplakin.
  • lipid vehicles and methods of the present invention can be used to deliver a broad range of pharmaceutical compositions and drugs.
  • the vehicles of the present invention carry small organic or inorganic compounds as bioactive agents.
  • suitable pharmaceuticals or bioactive agents include, but are not limited to, antimicrobials, antibiotics, antimycobacterial, antifungals, antivirals, neoplastic agents, agents affecting the immune response, blood calcium regulators, agents useful in glucose regulation, anticoagulants, antithrombotics, antihyperlipidemic agents, cardiac drugs, thyromimetic and antithyroid drugs, adrenergics, antihypertensive agents, cholinergics, anticholinergics, antispasmodics, antiulcer agents, skeletal and smooth muscle relaxants, prostaglandins, general inhibitors of the allergic response, antihistamines, local anesthetics, analgesics, narcotic antagonists, antitussives, sedative-hypnotic agents, anti
  • the bioactive agent will be an antineoplastic agent, such as vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, streptozotocin, and the like.
  • antineoplastic agent such as vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, streptozotocin, and the like.
  • Especially preferred antitumor agents include, for example, actinomycin D, vincristine, vinblastine, cystine arabinoside, anthracyclines, alkylative agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs.
  • Anticancer agents further include carcinostatic agents such as adriamycin, daunomycin, mitomycin, epirubicin, 5-FU, and aclacinomycin, toxins such as ricin A and diphtheria toxin, and antisense RNA.
  • Encapsulation of anticancer agent into lipome vehicles can be accomplished by hydration of the lipids with an aqueous solution of the anticancer agent.
  • Adriamycin, daunomycin, and epirubicin may be encapsulated into a liposome by means of a remote loading method that takes advantage of a pH gradient (D. M. Lawrence et al., 1989, Cancer Research 49:5922).
  • the lipid vehicles of the present invention can be used to deliver anti-infective agents.
  • the vehicles of the present invention can also be used for the selective delivery of other drugs including, but not limited to, local anesthetics, e.g., dibucaine and chlorpromazine; beta-adrenergic blockers, e.g., propranolol, timolol and labetolol; antihypertensive agents, e.g., clonidine and hydralazine; anti-depressants, e.g., imipramine, amitriptyline and doxepim; anticonversants, e.g., phenytoin; antihistamines, e.g., diphenhydramine, chlorphenirimine and promethazine; antibiotic/antibacterial agents, e.g., gentamycin, ciprofloxacin, and cefoxitin.
  • local anesthetics e.g., dibuca
  • antibiotics such as macrolides and lincosamines (e.g., lincomycin, erythromycin, dirithromycin, clindamycin, clarithromycin, and azithromycin); ample spectrum penicillins (e.g., ticarcillin, piperacillin, mezlocillin, carbenicillin indanyl, bacampicillin, ampicillin, and amoxicillin); penicillins and beta-lactamase inhibitors (e.g., amoxicillin-clavulanic acid, ampicillin-sulbactam, benzylpenicillin, cloxacillin, dicloxacillin, methicillin, oxacillin, penicillin G (benzathine, potassium, procaine), penicillin V, piperacillin plus tazobactam, and ticarcillin plus clavulanic acid); aminoglycosides (e.g., amikacin, gentamicin, kanamycin, neomycin, net
  • antifungal agents e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine and amphotericin B; antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, narcotics, and imaging agents.
  • antifungal agents e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine and amphotericin B
  • antiparasitic agents e.g., hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, narcotics, and imaging agents.
  • Those of skill in the art will know of
  • the lipid vehicles of the present invention deliver vanilloid components, e.g., resiniferatoxin, capsaicin, tinyatoxin, and related compounds. Additionally, the lipid vehicles may deliver toxins, e.g., botulinum toxins, such as botulinum toxin A, botulinum toxin B, botulinum toxin C, botulinum toxin D, botulinum toxin E, botulinum toxin F, and botulinum toxin G.
  • toxins e.g., botulinum toxins, such as botulinum toxin A, botulinum toxin B, botulinum toxin C, botulinum toxin D, botulinum toxin E, botulinum toxin F, and botulinum toxin G.
  • Lipid vehicles can be formulated as described herein, or by other methods known in the art (e.g., U.S. Pat. No. 6,334,999
  • a composition e.g., pharmaceutical composition
  • a pharmaceutically acceptable excipient e.g., carrier, or diluent
  • a bioactive agent e.g., nucleic acid, polypeptide, peptide, or antibody
  • drug e.g., resiniferatoxin, capsaicin, tinyatoxin, or other vanilloid compounds
  • toxin e.g., botulinum toxin
  • compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • Preferred carriers, excipients, and diluents of the invention comprise physiological saline (i.e., 0.9% NaCl).
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH-buffering agents, which enhance the effectiveness of the active ingredient.
  • a bioactive agent or drug can be formulated into the pharmaceutical composition as neutralized physiologically acceptable salt forms.
  • Suitable salts include the acid addition salts (i.e., formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • compositions and vehicles can be administered systemically by oral or parenteral routes.
  • parenteral routes of administration include subcutaneous, intravesical, intramuscular, intraperitoneal, intravenous, transdermal, inhalation, intranasal, intra-arterial, intrathecal, enteral, sublingual, or rectal.
  • Intravenous administration for example, can be performed by injection of a unit dose.
  • unit dose when used in reference to a pharmaceutical composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier.
  • the disclosed pharmaceutical compositions and vehicles can also be administered via pulmonary inhaler or mucoactive aerosol therapy (nasal spray; see, e.g., M. Fuloria and B. K. Rubin, 2000, Respir. Care 45:868-873; I. Gonda, 2000, J. Pharm. Sci. 89:940-945; R. Dhand, 2000, Curr. Opin. Pulm. Med. 6(1):59-70; B. K. Rubin, 2000, Respir. Care 45(6):684-94; S. Suarez and A. J. Hickey, 2000, Respir. Care. 45(6):652-66).
  • topical administration can be used.
  • the disclosed pharmaceutical compositions and vehicles are administered by intravesical instillation.
  • compositions can be administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of modulation required. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are specific for each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration.
  • Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration.
  • continuous intravenous infusions sufficient to maintain concentrations of 10 nM to 10 ⁇ M in the blood are contemplated.
  • An exemplary pharmaceutical formulation comprises: a peptide or polypeptide (5.0 mg/ml); sodium bisulfite USP (3.2 mg/ml); disodium edetate USP (0.1 mg/ml); and water for injection q.s.a.d. (1.0 ml).
  • pg means picogram
  • ng means nanogram
  • ⁇ g means microgram
  • mg means milligram
  • ⁇ l means microliter
  • ml means milliliter
  • I means L.
  • the present invention encompasses novel methods of treatment that utilize the disclosed lipid vehicles.
  • treatments are provided for cancers, infections, pain (e.g., neuropathic pain), and other conditions relating to the bladder, genitourinary tract, gastrointestinal tract, pulmonary system, and other organs or body systems.
  • treatments for urinary system components e.g., kidneys, ureters, bladders, sphincter muscles, and urethras.
  • urinary system components e.g., kidneys, ureters, bladders, sphincter muscles, and urethras.
  • Non-limiting examples of gastrointestinal organs include the esophagus, stomach, large intestine, and small intestine.
  • Pulmonary system organs include, among other organs, the trachea, lungs, bronchi, bronchioles, alveoli, and cilia.
  • Genitourinary tract organs include, but are not limited to, the bladder, kidney, urethra, ureter, prostate, penis, testes, seminiferous tubules, epididymis, vas deferens, seminal vesicles, bulbourethral (Cowper) glands, uterus, vagina, and fallopian tubes.
  • bladder conditions include spastic neurogenic bladder, hypotonic neurogenic bladder, and bladder hyperactivity, pain, irritation, inflammation, micturition pattern alteration, incontinence, infection, and cancer.
  • Bladder cancers suitable for treatment include, for example, transitional cell carcinomas, squamous cell carcinomas, and adenocarcinomas. Also included are conditions relating to IC and UDSD.
  • the present invention further encompasses methods of treating conditions associated with involuntary muscle contractions, including, but not limited to, tremor (voice, head, and limb tremor); palatal myoclonus; dysthyroid myopathy, hemifacial spasms; tics; strabismus (e.g., concomitant strabismus and vertical strabismus); nystagmus; eyelid entropion; myokymia; bruxism (TMJ); tardive dyskinetic syndrome, lateral rectus palsy; hyperkinesias following hypoglossal-facial anastomosis; myoclonus of spinal cord origin; voice defects (e.g., stuttering); painful rigidity; tension headaches; lumbosacral strain and back spasm (myofascial); radiculopathy with secondary muscle spasm; spasticity; IC, spastic bladder; UDSD; achalasia (esophage
  • Treatments of involuntary contractions may be directed to any muscle groups, including those associated with control of the eye(s), lip(s), tongue, mouth, jaw, head, neck, face, arm, hand, finger, leg, trunk, vagina, cervix, bladder, and sphincter (e.g., esophageal, cardiac, pyloric, ileocaecal, O'Beirne, anal, urethra, and bladder neck sphincters). Treatments are also provided for furrows of the face and neck, including frown lines and facial wrinkles.
  • sphincter e.g., esophageal, cardiac, pyloric, ileocaecal, O'Beirne, anal, urethra, and bladder neck sphincters. Treatments are also provided for furrows of the face and neck, including frown lines and facial wrinkles.
  • the methods of the present invention can be used to treat an animal, preferably a mammal, more preferably a human subject.
  • the disclosed methods comprise administering a lipid vehicle to a mammal suffering from one or more of these conditions.
  • the lipid vehicle carries a biological agent (e.g., nucleic acid, peptide, polypeptide, or antibody), drug (e.g., pain therapeutics, anticancer treatments, or antibiotics), or toxin (e.g., botulinum toxin).
  • a biological agent e.g., nucleic acid, peptide, polypeptide, or antibody
  • drug e.g., pain therapeutics, anticancer treatments, or antibiotics
  • toxin e.g., botulinum toxin
  • the nucleic acid can be an antisense oligonucleotide targeted against a DNA sequence in the pathogen that is essential for development, metabolism, or reproduction of the pathogen.
  • the nucleic acid maybe the normal DNA sequence
  • the lipid vehicle may be injected into the blood stream, directly into a tissue, into the peritoneum, instilled into the trachea, or converted to an aerosol, which the animal breathes.
  • a single intravenous injection of 100 micrograms of a mixture of DNA and DOTMA:dioleoylphosphatidylethanaolamine can be used to efficiently transfect all tissues (Zhu et al., 1993, Science 261:209-211). It is also possible to use a catheter to implant lipid vehicles in a blood vessel wall, which can result in successful transfection of several cell types, including endothelial and vascular smooth muscle cells.
  • aerosol delivery of a chloramphenicol acetyltransferase (CAT) expression plasmid complexed to cationic liposomes produces high-level, lung-specific CAT gene expression in vivo for at least 21 days (Stribling et al., 1992, Proc. Natl. Acad. Sci. USA 89:11277-11281).
  • CAT chloramphenicol acetyltransferase
  • Specific targeting moieties can be used with the vehicles of this invention to target specific cells or tissues.
  • the targeting moiety such as an antibody or antibody fragment, is attached to a hydrophilic polymer and is combined with the vehicle after vehicle formation.
  • a targeting moiety in combination with a vehicle provides the ability to conveniently customize the vehicle for delivery to specific cells and tissues.
  • the disclosed vehicles carrying drugs e.g., pain remedies, anticancer, or antibiotics
  • bioactive agents e.g., nucleic acids, polypeptides, peptides, or antibodies
  • cancer cells e.g., immune cells, and cells of the bladder, genitourinary tract, gastrointestinal tract, pulmonary system, or other body organs or systems.
  • immune cells e.g., B and T cells
  • cells of the bladder e.g., B and T cells
  • Such cells can be targeted using antibodies or antibody fragments against cell surface antigens, including various receptors or markers.
  • cancers are characterized by overexpression of cell surface markers such as HER2, which is expressed in breast cancer cells, or IL-13 receptor, which is expressed in gliomas (reviewed in, e.g., J. Baselga et al., 1997, Oncology (Huntingt) 11(3 Suppl 2):43-8; S. Menard et al., 2000, J. Cell. Physiol. 182(2):150-62; W. Debinski, 1998, Crit. Rev. Oncog. 9(34):255-68).
  • Certain urogenitary tract cancers are characterized by expression of the uroplakin marker (see, e.g., X.
  • neurons are characterized by the expression of NGF receptor (reviewed by, e.g., L. Tessarollo, 1998, Cytokine Growth Factor Rev. 9(2):125-37; E. C. Yuen EC, et al., 1996, Brain Dev. 18(5):362-8; S. B. McMahon, 1996, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 351(1338):431-40; G. Dechant et al., 1994, Prog. Neurobiol. 42(2):347-52).
  • targeting moieties such as anti-HER2, anti-IL-13 receptor, and anti-NGF receptor antibodies or antibody fragments can be used to deliver the vehicle to the cell of choice.
  • the bioactive agent and/or drug is thereby delivered to the specific cell type, providing a useful and specific therapeutic treatment.
  • Cationic lipid-assisted drug delivery can be accomplished in accordance with well-established methods.
  • drugs that are soluble in organic solvents such as chloroform
  • the drug and cationic lipid are mixed in solvents in which both are soluble, and the solvent is then removed under vacuum.
  • the lipid-drug residue is then dispersed in an appropriate aqueous solvent, e.g., sterile physiological saline.
  • the suspension is subjected to up to several freeze/thaw cycles.
  • the suspension is then sonicated, to reduce the coarseness of the dispersion or to reduce the particle size to 20-30 nm diameter. This will depend on whether large or small particle size is most efficacious in the desired application.
  • the vehicles of the present invention that carry a bioactive agent can be delivered in any suitable manner.
  • the lipid mixture to be used for the lipid dispersion or liposomes can be coated on the inside surface of a flask or tube by evaporating the solvent from a solution of the mixture.
  • the lipid mixture should be capable of forming vesicles having single or multiple lipid bilayer walls and encapsulating an aqueous core.
  • the aqueous phase containing the dissolved agent e.g., physiological saline solution
  • the lipid vehicles of the invention can be used with or without vanilloid (e.g., capsaicin) and/or botulinum toxin (e.g., botulinum toxin D), which can then be used alone or in combination with a chemotherapeutic agent, targeting antibody, or DNA construct designed for the treatment of bladder cancer.
  • lipid vehicles or lipid vehicles comprising vanilloid and/or botulinum toxin can be used to prevent, treat, or ameliorate pain or voiding dysfunction associated with bladder cancer.
  • Lipid-based treatments for bladder cancer that employ chemotherapeutic agents see, e.g., J. B. Bassett et al., 1986, J. Urol. 135(3):612-5; C.
  • lipid vehicles of the invention may be used with or without vanilloid (e.g., capsaicin) and/or botulinum toxin (e.g., botulinum toxin D), which can then be used alone or in combination with one or more antibacterial agents.
  • vanilloid e.g., capsaicin
  • botulinum toxin e.g., botulinum toxin D
  • lipid vehicles or lipid vehicles comprising vanilloid and/or botulinum toxin can be used to prevent, treat, or ameliorate pain or voiding dysfunction associated with a urinary system infection.
  • Lipid-based treatments for infection are generally known in the art, including those employing tetracycline and doxycycline (L. Sangare et al., 1999, J. Med. Microbiol. 48(7):689-93; L.
  • the suspension can be subjected to ultrasonic waves for a time necessary to reduce the liposomes to the desired average size. If large liposomes are desired, the suspension can be agitated by hand or on a vortex mixer until a uniform dispersion is obtained, i.e., until visually observable large particles are present.
  • the agent or drug in the aqueous phase is eliminated by dialysis or by passage through a gel-filtration chromatographic column (e.g., agarose) equilibrated with the aqueous phase containing all normal components except the agent or drug.
  • the lipid mixture used can contain cholesterol or natural lipids in addition to the liposome compounds of the present invention.
  • the liposome-drug aggregate may then be delivered in any suitable manner (see above).
  • a hyperactive bladder model in Sprague-Dawley rats was established by exposure to acetic acid, or protamine sulfate (PS) in potassium chloride (KCl) solution. This was followed by instillation of LP (liposomes) in saline (in the case of the former) or LP/KCl. CMG (continuous cystometrogram) changes were examined and results were compared with control (saline instillation), hyperactive bladder (acetic acid or PS/KCl) and treatment with LP.
  • PS protamine sulfate
  • KCl potassium chloride
  • Intravesical bladder pressure was recorded via a transurethral catheter in adult female Sprague-Dawley rats anesthetized with urethane (1.2 g/kg) administered by subcutaneous injection (sc). Some animals were pretreated with capsaicin (125 mg/kg, sc) four days prior to the experiments.
  • Continuous cystometrograms were performed by slowly filling the bladder (0.04 ml/min) with solutions of various composition including saline, acetic acid (0.1%), potassium chloride (KCl, 500 mM), protamine sulfate (PS, 10 mg/ml), LP, PS/KCl, or LP/KCl. Parameters measured included intercontraction interval (ICl), amplitude of bladder contractions, compliance, and micturition pressure threshold (PT).
  • ICl intercontraction interval
  • PT micturition pressure threshold
  • a transurethral bladder catheter (PE-50) was connected via a three-way stopcock to a pressure transducer and to a syringe pump. This was used to record intravesical pressure and to infuse solutions into the bladder.
  • a control CMG was performed by slowly filling the bladder with saline (0.04 ml/min) to elicit repetitive voiding. The parameters recorded were amplitude, pressure threshold (PT), compliance, and intercontraction interval (ICl) of reflex bladder contractions. Measurements in each animal represented the average of 3 to 5 bladder contractions.
  • the experimental design is illustrated in FIG. 1 .
  • the KCl concentration used was within the range of concentrations present in normal rat urine (M. Ohnishi et al., 2001, Toxicol. Appl. Pharmacol. 174:122-129).
  • LP were prepared as described by Kirby and Gregoriadis (C. J. Kirby and G. Gregoriadia, 1984, “A simple procedure for preparing liposomes capable of high encapsulation efficiency under mild conditions” Liposome Technology, G. Gregnoriadis, Ed., C.R.C. Press, Inc., Boca Raton, Fla., vol. 1, p.20). Briefly, LP were constructed as a 2:1 molar ratio of L-a-phosphatidylcholine and cholesterol (Sigma chemical Co., St. Louis, Mo.) to a final lipid concentration of 2 mg/ml in saline. Lipids in chloroform were dried down together in the proper ratio under nitrogen. The residues were reconstituted as LP in saline or 500 mM KCl by intense sonication. This lipid composition produced liposomes with no net charge.
  • Bladder contraction amplitude was significantly increased (23%) in one series (Table 1C), but not in the other series (Table 1B). However, taking the average of the two series, bladder contraction amplitude showed a significant increase (16%).
  • PT was not significantly changed.
  • the infusion fluid was switched to LP/KCl, after a delay of 10 to 20 min, the ICl was significantly increased (63%, from 2.7 ⁇ 1.0 to 4.4 ⁇ 1.2 min). Switching to KCl alone did not alter the ICl for periods as long as 120 min ( FIGS. 2E-2F ; Tables 1B-1C). PT was significantly increased after shifting to LP/KCl or KCl infusion.
  • ICl and compliance were significantly reduced by 75-84% (from 15.3 ⁇ 2.2 to 2.4 ⁇ 0.5 min or from 12.6 ⁇ 2.0 to 3.2 ⁇ 1.3 min) and 71-76% (from 0.296 ⁇ 0.040 to 0.071 ⁇ 0.016 ml/cm H 2 O or from 0.275 ⁇ 0.048 to 0.079 ⁇ 0.026 ml/cm H 2 O) in two series of experiments ( FIGS. 3A-3F ; Tables 2A-2B). Amplitude was less affected, showing a slight increase, and PT was not significantly changed.
  • bladder hyperactivity evoked by AA was delayed for 0.5-1 hour.
  • ICl and compliance were reduced in magnitude by 51% and 33% at 1 hour (from 21.0 ⁇ 2.4 to 10.2 ⁇ 3.0 min and from 0.226 ⁇ 0.033 to 0.152 ⁇ 0.028 ml/cm H 2 O), but at 2 hours was similar to the effect in untreated animals (78% decrease to 4.6 ⁇ 1.2 min and 64% decrease to 0.082 ⁇ 0.024 ml/cm H 2 O) (Table 3).
  • bladder afferents played a key role in the mechanism of action of AA in the induction of bladder hyperactivity.
  • Previous experiments have shown that infusion of AA into the bladder stimulates nociceptive afferent fibers, induces an inflammatory reaction and evokes a hyperactive bladder (Y. Yu and W. C. de Groat, 1998, Brain Res. 807:11-18; L. A. Birder and W. C. de Groat, 1992, J. Neurosci. 12:48784889; K. B. Thor and M. A. Muhlhauser, 1999, Am. J. Physiol. 277:R1002-1012).
  • PS which increases epithelial permeability (K. B. Thor and M. A. Muhlhauser, 1999, Am. J. Physiol. 277:R1002-1012), was used in the above experiments to increase the penetration of KCl through the urothelial barrier and induce a similar activation of afferent neurons.
  • PS treatment Prior to PS treatment, the same concentration of KCl did not alter voiding function.
  • PS alone did not elicit bladder hyperactivity.
  • PS was not acting as a primary bladder irritant, and that under normal conditions KCl in the bladder lumen would not alter the excitability of afferent nerves in the bladder wall.
  • combined exposure to these agents mimicked the condition observed in IC patients.
  • the surface glycosaminoglycan (GAG) layer has been proposed as a protective barrier that coats the transitional cell surface (J. I. Bade et al., 1997, J. Urol. 79:168-171; G. Hohlbrugger, 1995, J. Urol. 154:6-15; C. L. Parsons et al., 1980, Science 208:605-607).
  • a GAG layer defect has been suggested in a subset of IC patients (C. L. Parsons et al., 1991, J. Urol. 145:732-735; C. L. Parsons et al., 1994, Br. J. Urol. 73:504-507).
  • LP are comprised of phospholipids in a system of concentric closed membranes and are used as a carrier for drugs or DNA constructs (K. Reimer et al., 1997, Dermatology 195(suppl. 2):93-99; M. Nishikawa et al., 2001, Human Gene Therapy 12:861-870; G. Gregoriadis, 1976, New Eng. J. Med. 295:704-710).
  • LP-based compositions provide a high-moisture film for wounds and mediate wound healing without chronic inflammatory-reaction in the neodermal layer (K. Reimer et al., 1997, Dermatology 195(suppl. 2):93-99; M.
  • LP interacts with cells by stable absorption, endocytosis, lipid transfer, and fusion (R. B. Egerdie et al., J. Urol. 142:390-398).
  • administration of LP to the wounded urothelium can be used as a novel method for treating patients with hyperactive bladder, IC, or other urinary system disorders.
  • An animal model for acute hyperactive bladder in rats was developed using intravesical infusion of protamine sulfate (PS), an agent used to break down urothelial barrier function, and physiological concentrations of potassium chloride (KCl).
  • PS protamine sulfate
  • KCl potassium chloride
  • Continuous CMGs were performed in urethane-anesthetized female rats.
  • the bladder was filled (0.04 ml/min) with normal saline followed by intravesical infusion for a 60 minute period with a test solution comprising either KCl (100 or 500 mM) or PS (10 or 30 mg/ml). Following this, 10 mg/ml PS treated animals were infused intravesically with 100, 300, or 500 mM KCl. Some animals were pretreated with capsaicin (125 mg/ml, sc) four days before the experiments.
  • PE-50 tubing (Clay-Adams, Parsippany, N.J.) was inserted into the bladder through the urethra and connected via a three-way stopcock to a pressure transducer and to a syringe pump. This was used for recording intravesical pressure and for infusing solutions into the bladder.
  • a control CMG was performed by slowly filling the bladder with saline (0.04 ml/min) to elicit repetitive voiding.
  • the amplitude, pressure threshold (PT), compliance, and intercontraction interval (ICl) of reflex bladder contractions were recorded.
  • Pressure threshold (PT) represents the pressure that induces the initial bladder contraction.
  • PT has often been used as a parameter corresponding to afferent nerve activity for the induction of reflex bladder contractions. Measurements in each animal represented the average of 3 to 5 bladder contractions.
  • capsaicin dissolved in a vehicle containing 10% ethanol, 10% TWEEN®80, and 80% physiological saline, at a concentration of 20 mg/ml was given subcutaneously in divided doses on 2 consecutive days.
  • CMGs performed with 300 or 500 mM KCl infusion significantly changed the ICl (76.9 or 82.9% decrease), compliance (60 or 63.4% decrease), and contraction amplitude (23.7 or 21.4% increase).
  • the PT was not significantly altered.
  • the effect mediated by 300 mM KCl occurred after a delay of 20 to 30 min, whereas the effect mediated by 500 mM KCl occurred after a delay of about 10 to 15 min. This implied that higher concentrations of KCl yielded faster penetration.
  • One hour of infusion of 100 mM KCl did not produce significant changes in the CMG parameters.
  • hyperactive bladder from the sequential infusion of PS (10 mg/ml) and KCl (500 mM) was delayed by about 1 hour.
  • the changes in ICl and compliance were reduced by 36 and 55% after 2 hours of infusion (ICl decreased from 20.3 ⁇ 1.2 to 13.1 ⁇ 2.8 min; compliance decreased from 0.224 to 0.100 ml/cm H 2 O; Table 7).
  • infusion of normal saline did not induce micturition reflex. This indicated that micturition reflex was blocked by hexamethonium injection or pelvic nerve transection.
  • Infusion of KCl (500 mM) following PS (10 mg/ml) instillation decreased compliance by 55.9% (from 0.093 ⁇ 0.026 to 0.041 ⁇ 0.011 ml/cm H 2 O). This indicated that potassium affected the direct stimulation of the detrusor muscle and caused the decrease in compliance.
  • PS has been well established as a model for bladder injury. Previous experiments showed distension of the bladder for 45 min with 1 ml of 10 mg/ml PS (P. C. Stein et al., 1996, J. Urol. 155:1133-1138). It is known that prolonged over-distension of the bladder alters the properties of the bladder wall (S. Keay et al., 1999, J. Urol. 162:1487-1489; G. Hohlbrugger, 1995, J. Urol. 154:6-15). This may enhance the cytodestructive effects of PS and result in immediate urothelial sloughing (P. C. Stein et al., 1996, J. Urol. 155:1133-1138).
  • compositions for the treatment of urinary system conditions preferably include excipients, diluents, or carriers comprising physiological saline, as described in detail herein.
  • Intravesical vanilloid therapy has been used to treat detrusor hyperreflexia in SCI (spinal cord injury) and MS (multiple sclerosis) patients.
  • Capsaicin (CAP) treatment requires high concentrations of ethanol (30% or greater) to achieve an effective dose. This level of ethanol is tissue toxic, and may, by itself, cause hemorrhagic cystitis.
  • the lipoidal phase of liposomes (LP), concentric phospholipid bilayers, may provide an attractive alternative to high concentrations of ethanol.
  • liposomal delivery of CAP was tested in urethane anesthetized rats.
  • Open transurethral cystometry (0.04 ml/min) was performed under urethane anesthesia (1.2 g/kg) in 15 female S-D rats (250-300 g). Following a two-hour control period of saline infusion, the infusate was switched to either LP with 1 mM CAP (LP/CAP), or LP alone for 30 minutes followed by LP/CAP.
  • LPs were constructed as described in Example 1, above. Briefly, a 2:1 molar ratio of phosphatidylcholine and cholesterol, was dried down from chloroform solvent under nitrogen, with or without CAP. The resultant residue was brought into a saline suspension at 2 mg/ml total lipid by intense sonication.
  • LP alone had no effect on bladder contraction frequency (0.13 ⁇ 0.02 vs. 0.13 ⁇ 0.01 bladder contractions/min for control and LP, respectively ( FIG. 7 ).
  • LP/CAP resulted in a dramatic increase in bladder contraction frequency (1.11 ⁇ 0.08 bladder contractions/min, P ⁇ 0.0001) within minutes of beginning the infusion ( FIG. 7 ).
  • Bladder contraction frequency subsequently slowed and finally halted by 124 ⁇ 24 minutes.
  • LP are capable of highly effective delivery of at least one hydrophobic drug, CAP, as evidenced by a dramatic increase in bladder contraction frequency and subsequent desensitization. Moreover, LP alone had no effect on the micturition reflex in the unirritated state. In combination with other experiments that have demonstrated a protective effect of LPs, this suggested that the LP vehicle may partially protect against the compromise of urothelial barrier function due to the neuro-inflammatory response caused by irritants, such as CAP. This experiment indicates that LP for other drugs, such as antibiotics and cancer treatments. Description of the experiments in this example can also be found in Y.-C. Chuang et al., 100 th Annual Meeting American Urological Association (AUA), Abstract; 2002, J. Urol. 167:41A, which are hereby incorporated herein by reference.
  • botulinum toxin Btx
  • liposome injections on autonomic (bladder) innervation of the lower urinary tract were investigated as follows.
  • Liposomes were prepared as described in Example 1.
  • Female SD rats (250) were anesthetized with urethane (1.2 g/kg). Animals received intravesical liposomes plus instillation of Btx D (5.7 ng/gm body weight; Sigma, St Louis, Mo.). Control animals received no injections. All animals received tracheotomies, and treated animals were artificially respired. Transvesical catheters were inserted and, 6 hr after Btx injection, the bladder was harvested for strip studies.
  • bladder strips (20-30 g) were mounted in a double-jacketed organ bath at 36° C. in oxygenated Krebs solution. Electrical field stimulation was delivered through platinum electrodes positioned at the top and bottom using trains of 100 shocks at 20 Hz with maximal voltage every 100 sec. Strip fatigue was tested by trains applied every 20 sec. Fatigue amplitude and area, as well as recovery amplitude and area, were calculated as percent of the control value and compared between groups.

Abstract

The present invention relates to compositions and methods for the administration of lipid-based vehicles to treat various disorders, including bladder inflammation, infection, dysfunction, and cancer. In various aspects, the compositions and methods of the invention are useful for prolonged delivery of drugs, e.g., antibiotics, pain treatments, and anticancer agents, to the bladder, genitourinary tract, gastrointestinal system, pulmonary system, and other organs or body systems. In particular, the present invention relates to liposome-based delivery of vanilloid compounds, such as resiniferatoxin, capsaicin, or tinyatoxin, and toxins, such as botulinum toxin, for the treatment of bladder conditions, including pain, inflammation, incontinence, and voiding dysfunction. Further related are methods of using these vehicles alone or in conjunction with antibodies, e.g., uroplakin antibodies, to improve duration of liposome attachment, and provide a long-term intravesical drug delivery platform. The present invention specifically relates to antibody-coated liposomes that are useful for targeting specific receptors for drug, peptide, polypeptide, or nucleic acid delivery. In one particular aspect, the present invention relates to liposomes coated with antibodies against nerve growth factor (NGF) receptor and containing NGF antisense nucleic acids, which are used as a treatment for neurogenic bladder dysfunction.

Description

    RELATED APPLICATION
  • This application is a continuation-in-part of U.S. application Ser. No. 60/311,868 filed Aug. 13, 2001, which is incorporated by reference herein in its entirety.
  • FIELD OF THE INVENTION
  • The present invention relates to compositions and methods for the Instillation of lipid vehicles (e.g., micelles, microemulsions, macroemulsions, and liposomes) to treat various disorders, including bladder inflammation and dysfunction. The vehicles of the present invention are useful for prolonged delivery of drugs such as antibiotics and anticancer agents to the bladder, genitourinary tract, gastrointestinal system, pulmonary system, and other organs or body systems. Specifically, the present invention relates to liposome-based delivery of resiniferatoxin, capsaicin, tinyatoxin, and other vanilloid compounds for the treatment of bladder pain, inflammation, incontinence, and voiding dysfunction. Also related is liposome-based delivery of toxins, such as botulinum toxin, for the treatment of involuntary muscle contractions including those associated with urethral dyssynergia and bladder spasticity. The vehicles of the present invention are used alone or in conjunction with antibodies, e.g., uroplakin antibodies, that improve duration of liposome attachment, and provide a long-term intravesical delivery platform for drug, peptide, polypeptide, or nucleic acid delivery. In one aspect, the present invention relates to liposomes coated with antibodies against nerve growth factor (NGF) receptor and containing NGF antisense nucleic acids, which are useful as treatments for neurogenic bladder dysfunction.
  • BACKGROUND OF THE INVENTION
  • Neuropathic pain is thought to occur because of a sensitization in the peripheral and central nervous systems after an initial injury to the peripheral system (see N. Attal, 2000, Clin. J. Pain 16(3 Suppl):S118-30). Direct injury to the peripheral nerves as well as many systemic diseases including AIDS/HIV, Herpes Zoster, syphilis, diabetes, and various autoimmune diseases, can induce this disorder. Such pain is also associated with conditions of the bladder, including interstitial cystitis. Neuropathic pain is typically experienced as burning, shooting, and unrelenting in its intensity, and can sometimes be more debilitating that the initial injury or the disease process which induced it. Unfortunately, the few remedies that have been reported to alleviate this condition are effective in only a small percentage of patients.
  • Interstitial cystitis (IC) is characterized by bladder pain, irritative voiding symptoms, and sterile urine (see R. Doggweiler-Wiygul et al., 2000, Curr. Rev. Pain 4(2):137-41). In IC, the bladder wall shows inflammatory infiltration with mucosal ulceration and scarring that causes smooth muscle contraction, diminished urinary capacity, hematuria, and frequent, painful urination. Although the pathogenesis of IC is uncertain, it seems likely that a dysfunctional epithelium results in the transepithelial migration of solutes, such as potassium, which depolarizes sensory nerves, and produces the symptoms (C. L. Parsons et al., 1991, J. Urol. 145:732; C. L. Parsons et al., 1994, J. Urol. 73:504; G. Hohlbrugger, 1999, Br. J. Urol. 83(suppl. 2):22; C. L. Parsons et al., 1998, J. Urol. 159:1862). Previous reports have shown that IC patients have defects in the glycosaminoglycan (GAG) layers of the uroepithelium (C. L. Parsons et al., 1991, J. Urol. 145:732; C. L. Parsons et al., 1994, J. Urol. 73:504; G. Hohlbrugger, 1999, Br. J. Urol. 83(suppl. 2):22). Thus, therapies that restore the mucosal lining or surface GAG layer, e.g., administration of heparine, hyaluronic acid, or pentosanpolysulfate, can reduce the leakage of irritant and result in palliation of IC symptoms (see, e.g., C. L. Parsons et al., 1994, Br. J. Urol. 73:504; J. I. Bade et al., 1997, Br. J. Urol. 79:168; J. C. Nickel et al., 1998, J. Urol. 160:612).
  • Capsaicin is a homovanillic acid derivative (8-methyl-N-vanillyl-6-nonenamid). It is the active component of the red pepper of the genus Capsicum, and has been used in humans for topical treatment of cluster headache, herpes zoster, and vasomotor rhinitis (see P. Holzer, 1994, Pharmacol. Rev. 43:143; Sicuteri et al., 1988, Med. Sci. Res. 16-1079; Watson et al., 1988, Pain 33:333; Marabini et al., 1988, Regul. Pept. 22:1). In vitro capsaicin modulates cellular growth, collagenase synthesis, and prostaglandin secretion from rheumatoid arthritis synoviocytes (see Matucci-Cerinic et al., 1990, Ann. Rheum. Dis. 49:598). Capsaicin has also been shown to be immunomodulatory as indicated by its ability to modulate lymphocyte proliferation, antibody production, and neutrophil chemotaxis (see Nilsson et al., 1988, J. Immunopharmac. 10:747; Nilsson et al., 1991, J. Immunopharmac. 13:21; and Eglezos et al., 1990, J. Neuroimmunol. 26:131). These effects play an important role in the use of capsaicin for treatment of arthritis. In addition, capsaicin induces mitochondrial swelling, inhibits NADH oxidase, induces apoptosis of transformed cells, stimulates adenylate cyclase, activates protein kinase C, inhibits superoxide anion generation and alters the redox state of the cell.
  • The various effects of capsaicin are mediated through a specific cellular receptor referred to as a vanilloid receptor. This receptor is shared by resiniferatoxin, an alkaloid derived from plants of the genus Euphorbia. Resiniferatoxin is a structural homologue of capsaicin, and has been shown to mimic many of the actions of capsaicin. Resiniferatoxin is also structurally similar to phorbol esters (phorbol myristate acetate), which interact with distinct binding sites and activate protein kinase C (see Szallasi, et al., 1989, Neurosci. 30:515; and Szallasi and Blumberg, 1989, Neurosci. 30:515). Unlike resiniferatoxin, capsaicin has no homology to phorbol myristate acetate. However, capsaicin can activate protein kinase C, suggesting that such activation is not due entirely to the phorbol ester-like moiety on resiniferatoxin.
  • Capsaicin has been used as an experimental tool because of its selective action on the small diameter afferent nerve fibers, or C fibers, which mediate pain. From studies in animals, capsaicin appears to trigger C fiber membrane depolarization by opening cation selective channels for calcium and sodium. Although detailed mechanisms of action are not yet known, capsaicin mediated effects include: (I) activation of nociceptors in peripheral tissues; (ii) eventual desensitization of peripheral nociceptors to one or more stimulus modalities; (iii) cellular degeneration of sensitive unmyelinated C fiber afferents; (iv) activation of neuronal proteases; (v) blockage of axonal transport; and (vi) the decrease of the absolute number of C fibers without affecting the number of myelinated fibers.
  • Because of the ability of capsaicin to desensitize nociceptors in peripheral tissues, its potential analgesic effects have been assessed in various clinical trials. U.S. Pat. No. 5,431,914, issued Jul. 11, 1995, suggests that a topical preparation containing a concentration of capsaicin of about 0.01% to about 0.1% could be used to treat internal organ pathologies. U.S. Pat. No. 5,665,378, issued Sep. 9, 1997, discusses a transdermal therapeutic formulation comprising capsaicin, a non-steroidal anti-inflammatant, and pamadorm (a diuretic agent) where the composition is said to contain from about 0.001-5% by weight capsaicin and to be useful in treating the pain and discomfort associated with menstrual cramps, bloating, and/or muscular pain such as muscular back pain. Several studies have assessed intravesical capsaicin as a treatment for urge incontinence in patients with spinal detrusor hyperreflexia or bladder hypersensitivity disorders (see F. Cruz, 1998, Int. Urogynecol. J. Pelvic Floor Dysfunct. 9:214-220).
  • However, since capsaicin application itself frequently causes burning pain and hyperalgesia apart from the neuropathic pain being treated, patient compliance has been poor and the drop out rates during clinical trials have exceeded fifty percent. The spontaneous burning pain and heat hyperalgesia are believed to be due to intense activation and temporary sensitization of the peripheral nociceptors at the site of capsaicin application (primary hyperalgesia). Mechanical hyperalgesia evident in areas surrounding the site of topical application appears to originate from central sensitization of dorsal horn neurons involved in pain transmission (secondary hyperalgesia). Because of these side effects, the maximal capsaicin concentration used in previous human studies has usually been limited to 0.075%.
  • Dystonias are neurological movement disorders characterized by involuntary muscle contractions that force certain parts of the body into abnormal, sometime painful, movements or postures (see S. B. Bressman, 2000, Clin. Neuropharmacol. 23(5):239-51). Dystonia disorders cause uncontrolled movement and prolonged muscle contraction, which can result in spasms, twisting body motions, tremor, or abnormal posture. These movements may involve the entire body, or only an isolated area, such as the arms and legs, trunk, neck, eyelids, face, bladder sphincter, or vocal cords. Dystonias result from environmental or disease-related damage to the basal ganglia, birth injury, (particularly due to lack of oxygen), certain infections, reactions to certain drugs, heavy-metal or carbon monoxide poisoning, trauma, or stroke can cause dystonic symptoms. Dystonias can also be symptoms of other diseases, some of which may be hereditary.
  • Urinary detrusor-sphincter dyssynergia (UDSD; also called detrusor-external sphincter dyssynergia and urethral dyssynergia) is a specific type of neurological movement disorder (see H. Madersbacher, 1990, Paraplegia 28(4):217-29; J. T. Andersen et al., 1976, J. Urol. 116(4):493-5). UDSD is characterized by involuntary urinary sphincter spasms occurring simultaneously with bladder contractions. The lack of coordination between detrusor contraction and urethral relaxation causes urinary obstruction (i.e., partial or complete block of urination). As a result of UDSD, the bladder cannot empty completely. This creates a buildup of urinary pressure, and can lead to severe urinary tract damage and life-threatening consequences. UDSD results from lesions of the corticospinal tract, which are caused by spinal chord injury, multiple sclerosis, or related conditions.
  • Another neurological movement disorder is hyperactive (also called contracted; spastic) neurogenic bladder (see M. H. Beers and R. Berkow (eds), 1999, The Merck Manual of Diagnosis and Therapy, Section 17:Genitourinary Disorders, Chapter 216: Myoneurogenic Disorders). In hyperactive bladder, the bladder contracts more frequently than normal, due to instability and inappropriate contraction of detrusor muscles (see, e.g., C. F. Jabs et al., 2001, Int. Urogynecol. J. Pelvic Floor Dysfunct. 12(1):58-68; S. K. Swami and P. Abrams, 1996, Urol. Clin. North Am. 23(3):417-25). Hyperactive bladders can empty spontaneously and result in urinary incontinence (urge incontinence). In addition, the uncoordinated contraction between the bladder and bladder outlet (vesical neck or external urinary sphincter) can result in vesicoureteral reflux with concomitant renal damage. Hyperactive bladder is usually due to brain or suprasacral spinal cord damage. The most common cause is spinal cord injury from transverse myelitis or traumatic cord transection. Hyperactive bladder can also be caused by conditions such as anxiety, aging, infections (e.g., syphilis), diabetes mellitus, brain and spinal cord tumors, stroke, ruptured intervertebral disk, and demyelinating and degenerative diseases (e.g., multiple sclerosis and amyotrophic lateral sclerosis).
  • Botulinum toxins are zinc endopeptidases produced by the anaerobic bacterium Clostridium botulinum. Previously known as a cause of a serious and often fatal paralysis acquired through ingestion of contaminated food, botulinum neurotoxins are presently used in both therapeutic and cosmetic applications (see N. Mahant et al., 2000, J. Clin. Neurosci. 7(5):389-94; A. Carruthers and J. Carruthers, 2001, Semin. Cutan. Med. Surg. 20(2):71-84). In particular, these toxins are used in the treatment of conditions involving involuntary muscle spasms, frown lines, and facial wrinkles.
  • There are seven known serotypes of botulinum toxins (designated A-G). The serotypes differ in their cellular targets, potency, and duration of action, but all exert their paralytic effect by inhibiting acetylcholine release at the neuromuscular junction (see M. F. Brin, 1997, Muscle Nerve 20(suppl 6):S146-S168). Each serotype acts by cleaving one or more proteins involved in vesicle transport and membrane fusion. For example, botulinum toxin A is internalized by endocytosis at the axon terminal, where it is fully activated by disulfide reduction reactions, and it targets SNAP-25 (see M. F. Brin, 1997, Muscle Nerve 20(suppl 6):S146-S168). The extent of botulinum toxin-mediated paralysis depends on the dose, volume, and serotype employed. Botulinum toxin A causes reversible denervation atrophy that is typically terminated by axon sprouting within 2-6 months (see M. F. Brin, 1997, Muscle Nerve 20(suppl 6):S146-S168).
  • A major drawback of current botulinum toxin therapies is the development of antitoxin antibodies in patients. Antitoxin antibodies result in resistance to botulinum toxin, and the reduction or elimination of its therapeutic effect. It has been estimated that the prevalence of neutralizing antibodies among patients receiving chronic treatment at the higher doses for torticollis or spasticity is probably at least 3% (see M. F. Brin, 1997, Muscle Nerve 20(suppl 6):S146-S168). Patients with botulinum toxin A resistance may benefit from injections with other serotypes, including botulinum toxin B, C, and F. However, differences in the duration of the effects of the other serotypes can be significant, and cause dramatic reductions in treatment efficacy (see M. F. Brin, 1997, Muscle Nerve.20(suppl 6):S146-S168).
  • Liposomes are self-assembling structures comprising concentric amphipathic lipid (e.g., phospholipid) bilayers separated by aqueous compartments (see, e.g., K. Reimer et al., 1997, Dermatology 195(suppl. 2):93; M. Schafer-Korting et al., 1989, Dermatology 21:1271). In liposomes, the amphipathic lipid molecules comprise a polar headgroup region covalently linked to one or two non-polar acyl chains. The energetically unfavorable contact between the hydrophobic acyl chains and the aqueous solution surrounding the lipid molecules causes the polar headgroups and acyl chains to rearrange. The polar headgroups become oriented toward the aqueous solution, while the acyl chains orient towards the interior part of the bilayer. The lipid bilayer structure thereby comprises two opposing monolayers, wherein the acyl chains are shielded from contact with the surrounding medium.
  • Liposomes are excellent vehicles for drug delivery and gene therapy (K. Reimer et al., 1997, Dermatology 195(suppl. 2):93; T. Tsuruta et al., 1997, J. Urol. 157:1652; F. Szoka, 2000, Mol. Therapy 1:S2; G. Gregoriadis, 1976, New Eng. J. Med. 295:704). Previous studies have demonstrated that submucosal injection of liposomal doxorubicin into bladder wall provides an effective and safe treatment for bladder cancer with pelvic lymph node metastasis (I. Tsuruta et al., 1997, J. Urol. 157:1652). In a liposome-drug delivery system, an active ingredient, such as a drug, is encapsulated or entrapped in the liposome and then administered to the patient to be treated. Alternatively, if the active ingredient is lipophilic, it may be associated with the lipid bilayer. Active ingredients encapsulated by liposomes reduce toxicity, increase efficacy, or both. Notably, liposomes are thought to interact with cells by stable absorption, endocytosis, lipid transfer, and fusion (R. B. Egerdie et al., 1989, J. Urol. 142:390). In this way, liposomes comprise molecular films, which fuse with cells and provide optimal conditions for wound healing (K. Reimer et al., 1997, Dermatology 195(suppl. 2):93; M. Schafer-Korting et al., 1989, J. Am. Acad. Dermatol. 21:1271). Generally, liposomes have low antigenicity and can be used to encapsulate and deliver components that cause undesirable immune responses in patients (see A. Natsume et al., 2000, Jpn. J. Cancer Res. 91:363-367).
  • SUMMARY OF THE INVENTION
  • The present invention encompasses improved treatments for pain (e.g., neuropathic pain), pain-intensive disorders (e.g., IC), muscle contraction disorders (e.g., IC, hyperactive bladder, and UDSD), and related conditions by providing compositions and methods for the intravesical administration of lipid vehicles. Liposomes provide non-toxic vehicles for the delivery of lipophilic therapeutic agents that have irritative side effects (e.g., vanilloids such as capsaicin) or undesirable antigenicity (e.g., botulinum toxin). Advantageously, the disclosed lipid vehicles can be used simultaneously deliver and ameliorate irritation caused by irritating therapeutic agents. The vehicles can also be used to reduce or prevent antibody-mediated resistance to antigenic therapeutic agents. In addition, the disclosed lipid vehicles can be utilized as an intravesical drug delivery platform for antibiotic and anticancer agents in the bladder and other luminal organ systems, e.g., the distal colon and vagina.
  • The invention includes compositions comprising lipid vehicles (e.g., micelles, microemulsions, macroemulsions, and liposomes) for use as intravesical instillation vehicles for cells or tissues. Such vehicles may further include antibodies, for example, uroplakin or NGF receptor antibodies. These antibodies may be conjugated to the surface of the liposome, and act to target the liposome to specific cell: types and/or receptors. In addition, the vehicles may include compositions, including capsaicin, resiniferatoxin, tinyatoxin, and other vanilloids, which can be delivered to the cells. The vehicles may also include compositions comprising bioactive agents (e.g., antisense nucleic acids or peptides), drugs (e.g., pain therapeutics, anticancer treatments, or antibiotics), toxins (e.g., botulinum toxin), or other agents.
  • The present invention further encompasses methods of treating various disorders, e.g., defects or diseases of the genitourinary tract, gastrointestinal tract, pulmonary system, and other body systems, using the disclosed lipid vehicles. In particular, the disclosed vehicles can be administered via intravesical instillation to treat interstitial cystitis (IC), urinary detrusor-sphincter dyssynergia (UDSD), spastic neurogenic bladder, hyperactive bladder, or other conditions of the genitourinary system. The disclosed vehicles can also be administered intravesically to treat systemic infections and cancers, utilizing the unique interaction of the disclosed vehicles as a novel route for prolonged delivery of such therapies.
  • The invention also encompasses methods of treating pain (e.g., neuropathic pain) associated with cancers and/or disorders of the bladder, genitourinary tract, gastrointestinal tract, pulmonary system, and other body systems, using the disclosed lipid vehicles. In particular, the disclosed vehicles can be administered via intravesical instillation to treat pain associated with IC, or other conditions of the bladder, such as bladder infections and bladder cancer. In specific embodiments, these vehicles may comprise vanilloids, e.g., capsaicin, resiniferatoxin, or tinyatoxin, and may further comprise surface antibodies, e.g., uroplakin or NGF receptor antibodies, to target pain relief to the affected sites.
  • Further encompassed are methods of treating disorders associated with involuntary muscle contraction (e.g., dystonia, dyssynergia, and spasticity) affecting the genitourinary tract, gastrointestinal tract, pulmonary system, or other body systems, using the disclosed lipid vehicles. In one aspect, the disclosed vehicles can be administered via intravesical instillation to treat muscle contractions caused by IC, UDSD, spastic neurogenic bladder, or related conditions. The vehicles may be empty or may carry toxins, e.g., botulinum toxins, to deliver relief from muscle contractions at the affected sites.
  • Other applications and advantages afforded by the present invention will be apparent from the detailed description and exemplification hereinbelow.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • The appended drawings of the figures are presented to further describe the invention and to assist in its understanding through clarification of its various aspects.
  • FIG. 1 shows the experimental design for the studies described in Examples 1-2 (below).
  • FIGS. 2A-2F show CMG tracing results. Treatments included saline (control), protamine sulfate in potassium chloride (PS/KCl), and liposomes in KCl (LP/KCl) or KCl alone. PS/KCl elicited bladder hyperactivity. LP/KCl partly reversed the irritative effect of LP/KCl, and this reversal was maintained after switching to KCl. FIGS. 2B, 2D, and 2F show saline infusion (control), PS/KCl infusion, and KCl infusion, respectively, in the control animal. FIGS. 2A, 2C, and 2E show saline infusion (control), PS/KCl infusion, and liposomal infusion in the presence of maintenance KCl, respectively.
  • FIGS. 3A-3F show CMG tracing results. Treatments included saline (control), acetic acid (M), and liposomes (LP) or saline. AA elicited bladder hyperactivity. LP partly reversed the irritative effect of AA, and this reversal was maintained after switching to saline. FIGS. 3B, 3D, and 3F show saline infusion (control), AA infusion and saline infusion, respectively, in the control animal. FIGS. 3A, 3C, and 3E show saline infusion (control), AA infusion, and liposomal infusion in the presence of maintenance M, respectively.
  • FIGS. 4A-4D show CMG tracing results. Treatments included saline (control) and various concentrations of protamine sulfate (PS). High concentrations of PS induced bladder hyperactivity (decreased ICl), whereas low concentrations of PS produced no effect. FIG. 4A shows a control CMG measured before PS treatment. FIG. 4B shows a CMG measured during treatment with low concentrations of PS. FIG. 4C shows a control CMG measured before PS treatment. FIG. 4D shows a CMG measured during treatment with high concentrations of PS.
  • FIGS. 5A-5F show CMG tracing results. Treatments included saline (control) and various concentrations of potassium chloride (KCl) following one hour of PS (10 mg/ml). High concentrations of KCl induced bladder hyperactivity (decrease ICl), whereas low concentrations of KCl had no effect. FIG. 5A shows a control CMG measured before KCl treatment. FIG. 5B shows a CMG measured during treatment with 100 mM KCl. FIG. 5C shows a control CMG measured before KCl treatment. FIG. 5D shows a CMG measured during treatment with 300 mM KCl. FIG. 5E shows a control CMG measured before KCl treatment. FIG. 5F shows a CMG measured during treatment with 500 mM KCl.
  • FIGS. 6A-6B show CMG tracing results. Treatments included saline (control) and KCl (500 mM) infusion following PS (10 mg/ml) infusion in micturition reflex suppressed animals. KCl stimulated the detrusor muscle and decreased bladder compliance. FIG. 6A shows a control CMG measured before KCl treatment. FIG. 6B shows a CMG measured during KCl treatment.
  • FIG. 7 shows the efficacy of liposomal delivery of capsaicin utilizing bladder contraction frequency as a bioassay of the irritative effects of the vanilloid. Column 1: saline; Column 2: liposomes; Column 3: liposomes plus capsaicin. Inclusion of capsaicin into the liposomal preparation allowed for effective capsaicin delivery. The addition of saline or liposomes produced no change in bladder contraction frequency. The combination of liposome and capsaicin produced a significant increase in bladder contraction frequency.
  • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention relates to the administration of lipid vehicles to provide long-lasting drug delivery to diseased or dysfunctional cells, tissues, or body systems. In particular, the invention relates to treatments for urinary system components, e.g., kidneys, ureters, bladders, sphincter muscles, and urethras. Specifically encompassed are treatments for bladder irritation and irritation-induced bladder dysfunction. In accordance with the present invention, nonionic liposomes are formulated to act as a drug with prolonged efficacy for topical bladder instillation, and bladder-protective effects. The efficacy and protective effects of such formulations are unexpected and surprising results. Advantageously, the disclosed liposome vehicles can be used to simultaneously deliver and ameliorate irritation caused by irritating therapeutic agents, e.g., resiniferatoxin or other vanilloid agents. The disclosed methods of intravesical administration of liposomes provide novel treatments for IC patients. Such methods can also be employed for the treatment of other disorders of the urinary system, bladder, genitourinary tract, gastrointestinal tract, pulmonary system, and other body organs and systems, including cancers, infections, and spasticity.
    • Lipid Vehicles
  • The lipid vehicles of the present invention encompass micelles, microemulsions, macroemulsions, liposomes, and similar carriers. The term micelles refers to colloidal aggregates of amphipathic (surfactant) molecules that are formed at a well-defined concentration known as the critical micelle concentration. Micelles are oriented with the nonpolar portions at the interior and the polar portions at the exterior surface, exposed to water. The typical number of aggregated molecules in a micelle (aggregation number) is 50 to 100. As described herein, microemulsions are essentially swollen micelles, although not all micellar solution can be swollen to form microemulsion. Microemulsions are thermodynamically stable, are formed spontaneously, and contain particles that are extremely small. Droplet diameters in microemulsions typically range from 10-100 nm. In contrast, the term macroemulsions refers to droplets with diameters greater than 100 nm. As described herein, liposomes are closed lipid vesicles comprising lipid bilayers that encircle aqueous interiors. Liposomes typically have diameters of 25 nm to 1 μm (see, e.g., D. O. Shah (ed), 1998, Micelles, Microemulsions, and Monolayers: Science and Technology, Marcel Dekker; A. S. Janoff (ed), 1998, Liposomes: Rational Design, Marcel Dekker).
  • Lipid vehicles of the invention may carry a bioactive agent (e.g., a nucleic acid, polypeptide, peptide, or antibody molecule) or drug (e.g., one or more pepper extract compounds such as capsaicin, resiniferatoxin, tinyatoxin and other vanilloids, as well as antibiotics, anti-inflammatory agents, and antispasmodics). As used herein, the terms nucleic acid and polynucleotide are synonymous, and refer to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribonucleotides. The terms protein and polypeptide are synonymous as used herein, and refer to polymers comprising amino acid residues linked by peptide bonds. Peptides are defined as fragments or portions of polypeptides, preferably fragments or portions having at least one functional activity (e.g., binding, antigenic, or catalytic activity) as the complete polypeptide sequence (see, e.g., by H. Lodish et al., 1999, Molecular Cell Biology, W. H. Freedman and Sons, N.Y.; L. Stryer, 2001, Biochemistry, W. H. Freedman and Sons, N.Y.; B. Lewin, 1999, Genes VII, Oxford University Press).
  • In one preferred embodiment, the vehicle is a liposome formulation, and the drug is an organic or inorganic small molecule. The principal lipid of the vehicle is, preferably, phosphatidylcholine, but can include various natural (e.g., tissue derived L-α-phosphatidyl: egg yolk, heart, brain, liver, soybean) and/or synthetic (e.g., saturated and unsaturated 1,2-diacyl-SN-glycero-3-phosphocholines, 1-acyl-2-acyl-SN-glycero-3-phosphocholines, 1,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the same. Such lipids can be used alone, or in combination with a helper lipid. Preferred helper lipids are non-ionic or uncharged at physiological pH. Particularly preferred non-ionic lipids include, but are not limited to, cholesterol and DOPE (1,2-dioleolylglyceryl phosphatidylethanolamine), with cholesterol being most preferred. The molar ratio of a phospholipid to helper lipid can range from about 3:1 to about 1:1, more preferably from about 1.5:1 to about 1:1, and most preferably, the molar ratio is about 1:1.
  • A liposome used for the preparation of a vehicle of the invention is, in simplest form, composed of two lipid layers. The lipid layer may be a monolayer, or may be multilamellar and include multiple layers. Constituents of the liposome may include, for example, phosphatidylcholine, cholesterol, phosphatidylethanolamine, etc. Phosphatidic acid, which imparts an electric charge, may also be added. Exemplary amounts of these constituents used for the production of the liposome include, for instance, 0.3 to 1 mol, preferably 0.4 to 0.6 mol of cholesterol; 0.01 to 0.2 mol, preferably 0.02 to 0.1 mol of phosphatidylethanolamine; 0.0-0.4 mol, preferably 0-0.15 mol of phosphatidic acid per 1 mol of phosphatidylcholine.
  • Liposomes of the present invention can be constructed by well-known techniques (see, e.g., G. Gregoriadis (ed.), 1993, Liposome Technology Vols. 1-3, CRC Press, Boca Raton, Fla.). Lipids are typically dissolved in chloroform and spread in a thin film over the surface of a tube or flask by rotary evaporation. If liposomes comprised of a mixture of lipids are desired, the individual components are mixed in the original chloroform solution. After the organic solvent has been eliminated, a phase consisting of water optionally containing buffer and/or electrolyte is added and the vessel agitated to suspend the lipid. Optionally, the suspension is then subjected to ultrasound, either in an ultrasonic bath or with a probe sonicator, until the particles are reduced in size and the suspension is of the desired clarity. For transfection, the aqueous phase is typically distilled water and the suspension is sonicated until nearly clear, which requires several minutes depending upon conditions, kind, and quality of the sonicator. Commonly, lipid concentrations are 1 mg/ml of aqueous phase, but could be higher or lower by about a factor of ten.
  • Liposomes according to the invention optionally have one or more amphiphiles. The exact composition of the liposomes will depend on the particular circumstances for which they are to be used. Those of ordinary skill in the art will find it a routine matter to determine a suitable composition. The liposomes of the present invention comprise at least one compound of the present invention. In a preferred embodiment, the liposomes of the present invention consist essentially of a single type of phospholipid. In another preferred embodiment, the liposomes comprise mixtures of phospholipids. In yet another preferred embodiment, the liposomes of the present invention comprise one or more phospholipids in a mixture with one or more natural or synthetic lipids, e.g., cholesterol or DOPE.
  • Liposomes can be produced in accordance with established methods. For example, a mixture of the above-mentioned lipids, from which the solvents have been removed, can be emulsified by the use of a homogenizer, lyophilized, and melted to obtain multilamellar liposomes. Alternatively, unilamellar liposomes can be produced by the reverse phase evaporation method (Szoka and Papahadjopoulos, 1978, Proc. Natl. Acad. Sci. USA 75:4194-4198). Unilamellar vesicles can also be prepared by sonication or extrusion. Sonication is generally performed with a bath-type sonifier, such as a Branson tip sonifier (G. Heinemann Ultrashall und Labortechnik, Schwabisch Gmund, Germany) at a controlled temperature as determined by the melting point of the lipid. Extrusion may be carried out by biomembrane extruders, such as the Lipex Biomembrane Extruder (Northern Lipids Inc, Vancouver, British Columbia, Canada). Defined pore size in the extrusion filters may generate unilamellar liposomal vesicles of specific sizes. The liposomes can also be formed by extrusion through an asymmetric ceramic filter, such as a Ceraflow Microfilter (commercially available from the Norton Company, Worcester, Mass.).
  • Following liposome preparation, the liposomes that have not been sized during formation may be sized by extrusion to achieve a desired size range and relatively narrow distribution of liposome sizes. A size range of about 0.2-0.4 microns will allow the liposome suspension to be sterilized by filtration through a conventional filter (e.g., a 0.22 micron filter). The filter sterilization method can be carried out on a high throughput basis.
  • Several techniques are available for sizing liposomes to a desired size, including, ultrasonication, high-speed homogenization, and pressure filtration (M. J. Hope et al., 1985, Biochimica et Biophysica Acta 812:55; U.S. Pat. Nos. 4,529,561 and 4,737,323). Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles less than about 0.05 microns in size. Multilamellar vesicles can be recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns. The size of the liposomal vesicles may be determined by quasi-elastic light scattering (QELS) (see Bloomfield, 1981, Ann. Rev. Biophys. Bioeng. 10:421-450). Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.
  • Liposomes can be extruded through a small-pore polycarbonate membrane or an asymmetric ceramic membrane to yield a well-defined size distribution. Typically, a suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved. The liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size. For use in the present invention, liposomes have a size of about 0.05 microns to about 0.5 microns. More preferred are liposomes having a size of about 0.05 to about 0.2 microns.
  • Various conditions can be used to trigger the liposome to release its payload or active agent, including pH, ionic strength, controlled release and antibody attachment. Research related to pH-sensitive liposomes has focused principally on anionic liposomes comprised largely of phosphatidylethanolamine (PE) bilayers (see, Huang et al., 1989, Biochemistry 28:9508-9514; Duzgunes et al., 1990, “pH-Sensitive Liposomes” Membrane Fusion J. Wilschut and D. Hoekstra (eds.), Marcel-Decker Inc., New York, N.Y. pp. 713-730; Yatvin et al., 1980, Science, 210, 1253-1255). More recently, pH-sensitive cationic liposomes have been developed to mediate transfer of DNA into cells. For instance, researchers have described a series of amphiphiles with headgroups containing imidazole, methylimidazole, or aminopyridine moieties (see, Budker et al., 1996, Nature Biotech. 14:760-764). Also described are lipid molecules within liposome assemblies that are capable of structural reorganization upon a change in pH (see, e.g., U.S. Pat. No. 6,200,599 to Nantz et al.).
  • From the detailed description herein, it will be clear to those skilled in the art that the vehicles of the present invention are useful for both in vitro and in vivo applications. The vehicles of the present invention will find use for nearly any in vitro or in vivo application requiring delivery of bioactive agents (e.g., nucleic acids, peptides, polypeptides, or antibodies) and/or drugs (e.g., pain therapeutics, anticancer treatments, or antibiotics) into cells.
    • Nucleic Acids
  • In practicing the present invention, many conventional techniques in molecular biology, microbiology, and recombinant DNA may be employed. Such techniques are well known and are explained fully in, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; F. M. Ausubel et al. (eds), 1995, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, N.Y.; D. N. Glover (ed), 1985, DNA Cloning: A Practical Approach, Volumes I and II; M. L. Gait (ed), 1984, Oligonucleotide Synthesis; Hames and Higgins (eds), 1985, Nucleic Acid Hybridization; Hames and Higgins (eds), 1984, Transcription and Translation; Perbal, 1984, A Practical Guide to Molecular Cloning; The Series, Methods in Enzymology, Academic Press, Inc.; J. H. Miller and M. P. Calos (eds), 1987, Gene Transfer Vectors for Mammalian Cells, Cold Spring Harbor Laboratory; Wu and Grossman (eds), Methods in Enzymology, Vol. 154; Wu (ed), Methods in Enzymology, Vol. 155.
  • Nucleic acids of all types may be associated with the lipid vehicles of the present invention. In accordance with the present invention, nucleic acids may be single- or double-stranded molecules, i.e., DNA, RNA, or DNA-DNA, DNA-RNA or RNA-RNA hybrids, or protein nucleic acids (PNAs) formed by conjugating bases to an amino acid backbone. Nucleic acids may also be oligonucleotides such as antisense oligonucleotides, chimeric DNA-RNA polymers, and ribozymes, as well as modified versions of these nucleic acids wherein the modification may be in the base, the sugar moiety, the phosphate linkage, or in any combination thereof. The nucleic acids may comprise an essential gene or fragment thereof, in which the target cell or cells is deficient in some manner. This can occur where the gene is lacking or where the gene is mutated resulting in under- or over- expression. The nucleic acids can also comprise antisense oligonucleotides. Such antisense oligonucleotides may be constructed to inhibit expression of a target gene.
  • In one embodiment, DNA containing all or part of the coding sequence for a polypeptide, or a complementary sequence thereof, is incorporated into a vector and inserted into a lipid vehicle for gene therapy applications. In recent years, significant technological advances have been made in the area of gene therapy for both genetic and acquired diseases (Kay et al., 1997, Proc. Natl. Acad. Sci. USA, 94:12744-12746). Gene therapy can be defined as the transfer of DNA for therapeutic purposes. Improvement in gene transfer methods has allowed for development of gene therapy protocols for the treatment of diverse types of diseases. Gene therapy has also taken advantage of recent advances in the identification of new therapeutic genes, improvement in both viral and non-viral gene delivery systems, better understanding of gene regulation, and improvement in cell isolation and transplantation. Gene therapy can be carried out according to generally accepted methods as described by, for example, Friedman, 1991, Therapy for Genetic Diseases, Friedman, Ed., Oxford University Press, pages 105-121.
  • Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector may be used. Methods for introducing DNA into cells are known in the art, and the choice of method is within the competence of one skilled in the art (Robbins (ed), 1997, Gene Therapy Protocols, Human Press, N.J.). Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and non-viral transfer methods.
  • A number of viruses have been used as gene transfer vectors, including polyoma, i.e., SV40 (Madzak et al., 1992, J. Gen. Virol., 73:1533-1536), adenovirus (Berkner, 1992, Curr. Top. Microbiol. Immunol. 158:39-6; Berkner et al., 1988, Bio Techniques, 6:616-629; Gorziglia et al., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Natl. Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256), vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495- 499), adeno-associated virus (Muzyczka, 1992, Cuff. Top. Microbiol. Immunol., 158:91- 123; Ohi et al., 1990, Gene, 89:279-282), herpes viruses including HSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J. Virol., 66:2952-2965; Fink et al., 1992, Hum. Gene Ther., 3:11-19; Breakfield et al., 1987, Mol. Neurobiol., 1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199), and retroviruses of avian (Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J. Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol., 54:401- 407), and human origin (Page et al., 1990, J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739). Most human gene therapy protocols have been based on disabled murine retroviruses.
  • Non-viral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Graham et al., 1973, Virology, 52:456-467; Pellicer et al., 1980, Science, 209:1414-1422), mechanical techniques, for example microinjection (Anderson et al., 1980, Proc. Natl. Acad. Sci. USA, 77:5399-5403; Gordon et al., 1980, Proc. Natl. Acad. Sci. USA, 77:7380-7384; Brinster et al., 1981, Cell, 27:223-231; Constantini et al., 1981, Nature, 294:92-94), membrane fusion-mediated transfer via liposomes (Feigner et al., 1987, Proc. Natl. Acad. Sci. USA, 84:7413-7417; Wang et al., 1989, Biochemistry, 28:9508-9514; Kaneda et al., 1989, J. Biol. Chem., 264:12126-12129; Stewart et al., 1992, Hum. Gene Ther., 3:267-275; Nabel et al., 1990, Science, 249:1285-1288; Lim et al., 1992, Circulation, 83:2007-2011), and direct DNA uptake and receptor-mediated DNA transfer (Wolff et al., 1990, Science, 247:1465-1468; Wu et al., 1991, BioTechniques, 11:474485; Zenke et al., 1990, Proc. Natl. Acad. Sci. USA, 87:3655-3659; Wu et al., 1989, J. Biol. Chem., 264:16985-16987; Wolff et al., 1991, BioTechniques, 11:474-485; Wagner et al., 1991, Proc. Natl. Acad. Sci. USA, 88:4255-4259; Cotten et al., 1990, Proc. Natl. Acad. Sci. USA, 87:4033-4037; Curiel et al., 1991, Proc. Natl. Acad. Sci. USA, 88:8850-8854; Curiel et al., 1991, Hum. Gene Ther., 3:147-154).
  • In one approach, plasmid DNA is complexed with a polylysine-conjugated antibody specific to the adenovirus hexon protein, and the resulting complex is bound to an adenovirus vector. The trimolecular complex is then used to infect cells. The adenovirus vector permits efficient binding, internalization, and degradation of the endosome before the coupled DNA is damaged. In another approach, liposome/DNA is used to mediate direct in vivo gene transfer. While in standard liposome preparations the gene transfer process is non-specific, localized in vivo uptake and expression have been reported in tumor deposits, for example, following direct in situ administration (Nabel, 1992, Hum. Gene Ther., 3:399-410).
  • Suitable gene transfer vectors possess a promoter sequence, preferably a promoter that is cell-specific and placed upstream of the sequence to be expressed. The vectors may also contain, optionally, one or more expressible marker genes for expression as an indication of successful transfection and expression of the nucleic acid sequences contained in the vector. In addition, vectors can be optimized to minimize undesired immunogenicity and maximize long-term expression of the desired gene product(s) (see Nabe, 1999, Proc. Natl. Acad. Sci. USA 96:324-326). Moreover, vectors can be chosen based on cell-type that is targeted for treatment.
  • Illustrative examples of vector constructs for transfection or infection of the host cells include replication-defective viral vectors, DNA virus or RNA virus (retrovirus) vectors, such as adenovirus, herpes simplex virus and adeno-associated viral vectors. Adeno-associated virus vectors are single stranded and allow the efficient delivery of multiple copies of nucleic acid to the cell's nucleus. Preferred are adenovirus vectors. The vectors will normally be substantially free of any prokaryotic DNA and may comprise a number of different functional nucleic acid sequences. An example of such functional sequences may be a DNA region comprising transcriptional and translational initiation and termination regulatory sequences, including promoters (e.g., strong promoters, inducible promoters, and the like) and enhancers which are active in the host cells. Also included as part of the functional sequences is an open reading frame (polynucleotide sequence) encoding a protein of interest. Flanking sequences may also be included for site-directed integration. In some situations, the 5′-flanking sequence will allow homologous recombination, thus changing the nature of the transcriptional initiation region, so as to provide for inducible or non-inducible transcription to increase or decrease the level of transcription, as an example.
  • In general, an encoded and expressed polypeptide may be intracellular, i.e., retained in the cytoplasm, nucleus, or in an organelle, or may be secreted by the cell. For secretion, the natural signal sequence present in a polypeptide may be retained. When the polypeptide or peptide is a fragment of a protein, a signal sequence may be provided so that, upon secretion and processing at the processing site, the desired protein will have the natural sequence. Specific examples of coding sequences of interest for use in accordance with the present invention include the polypeptide-coding sequences disclosed herein.
  • A marker may be present for selection of cells containing the vector construct. The marker may be an inducible or non-inducible gene and will generally allow for positive selection under induction, or without induction, respectively. Examples of marker genes include neomycin, dihydrofolate reductase, glutamine synthetase, and the like. The vector employed will generally also include an origin of replication and other genes that are necessary for replication in the host cells, as routinely employed by those having skill in the art.
  • As an example, the replication system comprising the origin of replication and any proteins associated with replication encoded by a particular virus may be included as part of the construct. The replication system must be selected so that the genes encoding products necessary for replication do not ultimately transform the cells. Such replication systems are represented by replication-defective adenovirus (see G. Acsadi et al., 1994, Hum. Mol. Genet. 3:579-584) and by Epstein-Barr virus. Examples of replication defective vectors, particularly, retroviral vectors that are replication defective, are BAG, (see Price et al., 1987, Proc. Natl. Acad. Sci. USA, 84:156; Sanes et al., 1986, EMBO J., 5:3133). It will be understood that the final gene construct may contain one or more genes of interest, for example, a gene encoding a bioactive metabolic molecule. In addition, cDNA, synthetically produced DNA or chromosomal DNA may be employed utilizing methods and protocols known and practiced by those having skill in the art.
  • According to one approach for gene therapy, a vector containing an antisense sequence or encoding a polypeptide is directly injected into the recipient cells (in vivo gene therapy). Alternatively, cells from the intended recipients are explanted, genetically modified to contain the antisense or encode the polypeptide, and reimplanted into the donor (ex vivo gene therapy). An ex vivo approach provides the advantage of efficient viral gene transfer, which is superior to in vivo gene transfer approaches. In accordance with ex vivo gene therapy, the host cells are first transfected with engineered vectors containing at least one nucleic acid sequence, suspended in a physiologically acceptable carrier, excipient, or diluent such as saline or phosphate buffered saline, and the like; and then administered to the host. The desired protein and/or RNA is expressed by the injected cells. The introduced gene products are thereby utilized to treat or ameliorate a disorder that is related to altered expression or function of a gene.
  • In one particular embodiment, an antisense nucleic acid sequence is carried by a lipid vehicle of the invention. An antisense sequence can be wholly or partially complementary to a target nucleic acid, and can be DNA, or its RNA counterpart (i.e., wherein T residues of the DNA are U residues in the RNA counterpart). Antisense nucleic acids can be produced by standard techniques (see, for example, Shewmaker et al., U.S. Pat. No. 5,107,065).
  • An antisense nucleic acid may comprise a sequence complementary to a portion of a protein coding sequence. A portion, for example a sequence of 16 nucleotides, may be sufficient to inhibit expression of the protein. Or, an antisense nucleic acid or oligonucleotide, complementary to 5′ or 3′ untranslated regions, or overlapping the translation initiation codons (5′ untranslated and translated regions), of target genes, or genes encoding a functional equivalent can also be effective. Accordingly, antisense nucleic acids or oligonucleotides can be used to inhibit the expression of the gene encoded by the sense strand or the mRNA transcribed from the sense strand.
  • In addition, antisense nucleic acids and oligonucleotides can be constructed to bind to duplex nucleic acids either in the genes or the DNA:RNA complexes of transcription, to form stable triple helix-containing or triplex nucleic acids to inhibit transcription and/or expression of a gene (Frank-Kamenetskii, M. D. and Mirkin, S. M., 1995, Ann. Rev. Biochem. 64:65-95). Such oligonucleotides of the invention can be constructed using the base-pairing rules of triple helix formation and the nucleotide sequences of the target genes.
  • In preferred embodiments, at least one of the phosphodiester bonds of an antisense oligonucleotide has been substituted with a structure that functions to enhance the ability of the compositions to penetrate into the region of cells where the RNA whose activity is to be modulated is located. It is preferred that such substitutions comprise phosphorothioate bonds, methyl phosphonate bonds, or short chain alkyl or cycloalkyl structures. In accordance with other preferred embodiments, the phosphodiester bonds are substituted with structures which are, at once, substantially non-ionic and non-chiral, or with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in the practice of the invention.
  • Oligonucleotides may also include species that include at least some modified base forms. Thus, purines and pyrimidines other than those normally found in nature may be so employed. Similarly, modifications on the furanosyl portions of the nucleotide subunits may also be affected, as long as the essential tenets of this invention are adhered to. Examples of such modifications are 2′-O-alkyl- and 2′-halogen-substituted nucleotides. Some non-limiting examples of modifications at the 2′ position of sugar moieties which are useful in the present invention include OH, SH, SCH3, F, OCH3, OCN, O(CH2)n NH2 and O(CH2)n CH3, where n is from 1 to about 10. Such oligonucleotides are functionally interchangeable with natural oligonucleotides or synthesized oligonucleotides, which have one or more differences from the natural structure. All such analogs are comprehended by this invention so long as they function effectively to hybridize with a nucleic acid to inhibit the function thereof.
  • The antisense oligonucleotides in accordance with this invention preferably comprise from about 3 to about 50 subunits. It is more preferred that such oligonucleotides and analogs comprise from about 8 to about 25 subunits and still more preferred to have from about 12 to about 20 subunits. As defined herein, a subunit is a base and sugar combination suitably bound to adjacent subunits through phosphodiester or other bonds.
  • The antisense oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is available from several vendors, including PE Applied Biosystems (Foster City, Calif.). Any other means for such synthesis may also be employed, however, the actual synthesis of the oligonucleotides is well within the abilities of the practitioner. Also well-known are methods for preparing modified oligonucleotides, such as phosphorothioates and alkylated derivatives.
  • The oligonucleotides of this invention are designed to be hybridizable with target RNA (e.g., mRNA) or DNA. For example, an oligonucleotide (e.g., DNA oligonucleotide) that hybridizes to a mRNA molecule can be used to target the mRNA for RnaseH digestion. Alternatively, an oligonucleotide that hybridizes to the translation initiation site of an mRNA molecule can be used to prevent translation of the mRNA. In another approach, oligonucleotides that bind to double-stranded DNA can be administered. Such oligonucleotides can form a triplex construct and inhibit the transcription of the DNA. Triple helix pairing prevents the double helix from opening sufficiently to allow the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described (see, e.g., J. E. Gee et al., 1994, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.).
  • As non-limiting examples, antisense oligonucleotides may be targeted to hybridize to the following regions: mRNA cap region; translation initiation site; translational termination site; transcription initiation site; transcription termination site; polyadenylation signal; 3′ untranslated region; 5′ untranslated region; 5′ coding region; mid coding region; and 3′ coding region. Preferably, the complementary oligonucleotide is designed to hybridize to the most unique 5′ sequence of a gene, including any of about 15-35 nucleotides spanning the 5′ coding sequence. Appropriate oligonucleotides can be designed using OLIGO software (Molecular Biology Insights, Inc., Cascade, Colo.; http://www.oligo.net).
  • In accordance with the present invention, an antisense oligonucleotide can be synthesized, formulated as a pharmaceutical composition, and administered to a subject. The synthesis and utilization of antisense and triplex oligonucleotides have been previously described (e.g., H. Simon et al., 1999, Antisense Nucleic Acid Drug Dev. 9:527-31; F. X. Barre et al., 2000, Proc. Natl. Acad. Sci. USA 97:3084-3088; R. Elez et al., 2000, Biochem. Biophys. Res. Commun. 269:352-6; E. R. Sauter et al., 2000, Clin. Cancer Res. 6:654-60). Alternatively, expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population.
  • Methods that are well known to those skilled in the art can be used to construct recombinant vectors which will express a nucleic acid sequence that is complementary to a target gene. These techniques are described both in Sambrook et al., 1989 and in Ausubel et al., 1992. For example, gene expression can be inhibited by transforming a cell or tissue with an expression vector that expresses high levels of untranslatable sense or antisense sequences. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and even longer if appropriate replication elements included in the vector system.
  • Various assays may be used to test the ability of antisense oligonucleotides to inhibit gene expression. For example, mRNA levels can be assessed by Northern blot analysis (Sambrook et al., 1989; Ausubel et al., 1992; J. C. Alwine et al. 1977, Proc. Natl. Acad. Sci. USA 74:5350-5354; I. M. Bird, 1998, Methods Mol. Biol. 105:325-36), quantitative or semi-quantitative RT-PCR analysis (see, e.g., W. M. Freeman et al., 1999, Biotechniques 26:112-122; Ren et al., 1998, Mol. Brain Res. 59:256-63; J. M. Cale et al., 1998, Methods Mol. Biol. 105:351-71), or in situ hybridization (reviewed by A. K. Raap, 1998, Mutat. Res. 400:287-298). Alternatively, polypeptide levels can be measured, e.g., by western blot analysis, indirect immunofluorescence, or immunoprecipitation techniques (see, e.g., J. M. Walker, 1998, Protein Protocols on CD-ROM, Humana Press, Totowa, N.J.).
  • In specific embodiments, the lipid vehicles of the present invention carry nucleotide sequences encoding cytotoxins (e.g., diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), and the pertussis adenylate cyclase (CYA)), antisense nucleic acids (e.g., NGF antisense), ribozymes, labeled nucleic acids, and nucleic acids encoding tumor suppressor genes such as p53, p 110Rb, and p72.
  • NGF antisense nucleic acids have been described by, e.g., K. A. Chang et al., 1999, J. Mol. Neurosci. 12(1):69-74; C. Culmsee et al., 1999, Neurochem. Int. 35(1):47-57; F. Hallbook et al., 1997, Antisense Nucleic Acid Drug Dev. 7(2):89-100. Such antisense nucleic acids can be used with the lipid vehicles of the invention for treating NGF-related diseases, including disorders of the brain (e.g., Alzheimer's) (see, e.g., K. A. Chang et al., 1999, J. Mol. Neurosci. 12(1):69-74; R. Hellweg et al., 1998, Int. J. Dev. Neurosci. 16(7-8):787-94) and bladder (e.g., inflammation and dysfunction) (see, e.g., M. A. Vizzard, 2000, Exp. Neurol. 161(1):273-84; D. Oddiah et al., 1998, Neuroreport. 9(7):1455-8; M. C. Dupont et al., 1995, Adv. Exp. Med. Biol. 385:41-54).
  • Antibodies
  • Lipid vehicles of the present invention can be conjugated to antibodies, i.e., polyclonal and/or monoclonal antibodies, fragments thereof, or immunologic binding equivalents thereof. The term antibody is used both to refer to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities. Antibodies can include whole antibody molecules, hybrid antibodies, chimeric antibodies, and univalent antibodies. Also included are antibody fragments, including Fc, Fv, Fab1, and F(ab)2 fragments of antibodies.
  • Antibodies may be obtained from commercial sources, e.g., Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.; Advanced Targeting Systems, San Diego, Calif.; Connex GmbH (Martinsried, Germany), Covance Research Products, Cumberland, Va.; Pierce Endogen, Rockford, Ill.; DiaSorin, Stillwater, Minn.; and DAKO Corporation, Carpinteria, Calif. Alternatively, antibodies may be produced in an animal host (e.g., rabbit, goat, mouse, or other non-human mammal) by immunization with immunogenic components. Antibodies may also be produced by in vitro immunization (sensitization) of immune cells. The antibodies may also be produced in recombinant systems programmed with appropriate antibody-encoding DNA. Alternatively, the antibodies may be constructed by biochemical reconstitution of purified heavy and light chains.
  • An isolated polypeptide or portion thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation (see, e.g., E. Harlow and D. Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). A full-length polypeptide can be used or, alternatively, antigenic peptide portions can be used as immunogens. An antigenic peptide typically comprises at least 5 contiguous amino acid residues, and encompasses an epitope of a polypeptide such that an antibody raised against the peptide forms a specific immune complex with the peptide. The immunogenic polypeptides or peptides for use with the present invention may be isolated from cells or may be chemically synthesized.
  • An appropriate immunogenic preparation can contain, for example, a recombinantly produced polypeptide or a chemically synthesized polypeptide, or portions thereof. The preparation can further include an adjuvant or similar immunostimulatory agent. A number of adjuvants are known and used by those skilled in the art. Non-limiting examples of suitable adjuvants include incomplete Freund's adjuvant, mineral gels such as alum, aluminum phosphate, aluminum hydroxide, aluminum silica, and surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • Further examples of adjuvants include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3 hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/TWEEN®80 emulsion. A particularly useful adjuvant comprises 5% (wt/vol) squalene, 2.5% Pluronic L121 polymer and 0.2% polysorbate in phosphate buffered saline (Kwak et al., 1992, New Eng. J. Med. 327:1209-1215). Preferred adjuvants include complete BCG, Detox, (RIBI, Immunochem Research Inc.), ISCOMS, and aluminum hydroxide adjuvant (Superphos, Biosector). The effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against the immunogenic peptide.
  • Polyclonal antibodies to polypeptides can be prepared as described above by immunizing a suitable subject with an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique (see Kohler and Milstein, 1975, Nature 256:495-497; Brown et al., 1981, J. Immunol. 127:539-46; Brown et al., 1980, J. Biol. Chem. 255:4980-83; Yeh et al., 1976, PNAS 76:2927-31; and Yeh et al., 1982, Int. J. Cancer 29:269-75), the human B cell hybridoma technique (Kozbor et al., 1983, Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques.
  • The technology for producing hybridomas is well-known (see generally R. H. Kenneth, 1980, Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y.; E. A. Lerner, 1981, Yale J. Biol. Med., 54:387-402; M. L. Gefter et al., 1977, Somatic Cell Genet. 3:231-36). In general, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds to the polypeptides or peptides.
  • Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the corresponding polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).
  • Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al., 1991, Bio/Technology 9:1370-1372; Hay et al., 1992, Hum. Antibod. Hybridomas 3:81-85; Huse et al., 1989, Science 246:1275-1281; Griffiths et al., 1993, EMBO J 12:725-734; Hawkins et al., 1992, J. Mol. Biol. 226:889-896; Clarkson et al., 1991, Nature 352:624-628; Gram et al., 1992, PNAS 89:3576-3580; Garrad et al., 1991, Bio/Technology 9:1373-1377; Hoogenboom et al., 1991, Nuc. Acid Res. 19:4133-4137; Barbas et al., 1991, PNAS 88:7978-7982; and McCafferty et al., 1990, Nature 348:552-55.
  • Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies comprising both human and non-human portions, can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al., 1988, Science 240:1041-1043; Liu et al., 1987, PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; S. L. Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al., 1988, Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060.
  • Fv fragments of monoclonal antibodies may be produced in bacteria using single chain antibody technology (U.S. Pat. No. 4,946,778 and International Application No. WO 88/09344). In addition, Fv fragments can be genetically engineered to contain glycosylation sites. These engineered Fv fragments can then be produced in mammalian cells, to result in a fragment containing carbohydrate moieties. Fab or F(ab′)2 fragments of monoclonal antibodies may be produced by enzymatic cleavage of whole IgG which is produced by a hybridoma or a transfected cell lines (e.g., a myeloma or a cell line such as Chinese Hamster Ovary (CHO) cells), using pepsin or papain digestion, respectively.
  • The antibodies or antibody fragments can be conjugated to liposomes using conventional techniques (see, e.g., M. J. Ostro (ed.) 1987, Liposomes: from Biophysics to Therapeutics, Marcel Dekker, New York, N.Y.). One preferred method of preparing liposomes and conjugating immunoglobulins to their surface is described by Y. Ishimoto et al., 1984, J. Immunol Met. 75:351-360. In accordance with this method, multilamillar liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, and phosphotidylethanolamine are prepared. Purified fragments are then coupled to the phosphatidylethanolamine by the cross-linking agent N-hydroxysuccinimidyl 3-(2-pyridyidithio) propionate. The coupling of the antibody or fragment to the liposome is demonstrated by the release of a pre-trapped marker, e.g., carboxyfluorescence, from the liposomes. This release occurs upon incubation with a secondary antibody against the conjugated antibody, fragment, or complement.
  • The antibodies or antibody fragments can also be coupled to a liposome or another carrier of the invention via carbohydrate moieties. Such coupling can be used provided that the carbohydrate moiety is not in the hypervariable region or at the antibody binding sites. In this way, conjugation via the cross-linking with the carbohydrate will not affect binding, and the binding sites will still be available to bind to cell surface antigens. One preferred method for coupling antibodies or antibody fragments of the invention (other than Fv) to a polymer backbone or a liposome involves conjugation through the carbohydrate moieties in the constant regions. This maximizes the number of available antigen-binding sites. Methods for derivatizing sugar ring moieties to create hydrazide groups for coupling with antibody fragments (and antibodies) have been established (see J. D. Rodwell et al., 1986, Proc. Natl. Acad. Sci. USA 83:2632-36). Several immunoconjugates prepared in this way are in clinical studies or pending approval for routine clinical uses.
  • Binding of a monoclonal antibody to the surface of a liposome may also be accomplished by the formation of cross-linkage between phosphatidylethanolamine and the antibody using glutaraldehyde. Alternatively, a thiolated antibody can be allowed to react with a liposome comprising a lipid into which a maleimide group has been incorporated. Remaining maleimide groups on the surface of the liposome may be further reacted with a compound containing thiolated polyalkyleneglycol moiety. Thiolation of an antibody or antibody fragment may be achieved through use of N-succinimidyl-3-(2-pyridyidithio)propionate (SPDP), which is usually used for thiolation of protein, iminothiolane, or mercaptoalkylimidate. Alternatively, a dithiol group endogenous to an antibody may be reduced to form a thiol group. The latter method is preferred for maintaining antibody function. In accordance with another method, whole antibodies are treated with an enzyme such as pepsin to form F(ab)2 fragments, which are then reduced with dithiothreitol (DTT) to form Fab fragments, which results in the production of one to three thiol groups. The conjugation of the thiolated antibody to the maleimide group-containing liposome may be accomplished by reacting the components in a neutral buffer solution at pH 6.5-7.5 for 2-16 hours.
  • In specific embodiments, the lipid vehicles of the present invention are conjugated to antibodies or antibody fragments directed to NGF receptor or uroplakin.
  • Pharmaceutical Compositions
  • The lipid vehicles and methods of the present invention can be used to deliver a broad range of pharmaceutical compositions and drugs. In addition to the aforementioned nucleic acids, the vehicles of the present invention carry small organic or inorganic compounds as bioactive agents. Suitable pharmaceuticals or bioactive agents include, but are not limited to, antimicrobials, antibiotics, antimycobacterial, antifungals, antivirals, neoplastic agents, agents affecting the immune response, blood calcium regulators, agents useful in glucose regulation, anticoagulants, antithrombotics, antihyperlipidemic agents, cardiac drugs, thyromimetic and antithyroid drugs, adrenergics, antihypertensive agents, cholinergics, anticholinergics, antispasmodics, antiulcer agents, skeletal and smooth muscle relaxants, prostaglandins, general inhibitors of the allergic response, antihistamines, local anesthetics, analgesics, narcotic antagonists, antitussives, sedative-hypnotic agents, anticonvulsants, antipsychotics, anti-anxiety agents, antidepressant agents, anorexigenics, non-steroidal anti-inflammatory agents, steroidal anti-inflammatory agents, antioxidants, vaso-active agents, bone-active agents, antiarthritics, and diagnostic agents.
  • In certain preferred aspects, the bioactive agent will be an antineoplastic agent, such as vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, streptozotocin, and the like. Especially preferred antitumor agents include, for example, actinomycin D, vincristine, vinblastine, cystine arabinoside, anthracyclines, alkylative agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs. Anticancer agents further include carcinostatic agents such as adriamycin, daunomycin, mitomycin, epirubicin, 5-FU, and aclacinomycin, toxins such as ricin A and diphtheria toxin, and antisense RNA. Encapsulation of anticancer agent into lipome vehicles can be accomplished by hydration of the lipids with an aqueous solution of the anticancer agent. Adriamycin, daunomycin, and epirubicin may be encapsulated into a liposome by means of a remote loading method that takes advantage of a pH gradient (D. M. Lawrence et al., 1989, Cancer Research 49:5922).
  • In certain aspects, the lipid vehicles of the present invention can be used to deliver anti-infective agents. The vehicles of the present invention can also be used for the selective delivery of other drugs including, but not limited to, local anesthetics, e.g., dibucaine and chlorpromazine; beta-adrenergic blockers, e.g., propranolol, timolol and labetolol; antihypertensive agents, e.g., clonidine and hydralazine; anti-depressants, e.g., imipramine, amitriptyline and doxepim; anticonversants, e.g., phenytoin; antihistamines, e.g., diphenhydramine, chlorphenirimine and promethazine; antibiotic/antibacterial agents, e.g., gentamycin, ciprofloxacin, and cefoxitin. Also included are antibiotics such as macrolides and lincosamines (e.g., lincomycin, erythromycin, dirithromycin, clindamycin, clarithromycin, and azithromycin); ample spectrum penicillins (e.g., ticarcillin, piperacillin, mezlocillin, carbenicillin indanyl, bacampicillin, ampicillin, and amoxicillin); penicillins and beta-lactamase inhibitors (e.g., amoxicillin-clavulanic acid, ampicillin-sulbactam, benzylpenicillin, cloxacillin, dicloxacillin, methicillin, oxacillin, penicillin G (benzathine, potassium, procaine), penicillin V, piperacillin plus tazobactam, and ticarcillin plus clavulanic acid); aminoglycosides (e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, and tobramycin); and tetracyclines (e.g., tetracycline, oxytetracycline, minocycline, methacycline, doxycycline, and demedocycline). Further included are antifungal agents, e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine and amphotericin B; antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, narcotics, and imaging agents. Those of skill in the art will know of other agents suitable for use with the formulations and methods of the present invention.
  • In specific embodiments, the lipid vehicles of the present invention deliver vanilloid components, e.g., resiniferatoxin, capsaicin, tinyatoxin, and related compounds. Additionally, the lipid vehicles may deliver toxins, e.g., botulinum toxins, such as botulinum toxin A, botulinum toxin B, botulinum toxin C, botulinum toxin D, botulinum toxin E, botulinum toxin F, and botulinum toxin G. Lipid vehicles can be formulated as described herein, or by other methods known in the art (e.g., U.S. Pat. No. 6,334,999 to Gilbert et al.; U.S. Pat. No. 6,083,530 to Mayer et al.; U.S. Pat. No. 5,939,096 to Clerc et al.; U.S. Pat. No. 5,795,589 to Mayer et al.; U.S. Pat. No. 5,744,158 to Mayer et al.; and U.S. Pat. No. 5,616,341 to Mayer et al., which are incorporated herein by reference).
  • Preferably, a composition (e.g., pharmaceutical composition) includes, in admixture, a pharmaceutically acceptable excipient, carrier, or diluent, and one or more of a bioactive agent (e.g., nucleic acid, polypeptide, peptide, or antibody), drug (e.g., resiniferatoxin, capsaicin, tinyatoxin, or other vanilloid compounds), or toxin (e.g., botulinum toxin), as described herein, as an active ingredient. The preparation of pharmaceutical compositions that contain bioactive agents as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. Preferred carriers, excipients, and diluents of the invention comprise physiological saline (i.e., 0.9% NaCl). In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH-buffering agents, which enhance the effectiveness of the active ingredient.
  • A bioactive agent or drug can be formulated into the pharmaceutical composition as neutralized physiologically acceptable salt forms. Suitable salts include the acid addition salts (i.e., formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • The pharmaceutical compositions and vehicles can be administered systemically by oral or parenteral routes. Non-limiting parenteral routes of administration include subcutaneous, intravesical, intramuscular, intraperitoneal, intravenous, transdermal, inhalation, intranasal, intra-arterial, intrathecal, enteral, sublingual, or rectal. Intravenous administration, for example, can be performed by injection of a unit dose. The term unit dose when used in reference to a pharmaceutical composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier. The disclosed pharmaceutical compositions and vehicles can also be administered via pulmonary inhaler or mucoactive aerosol therapy (nasal spray; see, e.g., M. Fuloria and B. K. Rubin, 2000, Respir. Care 45:868-873; I. Gonda, 2000, J. Pharm. Sci. 89:940-945; R. Dhand, 2000, Curr. Opin. Pulm. Med. 6(1):59-70; B. K. Rubin, 2000, Respir. Care 45(6):684-94; S. Suarez and A. J. Hickey, 2000, Respir. Care. 45(6):652-66). In addition, topical administration can be used. Preferably, the disclosed pharmaceutical compositions and vehicles are administered by intravesical instillation.
  • Pharmaceutical compositions can be administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of modulation required. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are specific for each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration.
  • Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusions sufficient to maintain concentrations of 10 nM to 10 μM in the blood are contemplated. An exemplary pharmaceutical formulation comprises: a peptide or polypeptide (5.0 mg/ml); sodium bisulfite USP (3.2 mg/ml); disodium edetate USP (0.1 mg/ml); and water for injection q.s.a.d. (1.0 ml). As used herein, pg means picogram, ng means nanogram, μg means microgram, mg means milligram, μl means microliter, ml means milliliter, and I means L.
  • Further guidance in preparing pharmaceutical formulations is found in, e.g., Gilman et al. (eds), 1990, Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed., 1990, Mack Publishing Co., Easton, Pa.; Avis et al. (eds), 1993, Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, New York; Lieberman et al. (eds), 1990, Pharmaceutical Dosage Forms: Disperse Systems, Dekker, N.Y.
    • Therapeutic Applications
  • In various aspects, the present invention encompasses novel methods of treatment that utilize the disclosed lipid vehicles. In particular, treatments are provided for cancers, infections, pain (e.g., neuropathic pain), and other conditions relating to the bladder, genitourinary tract, gastrointestinal tract, pulmonary system, and other organs or body systems. Specifically encompassed are treatments for urinary system components, e.g., kidneys, ureters, bladders, sphincter muscles, and urethras. Non-limiting examples of gastrointestinal organs include the esophagus, stomach, large intestine, and small intestine. Pulmonary system organs include, among other organs, the trachea, lungs, bronchi, bronchioles, alveoli, and cilia. Genitourinary tract organs include, but are not limited to, the bladder, kidney, urethra, ureter, prostate, penis, testes, seminiferous tubules, epididymis, vas deferens, seminal vesicles, bulbourethral (Cowper) glands, uterus, vagina, and fallopian tubes. Non-limiting examples of bladder conditions include spastic neurogenic bladder, hypotonic neurogenic bladder, and bladder hyperactivity, pain, irritation, inflammation, micturition pattern alteration, incontinence, infection, and cancer. Bladder cancers suitable for treatment include, for example, transitional cell carcinomas, squamous cell carcinomas, and adenocarcinomas. Also included are conditions relating to IC and UDSD.
  • The present invention further encompasses methods of treating conditions associated with involuntary muscle contractions, including, but not limited to, tremor (voice, head, and limb tremor); palatal myoclonus; dysthyroid myopathy, hemifacial spasms; tics; strabismus (e.g., concomitant strabismus and vertical strabismus); nystagmus; eyelid entropion; myokymia; bruxism (TMJ); tardive dyskinetic syndrome, lateral rectus palsy; hyperkinesias following hypoglossal-facial anastomosis; myoclonus of spinal cord origin; voice defects (e.g., stuttering); painful rigidity; tension headaches; lumbosacral strain and back spasm (myofascial); radiculopathy with secondary muscle spasm; spasticity; IC, spastic bladder; UDSD; achalasia (esophageal); pelvirectal spasms (anismus and vaginismus); segmental dystonia, focal dystonia (e.g., blepharospasm (lid apraxia); oromandiibular distonia, facial dystonia, lingual dystonia, cervical dystonia (torticollis) and spasticity; laryngeal dystonia (spasmodic dysphonia; adductor spasmodic dysphonia, and abductor spasmodic dysphonia); task-specific dystonia (occupational cramps, such as writer's cramps); idiopathic and secondary focal distonia); and other spastic disorders. Treatments of involuntary contractions may be directed to any muscle groups, including those associated with control of the eye(s), lip(s), tongue, mouth, jaw, head, neck, face, arm, hand, finger, leg, trunk, vagina, cervix, bladder, and sphincter (e.g., esophageal, cardiac, pyloric, ileocaecal, O'Beirne, anal, urethra, and bladder neck sphincters). Treatments are also provided for furrows of the face and neck, including frown lines and facial wrinkles.
  • The methods of the present invention can be used to treat an animal, preferably a mammal, more preferably a human subject. The disclosed methods comprise administering a lipid vehicle to a mammal suffering from one or more of these conditions. In one aspect, the lipid vehicle carries a biological agent (e.g., nucleic acid, peptide, polypeptide, or antibody), drug (e.g., pain therapeutics, anticancer treatments, or antibiotics), or toxin (e.g., botulinum toxin). For example, if the disease is the result of infection by a pathogen, the nucleic acid can be an antisense oligonucleotide targeted against a DNA sequence in the pathogen that is essential for development, metabolism, or reproduction of the pathogen. As another example, if the disease is related to a genetic defect (i.e., wherein certain endogenous DNA is missing or has been mutated), resulting in under- or over-expression, the nucleic acid maybe the normal DNA sequence.
  • Several methods of in vivo lipofection have been reported. In the case of whole animals, the lipid vehicle may be injected into the blood stream, directly into a tissue, into the peritoneum, instilled into the trachea, or converted to an aerosol, which the animal breathes. For example, a single intravenous injection of 100 micrograms of a mixture of DNA and DOTMA:dioleoylphosphatidylethanaolamine can be used to efficiently transfect all tissues (Zhu et al., 1993, Science 261:209-211). It is also possible to use a catheter to implant lipid vehicles in a blood vessel wall, which can result in successful transfection of several cell types, including endothelial and vascular smooth muscle cells. In particular, aerosol delivery of a chloramphenicol acetyltransferase (CAT) expression plasmid complexed to cationic liposomes produces high-level, lung-specific CAT gene expression in vivo for at least 21 days (Stribling et al., 1992, Proc. Natl. Acad. Sci. USA 89:11277-11281). One representative method for aerosol delivery has been performed for as follows: 1) 6 mg plasmid DNA and 12 μM DOTMA/DOPE liposomes were each diluted to 8 ml with water and mixed; 2) equal volumes were placed into two Acorn I nebulizers (Marquest, Englewood, Colo.); 3) animals were loaded into an Intox small-animal exposure chamber (Albuquerque) and an air flow rate of 4 L/min is used to generate the aerosol (about 90 min were required to aerosolize this volume); 4) the animals were removed from the chamber for 1-2 hr, and the procedure was repeated.
  • Specific targeting moieties can be used with the vehicles of this invention to target specific cells or tissues. In one embodiment, the targeting moiety, such as an antibody or antibody fragment, is attached to a hydrophilic polymer and is combined with the vehicle after vehicle formation. Thus, the use of a targeting moiety in combination with a vehicle provides the ability to conveniently customize the vehicle for delivery to specific cells and tissues. In specific embodiments, the disclosed vehicles carrying drugs (e.g., pain remedies, anticancer, or antibiotics) and/or bioactive agents (e.g., nucleic acids, polypeptides, peptides, or antibodies) can be specifically targeted to cancer cells, immune cells (e.g., B and T cells), and cells of the bladder, genitourinary tract, gastrointestinal tract, pulmonary system, or other body organs or systems. Such cells can be targeted using antibodies or antibody fragments against cell surface antigens, including various receptors or markers.
  • For example, many cancers are characterized by overexpression of cell surface markers such as HER2, which is expressed in breast cancer cells, or IL-13 receptor, which is expressed in gliomas (reviewed in, e.g., J. Baselga et al., 1997, Oncology (Huntingt) 11(3 Suppl 2):43-8; S. Menard et al., 2000, J. Cell. Physiol. 182(2):150-62; W. Debinski, 1998, Crit. Rev. Oncog. 9(34):255-68). Certain urogenitary tract cancers are characterized by expression of the uroplakin marker (see, e.g., X. Xu et al., 2001, Cancer 93(3):216-21; J. J. Lu et al., 2000, Clin. Cancer Res. 6(8):3166-71; U. Kaufmann et al., 2000, Am. J. Clin. Pathol. 113(5):683-7; S. M. Li et al., 1999, J. Urol. 162(3 Pt 1):931-5; I. Yuasa et al., 1999, Int. J. Urol. 6(6):286-92; I. Yuasa et al., 1998, Jpn. J. Cancer Res. 89(9):879-82; R. L. Wu et al., 1998, Cancer Res. 58(6):1291-7). In addition, neurons are characterized by the expression of NGF receptor (reviewed by, e.g., L. Tessarollo, 1998, Cytokine Growth Factor Rev. 9(2):125-37; E. C. Yuen EC, et al., 1996, Brain Dev. 18(5):362-8; S. B. McMahon, 1996, Philos. Trans. R. Soc. Lond. B. Biol. Sci. 351(1338):431-40; G. Dechant et al., 1994, Prog. Neurobiol. 42(2):347-52). Thus, targeting moieties such as anti-HER2, anti-IL-13 receptor, and anti-NGF receptor antibodies or antibody fragments can be used to deliver the vehicle to the cell of choice. The bioactive agent and/or drug is thereby delivered to the specific cell type, providing a useful and specific therapeutic treatment.
  • Cationic lipid-assisted drug delivery can be accomplished in accordance with well-established methods. For drugs that are soluble in organic solvents, such as chloroform, the drug and cationic lipid are mixed in solvents in which both are soluble, and the solvent is then removed under vacuum. The lipid-drug residue is then dispersed in an appropriate aqueous solvent, e.g., sterile physiological saline. Optionally, the suspension is subjected to up to several freeze/thaw cycles. The suspension is then sonicated, to reduce the coarseness of the dispersion or to reduce the particle size to 20-30 nm diameter. This will depend on whether large or small particle size is most efficacious in the desired application. For some applications, it may be useful to generate extruded liposomes by forming the suspension through a filter with pores of 100 nm diameter or smaller. In addition, it may be useful to include cholesterol or natural phospholipids in the mixture to generate lipid-drug aggregates.
  • The vehicles of the present invention that carry a bioactive agent can be delivered in any suitable manner. For agents that are soluble in aqueous solution and insoluble in organic solvents, the lipid mixture to be used for the lipid dispersion or liposomes can be coated on the inside surface of a flask or tube by evaporating the solvent from a solution of the mixture. In general, the lipid mixture should be capable of forming vesicles having single or multiple lipid bilayer walls and encapsulating an aqueous core. The aqueous phase containing the dissolved agent (e.g., physiological saline solution) can then be added to the lipid, agitated to generate a suspension, and then optionally frozen and thawed up to several times.
  • In particular embodiments, the lipid vehicles of the invention can be used with or without vanilloid (e.g., capsaicin) and/or botulinum toxin (e.g., botulinum toxin D), which can then be used alone or in combination with a chemotherapeutic agent, targeting antibody, or DNA construct designed for the treatment of bladder cancer. Specifically, lipid vehicles or lipid vehicles comprising vanilloid and/or botulinum toxin can be used to prevent, treat, or ameliorate pain or voiding dysfunction associated with bladder cancer. Lipid-based treatments for bladder cancer that employ chemotherapeutic agents (see, e.g., J. B. Bassett et al., 1986, J. Urol. 135(3):612-5; C. P. Dinney et al., 1995, J. Interferon Cytokine Res. 15(6):585-92; T. Tsuruta et al., 1997, J. Urol. 1997 157(5):1652-4; H Kiyokawa et al., 1999, J. Urol. 161(2):665-7), targeting antibodies (see, e.g., J. Morgan et al., 1994, Photochem. Photobiol. 60(5):486-96; A. Aicher et al., 1994, Urol. Res. 22(1):25-32), and DNA constructs (e.g., Y. Horiguchi et al., 2000, Gene Ther. 7(10):844-51; L. A. Larchian et al., 2000, Clin. Cancer Res. 6(7):2913-20; M. Cemazar et al., 2002, Cancer Gene Ther. 9(4):399406) are known in the art.
  • In another embodiment, lipid vehicles of the invention may be used with or without vanilloid (e.g., capsaicin) and/or botulinum toxin (e.g., botulinum toxin D), which can then be used alone or in combination with one or more antibacterial agents. Specifically, lipid vehicles or lipid vehicles comprising vanilloid and/or botulinum toxin can be used to prevent, treat, or ameliorate pain or voiding dysfunction associated with a urinary system infection. Lipid-based treatments for infection are generally known in the art, including those employing tetracycline and doxycycline (L. Sangare et al., 1999, J. Med. Microbiol. 48(7):689-93; L. Sangare et al., 1998, J. Antimicrob. Chemother. 42(6):831-4); tobramycin (C. Beaulac et al., 1999, J. Drug Target. 7(1):3341); gentamycin and ceftazidime (R. M. Schiffelers et al., 2001, Int. J. Pharm. 214(1-2):103-5; R. M. Schiffelers et al., 2001, J. Pharm. Exp. Ther. 298(1)369-75); anthracycline (N. Dos Santos et al., 2002, Biochem. Biophys. Acta 1561(2):188-201); ciprofloxacin (B. Wiechens et al., 1999, Ophthalmologica 213(2):120-8), and other anti-infectives.
  • To generate small liposomes the suspension can be subjected to ultrasonic waves for a time necessary to reduce the liposomes to the desired average size. If large liposomes are desired, the suspension can be agitated by hand or on a vortex mixer until a uniform dispersion is obtained, i.e., until visually observable large particles are present. For vehicles comprising a bioactive agent or drug, alone, the agent or drug in the aqueous phase is eliminated by dialysis or by passage through a gel-filtration chromatographic column (e.g., agarose) equilibrated with the aqueous phase containing all normal components except the agent or drug. The lipid mixture used can contain cholesterol or natural lipids in addition to the liposome compounds of the present invention. The liposome-drug aggregate may then be delivered in any suitable manner (see above).
  • Pain Treatments
  • The present invention includes but is not limited to the following embodiments:
      • A method of treating pain in an organ in a mammalian subject which comprises administering to the subject a pharmaceutical composition comprising a lipid vehicle in an amount effective to treat the condition.
      • The method of the preceding embodiment, wherein the lipid vehicle is a liposome.
      • The method of any one of the preceding embodiments, wherein the organ is a genitourinary tract organ.
      • The method of any one of the preceding embodiments, wherein the genitourinary tract organ is selected from the group consisting of a bladder, kidney, urethra, ureter, prostate, penis, testes, seminiferous tubules, epididymis, vas deferens, seminal vesicles, bulbourethral glands, uterus, vagina, and fallopian tubes.
      • The method of any one of the preceding embodiments, wherein the organ is a gastrointestinal tract organ.
      • The method of any one of the preceding embodiments, wherein the gastrointestinal tract organ is selected from the group consisting of esophagus, stomach, large intestine, and small intestine.
      • The method of any one of the preceding embodiments, wherein the pain is associated with a condition selected from the group consisting of infection, inflammation, irritation, cancer, and spasticity.
      • The method of any one of the preceding embodiments, wherein the lipid vehicle is administered using a method selected from the group consisting of intravesical instillation, intravenous, topical, nasal spray, pulmonary inhaler, and oral administration.
      • The method of any one of the preceding embodiments, wherein the lipid vehicle further comprises a vanilloid compound.
      • The method of any one of the preceding embodiments, wherein the vanilloid is selected from the group consisting of capsaicin, resiniferatoxin, and tinyatoxin.
      • The method of any one of the preceding embodiments, wherein the lipid vehicle is a liposome.
      • The method of any one of the preceding embodiments, wherein the organ is a genitourinary tract organ.
      • The method of any one of the preceding embodiments, wherein the genitourinary tract organ is selected from the group consisting of a bladder, kidney, urethra, ureter, prostate, penis, testes, seminiferous tubules, epididymis, vas deferens, seminal vesicles, bulbourethral (Cowper) glands, uterus, vagina, and fallopian tubes.
      • The method of any one of the preceding embodiments, wherein the organ is a gastrointestinal tract organ.
      • The method of any one of the preceding embodiments, wherein the gastrointestinal tract organ is selected from the group consisting of esophagus, stomach, large intestine, and small intestine.
      • The method of any one of the preceding embodiments, wherein the organ is a pulmonary system organ.
      • The method of any one of the preceding embodiments, wherein the pulmonary system organ is selected from the group consisting of trachea, lungs, bronchi, bronchioles, alveoli, and cilia.
      • The method of any one of the preceding embodiments, wherein the lipid vehicle is administered by a method selected from the group consisting of intravesical instillation, intravenous, topical, nasal spray, pulmonary inhaler, and oral administration.
      • The method of any one of the preceding embodiments, wherein the carrier, excipient, or diluent comprises physiological saline.
  • Muscle Treatments
  • The present invention includes but is not limited to the following embodiments:
      • A method of treating involuntary muscle contraction in a mammalian subject which comprises administering to the subject a pharmaceutical composition comprising a lipid vehicle carrying botulinum toxin in an amount effective to treat the contraction.
      • The method of the preceding embodiment, wherein the lipid vehicle is a liposome.
      • The method of any one of the preceding embodiments, wherein the botulinum toxin is selected from the group consisting of botulinum toxins A through G.
      • The method of any one of the preceding embodiments, wherein the involuntary muscle contraction affects a body part selected from the group consisting of the eye, lip, tongue, mouth, jaw, head, neck, face, arm, hand, finger, leg, trunk, vagina, cervix, and bladder.
      • The method of any one of the preceding embodiments, wherein the involuntary muscle contraction affects a sphincter selected from the group consisting of esophageal, cardiac, pyloric, ileocaecal, O'Beirne, anal, urethra, and bladder neck sphincters.
      • The method of any one of the preceding embodiments, wherein the involuntary muscle contraction is associated with a condition selected from the group consisting of tremors, hemifacial spasms, tics, strabismus, nystagmus, eyelid entropion, myokymia, bruxism, tardive dyskinetic syndrome, lateral rectus palsy, stuttering, painful rigidity, tension headache, back spasm, radiculopathy, spasticity, spastic bladder, urinary detrusor-sphincter dyssynergia, achalasia, anismus, vaginismus, segmental dystonia, idiopathic dystonia, and secondary focal distonia.
      • The method of any one of the preceding embodiments, wherein the involuntary muscle contraction is associated with a focal dystonia selected from the group consisting of blepharospasm, oromandiibular distonia, facial dystonia, lingual dystonia, cervical dystonia, torticollis, spasmodic dysphonia, and task-specific dystonia.
      • The method of any one of the preceding embodiments, wherein the lipid vehicle is administered by a method selected from the group consisting of intravesical instillation, intravenous, topical, nasal spray, pulmonary inhaler, and oral administration.
      • The method of any one of the preceding embodiments, wherein the carrier, excipient, or diluent comprises physiological saline.
    EXAMPLES
  • The examples as set forth herein are meant to exemplify the various aspects of the present invention and are not intended to limit the invention in any way.
  • Example 1
  • A hyperactive bladder model in Sprague-Dawley rats was established by exposure to acetic acid, or protamine sulfate (PS) in potassium chloride (KCl) solution. This was followed by instillation of LP (liposomes) in saline (in the case of the former) or LP/KCl. CMG (continuous cystometrogram) changes were examined and results were compared with control (saline instillation), hyperactive bladder (acetic acid or PS/KCl) and treatment with LP.
  • Materials and Methods
  • Intravesical bladder pressure was recorded via a transurethral catheter in adult female Sprague-Dawley rats anesthetized with urethane (1.2 g/kg) administered by subcutaneous injection (sc). Some animals were pretreated with capsaicin (125 mg/kg, sc) four days prior to the experiments. Continuous cystometrograms (CMGs) were performed by slowly filling the bladder (0.04 ml/min) with solutions of various composition including saline, acetic acid (0.1%), potassium chloride (KCl, 500 mM), protamine sulfate (PS, 10 mg/ml), LP, PS/KCl, or LP/KCl. Parameters measured included intercontraction interval (ICl), amplitude of bladder contractions, compliance, and micturition pressure threshold (PT).
  • Animal Preparation
  • Thirty-four female Sprague-Dawley rats (250-300 g) were used in this study. Animals were anesthetized with urethane (1.2 g/kg, sc). Body temperature was maintained in the physiological range using a heating lamp.
  • Cystometrogram (CMG)
  • A transurethral bladder catheter (PE-50) was connected via a three-way stopcock to a pressure transducer and to a syringe pump. This was used to record intravesical pressure and to infuse solutions into the bladder. A control CMG was performed by slowly filling the bladder with saline (0.04 ml/min) to elicit repetitive voiding. The parameters recorded were amplitude, pressure threshold (PT), compliance, and intercontraction interval (ICl) of reflex bladder contractions. Measurements in each animal represented the average of 3 to 5 bladder contractions.
  • Induction of a Hyperactive Bladder
  • After performing control CMGs with saline infusion, five intravesical infusion experiments were performed in parallel: (1) infusion of protamine sulfate (PS) (Sigma Chemical Co.; 10 mg/ml) for one hour (N=6) to increase epithelial permeability; (2) infusion of KCl (500 mM) for one hour, then infusion with PS/KCl for another one hour, followed by infusion of either KCl for two hours (N=6) or LP/KCl for two hours (N=6); (3) infusion of acetic acid (AA) (0.1%) for one hour, followed by infusion of saline (N=6) or LP (N=6) for two hours; (4) infusion of LP for one hour, followed by infusion of AA for one hour (N=6); and (5) infusion of AA (N=4) for two hours in animals subcutaneously injected with capsaicin (125 mg/kg in 10% ethanol, 10% TWEEN®80, 80% saline) four days before the experiment (C. L. Cheng et al., 1993, Am. J. Physiol. 265:R132-138). The experimental design is illustrated in FIG. 1. The KCl concentration used was within the range of concentrations present in normal rat urine (M. Ohnishi et al., 2001, Toxicol. Appl. Pharmacol. 174:122-129).
  • Preparation of Liposomes (LP)
  • LP were prepared as described by Kirby and Gregoriadis (C. J. Kirby and G. Gregoriadia, 1984, “A simple procedure for preparing liposomes capable of high encapsulation efficiency under mild conditions” Liposome Technology, G. Gregnoriadis, Ed., C.R.C. Press, Inc., Boca Raton, Fla., vol. 1, p.20). Briefly, LP were constructed as a 2:1 molar ratio of L-a-phosphatidylcholine and cholesterol (Sigma chemical Co., St. Louis, Mo.) to a final lipid concentration of 2 mg/ml in saline. Lipids in chloroform were dried down together in the proper ratio under nitrogen. The residues were reconstituted as LP in saline or 500 mM KCl by intense sonication. This lipid composition produced liposomes with no net charge.
  • Statistical Analysis
  • Statistical analyses were performed using Student's t test for paired or unpaired data, where applicable. A p-value less than 0.05 was considered significant. All data are expressed in Example 2 (below) as means±S.E (standard error).
  • Example 2
  • The results of the experiments described in Example 1 are presented herein below. In summary, ICl (intercontraction interval) was decreased after exposure to acetic acid (79.8% decrease) or PS/KCl (81% decrease). However, ICl was not changed by LP, PS, or KCl alone. The decrease in ICl was partly reversed after infusion of LP (172.8% increase) or LP/KCl (63% increase), but was not significantly changed after by saline or KCl administration. Pretreatment with capsaicin delayed the onset of the irritative effects of acetic acid by approximately 30 to 60 min, but did not change the magnitude after two hours of infusion.
  • CMGs in PS/KCl Infusion Group
  • As shown in Tables 1A-1C, infusion of PS (10 mg/ml) or KCl (500 mM) alone did not significantly change the CMGs. However infusion of PS/KCl provided an irritative effect after a delay of 30 to 40 min (FIGS. 2B, 2D). The ICl and compliance were significantly reduced by 79-83% (from 15.8±1.4 to 2.7±1.0 min or from 16.3±1.5 to 3.4±0.7 min) and 58-75% (from 0.284±0.028 to 0.070±0.019 ml/cm H2O or from 0.226±0.050 to 0.096±0.037 ml/cm H2O) in two series of experiments.
  • Bladder contraction amplitude was significantly increased (23%) in one series (Table 1C), but not in the other series (Table 1B). However, taking the average of the two series, bladder contraction amplitude showed a significant increase (16%). PT was not significantly changed. When the infusion fluid was switched to LP/KCl, after a delay of 10 to 20 min, the ICl was significantly increased (63%, from 2.7±1.0 to 4.4±1.2 min). Switching to KCl alone did not alter the ICl for periods as long as 120 min (FIGS. 2E-2F; Tables 1B-1C). PT was significantly increased after shifting to LP/KCl or KCl infusion. Compliance was not significantly changed after shifting to LP/KCl infusion, but was further reduced (from 0.096±0.037 to 0.043±0.014 ml/cm H2O) after shifting to KCl infusion.
    TABLE 1A
    Effects of saline and protamine sulfate (PS) on CMG parameters
    ICI Compliance PT Amplitude
    (min) (ml/cm H2O) (cm H2O) (cm H2O)
    Saline 13.8 ± 3.1 0.197 ± 0.030 7.6 ± 0.7 23.5 ± 1.7
    PS (10 mg/ml) 14.9 ± 1.7 0.210 ± 0.031 5.9 ± 0.9 27.5 ± 0.7

    Parameters included intercontraction interval (ICI), compliance, pressure threshold (PT), and amplitude. No statistically significant differences were observed between saline and 1 hour of treatment with PS.

    Values are means ± S.E., N = 6.
  • TABLE 1B
    Effects of saline, KCl, PS/KCl, and LP/KCl on CMG parameters
    ICI Compliance PT Amplitude
    (min) (ml/cm H2O) (cm H2O) (cm H2O)
    Saline 15.6 ± 2.1 0.270 ± 0.020 7.3 ± 0.8 26.5 ± 1.1
    KCl 15.8 ± 1.4 0.284 ± 0.028 8.2 ± 0.9 28.8 ± 1.2
    PS/KCl  2.7 ± 1.0*  0.070 ± 0.019* 6.7 ± 0.7 31.2 ± 1.0
    LP/KCl  4.4 ± 1.2* 0.065 ± 0.029 10.3 ± 0.6* 31.3 ± 2.8

    Parameters included intercontraction interval (ICI), compliance, pressure threshold (PT), and amplitude.

    Values are means ± S.E., N = 6.

    *P < 0.05, in comparison with pre-treatment.
  • TABLE 1C
    Effects of saline, KCl, PS/KCl, and KCl on CMG parameters
    ICI Compliance PT Amplitude
    (min) (ml/cm H2O) (cm H2O) (cm H2O)
    Saline 18.0 ± 2.0 0.270 ± 0.022  6.3 ± 0.7 25.7 ± 1.6
    KCl 16.3 ± 1.5 0.226 ± 0.050  6.8 ± 0.6 28.8 ± 2.2
    PS/KCl  3.4 ± 0.7* 0.096 ± 0.037* 7.3 ± 0.4  35.3 ± 2.6*
    KCl  2.9 ± 0.5 0.043 ± 0.014* 11.8 ± 1.6* 33.0 ± 2.3

    Parameters included intercontraction interval (ICI), compliance, pressure threshold (PT), and amplitude.

    Values are means ± S.E., N = 6.

    *P < 0.05, in comparison with pre-treatment.
  • CMGs in AA Infusion Group
  • The irritative effect of AA was evident at about 20 to 30 min following infusion. ICl and compliance were significantly reduced by 75-84% (from 15.3±2.2 to 2.4±0.5 min or from 12.6±2.0 to 3.2±1.3 min) and 71-76% (from 0.296±0.040 to 0.071±0.016 ml/cm H2O or from 0.275±0.048 to 0.079±0.026 ml/cm H2O) in two series of experiments (FIGS. 3A-3F; Tables 2A-2B). Amplitude was less affected, showing a slight increase, and PT was not significantly changed. Upon subsequent infusion of LP, ICl and compliance were significantly increased (179%, from 2.4±0.5 to 6.7±1.5 min; and 38%, from 0.071±0.016 ml/cm H2O to 0.114±0.020 ml/cm H2O) after approximately 10 to 20 min (FIGS. 3E-3F; Tables 2A-2B). This increase persisted for as long as 120 min after switching to infusion of saline. LP infusion alone for 1 hour did not change the micturition reflex in untreated animals (Table 2C); and the effect of an AA infusion was not reduced by prior intravesical administration of LP.
    TABLE 2A
    Effects of saline, acetic acid (AA), and liposomes (LP) on CMG
    parameters
    ICI Compliance PT Amplitude
    (min) (ml/cm H2O) (cm H2O) (cm H2O)
    Saline 15.3 ± 2.2  0.296 ± 0.040  6.6 ± 0.9 28.7 ± 1.7
    AA 2.4 ± 0.5* 0.071 ± 0.016* 8.3 ± 0.9 36.8 ± 6.5
    LP 6.7 ± 1.5* 0.114 ± 0.020* 10.2 ± 1.8  30.8 ± 6.5

    Parameters included intercontraction interval (ICI), compliance, pressure threshold (PT), and amplitude.

    Values are means ± S.E., N = 6.

    *P < 0.05, in comparison with pre-treatment.
  • TABLE 2B
    Effects of saline, AA, and saline on CMG parameters
    ICI Compliance PT Amplitude
    (min) (ml/cm H2O) (cm H2O) (cm H2O)
    Saline 12.6 ± 2.0 0.275 ± 0.048 7.2 ± 1.0 28.2 ± 3.2
    AA  2.9 ± 0.9*  0.079 ± 0.026* 9.1 ± 1.4 37.0 ± 7.0
    Saline  3.2 ± 1.3 0.063 ± 0.011 8.6 ± 1.2  28.2 ± 7.0*

    Parameters included intercontraction interval (ICI), compliance, pressure threshold (PT), and amplitude.

    Values are means ± S.E., N = 6.

    *P < 0.05, in comparison with pre-treatment.
  • TABLE 2C
    Effects of saline, LP, and AA on CMG parameters
    ICI Compliance PT Amplitude
    (min) (ml/cm H2O) (cm H2O) (cm H2O)
    Saline 14.7 ± 3.0 0.254 ± 0.028 7.0 ± 0.8 29.8 ± 2.4
    LP 16.0 ± 1.9 0.255 ± 0.023 6.7 ± 0.6 30.0 ± 2.3
    AA  3.1 ± 0.8*  0.071 ± 0.022* 6.7 ± 0.7  38.0 ± 1.3*

    Parameters included intercontraction interval (ICI), compliance, pressure threshold (PT), and amplitude.

    Values are means ± S.E., N = 6.

    *P < 0.05, in comparison with pre-treatment.
  • CMGs in Animals Pretreated with Capsaicin
  • In capsaicin pretreated animals, bladder hyperactivity evoked by AA was delayed for 0.5-1 hour. ICl and compliance were reduced in magnitude by 51% and 33% at 1 hour (from 21.0±2.4 to 10.2±3.0 min and from 0.226±0.033 to 0.152±0.028 ml/cm H2O), but at 2 hours was similar to the effect in untreated animals (78% decrease to 4.6±1.2 min and 64% decrease to 0.082±0.024 ml/cm H2O) (Table 3). In addition, the reduction of ICl (10.2±3.0 min) after 1 hour application of AA in capsaicin-pretreated rats (Table 3) was significantly longer (p<0.05) compared to the ICl (2.9±0.9 min) measured within 1 hour of application in untreated rats (Table 2B). This indicated that C-fiber desensitization by capsaicin pretreatment suppressed AA-induced bladder hyperactivity and delayed the onset of AA-induced hyperactivity.
    TABLE 3
    Effects of saline & AA on CMG parameters in capsaicin pretreated
    rats
    ICI Compliance PT Amplitude
    (min) (ml/cm H2O) (cm H2O) (cm H2O)
    Saline 21.0 ± 2.4  0.226 ± 0.033  7.9 ± 0.8 21.1 ± 1.3
    AA (1 hr) 10.2 ± 3.0* 0.152 ± 0.028* 6.7 ± 0.2 20.9 ± 2.5
    AA (2 hr) 4.6 ± 1.2 0.082 ± 0.024* 6.7 ± 0.7 23.6 ± 5.7

    Parameters included intercontraction interval (ICI), compliance, pressure threshold (PT), and amplitude.

    Values are means ± S.E., N = 4.

    *P < 0.05, in comparison with pre-treatment.
  • SUMMARY
  • The sum of the results from Examples 1-2 indicated that 1) intravesical administration of LP suppressed chemically-induced bladder hyperactivity; and 2) low-dose PS treatment in the presence of physiological KCl produced sustained bladder hyperactivity. The administration of LP thereby represents new treatment approaches for a damaged or leaky urothelium, while low-dose PS provides a pharmacological model for examination of drugs that might restore the leaky urothelium. In addition, it is of interest that AA-induced hyperactivity was also reduced by LP. This indicated that chemically-induced irritation/inflammation of the bladder mucosa, as well as direct breakdown of the urothelial barrier by PS, could both be reversed by LP. Without wising to be bound by theory, it is possible that the effects of LP, as observed above, were mediated by the production of a film on the urothelium that reduced the influx of irritants. It is also possible that LP stabilized neuronal membranes and reduced the hyper-excitability of afferent receptors.
  • From these experiments, it became clear that bladder afferents played a key role in the mechanism of action of AA in the induction of bladder hyperactivity. Previous experiments have shown that infusion of AA into the bladder stimulates nociceptive afferent fibers, induces an inflammatory reaction and evokes a hyperactive bladder (Y. Yu and W. C. de Groat, 1998, Brain Res. 807:11-18; L. A. Birder and W. C. de Groat, 1992, J. Neurosci. 12:48784889; K. B. Thor and M. A. Muhlhauser, 1999, Am. J. Physiol. 277:R1002-1012). The stimulation of silent C-fibers has been implicated to play a central role in the pathogenesis of some hyperactive bladders; whereas A-δ afferents are usually thought to be primarily responsible for triggering normal voiding function (Y. Yu and W. C. de Groat, 1998, Brain Res. 807:11-18 13; C. L. Cheng et al., 1993, Am. J. Physiol. 265:R132-138; K. B. Thor and M. A. Muhlhauser, 1999, Am. J. Physiol. 277:R1002-1012). However, as shown above, capsaicin pretreatments at a dose known to desensitize C fiber bladder afferents delayed and reduced the effect of intravesical AA. This suggested that sensitization of myelinated A-δ afferents may also play a role in bladder hyperactivity induced by AA. The concentration of AA used in these experiments was 0.1%, which could dissolve the GAG layer, damage the urothelial barrier, and facilitate deeper penetration of irritant (M. Leppilahti et al., 1999, Urol. Res. 27:272-276). AA could also produce bladder hyperactivity through the VR1 receptors or proton sensitive channels (J. M. Welch et al., 2000, Proc. Natl. Acad. Sci. USA 97(25):13889-13894).
  • The prevailing theories for the pathogenesis of IC describe a leaky and dysfunctional urothelium that allows transepithelial migration of irritants such as potassium into the deep layers of bladder wall. There, the irritants depolarize afferent nerves and induce abnormal sensations as well as frequent voiding (C. L. Parsons et al., 1991, J. Urol. 145:732-735; C. L. Parsons et al., 1994, Br. J. Urol. 73:504-507; G. Hohlbrugger, 1999, Br. J. Urol. 83(suppl. 2):22-28; J. I. Bade et al., 1997, Br. J. Urol. 79:168-171). PS, which increases epithelial permeability (K. B. Thor and M. A. Muhlhauser, 1999, Am. J. Physiol. 277:R1002-1012), was used in the above experiments to increase the penetration of KCl through the urothelial barrier and induce a similar activation of afferent neurons. Prior to PS treatment, the same concentration of KCl did not alter voiding function. In addition, PS alone did not elicit bladder hyperactivity. Thus, it seems reasonable to assume that PS was not acting as a primary bladder irritant, and that under normal conditions KCl in the bladder lumen would not alter the excitability of afferent nerves in the bladder wall. However, combined exposure to these agents (see above) mimicked the condition observed in IC patients.
  • The use of PS/KCl, as described above, resulted in a decrease in ICl and compliance, an increase in bladder contraction amplitude, and no change in PT. High concentration of potassium has been used as a provocative test for IC patients (C. L. Parsons et al., 1998, J. Urol. 159:1862-1867). A previous study has shown that a high concentration of potassium triggers C-afferent fibers and causes further release of neurotransmitters or neuromodulators (J. Morrison et al., 1999, Scand. J. Urol. Nephrol. suppl 201:73-75). Subsequently, potassium induces the depolarization of detrusor muscle and provokes muscle contraction or tissue damage (G. Hohlbrugger, 1999, Br. J. Urol. 83(suppl. 2):22-28; C. L. Parsons et al., 1998, J. Urol. 159:1862-1867). The acute exposure of high concentration of potassium to the detrusor causes a decrease in bladder compliance and capacity (P. C. Stein et al., 1996, J. Urol. 155:1133-1138). In agreement with this, the experiments described above showed that ICl and compliance were decreased, but PT was not changed. However, it is known that high concentrations of potassium can irritate the urethra and cause high outlet resistance (G. Hohlbrugger, 1999, Br. J. Urol. 83(suppl. 2):22-28). Consistent with this, bladder contraction amplitude was elevated in the combination of our two series shown above.
  • The surface glycosaminoglycan (GAG) layer has been proposed as a protective barrier that coats the transitional cell surface (J. I. Bade et al., 1997, J. Urol. 79:168-171; G. Hohlbrugger, 1995, J. Urol. 154:6-15; C. L. Parsons et al., 1980, Science 208:605-607). A GAG layer defect has been suggested in a subset of IC patients (C. L. Parsons et al., 1991, J. Urol. 145:732-735; C. L. Parsons et al., 1994, Br. J. Urol. 73:504-507). LP are comprised of phospholipids in a system of concentric closed membranes and are used as a carrier for drugs or DNA constructs (K. Reimer et al., 1997, Dermatology 195(suppl. 2):93-99; M. Nishikawa et al., 2001, Human Gene Therapy 12:861-870; G. Gregoriadis, 1976, New Eng. J. Med. 295:704-710). LP-based compositions provide a high-moisture film for wounds and mediate wound healing without chronic inflammatory-reaction in the neodermal layer (K. Reimer et al., 1997, Dermatology 195(suppl. 2):93-99; M. Schafer-Korting et al., 1989, J. Am. Acad. Dermatol. 21:1271-1275). Other investigators have suggested that LP interacts with cells by stable absorption, endocytosis, lipid transfer, and fusion (R. B. Egerdie et al., J. Urol. 142:390-398). As demonstrated herein, administration of LP to the wounded urothelium can be used as a novel method for treating patients with hyperactive bladder, IC, or other urinary system disorders.
  • Example 3
  • An animal model for acute hyperactive bladder in rats was developed using intravesical infusion of protamine sulfate (PS), an agent used to break down urothelial barrier function, and physiological concentrations of potassium chloride (KCl).
  • Materials and Methods
  • Continuous CMGs were performed in urethane-anesthetized female rats. The bladder was filled (0.04 ml/min) with normal saline followed by intravesical infusion for a 60 minute period with a test solution comprising either KCl (100 or 500 mM) or PS (10 or 30 mg/ml). Following this, 10 mg/ml PS treated animals were infused intravesically with 100, 300, or 500 mM KCl. Some animals were pretreated with capsaicin (125 mg/ml, sc) four days before the experiments.
  • Animal Preparation
  • The study was performed on 40 female Sprague-Dawley rats weighing 250-300 gm. Animals were anesthetized with 1.2 gm/kg urethane injected subcutaneously. Body temperature was maintained in the physiological range using a heating lamp.
  • Cystometrogram (CMG)
  • PE-50 tubing (Clay-Adams, Parsippany, N.J.) was inserted into the bladder through the urethra and connected via a three-way stopcock to a pressure transducer and to a syringe pump. This was used for recording intravesical pressure and for infusing solutions into the bladder. A control CMG was performed by slowly filling the bladder with saline (0.04 ml/min) to elicit repetitive voiding. The amplitude, pressure threshold (PT), compliance, and intercontraction interval (ICl) of reflex bladder contractions were recorded. Pressure threshold (PT) represents the pressure that induces the initial bladder contraction. PT has often been used as a parameter corresponding to afferent nerve activity for the induction of reflex bladder contractions. Measurements in each animal represented the average of 3 to 5 bladder contractions.
  • Induction of Hyperactive Bladder
  • After performing control CMGs with saline infusion, three intravesical infusion experiments were performed in parallel: (1) infusion of KCl (100 or 500 mM in saline) for one hour (N=4 in each group); (2) infusion of protamine sulfate (PS) (Sigma Chemical Co.; 30 mg/ml in saline) for one hour (N=6); and (3) infusion of PS (10 mg/ml) for one hour, followed by 100, 300, or 500 mM KCl for one hour (N=6, in each subgroup). In four animals, capsaicin dissolved in a vehicle containing 10% ethanol, 10% TWEEN®80, and 80% physiological saline, at a concentration of 20 mg/ml was given subcutaneously in divided doses on 2 consecutive days. The doses included 25 and 50 mg/kg at a 12-hour interval on the first day and 50 mg/kg on the second day, as previously described (C. L. Cheng et al., 1993, Am. J. Physiol. 265:R132-138). All injections were performed under halothane anesthesia. Four days after the last dosage of capsaicin, the animals were anesthetized and treated with intravesical administration of PS (10 mg/ml) for 1 hour followed by KCl (500 mM) infusion.
  • Suppression of Micturition Reflex
  • To evaluate the direct effects of potassium on detrusor muscle, micturition reflex was blocked in 4 animals by either intravenous hexamethonium injection (25 mg/kg) (N=2) or transection of bilateral pelvic nerves (N=2).
  • Statistical Analysis
  • Statistical analyses were performed using Student's t test for paired or unpaired data, as applicable, with P<0.05 considered significant. Quantitative data are expressed in Example 4 (below) as means plus or minus standard error.
  • Example 4
  • The results of the experiments described in Example 3 are presented herein below. In summary, the intravesical administration of high concentrations of PS (30 mg/ml) produced irritative effects with decreases in intercontraction interval (ICl decreased by 80.6%). This was not observed with administration of KCl (100 or 500 mM) or a low concentration of PS (10 mg/ml). Following infusion of a low concentration of PS, infusion of 300 or 500 mM KCl produced irritative effects (ICl decreased by 76.9 or 82.9%, respectively). The onset of irritation occurred more rapidly following 500 mM KCl (10 to 15 min) than with 300 mM KCl (20 to 30 min). Capsaicin pretreatment delayed the onset (approximate 60 min) and reduced the magnitude (ICl decreased by 35.5%) of irritative effects.
  • CMGs in Various Infusion Group
  • As shown in Table 4, the CMG parameters during infusion of 100 mM or 500 mM KCl alone were not significantly different from those during saline administration. These results indicated that the bladder barrier function was not affected by control conditions. During a one hour period of instillation with 10 mg/ml PS, there was no significant change in comparison with saline administration (FIGS. 4A-4D; Table 5). These results indicated that low-dose PS was not by itself a bladder irritant. However, instillation of 30 mg/ml PS into bladder resulted in an irritative effect (ICl decreased by 80.6%, compliance decreased by 63.6%, and PT increased by 36.8%) after a delay of 40 to 45 min (FIGS. 4A-4D; Table 5).
    TABLE 4
    Effects of 100 mM and 500 mM KCl on CMG parameters
    saline Post saline Post
    (control; N = 4) 100 mM KCl (control; N = 4) 500 mM KCl
    PT (cm H2O) 7.2 ± 0.5 5.9 ± 0.5 7.3 ± 0.8 8.2 ± 0.9
    Amplitude (cm H2O) 25.5 ± 1.4  28.2 ± 0.5  26.5 ± 1.1  28.8 ± 1.2 
    Compliance (ml/cm H2O) 0.206 ± 0.013 0.205 ± 0.032 0.268 ± 0.013 0.237 ± 0.023
    ICI (min) 13.9 ± 2.4  13.8 ± 2.0  15.6 ± 2.1  15.8 ± 2.4 

    Parameters included volume pressure threshold (PT), amplitude, compliance, and intercontraction interval (ICI). No statistically significant differences were observed between 100 and 500 mM KCl treatment (N = 4 in each group).

    Values are means ± S.E.
  • TABLE 5
    Effects of 10 and 30 mg/ml PS on CMG parameters
    saline Post saline Post
    (control; N = 18) 10 mg/ml PS (control; N = 6) 30 mg/ml PS
    PT (cm H2O) 4.2 ± 0.8 3.4 ± 0.9 5.7 ± 0.6 7.8 ± 1.1*
    Amplitude (cm H2O) 24.1 ± 2.4  25.3 ± 2.0  27.7 ± 0.8  28.3 ± 3.1 
    Compliance (ml/cm H2O) 0.169 ± 0.012 0.176 ± 0.013 0.228 ± 0.024 0.083 ± 0.022*
    ICI (min) 16.8 ± 0.9  17.8 ± 0.7  13.9 ± 2.1  2.7 ± 0.7*

    N = 4 in each group. Parameters included volume pressure threshold (PT), amplitude, compliance, and intercontraction interval (ICI).

    Values are means ± S.E.

    *P < 0.05, in comparison with control.
  • Effect of KCl Following Low Concentrations of PS (10 mg/ml) Infusion
  • As shown in FIGS. 5A-5F and Table 6, CMGs performed with 300 or 500 mM KCl infusion significantly changed the ICl (76.9 or 82.9% decrease), compliance (60 or 63.4% decrease), and contraction amplitude (23.7 or 21.4% increase). However, the PT was not significantly altered. The effect mediated by 300 mM KCl occurred after a delay of 20 to 30 min, whereas the effect mediated by 500 mM KCl occurred after a delay of about 10 to 15 min. This implied that higher concentrations of KCl yielded faster penetration. One hour of infusion of 100 mM KCl did not produce significant changes in the CMG parameters.
    TABLE 6
    Effects of 100, 300 and 500 mM KCl on CMG following 10 mg/ml PS
    saline Post saline Post saline Post
    (control; N = 6) 100 mM KCl (control; N = 6) 300 mM KCl (control; N = 6) 500 mM KCl
    PT 3.1 ± 1.2 3.0 ± 1.3 2.9 ± 0.6 3.3 ± 0.9 4.0 ± 0.9 5.4 ± 1.0
    (cm H2O)
    Amplitude 26.8 ± 3.7  28.2 ± 3.7  25.3 ± 0.9  31.3 ± 0.7* 24.3 ± 2.1  29.5 ± 3.4*
    (cm H2O)
    Compliance 0.161 ± 0.022 0.157 ± 0.022 0.165 ± 0.027  0.066 ± 0.012* 0.205 ± 0.018  0.075 ± 0.009*
    (ml/cm H2O)
    ICI 17.2 ± 1.3  18.8 ± 2.1  17.4 ± 2.0   5.2 ± 1.7* 18.8 ± 1.7   3.2 ± 1.3*
    (min)

    N = 6 in each group.

    Parameters included volume pressure threshold (PT), amplitude, and intercontraction interval (ICI).

    Statistically significant differences were observed between control and 300/500 mM KCl treatment.

    Values are means ± S.E.

    *P < 0.05, in comparison with control.
  • CMGs in Animals Pretreated with Capsaicin
  • In capsaicin pretreated animals, hyperactive bladder from the sequential infusion of PS (10 mg/ml) and KCl (500 mM) was delayed by about 1 hour. In addition, the changes in ICl and compliance were reduced by 36 and 55% after 2 hours of infusion (ICl decreased from 20.3±1.2 to 13.1±2.8 min; compliance decreased from 0.224 to 0.100 ml/cm H2O; Table 7). After sequential infusion of PS (10 mg/ml) and KCl (500 mM) in capsaicin-pretreated rats (Table 7), the ICl (13.1±2.8 min) was significantly longer (p<0.05) than the ICl (3.2±1.3 min) following intravesical application of PS (10 mg/ml) and KCl (500 mM) in untreated rats (Table 6). This indicated that C-fiber desensitization by capsaicin pretreatment suppressed PS/KCl-induced bladder hyperactivity.
    TABLE 7
    Effects of KCl (500 mM) on CMG following one hour infusion of PS
    (10 mg/ml) in capsaicin pretreated animals
    Saline (control; N = 4) KCl (1 hr) KCl (2 hr)
    PT (cm H2O) 8.4 ± 0.9 5.8 ± 0.5 7.6 ± 1.7
    Amplitude (cm H2O) 18.3 ± 1.5  20.3 ± 0.3  16.0 ± 1.7 
    Compliance (ml/cm H2O) 0.244 ± 0.007 0.218 ± 0.013  0.100 ± 0.016*
    ICI (min) 20.3 ± 1.2  21.3 ± 2.8  13.1 ± 2.8*

    N = 4 in each group. Parameters included volume pressure threshold (PT), amplitude, compliance, and intercontraction interval (ICI).

    Values are means ± S.E.

    *P < 0.05, in comparison with control.
  • CMGs in Micturition Reflex Suppressed Animals
  • As shown in FIGS. 6A-6B, infusion of normal saline did not induce micturition reflex. This indicated that micturition reflex was blocked by hexamethonium injection or pelvic nerve transection. Infusion of KCl (500 mM) following PS (10 mg/ml) instillation decreased compliance by 55.9% (from 0.093±0.026 to 0.041±0.011 ml/cm H2O). This indicated that potassium affected the direct stimulation of the detrusor muscle and caused the decrease in compliance.
  • SUMMARY
  • In summary, the results from Examples 3-4 demonstrated that 1) low dose PS (10 mg/ml) was not a bladder irritant, but a noncytotoxic affront to urothelial barrier function; and 2) the use of “physiological” normal saline, versus more appropriately physiological 300 or 500 mM KCl (M. Ohnishi et al., 2001, Toxicol. Appl. Pharmacol. 174:122-129; J. Morrison et al., 1999, Scand. J. Urol. Nephrol. suppl 201:73-75) for cystometry affected the function of the lower urinary tract in animal models of hyperactive bladder. It has been postulated that a critical component of IC is a leaky urothelium (C. L. Parsons et al., 1991, J. Urol. 145:732-735; C. L. Parsons et al., 1994, Br. J. Urol. 73:504-507; S. Keay et al., 1999, J. Urol. 162:1487-1489). A compromised urothelial barrier is believed to result in an influx of highly concentrated, noxious substances that are normally passed through the urinary tract without reabsorption (G. Hohlbrugger, 1999, Br. J. Urol. 83:22-28; C. L. Parsons et al., 1998, J. Urol. 159:1862-1867). Where the urothelial barrier is broken down, these substances can cross back into the bladder, where they stimulate activity of resident C-fiber afferents. This transmits pain sensations and causes sensory symptoms (C. L. Parsons et al., 1998, J. Urol. 159:1862-1867). According to one theory, the influx of concentrated potassium from the urine to the submucosal region depolarizes bladder wall sensory afferents, and initiates hyperactive bladder (G. Hohlbrugger, 1999, Br. J. Urol. 83:22-28; C. L. Parsons et al., 1998, J. Urol. 159:1862-1867).
  • The penetration of high concentrations of potassium through a leaky urothelium is also known to directly stimulate detrusor muscle and contribute to the decrease in bladder compliance (G. Hohlbrugger, 1999, Br. J. Urol. 83:22-28; G. Hohlbrugger, 1995, J. Urol. 154:6-15). The methods described herein block the micturition reflex by an autonomic ganglion blockade (hexamethonium) or a pelvic nerve transection, but decreases are still observed in the compliance after intravesical infusion of KCl following PS treatment. This provides evidence of the stimulation of detrusor by high concentration of potassium. As shown herein, ICl was decreased, but PT was not changed. High concentration of potassium has been known to irritate the bladder neck and cause high outlet resistance (G. Hohlbrugger, 1999, Br. J. Urol. 83:22-28). Consistent with this, the bladder contraction amplitude was elevated in some of the results shown above.
  • The use of PS has been well established as a model for bladder injury. Previous experiments showed distension of the bladder for 45 min with 1 ml of 10 mg/ml PS (P. C. Stein et al., 1996, J. Urol. 155:1133-1138). It is known that prolonged over-distension of the bladder alters the properties of the bladder wall (S. Keay et al., 1999, J. Urol. 162:1487-1489; G. Hohlbrugger, 1995, J. Urol. 154:6-15). This may enhance the cytodestructive effects of PS and result in immediate urothelial sloughing (P. C. Stein et al., 1996, J. Urol. 155:1133-1138). However, in the open CMG method described herein, one hour exposure of urothelium to the same concentration of PS did not cause obvious changes in CMG. Yet, urothelial barrier function was compromised, and resulted in influx of high concentrations of potassium and bladder stimulation. Prior to PS treatment, the same concentration of potassium did not induce a hyperactive bladder. These data support the idea that abnormal epithelial permeability with an addition of high concentration of potassium in the urine is a key component to induce the symptoms of bladder hypersensitivity. It is hypothesized that without mechanical destruction of urothelium, prolong exposure of PS might still lead to a subtle change but breakdown the barrier function. This model might elucidate the mechanism involved in the potassium test for the diagnosis of IC (C. L. Parsons et al., 1998, J. Urol. 159:1862-1867).
  • Other investigators have reported that intravesical instillation of 150 mM KCl through a pair of bladder dome catheter at a rate of 0.250 ml/min to a maximal pressure of 30 mm Hg can excite the afferent activity in hypogastric nerves, but is rarely detected in pelvic nerves (N. G. Moss et al., 1997, Am. J. Physiol. 272:R695-703). In these experiments, the average bladder volume used was 1.5 ml. This is 2 to 3 fold of normal bladder capacity in the rats and can result in over-distension of the bladder (N. G. Moss et al., 1997, Am. J. Physiol. 272:R695-703; M. Leppilahti et al., 1999, Urol. Res. 27:272-276; Y. C. Chuang et al., 2001, J. Urol. 165:975-979). Additionally, previous experiments showed the reduction of bladder capacity after a period of delay in a closed CMG method with intravesical isotonic KCl treatment (G. Hohlbrugger and P. Lentsch, 1985, Eur. Urol. 11:127-130). These effects were enhanced by pretreatment with 50% DMSO (G. Hohlbrugger and P. Lentsch, 1985, Eur. Urol. 11:127-130). However, the experiments described above showed that continuous infusion of KCl (500 mM) for 1 hour without pretreatment with PS did not induce significant bladder irritation. It is possible that altering the urothelium properties by either over-distension or DMSO instillation could increase the bladder permeability and induce afferent firing and hyperactive bladder by KCl administration.
  • In conclusion, the use of 300 or 500 mM KCl for cystometry, versus physiological saline, affects the function of the lower urinary tract in animal models of hyperactive bladder. Accordingly, pharmaceutical compositions for the treatment of urinary system conditions preferably include excipients, diluents, or carriers comprising physiological saline, as described in detail herein.
  • Example 5
  • Intravesical vanilloid therapy has been used to treat detrusor hyperreflexia in SCI (spinal cord injury) and MS (multiple sclerosis) patients. Capsaicin (CAP) treatment requires high concentrations of ethanol (30% or greater) to achieve an effective dose. This level of ethanol is tissue toxic, and may, by itself, cause hemorrhagic cystitis. The lipoidal phase of liposomes (LP), concentric phospholipid bilayers, may provide an attractive alternative to high concentrations of ethanol. In an attempt to address this possibility, liposomal delivery of CAP was tested in urethane anesthetized rats.
  • Methods
  • Open transurethral cystometry (0.04 ml/min) was performed under urethane anesthesia (1.2 g/kg) in 15 female S-D rats (250-300 g). Following a two-hour control period of saline infusion, the infusate was switched to either LP with 1 mM CAP (LP/CAP), or LP alone for 30 minutes followed by LP/CAP. The efficacy of CAP delivery was determined by the onset time of initial evidence of bladder irritation and subsequent desensitization, and bladder contraction frequency. LPs were constructed as described in Example 1, above. Briefly, a 2:1 molar ratio of phosphatidylcholine and cholesterol, was dried down from chloroform solvent under nitrogen, with or without CAP. The resultant residue was brought into a saline suspension at 2 mg/ml total lipid by intense sonication.
  • Results
  • LP alone had no effect on bladder contraction frequency (0.13±0.02 vs. 0.13±0.01 bladder contractions/min for control and LP, respectively (FIG. 7). However, LP/CAP resulted in a dramatic increase in bladder contraction frequency (1.11±0.08 bladder contractions/min, P<0.0001) within minutes of beginning the infusion (FIG. 7). Bladder contraction frequency subsequently slowed and finally halted by 124±24 minutes.
  • Conclusions
  • LP are capable of highly effective delivery of at least one hydrophobic drug, CAP, as evidenced by a dramatic increase in bladder contraction frequency and subsequent desensitization. Moreover, LP alone had no effect on the micturition reflex in the unirritated state. In combination with other experiments that have demonstrated a protective effect of LPs, this suggested that the LP vehicle may partially protect against the compromise of urothelial barrier function due to the neuro-inflammatory response caused by irritants, such as CAP. This experiment indicates that LP for other drugs, such as antibiotics and cancer treatments. Description of the experiments in this example can also be found in Y.-C. Chuang et al., 100th Annual Meeting American Urological Association (AUA), Abstract; 2002, J. Urol. 167:41A, which are hereby incorporated herein by reference.
  • Example 6
  • The effects of botulinum toxin (Btx) and liposome injections on autonomic (bladder) innervation of the lower urinary tract were investigated as follows.
  • Methods
  • Liposomes were prepared as described in Example 1. Female SD rats (250) were anesthetized with urethane (1.2 g/kg). Animals received intravesical liposomes plus instillation of Btx D (5.7 ng/gm body weight; Sigma, St Louis, Mo.). Control animals received no injections. All animals received tracheotomies, and treated animals were artificially respired. Transvesical catheters were inserted and, 6 hr after Btx injection, the bladder was harvested for strip studies.
  • For contractility experiments, bladder strips (20-30 g) were mounted in a double-jacketed organ bath at 36° C. in oxygenated Krebs solution. Electrical field stimulation was delivered through platinum electrodes positioned at the top and bottom using trains of 100 shocks at 20 Hz with maximal voltage every 100 sec. Strip fatigue was tested by trains applied every 20 sec. Fatigue amplitude and area, as well as recovery amplitude and area, were calculated as percent of the control value and compared between groups.
  • Results
  • Mean in vitro recovery amplitude of liposomes plus Btx was 68% that of the control value. It was concluded that liposomes carrying Btx significantly decreased bladder contractility.
  • The contents of all patents, patent applications, published articles, books, reference manuals, texts and abstracts cited herein are hereby incorporated by reference in their entirety to more fully describe the state of the art to which the present invention pertains.
  • The present invention has been described in detail including the preferred embodiments thereof. However, it will be appreciated by those skilled in the art, upon consideration of this disclosure, that modifications and improvements may be made thereon without departing from the spirit and scope of the invention as set forth in the description and claims.

Claims (6)

1-59. (canceled)
60. A method of treating bladder conditions comprising administering via intravesical instillation, to a mammalian subject in need thereof, an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises:
a) a non-ionic lipid vehicle, optionally containing an additional drug therein; and
b) a physiologically acceptable carrier, excipient, or diluent.
61. The method of treating bladder conditions according to claim 60, wherein said non-ionic lipid vehicle contains an additional drug therein.
62. The method of treating bladder conditions according to claim 61, wherein said drug comprises a botulinum toxin or a vanilloid.
63. The method of treating bladder conditions according to claim 62, wherein said drug is selected from the group consisting of a botulinum toxin, resiniferatoxin, capsaicin and tinyatoxin.
64. The method of treating bladder conditions according to claim 63, wherein said drug is administered in an amount effective to treat a condition selected from the group consisting of bladder pain, bladder inflammation, incontinence, voiding disfunction, urethral dyssynergia and bladder spasticity.
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US12/651,075 US20100104631A1 (en) 2001-08-13 2009-12-31 Method of treatment for bladder dysfunction
US13/350,326 US20120128762A1 (en) 2005-07-20 2012-01-13 Sphingomyelin Liposomes for the Treatment of Hyperactive Bladder Disorders
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070122466A1 (en) * 2001-08-13 2007-05-31 University Of Pittsburgh Sphingomyelin liposomes for the treatment of hyperactive bladder disorders
WO2008147807A2 (en) 2007-05-23 2008-12-04 Amcol International Corporation Cholesterol-interacting layered phyllosilicates and methods of reducing hypercholesteremia in a mammal
US20100303932A1 (en) * 2009-05-28 2010-12-02 Baskaran Thyagarajan Method for Attenuating the Effects of Botulinum Toxin
US20110117026A1 (en) * 2008-05-19 2011-05-19 Yu-Chen Tseng Methods and compositions for the delivery of bioactive compounds
US20120189677A1 (en) * 2011-01-20 2012-07-26 Stephen Tonge Formulations
US8728473B2 (en) 2010-12-01 2014-05-20 Alderbio Holdings Llc Methods of preventing or treating pain using anti-NGF antibodies
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US10010439B2 (en) 2010-06-13 2018-07-03 Synerz Medical, Inc. Intragastric device for treating obesity
US10413436B2 (en) 2010-06-13 2019-09-17 W. L. Gore & Associates, Inc. Intragastric device for treating obesity
US10420665B2 (en) 2010-06-13 2019-09-24 W. L. Gore & Associates, Inc. Intragastric device for treating obesity
US10779980B2 (en) 2016-04-27 2020-09-22 Synerz Medical, Inc. Intragastric device for treating obesity

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013116822A1 (en) * 2012-02-02 2013-08-08 Lipella Pharmaceuticals, Inc. Methods and compositions for treating gastric disorders
US20100104631A1 (en) * 2001-08-13 2010-04-29 Lipella Pharmaceuticals Inc. Method of treatment for bladder dysfunction
US20030059375A1 (en) * 2001-08-20 2003-03-27 Transave, Inc. Method for treating lung cancers
US6921538B2 (en) * 2002-05-10 2005-07-26 Allergan, Inc. Therapeutic treatments for neuropsychiatric disorders
US7691394B2 (en) 2002-05-28 2010-04-06 Botulinum Toxin Research Associates, Inc. High-potency botulinum toxin formulations
WO2004010934A2 (en) * 2002-07-29 2004-02-05 Rajiv Doshi Methods for the use of neurotoxin in the treatment of urologic disorders
US9186322B2 (en) * 2002-08-02 2015-11-17 Insmed Incorporated Platinum aggregates and process for producing the same
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WO2004037270A1 (en) * 2002-10-25 2004-05-06 Vasogen Ireland Limited Cyclooxygenase regulation with pg liposomes
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US7879351B2 (en) * 2002-10-29 2011-02-01 Transave, Inc. High delivery rates for lipid based drug formulations, and methods of treatment thereof
US7544369B2 (en) * 2002-10-29 2009-06-09 Transave, Inc. Sustained release of antiinfectives
US7718189B2 (en) 2002-10-29 2010-05-18 Transave, Inc. Sustained release of antiinfectives
ATE426408T1 (en) * 2002-12-20 2009-04-15 Botulinum Toxin Res Ass Inc PHARMACEUTICAL COMPOSITIONS CONTAINING BOTULINUM TOXIN AND HUMAN SERUM ALBUMINE
EP1613297B1 (en) * 2003-04-14 2007-06-06 LTS Lohmann Therapie-Systeme AG Therapeutic patch with polysiloxane matrix comprising capsaicin
US7216038B2 (en) 2003-09-11 2007-05-08 Franco Vitaliano Quantum information processing elements and quantum information processing platforms using such elements
US7219018B2 (en) * 2003-09-11 2007-05-15 Franco Vitaliano Quantum information processing elements and quantum information processing platforms using such elements
US7219017B2 (en) 2003-09-11 2007-05-15 Franco Vitaliano Quantum information processing elements and quantum information processing platforms using such elements
GB0328060D0 (en) * 2003-12-04 2004-01-07 Sod Conseils Rech Applic Botulinum toxin treatment
US7979150B2 (en) * 2003-12-05 2011-07-12 The Regents Of The University Of Michigan Biodegradable/bioresorbable tissue augmentation/reconstruction device
US7393924B2 (en) * 2004-01-06 2008-07-01 Franco Vitaliano Smart bio-nanoparticle elements
PT2985039T (en) * 2004-03-03 2018-11-09 Revance Therapeutics Inc Topical application and transdermal delivery of botulinum toxins
US20060116336A1 (en) * 2004-03-17 2006-06-01 American Pharmaceutical Partners, Inc. Lyophilized azithromycin formulation
US7468428B2 (en) * 2004-03-17 2008-12-23 App Pharmaceuticals, Llc Lyophilized azithromycin formulation
WO2005089448A2 (en) * 2004-03-18 2005-09-29 Transave, Inc. Administration of cisplatin by inhalation
US20070065522A1 (en) * 2004-03-18 2007-03-22 Transave, Inc. Administration of high potency platinum compound formulations by inhalation
SG115845A1 (en) * 2004-03-31 2005-10-28 Univ Singapore Modulation of trip-br function and method of treating proliferative disorders
AT413646B (en) * 2004-04-02 2006-04-15 Hammer Johann Dr USE OF CAPSAICIN AND / OR CAPSAICINOIDES
CA2566174A1 (en) * 2004-05-21 2005-12-01 Transave, Inc. Treatment of lung diseases and pre-lung disease conditions
US7993578B2 (en) * 2004-10-29 2011-08-09 Depuy Spine, Inc. Methods and kits for aseptic filling of products
AU2005306802A1 (en) * 2004-11-08 2006-05-26 Transave, Inc. Methods of treating cancer with lipid-based platinum compound formulations administered intraperitoneally
ATE489086T1 (en) * 2004-12-22 2010-12-15 Mestex Ag MIXTURE OF A VANILLOID RECEPTOR AGONIST WITH A NERVE REGENERATION-INHIBITING SUBSTANCE, USE OF IT FOR THE PRODUCTION OF A PAIN RELIEF AND METHOD FOR APPLYING THIS AGENT
US7727537B2 (en) 2005-02-14 2010-06-01 Dpm Therapeutics Corp. Stabilized compositions for topical administration and methods of making same
EP1853303B1 (en) * 2005-02-14 2015-12-02 DPM Therapeutics Corporation Stabilized compositions for topical administration and methods of making same
US7838011B2 (en) 2005-02-14 2010-11-23 Pankaj Modi Stabilized protein compositions for topical administration and methods of making same
WO2006130978A1 (en) 2005-06-06 2006-12-14 The University Of British Columbia Polymer-based serum albumin substitute
EP1741444A1 (en) * 2005-07-05 2007-01-10 Jerini AG Kinin antagonists for treating bladder dysfunction
CN101384247B (en) * 2005-07-18 2013-05-22 麻萨诸塞州洛厄尔大学 Compositions and methods for making and using nanoemulsions
EP1933813A4 (en) * 2005-10-11 2013-02-27 Univ Pittsburgh Sphingomyelin liposomes for the treatment of hyperactive bladder disorders
EP2395012B8 (en) 2005-11-02 2018-06-06 Arbutus Biopharma Corporation Modified siRNA molecules and uses thereof
US9107824B2 (en) 2005-11-08 2015-08-18 Insmed Incorporated Methods of treating cancer with high potency lipid-based platinum compound formulations administered intraperitoneally
US20070190181A1 (en) * 2005-11-08 2007-08-16 Pilkiewicz Frank G Methods of treating cancer with lipid-based platinum compound forumulations administered intravenously
US20070190180A1 (en) * 2005-11-08 2007-08-16 Pilkiewicz Frank G Methods of treating cancer with high potency lipid-based platinum compound formulations administered intravenously
US20070190182A1 (en) * 2005-11-08 2007-08-16 Pilkiewicz Frank G Methods of treating cancer with high potency lipid-based platinum compound formulations administered intraperitoneally
US20070122350A1 (en) * 2005-11-30 2007-05-31 Transave, Inc. Safe and effective methods of administering therapeutic agents
US9486408B2 (en) 2005-12-01 2016-11-08 University Of Massachusetts Lowell Botulinum nanoemulsions
HUE054852T2 (en) * 2005-12-01 2021-10-28 Univ Massachusetts Lowell Botulinum nanoemulsions
CA2631872C (en) 2005-12-08 2014-04-01 Transave, Inc. Lipid-based compositions of antiinfectives for treating pulmonary infections and methods of use thereof
US8703179B2 (en) 2006-05-11 2014-04-22 Kimberly-Clark Worldwide, Inc. Mucosal formulation
US7282224B1 (en) * 2006-06-09 2007-10-16 Guthy-Renker Corporation Pain relief composition
US7943666B2 (en) 2006-07-24 2011-05-17 Trinity Laboratories, Inc. Esters of capsaicin for treating pain
US20080107747A1 (en) * 2006-10-23 2008-05-08 Roederer Joy E Pain relief composition
KR101518077B1 (en) 2006-12-01 2015-05-28 안테리오스, 인코퍼레이티드 Peptide nanoparticles and uses therefor
KR20090106493A (en) 2006-12-01 2009-10-09 안테리오스, 인코퍼레이티드 Micellar nanoparticles comprising botulinum toxin
US20100196455A1 (en) * 2007-05-04 2010-08-05 Transave, Inc. Compositions of Multicationic Drugs for Reducing Interactions with Polyanionic Biomolecules and Methods of Use Thereof
US9333214B2 (en) 2007-05-07 2016-05-10 Insmed Incorporated Method for treating pulmonary disorders with liposomal amikacin formulations
WO2008137917A1 (en) * 2007-05-07 2008-11-13 Transave, Inc. Method of treating bacterial infections with antibacterial formulations
US9114081B2 (en) 2007-05-07 2015-08-25 Insmed Incorporated Methods of treating pulmonary disorders with liposomal amikacin formulations
US9119783B2 (en) 2007-05-07 2015-09-01 Insmed Incorporated Method of treating pulmonary disorders with liposomal amikacin formulations
WO2008151022A2 (en) 2007-05-31 2008-12-11 Anterios, Inc. Nucleic acid nanoparticles and uses therefor
CN105147594A (en) * 2007-11-30 2015-12-16 恩德制药解决方案公司 Compositions and methods for the treatment of bladder cancer
RU2570559C2 (en) * 2007-12-17 2015-12-10 Пфайзер Лимитед Treatment of interstitial cystitis
AU2008342535B2 (en) 2007-12-27 2015-02-05 Arbutus Biopharma Corporation Silencing of polo-like kinase expression using interfering RNA
CN102065841A (en) * 2008-04-04 2011-05-18 里培拉药品公司 Treatment of bladder dysfunction using liposomal botulinum toxin
AU2009236219B8 (en) 2008-04-15 2015-06-25 Arbutus Biopharma Corporation Silencing of CSN5 gene expression using interfering RNA
CN105147608B (en) * 2008-06-26 2019-12-10 安特里奥公司 Dermal delivery
US20100086585A1 (en) * 2008-10-06 2010-04-08 Henry John Smith Antinuclear antibody utilized as a targeting agent for pharmaceutical compounds used in the treatment of cancer and other diseases
US9066851B2 (en) 2008-12-04 2015-06-30 Botulinum Toxin Research Associates, Inc. Extended length botulinum toxin formulation for human or mammalian use
WO2010078403A2 (en) * 2008-12-30 2010-07-08 Lipella Pharmaceuticals Inc. Methods and compositions for diagnosing urological disorders
US11235062B2 (en) * 2009-03-06 2022-02-01 Metaqor Llc Dynamic bio-nanoparticle elements
US11096901B2 (en) 2009-03-06 2021-08-24 Metaqor Llc Dynamic bio-nanoparticle platforms
US20110274745A1 (en) * 2009-11-10 2011-11-10 Lipella Pharmaceuticals Inc. Instillation of liposomal formulation of sirna and antisense oligonucleotides
JP5914366B2 (en) 2010-03-01 2016-05-11 ザ ユニヴァーシティ オヴ ブリティッシュ コロンビア Derivatized hyperbranched polyglycerols
CA2801066C (en) 2010-06-02 2021-02-09 Alnylam Pharmaceuticals, Inc. Compositions and methods directed to treating liver fibrosis
WO2011160062A2 (en) 2010-06-17 2011-12-22 The Usa As Represented By The Secretary, National Institutes Of Health Compositions and methods for treating inflammatory conditions
WO2012079046A2 (en) 2010-12-10 2012-06-14 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of klf-1 and bcl11a genes
US9193973B2 (en) 2010-12-10 2015-11-24 Alynylam Pharmaceuticals, Inc. Compositions and methods for increasing erythropoietin (EPO) production
EP2670843B1 (en) 2011-02-03 2014-10-29 The Government Of The U.S.A, As Represented By The Secretary, Dept. Of Health And Human Services Multivalent vaccines for rabies virus and filoviruses
CA2831284C (en) 2011-03-29 2023-12-12 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of tmprss6 gene
EP3354343B1 (en) 2011-06-02 2023-09-06 President and Fellows of Harvard College Methods and uses for ex vivo tissue culture systems
CN112961855A (en) 2011-06-21 2021-06-15 阿尔尼拉姆医药品有限公司 Angiopoietin-like 3(ANGPTL3) iRNA compositions and methods of use thereof
EP2723865B1 (en) 2011-06-21 2019-03-27 Alnylam Pharmaceuticals, Inc. METHODS FOR DETERMINING ACTIVITY OF RNAi IN A SUBJECT
EP3366312A1 (en) 2011-06-23 2018-08-29 Alnylam Pharmaceuticals, Inc. Serpina 1 sirnas: compositions of matter and methods of treatment
AU2012275841A1 (en) 2011-06-27 2014-01-16 The Jackson Laboratory Methods and compositions for treatment of cancer and autoimmune disease
US20140328811A1 (en) 2011-08-01 2014-11-06 Alnylam Pharmaceuticals, Inc. Method for improving the success rate of hematopoietic stem cell transplants
KR20140103122A (en) 2011-11-17 2014-08-25 넨키 인스티튜트 오브 익스페리멘탈 바이올로지 Compositions and methods for treating glioma
EP2780456A1 (en) 2011-11-17 2014-09-24 The U.S.A. as represented by the Secretary, Department of Health and Human Services Therapeutic rna switches compositions and methods of use
US9035039B2 (en) 2011-12-22 2015-05-19 Protiva Biotherapeutics, Inc. Compositions and methods for silencing SMAD4
US9133461B2 (en) 2012-04-10 2015-09-15 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of the ALAS1 gene
US9393291B2 (en) 2012-04-12 2016-07-19 Botulinum Toxin Research Associates, Inc. Use of botulinum toxin for the treatment of cerebrovascular disease, renovascular and retinovascular circulatory beds
US9127274B2 (en) 2012-04-26 2015-09-08 Alnylam Pharmaceuticals, Inc. Serpinc1 iRNA compositions and methods of use thereof
US20150272880A1 (en) 2012-05-21 2015-10-01 Insmed Incorporated Systems for treating pulmonary infections
WO2014039533A2 (en) 2012-09-04 2014-03-13 Eleison Pharmaceuticals, Llc Preventing pulmonary recurrence of cancer with lipid-complexed cisplatin
SG10202011046RA (en) 2012-09-21 2020-12-30 Intensity Therapeutics Inc Method of treating cancer
CN104884047A (en) 2012-11-29 2015-09-02 英斯梅德股份有限公司 Stabilized vancomycin formulations
EP4083209A1 (en) 2012-12-05 2022-11-02 Alnylam Pharmaceuticals, Inc. Pcsk9 irna compositions and methods of use thereof
NZ712336A (en) 2013-03-14 2020-03-27 Alnylam Pharmaceuticals Inc Complement component c5 irna compositions and methods of use thereof
WO2014160871A2 (en) 2013-03-27 2014-10-02 The General Hospital Corporation Methods and agents for treating alzheimer's disease
WO2014182661A2 (en) 2013-05-06 2014-11-13 Alnylam Pharmaceuticals, Inc Dosages and methods for delivering lipid formulated nucleic acid molecules
PT2999785T (en) 2013-05-22 2018-07-09 Alnylam Pharmaceuticals Inc Serpina1 irna compositions and methods of use thereof
IL293657A (en) 2013-05-22 2022-08-01 Alnylam Pharmaceuticals Inc Tmprss6 irna compositions and methods of use thereof
EP3007709B1 (en) 2013-06-14 2021-12-08 Akamara Therapeutics, Inc. Lipid-based platinum compounds and nanoparticles
WO2015042170A1 (en) 2013-09-17 2015-03-26 Wayne State University Compositions and uses of combinations of dim-related indoles and selected anti-androgen compounds
CN105793423A (en) 2013-10-02 2016-07-20 阿尔尼拉姆医药品有限公司 Compositions and methods for inhibiting expression of the LECT2 gene
SG10201910929QA (en) 2013-10-04 2020-01-30 Alnylam Pharmaceuticals Inc Compositions and methods for inhibiting expression of the alas1 gene
US10639278B2 (en) 2013-10-22 2020-05-05 Lipella Pharmaceuticals, Inc. Delivery of agents using metastable liposomes
JP6710638B2 (en) 2013-12-12 2020-06-17 アルナイラム ファーマシューティカルズ, インコーポレイテッドAlnylam Pharmaceuticals, Inc. Complement component iRNA composition and method of using the same
KR20230152154A (en) 2014-02-11 2023-11-02 알닐람 파마슈티칼스 인코포레이티드 KETOHEXOKINASE (KHK) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
WO2015175510A1 (en) 2014-05-12 2015-11-19 Alnylam Pharmaceuticals, Inc. Methods and compositions for treating a serpinc1-associated disorder
AU2015258947B2 (en) 2014-05-15 2020-04-09 Insmed Incorporated Methods for treating pulmonary non-tuberculous mycobacterial infections
SG10202104570TA (en) 2014-05-22 2021-06-29 Alnylam Pharmaceuticals Inc Angiotensinogen (agt) irna compositions and methods of use thereof
US11484580B2 (en) 2014-07-18 2022-11-01 Revance Therapeutics, Inc. Topical ocular preparation of botulinum toxin for use in ocular surface disease
US9901627B2 (en) 2014-07-18 2018-02-27 Revance Therapeutics, Inc. Topical ocular preparation of botulinum toxin for use in ocular surface disease
WO2016040589A1 (en) 2014-09-12 2016-03-17 Alnylam Pharmaceuticals, Inc. Polynucleotide agents targeting complement component c5 and methods of use thereof
US10415037B2 (en) 2014-10-02 2019-09-17 Arbutus Biopharma Corporation Compositions and methods for silencing hepatitis B virus gene expression
JOP20200115A1 (en) 2014-10-10 2017-06-16 Alnylam Pharmaceuticals Inc Compositions And Methods For Inhibition Of HAO1 (Hydroxyacid Oxidase 1 (Glycolate Oxidase)) Gene Expression
EP3207138B1 (en) 2014-10-17 2020-07-15 Alnylam Pharmaceuticals, Inc. Polynucleotide agents targeting aminolevulinic acid synthase-1 (alas1) and uses thereof
WO2016064882A1 (en) 2014-10-20 2016-04-28 The Children's Medical Center Corporation Sustained and reversible oral drug delivery systems
WO2016069694A2 (en) 2014-10-30 2016-05-06 Alnylam Pharmaceuticals, Inc. Polynucleotide agents targeting serpinc1 (at3) and methods of use thereof
JOP20200092A1 (en) 2014-11-10 2017-06-16 Alnylam Pharmaceuticals Inc HEPATITIS B VIRUS (HBV) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
WO2016081444A1 (en) 2014-11-17 2016-05-26 Alnylam Pharmaceuticals, Inc. Apolipoprotein c3 (apoc3) irna compositions and methods of use thereof
TW201702218A (en) 2014-12-12 2017-01-16 美國杰克森實驗室 Compositions and methods relating to the treatment of cancer, autoimmune disease, and neurodegenerative disease
US11400139B2 (en) 2015-01-20 2022-08-02 The Children's Medical Center Corporation Anti-NET compounds for treating and preventing fibrosis and for facilitating wound healing
EP3256587A2 (en) 2015-02-13 2017-12-20 Alnylam Pharmaceuticals, Inc. Patatin-like phospholipase domain containing 3 (pnpla3) irna compositions and methods of use thereof
WO2016164746A1 (en) 2015-04-08 2016-10-13 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of the lect2 gene
WO2016168286A1 (en) 2015-04-13 2016-10-20 Alnylam Pharmaceuticals, Inc. Angiopoietin-like 3 (angptl3) irna compositions and methods of use thereof
CN115322991A (en) 2015-05-06 2022-11-11 阿尔尼拉姆医药品有限公司 Factor XII, kallikrein B, plasma Freuey factor 1 and kininogen 1 iRNA compositions and methods of use thereof
WO2016197132A1 (en) 2015-06-04 2016-12-08 Protiva Biotherapeutics Inc. Treating hepatitis b virus infection using crispr
EP3307316A1 (en) 2015-06-12 2018-04-18 Alnylam Pharmaceuticals, Inc. Complement component c5 irna compositions and methods of use thereof
EP3310918B1 (en) 2015-06-18 2020-08-05 Alnylam Pharmaceuticals, Inc. Polynucleotide agents targeting hydroxyacid oxidase (glycolate oxidase, hao1) and methods of use thereof
WO2016209862A1 (en) 2015-06-23 2016-12-29 Alnylam Pharmaceuticals, Inc. Glucokinase (gck) irna compositions and methods of use thereof
US10494632B2 (en) 2015-07-10 2019-12-03 Alnylam Pharmaceuticals, Inc. Insulin-like growth factor binding protein, acid labile subunit (IGFALS) compositions and methods of use thereof
US20180208932A1 (en) 2015-07-29 2018-07-26 Arbutus Biopharma Corporation Compositions and methods for silencing hepatitis b virus gene expression
CN108368507B (en) 2015-09-02 2022-03-22 阿尔尼拉姆医药品有限公司 iRNA compositions of programmed cell death 1 ligand 1(PD-L1) and methods of use thereof
EP3350328A1 (en) 2015-09-14 2018-07-25 Alnylam Pharmaceuticals, Inc. Polynucleotide agents targeting patatin-like phospholipase domain containing 3 (pnpla3) and methods of use thereof
WO2017048843A1 (en) 2015-09-14 2017-03-23 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of the alas1 gene
WO2017062875A1 (en) 2015-10-08 2017-04-13 The Children's Medical Center Corporation Compositions and methods for on-demand high-efficiency triggerable anesthesia
WO2017064657A1 (en) 2015-10-16 2017-04-20 Invictus Oncology Pvt. Ltd. Fluorescent anticancer platinum drugs
IL259795B1 (en) 2015-12-07 2023-12-01 Genzyme Corp Methods and compositions for treating a serpinc1-associated disorder
JP2018536689A (en) 2015-12-10 2018-12-13 アルナイラム ファーマシューティカルズ, インコーポレイテッドAlnylam Pharmaceuticals, Inc. Sterol regulatory element binding protein (SREBP) chaperone (SCAP) iRNA compositions and methods of use thereof
US20170246262A1 (en) 2016-02-25 2017-08-31 Applied Biological Laboratories, Inc. Compositions and methods for protecting against airborne pathogens and irritants
US20170360815A1 (en) 2016-02-25 2017-12-21 Applied Biological Laboratories, Inc. Compositions and methods for protecting against airborne pathogens and irritants
EP4104854A3 (en) 2016-04-04 2023-03-08 The United States of America as represented by the Secretary of the Department of Health and Human Services Multivalent vaccines for rabies virus and coronaviruses
MA45295A (en) 2016-04-19 2019-02-27 Alnylam Pharmaceuticals Inc HIGH DENSITY LIPOPROTEIN BINDING PROTEIN (HDLBP / VIGILINE) RNA COMPOSITION AND METHODS FOR USING THEM
US20190256845A1 (en) 2016-06-10 2019-08-22 Alnylam Pharmaceuticals, Inc. COMPLEMENT COMPONENT C5 iRNA COMPOSITIONS AND METHODS OF USE THEREOF FOR TREATING PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)
MX2019005833A (en) 2016-11-21 2019-10-30 Eirion Therapeutics Inc Transdermal delivery of large agents.
TW202313978A (en) 2016-11-23 2023-04-01 美商阿尼拉製藥公司 Serpina1 irna compositions and methods of use thereof
TWI790217B (en) 2016-12-16 2023-01-21 美商阿尼拉製藥公司 METHODS FOR TREATING OR PREVENTING TTR-ASSOCIATED DISEASES USING TRANSTHYRETIN (TTR) iRNA COMPOSITIONS
WO2019014530A1 (en) 2017-07-13 2019-01-17 Alnylam Pharmaceuticals Inc. Lactate dehydrogenase a (ldha) irna compositions and methods of use thereof
SG11202002940QA (en) 2017-11-01 2020-04-29 Alnylam Pharmaceuticals Inc Complement component c3 irna compositions and methods of use thereof
EP3710587A1 (en) 2017-11-16 2020-09-23 Alnylam Pharmaceuticals, Inc. Kisspeptin 1 (kiss1) irna compositions and methods of use thereof
WO2019100039A1 (en) 2017-11-20 2019-05-23 Alnylam Pharmaceuticals, Inc. Serum amyloid p component (apcs) irna compositions and methods of use thereof
MX2020006012A (en) 2017-12-18 2020-09-14 Alnylam Pharmaceuticals Inc High mobility group box-1 (hmgb1) irna compositions and methods of use thereof.
US11116561B2 (en) * 2018-01-24 2021-09-14 Medtronic Ardian Luxembourg S.A.R.L. Devices, agents, and associated methods for selective modulation of renal nerves
JP2021519777A (en) 2018-03-30 2021-08-12 インスメッド インコーポレイテッド Continuous manufacturing method of liposome drugs
AR115960A1 (en) 2018-08-16 2021-03-17 Alnylam Pharmaceuticals Inc COMPOSITIONS AND METHODS TO INHIBIT THE EXPRESSION OF THE LECT2 GENE
CN112424355A (en) 2018-09-18 2021-02-26 阿尔尼拉姆医药品有限公司 Ketohexokinase (KHK) iRNA compositions and methods of use thereof
US10913951B2 (en) 2018-10-31 2021-02-09 University of Pittsburgh—of the Commonwealth System of Higher Education Silencing of HNF4A-P2 isoforms with siRNA to improve hepatocyte function in liver failure
EP4013870A1 (en) 2019-08-13 2022-06-22 Alnylam Pharmaceuticals, Inc. Small ribosomal protein subunit 25 (rps25) irna agent compositions and methods of use thereof
BR112022003860A2 (en) 2019-09-03 2022-08-16 Alnylam Pharmaceuticals Inc COMPOSITIONS AND METHODS FOR INHIBITING THE EXPRESSION OF THE LECT2 GENE
WO2021067747A1 (en) 2019-10-04 2021-04-08 Alnylam Pharmaceuticals, Inc. Compositions and methods for silencing ugt1a1 gene expression
WO2021087325A1 (en) 2019-11-01 2021-05-06 Alnylam Pharmaceuticals, Inc. Compositions and methods for silencing dnajb1-prkaca fusion gene expression
JP2023500661A (en) 2019-11-01 2023-01-10 アルナイラム ファーマシューティカルズ, インコーポレイテッド HUNTINGTIN (HTT) iRNA AGENT COMPOSITIONS AND METHODS OF USE THEREOF
TW202140509A (en) 2019-12-13 2021-11-01 美商阿尼拉製藥公司 Human chromosome 9 open reading frame 72 (c9orf72) irna agent compositions and methods of use thereof
WO2021154941A1 (en) 2020-01-31 2021-08-05 Alnylam Pharmaceuticals, Inc. Complement component c5 irna compositions for use in the treatment of amyotrophic lateral sclerosis (als)
TW202140786A (en) 2020-02-10 2021-11-01 美商艾爾妮蘭製藥公司 Compositions and methods for silencing vegf-a expression
WO2021178607A1 (en) 2020-03-05 2021-09-10 Alnylam Pharmaceuticals, Inc. Complement component c3 irna compositions and methods of use thereof for treating or preventing complement component c3-associated diseases
EP4121534A1 (en) 2020-03-18 2023-01-25 Alnylam Pharmaceuticals, Inc. Compositions and methods for treating subjects having a heterozygous alanine-glyoxylate aminotransferase gene (agxt) variant
WO2021195307A1 (en) 2020-03-26 2021-09-30 Alnylam Pharmaceuticals, Inc. Coronavirus irna compositions and methods of use thereof
US20230190785A1 (en) 2020-03-30 2023-06-22 Alnylam Pharmaceuticals, Inc. Compositions and methods for silencing dnajc15 gene expression
JP2023520582A (en) 2020-04-06 2023-05-17 アルナイラム ファーマシューティカルズ, インコーポレイテッド Compositions and methods for silencing MYOC expression
EP4133076A1 (en) 2020-04-07 2023-02-15 Alnylam Pharmaceuticals, Inc. Angiotensin-converting enzyme 2 (ace2) irna compositions and methods of use thereof
WO2021207189A1 (en) 2020-04-07 2021-10-14 Alnylam Pharmaceuticals, Inc. Compositions and methods for silencing scn9a expression
WO2021206922A1 (en) 2020-04-07 2021-10-14 Alnylam Pharmaceuticals, Inc. Transmembrane serine protease 2 (tmprss2) irna compositions and methods of use thereof
BR112022021813A2 (en) 2020-04-27 2023-01-17 Alnylam Pharmaceuticals Inc APOLIPOPROTEIN AND (APOE) IRNA AGENT COMPOSITIONS AND METHODS OF USE THEREOF
WO2021237097A1 (en) 2020-05-21 2021-11-25 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting marc1 gene expression
WO2021252557A1 (en) 2020-06-09 2021-12-16 Alnylam Pharmaceuticals, Inc. Rnai compositions and methods of use thereof for delivery by inhalation
WO2022066847A1 (en) 2020-09-24 2022-03-31 Alnylam Pharmaceuticals, Inc. Dipeptidyl peptidase 4 (dpp4) irna compositions and methods of use thereof
EP4225917A1 (en) 2020-10-05 2023-08-16 Alnylam Pharmaceuticals, Inc. G protein-coupled receptor 75 (gpr75) irna compositions and methods of use thereof
WO2022087041A1 (en) 2020-10-21 2022-04-28 Alnylam Pharmaceuticals, Inc. Methods and compositions for treating primary hyperoxaluria
EP4232582A1 (en) 2020-10-23 2023-08-30 Alnylam Pharmaceuticals, Inc. Mucin 5b (muc5b) irna compositions and methods of use thereof
TW202237150A (en) 2020-12-01 2022-10-01 美商艾拉倫製藥股份有限公司 Methods and compositions for inhibition of hao1 (hydroxyacid oxidase 1 (glycolate oxidase)) gene expression
TWI760996B (en) * 2020-12-25 2022-04-11 姜秉均 Nanoparticle containing hydrophobic substance and manufacturing process and use thereof
JP2024508714A (en) 2021-02-12 2024-02-28 アルナイラム ファーマシューティカルズ, インコーポレイテッド Superoxide dismutase 1 (SOD1) iRNA composition and method for using the same for treating or preventing superoxide dismutase 1- (SOD1-) related neurodegenerative diseases
CN117222739A (en) 2021-02-25 2023-12-12 阿尔尼拉姆医药品有限公司 Prion protein (PRNP) IRNA compositions and methods of use thereof
WO2022192519A1 (en) 2021-03-12 2022-09-15 Alnylam Pharmaceuticals, Inc. Glycogen synthase kinase 3 alpha (gsk3a) irna compositions and methods of use thereof
WO2022212231A2 (en) 2021-03-29 2022-10-06 Alnylam Pharmaceuticals, Inc. Huntingtin (htt) irna agent compositions and methods of use thereof
WO2022245583A1 (en) 2021-05-18 2022-11-24 Alnylam Pharmaceuticals, Inc. Sodium-glucose cotransporter-2 (sglt2) irna compositions and methods of use thereof
WO2022256290A2 (en) 2021-06-04 2022-12-08 Alnylam Pharmaceuticals, Inc. HUMAN CHROMOSOME 9 OPEN READING FRAME 72 (C9ORF72) iRNA AGENT COMPOSITIONS AND METHODS OF USE THEREOF
TW202333748A (en) 2021-07-19 2023-09-01 美商艾拉倫製藥股份有限公司 Methods and compositions for treating subjects having or at risk of developing a non-primary hyperoxaluria disease or disorder
WO2023076450A2 (en) 2021-10-29 2023-05-04 Alnylam Pharmaceuticals, Inc. HUNTINGTIN (HTT) iRNA AGENT COMPOSITIONS AND METHODS OF USE THEREOF
WO2023141314A2 (en) 2022-01-24 2023-07-27 Alnylam Pharmaceuticals, Inc. Heparin sulfate biosynthesis pathway enzyme irna agent compositions and methods of use thereof

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4708861A (en) * 1984-02-15 1987-11-24 The Liposome Company, Inc. Liposome-gel compositions
US4932936A (en) * 1988-01-29 1990-06-12 Regents Of The University Of Minnesota Method and device for pharmacological control of spasticity
US5298019A (en) * 1990-08-21 1994-03-29 Associated Synapse Biologics Controlled administration of chemodenervating pharmaceuticals
US5698549A (en) * 1994-05-12 1997-12-16 Uva Patent Foundation Method of treating hyperactive voiding with calcium channel blockers
US5763399A (en) * 1991-07-10 1998-06-09 C.R. Bard, Inc. Composition and method for revitalizing scar tissue
US5837694A (en) * 1993-10-18 1998-11-17 The Walter And Eliza Hall Institute Of Medical Research Method for enhancing neurone survival and agents useful for same
US6207180B1 (en) * 1997-04-03 2001-03-27 Thomas B. Ottoboni Intravesical drug delivery
US6239180B1 (en) * 1995-11-08 2001-05-29 The Regents Of The University Of California Transdermal therapeutic device and method with capsaicin and capsaicin analogs
US6239267B1 (en) * 1998-03-10 2001-05-29 Smithkline Beecham Plc VANILREP1 polynucleotides and VANILREP1 polypeptides
US6271211B1 (en) * 1997-02-13 2001-08-07 Albert Einstein College Of Medicine Of Yeshiva University Gene therapy for regulating penile smooth muscle tone
US20010018415A1 (en) * 1993-12-28 2001-08-30 Aoki K. Roger Method for treating tardive dyskinesia with botulinum toxin type B
US6365164B1 (en) * 1997-07-15 2002-04-02 University Technology Corporation Use of neurotoxin therapy for treatment of urologic and related disorders

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1165238A (en) * 1980-03-12 1984-04-10 Demetrios P. Papahadjopoulos Activated liposomes and method

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4708861A (en) * 1984-02-15 1987-11-24 The Liposome Company, Inc. Liposome-gel compositions
US4932936A (en) * 1988-01-29 1990-06-12 Regents Of The University Of Minnesota Method and device for pharmacological control of spasticity
US5298019A (en) * 1990-08-21 1994-03-29 Associated Synapse Biologics Controlled administration of chemodenervating pharmaceuticals
US5763399A (en) * 1991-07-10 1998-06-09 C.R. Bard, Inc. Composition and method for revitalizing scar tissue
US5837694A (en) * 1993-10-18 1998-11-17 The Walter And Eliza Hall Institute Of Medical Research Method for enhancing neurone survival and agents useful for same
US20010018415A1 (en) * 1993-12-28 2001-08-30 Aoki K. Roger Method for treating tardive dyskinesia with botulinum toxin type B
US5698549A (en) * 1994-05-12 1997-12-16 Uva Patent Foundation Method of treating hyperactive voiding with calcium channel blockers
US6239180B1 (en) * 1995-11-08 2001-05-29 The Regents Of The University Of California Transdermal therapeutic device and method with capsaicin and capsaicin analogs
US6271211B1 (en) * 1997-02-13 2001-08-07 Albert Einstein College Of Medicine Of Yeshiva University Gene therapy for regulating penile smooth muscle tone
US6207180B1 (en) * 1997-04-03 2001-03-27 Thomas B. Ottoboni Intravesical drug delivery
US6365164B1 (en) * 1997-07-15 2002-04-02 University Technology Corporation Use of neurotoxin therapy for treatment of urologic and related disorders
US6239267B1 (en) * 1998-03-10 2001-05-29 Smithkline Beecham Plc VANILREP1 polynucleotides and VANILREP1 polypeptides

Cited By (18)

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Publication number Priority date Publication date Assignee Title
US20070122466A1 (en) * 2001-08-13 2007-05-31 University Of Pittsburgh Sphingomyelin liposomes for the treatment of hyperactive bladder disorders
US8110217B2 (en) 2001-08-13 2012-02-07 University Of Pittsburgh Sphingomyelin liposomes for the treatment of hyperactive bladder disorders
WO2008147807A2 (en) 2007-05-23 2008-12-04 Amcol International Corporation Cholesterol-interacting layered phyllosilicates and methods of reducing hypercholesteremia in a mammal
EP2431043A1 (en) 2007-05-23 2012-03-21 Amcol International Corporation Cholesterol-interacting layered phyllosilicates for suppressing gastrointestinal cholesterol absorption
US20110117026A1 (en) * 2008-05-19 2011-05-19 Yu-Chen Tseng Methods and compositions for the delivery of bioactive compounds
US20100303932A1 (en) * 2009-05-28 2010-12-02 Baskaran Thyagarajan Method for Attenuating the Effects of Botulinum Toxin
US9526648B2 (en) 2010-06-13 2016-12-27 Synerz Medical, Inc. Intragastric device for treating obesity
US10010439B2 (en) 2010-06-13 2018-07-03 Synerz Medical, Inc. Intragastric device for treating obesity
US10413436B2 (en) 2010-06-13 2019-09-17 W. L. Gore & Associates, Inc. Intragastric device for treating obesity
US10420665B2 (en) 2010-06-13 2019-09-24 W. L. Gore & Associates, Inc. Intragastric device for treating obesity
US10512557B2 (en) 2010-06-13 2019-12-24 W. L. Gore & Associates, Inc. Intragastric device for treating obesity
US11135078B2 (en) 2010-06-13 2021-10-05 Synerz Medical, Inc. Intragastric device for treating obesity
US11351050B2 (en) 2010-06-13 2022-06-07 Synerz Medical, Inc. Intragastric device for treating obesity
US11596538B2 (en) 2010-06-13 2023-03-07 Synerz Medical, Inc. Intragastric device for treating obesity
US11607329B2 (en) 2010-06-13 2023-03-21 Synerz Medical, Inc. Intragastric device for treating obesity
US8728473B2 (en) 2010-12-01 2014-05-20 Alderbio Holdings Llc Methods of preventing or treating pain using anti-NGF antibodies
US20120189677A1 (en) * 2011-01-20 2012-07-26 Stephen Tonge Formulations
US10779980B2 (en) 2016-04-27 2020-09-22 Synerz Medical, Inc. Intragastric device for treating obesity

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