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WO2006110623A2 - Toxine cholerique ciblee pour le traitement de la douleur persistante ou chronique - Google Patents

Toxine cholerique ciblee pour le traitement de la douleur persistante ou chronique Download PDF

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Publication number
WO2006110623A2
WO2006110623A2 PCT/US2006/013239 US2006013239W WO2006110623A2 WO 2006110623 A2 WO2006110623 A2 WO 2006110623A2 US 2006013239 W US2006013239 W US 2006013239W WO 2006110623 A2 WO2006110623 A2 WO 2006110623A2
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cta
substance
conjugate
cholera toxin
cells
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WO2006110623A3 (fr
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Robert M. Caudle
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University of Florida
University of Florida Research Foundation Inc
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University of Florida
University of Florida Research Foundation Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Pain management and treatments range from analgesics to using relaxation and imagery as a tool for distraction 4 . Some methods are more effective than others. Many of the medications used for pain management have negative side effects.
  • Opioids are used for treatment of chronic pain, especially morphine which is commonly found in hospital settings. Although this narcotic induces sedation as well as pain relief it has many negative side effects. In addition to it being very addicting, morphine has frequently been associated with side effects such as nausea, vomiting, and constipation 4 . Studies conducted have also demonstrated the immunosuppressive effect of morphine, weakening the immune system of patients consuming the substance 8 . Long term use of the narcotic can severely damage a person's immune system increasing their risk of infection and other ailments. Other experiments have found that in smaller doses morphine's negative side effects can be minimized, but are none-the-less evident. Limiting the dosage unfortunately resulted in less pain blockage 4 . Morphine has helped those suffering to successfully cope with chronic pain, but due to its array of negative side effects and limitations other methods must be investigated.
  • FIG. 1 Synthesis of SP-CTA.
  • A Schematic representation of the procedure used to synthesize SP-CTA.
  • B Western blots of final SP-CTA product. SP-CTA and the filtrate from the Centricon Plus-20 concentrating tubes (Wash) were run on western blots and probed with antibodies to substance P and the catalytic subunit of cholera toxin (CTA). The SP-CTA product reacted with both antibodies.
  • Figure 2 In situ evaluation of SP-CTA. A. CHO cells stably transfected with the
  • NKl receptor were treated with either SP-CTA (lig/ml), CTA (lig/ml) or were not exposed to any agents (Control). The cells were cultured over night in these solutions and then immunocytochemistry was performed on the cells using an antibody to CTA and Rhodamine-tyramide amplification.
  • B. Concentration response relationship for SP-CTA on cAMP production. CHO-NKl cells were exposed to the indicated concentrations of SP-CTA for 1 hour. The cells were cultured for another 48 hours following washout of the SP-CTA with fresh culture media. The cells were harvested and cAMP content was measured using a commercial cAMP assay. (N 5 100mm plates/ concentration) C.
  • FIG. 4 Effect of intrathecal SP-CTA on thermal nociception in rats.
  • the invention relates to engineering of a compound involving conjugating a cholera toxin subunit A to a targeting molecule (TM) that assists in targeting the cholera toxin to a specific g-protein coupled receptor on neurons, or other cell types.
  • Targeting molecules may include but are not limited to, substance P, an opioid, and CGRP 5 or any peptide for which there is a g-protein coupled receptor.
  • Cholera toxin (CT) is a bacterial toxin secreted by Vibrio cholerae and comprising A and B subunits. The A subunit is the catalytic molecule and contributes to intracellular toxicity and the B subunit is required for binding of CT to a cell surface receptor.
  • ctxA and ctxB The structural genes encoding A and B subunits are designated as ctxA and ctxB respectively, (see, e.g., Kaper and Srivastava, Indian J. Med. Res.95:163-7 (1992); Field, Am. J. Clin. Nutr. (l):189-96 (1979); and Van Heyningen et al., Ciba Found Symp. 1976;(42):73-88 (1976)
  • Cholera toxin subunit A (CTA) is responsible for the toxic effect induced by this toxin. It is an ADP-ribosyltransferase that disrupts the proper signaling of G protein activity.
  • the nontoxic B subunit of cholera toxin, choleragenoid is responsible for the uptake and transport of the toxin into the axon and cell bodies 1 .
  • the A subunit of cholera toxin is preferably, though not necessarily used, because of its ability to alter cellular activity and function without cellular death.
  • a targeting molecule can be attached to Cholera toxin subunit A through a chemical bond, or the composition can be prepared as a chimera using techniques of recombinant DNA.
  • the conjugate can be used to specifically target cells having receptors to which the targeting molecule binds and inducing a cellular response in such cells.
  • the invention provides a fusion protein comprising the amino acid sequence encoding a target molecule and Cholera toxin subunit A.
  • This invention also provides a recombinant nucleic acid molecule comprising an isolated nucleic acid molecule encoding targeting molecule and cholera toxin subunit A. See U.S. Patent Nos.
  • a CTA-TM conjugate comprises a CTA protein and a TM, wherein said protein or TM include a polypeptide at least 80%, or at least 85% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to CTA and at least one of the TMs described herein, respectively.
  • % similarity for two polypeptides, is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) and the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) to find the best segment of similarity between two sequences.
  • a protein having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a CTA protein is intended that the amino acid sequence of the protein is identical to the reference sequence except that the protein sequence may include a ratio of up to five amino acid alterations per each 100 amino acids of the reference amino acid of the CTA.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • CTA or TMs, or fragments thereof can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis 53711).
  • Bestfit program Wiconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis 53711.
  • Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
  • the invention relates to engineering of a compound involving conjugating Substance P to a cholera toxin (SP-CTA) that suppresses pain cells while minimizing cell death.
  • SP-CTA cholera toxin
  • the term "Substance P" as used herein refers to Substance P and suitable analog's thereof. See for example, U.S. Patent Publication 2004/0253248.
  • Cholera toxin binds specifically to the surface receptors, GM-I glanglioside, of neurons. Cholera toxin is typically used to uncouple G-protein and increase cyclic Adenosine Monophosphate concentration within cells 5 .
  • the subject invention pertains to a method for reducing pain in a subject comprising administering to the subject a therapeutically effective amount of the conjugate SP-CTA.
  • the subject invention pertains to a method of treating a NK- lR-associated disorder in a subject, which comprises administering to the subject an amount of the pharmaceutical composition comprising a therapeutically effective amount of the conjugate comprising SP-CTA and a pharmaceutically acceptable carrier thereby treating the disorder associated with the NK-IR.
  • disorders or diseases include but are not limited to: respiratory conditions (e.g. asthma, allergic rhinitis), ophthalmic conditions (e.g. conjunctivitis), cutaneous conditions (e.g. allergic dermatitis, dermatitis by contact, psoriasis), intestinal conditions (e.g.
  • ulcerative colitis Crohn's disease
  • gastrointestinal tract central nervous system disorders such as anxiety and psychosis, inflammatory diseases such as rheumatoid arthritis and inflammatory bowel diseases, as well as pain in any of the aforesaid conditions, including migraine.
  • disorders or diseases include but are not limited to: Alzheimer's disease, multiple sclerosis, attenuation of morphine withdrawal, cardiovascular changes, oedema, such as oedema caused by the ⁇ nal injury, chronic inflammatory diseases such as rheumatoid arthritis, asthma/bronchial hyperactivity and other respiratory diseases including allergic rhinitis, inflammatory diseases of the gut including ulcerative colitis and Crohn's disease, ocular injury and ocular inflammatory diseases, proliferative vitreoretinopathy, irritable bowel syndrome and disorders of bladder function including cystitis and bladder detrusor hyperrefiexia, demyelinating diseases such as multiple sclerosis and amyotrophic lateral sclerosis, asthmatic disease, small cell carcinomas, in particular small cell lung cancer, depression, dysthymic disorders, chronic obstructive airways disease, hypersensitivity disorders such as poison ivy, vasospastic diseases such as angina and Reynauldis disease, fibrosing
  • mi OeTmV s diseased and other neuronatholoeical disorders such as peripheral neuropathy inflammatory diseases such as inflammatory bowel disease, irritable bowel syndrome, psoriasis, fibrositis, ocular inflammation, osteoarthritis and rheumatoid arthritis, allergies such as eczema and rhinitis; hypersensitivity disorders such as poison ivy; ophthalmic diseases such as conjunctivitis, vernal conjunctivitis, dry eye syndrome, and the like; cutaneous diseases such as contact dermatitis, atopic dermatitis, urticaria, and other eczematoid dermatitis; oedema; such as oedema caused by thermal injury; addition disorders such as alcoholism; stress related somatic disorders; reflex sympathetic dystrophy such as shoulder/hand syndrome; dysthymic disorders; neuropathy, such as diabetic or peripheral neuropathy and chemotherapy-induced neuropathy; postherpetic and other neuralgias
  • CTA is conjugated with other targeting molecules that assist in targeting CTA to g-protein receptors.
  • targeting molecules include, but are not limited to, endorphins, and particularly in the case of an analgesic, beta-endorphin (Fries, DS (2002), Opioid Analgesics, In Williams DA, Lemke TL, Foye's Principles of Medicinal Chemistry (5 ed.), Philadelphia: Lippincott Williams & Wilkins), enkephalins (particularly Met enkephalin and Leu-enkephalin, [Met] -enkephalin is Tyr-Gly-Gly-Phe-Met and[Leu] -enkephalin has Leu in place of Met), endomorphins (e.g., endomorphin-1; Tyr-Pro-T ⁇ -Phe-NH2, and endomorphin-2; Tyr-Pro-Phe- Phe-NH2) and the dynorphins.
  • endorphins e.g., endomorph
  • targeting molecules specific to G-protein receptors is provided in Br J Pharmacol 144: S4-S62 (2005).
  • Other targeting molecules that are contemplated for conjugation to CTA include, but are not limited to, free compound serotonin, bradykinin, bombesin, calcitonin, cholecystokinin, neurotensin, glucagon, secretin, somatostatin, motilin, vasopressin, oxytocin, prolactin, thyrotropin, an angiotensin, galanin, neuropeptide Y, thyrotropin-releasing hormone, gonadotropnin-releasing hormone, growth hormone-releasing hormone, luteinizing hormone, glucosylamine, lactylamine, leucine, glutamate and amino cholines.
  • Coupled as used herein is intended to mean that the coupled moieties are directly bonded to each other, or indirectly bonded to each other, such as by a linker.
  • linkage as used herein is intended to mean a bond or group formed by chemical reaction between two moieties such that the moieties are covalently coupled.
  • Methods for the preparation of a linkage such as an amide bond are described in Houben-Weyl, Methoden der organischen Chemie (Methods of Organic Chemistry), Volume 15/2; Bodanszky et al., in "Peptide Synthesis", E. Gross & J. Meienhofer (Eds), Academic Press, Y. Wiley, New York, 1976. Further reactions are detailed in R. C. Larock, Comprehensive Organic Transformations: A Guide to Functional Group Preparations, Wiley- VCH, 2nd ed., 1999.
  • the subjects to be treated or whose tissue may be used herein may be a mammal, or more specifically a human, horse, pig, rabbit, dog, cat, monkey, or rodent.
  • the si ⁇ hiprt i ⁇ 5 a human [026]
  • the invention includes the pharmaceutically acceptable salts and complexes of all the compounds described herein.
  • the salts include but are not limited to the following acids and bases. Examples of suitable inorganic acids include, but are not limited to: hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and boric acid.
  • suitable organic acids include but are not limited to: acetic acid, trifluoroacetic acid, formic acid, oxalic acid, malonic acid, succinic acid, tartaric acid, maleic acid, fumaric acid, methanesulfonic acid, trifluoromethanesulfonic acid, benzoic acid, glycolic acid, lactic acid, citric acid and mandelic acid.
  • suitable inorganic bases include, but are not limited to: ammonia, hydroxyethylamine and hydrazine.
  • Suitable organic bases include, but are not limited to, methylamine, ethylamine, trimethylamine, triethylamine, ethylenediamine, hydroxyethylamine, morpholine, piperazine and guanidine.
  • the invention further provides for the hydrates and polymorphs of all of the compounds described herein.
  • the pharmaceutical carrier may be a liquid and the pharmaceutical composition would be in the form of a solution.
  • the pharmaceutically acceptable carrier is a solid and the pharmaceutical composition is in the form of a powder or tablet.
  • the pharmaceutical carrier is a gel and the pharmaceutical composition is in the form of a suppository or cream.
  • the compound may be formulated as part of a pharmaceutically acceptable transdermal patch.
  • a solid carrier can include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or table- disintegrating agents, it can also be an encapsulating material.
  • the carrier is a finely divided solid that is in admixture with the finely divided active ingredient.
  • the active- ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredient.
  • Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fats.
  • the liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators.
  • suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g.
  • cellulose derivatives preferably sodium carboxymethyl cellulose solution
  • alcohols including monohydric and polyhydric alcohols, e.g. glycols
  • nnri nils e. ⁇ fr ⁇ r.tinnaterl r.nr.nrmt nil and aranhis nil ⁇
  • the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate.
  • Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration.
  • the liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant, which are useful for intranasal administration.
  • Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized for intramuscular, intrathecal, intratracheal, epidural, intraperitoneal or subcutaneous injections. Sterile solutions can also be administered intravenously.
  • the compounds may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
  • Carriers are intended to include necessary and inert binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes and coatings.
  • the pharmaceutical composition further comprises a cytokine.
  • cytokines include but are not limited: transforming growth factor beta, epidermal growth factor family, fibroblast growth factors, hepatocyte growth factor, insulin-like growth factors, B-nerve growth factor, platelet-derived growth factor, vascular endothelial growth factor, interleukin 1, IL-I receptor antagonist, interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6, IL-6 soluble receptor, interleukin 7, interleukin 8, interleukin 9, interleukin 10, interleukin 11, interleukin 12, interleukin 13, angiogenin, chemokines, colony stimulating factors, granulocyte-macrophage colony stimulating factors, erythropoietin, interferon, interferon gamma, leukemia inhibitory factor, oncostatin M, pleiotrophin, secretory leukocyte protease inhibitor, stem cell factor, tumor
  • the compound can be administered in the form of a sterile solution or suspension containing other solutes or suspending agents, for example, enough saline or glucose to make the solution isotonic, bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • a sterile solution or suspension containing other solutes or suspending agents, for example, enough saline or glucose to make the solution isotonic, bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • suitable pharmaceutical carriers include any of the standard pharmaceutically accepted carriers known to those of ordinary skill in the art.
  • suitable pharmaceutical carriers include, but are not limited to, phosphate buffered saline solution, water, emulsions such as oil/water emulsions or a triglyceride emulsion, various types of wetting agents, tablets, coated tablets and capsules.
  • a suitable pharmaceutically acceptable carrier may be selected taking into account the chosen mode of administration.
  • the pharmaceutical compositions may also include suitable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers.
  • the resulting pharmaceutical compositions may be liquids or lyophilized or otherwise dried formulations.
  • suitable diluents include, but are not limited to, Tris-HCL, Tris- acetate and Tris-phosphate.
  • the diluents employed may vary in their buffer contents pH and/or ionic strength.
  • additives examples include, but are not limited to: albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Plurpnic F68, bile acid salts), solubilizing agents (e.g., Thimerosal, benzyl alcohol), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparation of polymeric compounds such as polylactic acid, polyglycolic acid, polyvinyl pyrrolidone, etc.
  • solubilizing agents e.g., Thimerosal, benzyl alcohol
  • bulking substances or tonicity modifiers e.g., lactose, mannitol
  • covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particul
  • compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of the compounds.
  • antioxidants e.g., ascorbic acid
  • low molecular weight polypeptides i.e., polyarginine or tripeptide
  • proteins such as serum albumin, gelatin, or immunoglobulins
  • amino acids such as glycine, glutamine acid, aspartic acid, or arginine
  • chelating agents such as EDTA
  • sugar alcohols such as mannitol or sorbitol.
  • Controlled or sustained release compositions include formulation of lipophilic deposits (e.g., fatty acids, waxes, oils).
  • particulate compositions coated with polymers e.g., poloxamers or poloxamines
  • Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular compound in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors depending on the particular subject being treated, including subject age, weight, gender, diet and time of administration, will result in a need to adjust dosages.
  • Administration of the compound may be effected continuously or intermittently, in any treatment regimen, the composition may be administered to a patient either singly or in a cocktail containing two or more targeted toxins, other therapeutic agents, compositions, or the like, including, but not limited to, immunosuppressive agents, tolerance-inducing agents, potentiators and side-effect relieving agents.
  • immunosuppressive agents useful in suppressing allergic reactions of a host.
  • Preferred immunosuppressive agents include prednisone, prednisolone, DECADRON (Merck, Sharp & Dohme, West Point, Pa.), cyclophosphamide, cyclosporine, 6-mercaptopurine, methotrexate, azathioprine and i.v. gamma p-inbiilin or their combination.
  • Preferred notentiators include monensin. ammonium chloride. perhexiline, verapamil, amantadine, and chloroquine. All of is these agents are administered in generally-accepted efficacious dose ranges such as those disclosed in the Physician's Desk Reference, 41st Ed., Publisher Edward R. Barnhart, NJ. (1987).
  • an appropriate dosage level will generally be about 0.001 to 50 mg per kg patient body weight per day that can be administered in single or multiple doses.
  • the dosage level will be about 0.005 to about 25 mg/kg, per day; more preferably about 0.01 to about 10 mg/kg per day; and even more preferably about 0.05 to about 1 mg/kg per day.
  • Intrathecal injections were performed under isoflurane anesthesia via lumbar puncture between L4 and L5. All animal procedures in this project were reviewed and approved by the University of Florida's Institutional Animal Care and Use Committee.
  • CTA was purchased from List Biological Laboratories inc. (Cambell, CA). CTA has two cysteine residues in the C-terminal region 15,27 therefore these cysteine residues were used to attach substance P to CTA.
  • the synthesis of SP-CTA was accomplished using a modification of Pierce Biotechnology inc.'s maleimide protein cross-linking procedure. The synthesis was carried out in two stages.
  • the first stage was to link maleimide to the N-terminus of substance P by combining a 5 fold excess of Sulfosuccinimidyl 4-N-maleimidomethyl cyclohexane-1-carboxylate (Sulfo-SMCC) with substance P in phosphate buffered saline (PBS, pH 7.4). The mixture was incubated at room temperature for 1 hour. The substance P maleimide conjugate was separated from unreacted Sulfo- SMCC using a Sephadex G-10 (30 X 1.5 cm) column eluted with PBS.
  • Sulfo-SMCC Sulfosuccinimidyl 4-N-maleimidomethyl cyclohexane-1-carboxylate
  • the substance P maleimide conjugate was linked to the two cysteines on CTA by adding a 10 fold excess of the conjugate to CTA in PBS. This mixture was then incubated at room temperature for another hour. The SP-CTA was then separated from the unreacted substance P maleimide conjugate, washed with PBS three times and concentrated using Centricon Plus-20 filters. A sample of the final product was evaluated by western blots. Briefly, the sample was run on 4-20% PAGE gels, transferred to PVDF membranes and then probed with antibodies to either substance P or the catalytic subunit of cholera toxin. A secondary antibody coupled to horse radish peroxidase and enhanced chemiluminescence were used to visualize the bands. Figure 1 illustrates the synthetic pathway as well as western blots of the final product.
  • CHO-NKl Chinese Hamster Ovary cells stably expressing NKl receptors (CHO-NKl) (a generous gift from Dr. James Krause, Neurogen Corp.22) were plated on 100 mm plates for cAMP assays or 13 mm cover slips in 24 well culture plates for immunocytochemistry experiments. The cultures were grown in F12K media, 10% Fetal Bovine Serum, 1% L-glutamine, 1% penicillin- Streptomyosin, 25mM Hepes buffer, and G418 (500ig/ml). The cells were cultured at 37oC in a 5% CO2 atmosphere.
  • Enzyme Immunoassay was used according to the manufacturer's instructions. Briefly, the media on the cell cultures was removed and the cells were washed once with PBS (pH 7.4). The PBS was removed and 1 ml 0.1 M HCL was added to the cells. The cells were scraped from the plates into the HCL solution, sonicated and centrifuged (600 g, 10 minutes, 5°C). The protein in each sample was measured using Bio-Rad's (Hercules, CA) protein assay. The cAMP was acetylated with the addition of 100 ⁇ l of the kit's acetic anhydride solution.
  • a 100 ⁇ l sample was then neutralized with 50 ⁇ l of the kit's neutralizing buffer and the samples were added to the kit's 96 well plates that were pre- absorbed with antibodies to cAMP.
  • a standard curve and controls were set up as suggested by the manufacturer.
  • a cAMP- alkaline phosphatase conjugate 50 ⁇ l was added to the wells and the solution was incubated for 2 hours.
  • the plates were then washed 3 times and 200 ⁇ l of p-nitrophenyl phosphate solution (substrate) was added to each well and the plates were incubated for 1 hour.
  • the reaction was stopped with 50 ⁇ l 0.1 M HCL and the plate was read at 405 nm.
  • the concentration of cAMP in the samples was extrapolated from the data collected for the cAMP standards and expressed as the number of moles of cAMP per mg protein.
  • Rats were euthanized with pentobarbital and immediately transcardially perfused with ice cold PBS and then ice cold 4% paraformaldehyde in phosphate buffered saline (PBS)(pH 7.4). Cell cultures on cover slips were washed with PBS and fixed with 4% paraformaldehyde in PBS. The spinal cords were removed and post fixed overnight in 4% paraformaldehyde in PBS. The tissue was cryoprotected in 30% sucrose, mounted and sectioned in a cryostat (-20oC)(10-20 ⁇ m) and mounted on slides. The sections or cells were then blocked with 3% normal goat serum for 60 minutes with 0.75% triton X-100.
  • PBS phosphate buffered saline
  • the primary antibody was then added to the blocking solution (1:500-5,000) and the sections were incubated for 48 hours at 4oC.
  • the sections or cells were washed (8 X 5 mins) in PBS. Following the wash the sections or cells were incubated for 1 hour at room temperature in PBS with a secondary antibody that was coupled to horse radish peroxidase.
  • the sections were washed (8 X 5 mins) and then treated with diaminobenzidine.
  • the cultured cells were washed similarly and labeled using rhodamine labeled tyramide as described by the manufacturer (NEN, Boston, MA).
  • Thermal nociception was measured using the method of Hargreaves et al. . Briefly, the rats were placed on a clear glass surface and allowed 15 minutes to accommodate to the enclosure. An infrared light was directed onto a hind paw's plantar surface approximately in the middle of the foot. The latency for the animal to remove its foot from the path of the light was used as the dependent measure for thermal sensitivity.
  • the neuropeptide substance P was coupled to the catalytic subunit of cholera toxin (CTA) using the bifunctional linking agent sulfosuccinimidyl 4-N-maleimidomethyl cyclohexane-1- carboxylate (Sulfo-SMCC) as indicated in figure IA. Briefly, the Sulfo-SMCC was reacted with the N-terminal amine of substance P to form an amide linkage to the maleimide group. The substance P - maleimide was then conjugated to CTA through two cysteine residues in the C-terminal region of the CTA protein. The final product was washed and concentrated by centrifugation in Centricon filters with a cutoff of 5kd.
  • CTA cholera toxin
  • Sulfo-SMCC bifunctional linking agent sulfosuccinimidyl 4-N-maleimidomethyl cyclohexane-1- carboxylate
  • SP-CTA was tested on Chinese Hamster Ovary cells that were stably transfected with the NKl receptor 22 (CHO-NKl). To verify selective uptake of SP-CTA by the cells, the cells were incubated over night in either SP-CTA (1 ⁇ g/ml) or CTA (1 ⁇ g/ml) alone. The CHO-NKl cells were then fixed and prepared for immunocytochemistry with antibodies to CTA using a rhodamine- tyramide amplification system. As illustrated in figure 2A only the SP-CTA treated cells demonstrated an uptake of CTA indicating that linkage of CTA to substance P was required for the conjugate to be internalized.
  • FIG. 2B demonstrates the concentration response relationship for SP-CTA when the SP-CTA is applied for 1 hour and the cAMP was measured after culturing the cells for an additional 48 hours.
  • the time course of SP-CTA' s effect on cAMP production was evaluated by treating the cells for 1 hour with 100 ng/ml SP-CTA and then harvesting the cells for cAMP analysis 1, 2, 3 and 4 days following exposure to the conjugate.
  • SP-CTA' s effects on cAMP peaked at 1 day in the CHO-NKl cells and remained significantly elevated for 3 days.
  • Figure 2D demonstrates that 48 hours following a 4 hour exposure to 100 ng/ml of either substance P, CTA or SP-CTA only SP-CTA treated CHO-NKl cells produced an increase in cAMP production.
  • 100 ng/ml of substance P, CTA and SP-CTA remained in the culture media for the full 48 hours the substance P treated cells and the SP-CTA treated cells had significantly elevated levels of cAMP, whereas the CTA treated cells did not differ from control cells ( Figure 2E).

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Abstract

L'invention concerne un composé fabriqué par conjugaison d'une toxine cholérique (ou d'une sous-unité de celle-ci) à une molécule de ciblage facilitant le ciblage de la toxine cholérique sur un récepteur couplé aux protéines G spécifique sur des neurones. Les molécules de ciblage de l'invention peuvent comprendre, entre autres, une substance P, un opioïde et CGRP, ou un peptide quelconque pour lequel il existe un récepteur couplé aux protéines G. Plus particulièrement, l'invention se rapporte à la fabrication d'un composé par conjugaison d'une substance P à une toxine cholérique, ce composé supprimant la douleur chez un sujet tout en réduisant la mort cellulaire des neurones.
PCT/US2006/013239 2005-04-09 2006-04-10 Toxine cholerique ciblee pour le traitement de la douleur persistante ou chronique Ceased WO2006110623A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017011738A1 (fr) * 2015-07-16 2017-01-19 Saint Louis University Antagonistes de gpr160 pour le traitement de la douleur

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US4664911A (en) * 1983-06-21 1987-05-12 Board Of Regents, University Of Texas System Immunotoxin conjugates employing toxin B chain moieties
US6022950A (en) * 1984-06-07 2000-02-08 Seragen, Inc. Hybrid molecules having translocation region and cell-binding region
US5169933A (en) * 1988-08-15 1992-12-08 Neorx Corporation Covalently-linked complexes and methods for enhanced cytotoxicity and imaging
US6545126B1 (en) * 1999-03-18 2003-04-08 Wisconsin Alumni Research Foundation Chimeric toxins

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017011738A1 (fr) * 2015-07-16 2017-01-19 Saint Louis University Antagonistes de gpr160 pour le traitement de la douleur

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