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Title:  Methods for identifying an analog that promotes nerve regeneration
United States Patent: 
7,282,340
Issued: 
October 16, 2007

Inventors: 
Gold; Bruce G. (West Linn, OR)
Assignee: 
Oregon Health Sciences University (Portland, OR)
Appl. No.: 
10/943,506
Filed: 
September 17, 2004


 

Pharm Bus Intell & Healthcare Studies


Abstract

Analogs of FK506 that do not bind FKBP-12 have been found to effectively promote nerve cell growth and regeneration, thereby speeding functional recovery of damaged nervous tissue and axonal regeneration without causing immunosuppression.

SUMMARY OF THE INVENTION

FK506 analogs that do not bind FKBP-12 surprisingly have neurotrophic activity, displaying effectiveness in promoting nerve regeneration and functional recovery and stimulating neuritic outgrowth, for example.

According to one embodiment of the invention, pharmaceutical compositions are provided that comprise an amount of a non-FKBP-12-binding ("non-binding") FK506 analog that is effective in stimulating nerve cell growth and a pharmaceutically acceptable excipient, for example, a composition comprising a unit dose of the non-binding FK506 analog. Such compositions can also include other active ingredients, e.g., one or more neurotrophic factors including, but not limited to, neurotrophic growth factor (NGF), insulin growth factor (IGF-1), acidic or basic fibroblast growth factor (aFGF and bFGF, respectively), platelet-derived growth factor (PDGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factors (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), and neurotrophin 4/5 (NT 4/5). A preferred neurotrophic compound is NGF.

According to another embodiment of the invention, therapeutic methods are provided that comprise administering to a patient a pharmaceutical composition comprising a non-binding FK506 analog. For example, such methods can include administering an amount of such a composition that is effective in stimulating nerve cell growth.

According to another embodiment of the invention, methods for stimulating neurite outgrowth are provided that comprise contacting a nerve cell with a composition comprising a neurotrophic amount of a non-binding FK506 compound.

According to another embodiment of the invention, methods of drug discovery are provided that comprise assaying FK506 analogs for binding to FKBP-12, selecting an FK506 analog that does not bind FKBP-12, and assaying the selected FK506 analog for activity in promoting nerve cell growth.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, FK506 analogs that do not bind FKBP-12 have been found to effectively speed functional recovery of damaged nervous tissue and axonal regeneration. This discovery indicates that cyclophilin A-mediated calcineurin inhibition, which is dependent upon FKBP-12 binding, probably does not play a role in the ability of FK506 to speed nerve regeneration and that the nerve regenerative and immunosuppressive effects of FK506 arise via distinct mechanisms. The efficacy of FK506 and related compounds in promoting neurite outgrowth does not necessarily involve FKBP-12 inhibition.

Also encompassed are pharmaceutically acceptable derivatives of the FK506 analogs, including, but not limited to, any pharmaceutically acceptable salt, ester, salt of an ester, or any other derivative which, upon administration to a patient, is capable of providing directly or indirectly a non-binding FK506 analog or a metabolite or residue thereof that has the desired neurotrophic activity. Included within the scope of the invention are enantiomers, the racemic form, and diastereoisomeric mixtures. Enantiomers and diastereoisomers can be separated by conventional methods.

A "non-binding FK506 analog" is defined as an FK506 analog that does not bind to FKBP-12. Preferably, such FK506 analogs bind FKBP-12 with an apparent K.sub.d of greater than 10 .mu.M as measured using well-known assays, and preferably greater than 30 .mu.M, and more preferably greater than 100 .mu.M. Values for the apparent K.sub.d can be determined, for example, by a competitive LH-20 binding assay performed as described, for example, in:Harding et al., Nature 341:758-760, 1989 (using 32-[1-.sup.14C]-benzoyl FK506 as a reporting ligand; Siekierka et al., Nature 341:755-757, 1989, using [.sup.3H]dihydro-FK506 as a reporting ligand); and U.S. Pat. No. 5,654,332.

Alternatively, a "non-binding FK506 analog" is defined as an FK506 analog that does not significantly inhibit FKBP-12 rotomase activity when administered to a patient at dosage levels of about 0.01 to about 100 mg/kg body weight/day. Assays for inhibition of FKBP-12 rotamase activity are described in Harding et al. (Nature 341:758-760, 1989), Siekierka et al., Nature 341:755-757, 1989, and U.S. Pat. No. 5,654,332, for example. Chymotrypsin is able to cleave p-nitroanilide from the trans form of the artificial substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, but not from the cis form. The assays of Harding et al. and Siekierka et al. employ a reaction mixture that includes the cis form of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, FKBP-12, a test compound, and chymotrypsin, and spectrophotometrically measure the release of p-nitroanilide as a result of isomerization of the substrate.

Formulae I-VI above represent compounds that have a wide range of binding affinities for FKBP-12. Non-binding FK506 analogs can be readily identified by well-known assays for FKBP-12 binding or rotamase activity. Non-binding compounds can then be readily assessed for activity in promoting regeneration of nerve cells by the in vitro and in vivo assays discussed in the Examples below. The non-binding FK506 analogs are non-immunosuppressive, as can be demonstrated by well-known assays, e.g., as discussed in U.S. Pat. No. 5,516,797, WO 92/21313, WO 92/19593, and WO 92/04370.

FK506 analogs can be used in the form of salts preferably derived from inorganic or organic acids and bases, including, but not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include, but are not limited to, ammonium salts, alkali metal salts (such as sodium and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts), salts with organic bases (such as dicyclohexylamine salts), N-methyl-D-glucamine, and salts with amino acids (such as arginine, lysine, etc.). Basic nitrogen-containing groups can be quaternized, e.g., with such agents as lower alkyl halides (such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (such as dimethyl, diethyl, dibutyl, an diamyl sulfates), long-chain halides (such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), aralkyl halides (such as benzyl and phenethyl bromides), etc. Water or oil-soluble or dispersible products are produced thereby.

As would be apparent to the person of ordinary skill in the art, the nonbinding FK506 analogs can be modified by appending appropriate functionalities by well-known methods to enhance selected biological properties, including increasing penetration of the analogs into a given cellular compartment (e.g., blood, lymphatic system, central nervous system, etc.), increase oral availability, increase solubility to permit administration by injection, alter metabolism, and alter rate of excretion, for example.

Preferably, the FK506 analogs have a molecular weight below about 750 atomic mass units (a.m.u.) (as the parent compound, although the salts of such compounds can have higher molecular weights).

An "effective amount" of a composition according to the invention is an amount sufficient to achieve a statistically significant promotion of nerve cell growth or regeneration compared to a control. Nerve cell growth or regeneration can be readily assessed using an in vitro assay, e.g., the assay described in the Examples below. Alternatively, nerve cell growth or regeneration can be determined in an in vivo assay or by direct or indirect signs of nerve cell growth and regeneration in a patient. Preferably, the increase in nerve cell growth or regeneration is at least 10%, preferably at least 30%, and most preferably 50% or more compared to a control. Preferred dosage levels are between about 0.1 to about 400 mg/kg per day of the FK506 analog.

Therapeutic and Prophylactic Uses

FK506 analogs can be periodically administered to a mammal, including a human patient in need of such treatment, to promote neuronal regeneration and functional recovery and to stimulate neurite outgrowth and thereby to treat various neuropathological states, including damage to peripheral nerves and the central nervous system caused by physical injury (e.g., spinal cord injury and trauma, sciatic crush, and facial nerve crush), disease (e.g., diabetic neuropathy), cancer chemotherapy (e.g., by vinca alkaloids and doxorubicin), brain damage associated with stroke and ischemia associated with stroke, and neurological disorders including, but not limited to, various peripheral neuropathic and neurological disorders related to neurodegeneration including, but not limited to: trigeminal neuralgia, glossopharyngeal neuralgia, Bell's palsy, myasthenia gravis, muscular dystrophy, amyotrophic lateral sclerosis, progressive muscular atrophy, progressive bulbar inherited muscular atrophy, herniated, ruptured or prolapsed vertebral disk syndromes, cervical spondylosis, plexus disorders, thoracic outlet destruction syndromes, peripheral neuropathies such as those caused by lead, dapsone, ticks, porphyria, Gullain-Barre syndrome, Alzheimer's disease, Parkinson's disease, and Huntington's chorea.

In addition, pharmaceutical compositions according to the present invention display a wide range of other therapeutic or prophylactic properties, including, treatment of stroke (see, e.g., Sharkey and Butcher, Nature 371:336-339, 1994, Vagita et al., Life Sciences 59:1643-1650, 1996; Tokime et al., Neurosci. Lett. 206:81-84, 1996; Drake et al., Acta. Physiol. Scand 158:155-159, 1996; and Kuroda et al., Neurosci. Res. Comm. 19:83-90,1996), AIDS dementia (see, e.g., Dawson and Dawson, Adv. Neuroimmunol. 4:167-173, 1994; and Sekigawa et al., J. Clin. Immunol. 15:312-317, 1995); hair growth (Yamamoto et al., J. Investig. Dermatol. 102:160-164, 1994; Jiang et al., J. Investig. Dermatol. 104:523-525, 1995); and connective tissue disorders (see e.g., Steinmann et al., J. Biol. Chem. 266:1299-1303, 1991), and as a male contraceptive (see e.g., Hisatomi et al., Toxicology 109:75-83, 1996).

Pharmaceutical Formulations

Pharmaceutical formulations according to the present invention encompass formulations comprising (1) an amount (for example, a unit dosage) of a non-binding FK506 analog together with (2) one or more well-known non-toxic pharmaceutically acceptable excipients, including carriers, diluents, and/or adjuvants, and optionally (3) one or more biologically active ingredients. Standard pharmaceutical formulation techniques are used, such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (latest edition).

A pharmaceutical formulation according to the invention can include a single non-binding FK506 analog or combination of a non-binding FK506 analog with one or biologically active ingredients, including, but not limited to: (1) one or more non-binding FK506 analogs; (2) one or more FKBP-12-binding FK506 analogs; (3) one or more other neurotrophic agents, including, for example, NGF, IGF-1, aFGF, bFGF, PDGF, BDNF, CNTF, GDNF, NT-3, and NT 4/5; and so on.

It is preferred that the pharmaceutical formulation includes an amount of a neurotrophic agent(s), preferably NGF, such that the patient receives a dosage of between about 0.01 to 100 .mu.g/kg body weight/day of the neurotrophic agent, or that the neurotrophic agent be administered separately, e.g., in separate single or multiple dosage forms, preferably concurrently, consecutively, or within less than about five hours of each other.

The compositions can be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions (e.g., eye or ear drops, throat or nasal sprays, etc.), transdermal patches, and other forms known in the art.

Such pharmaceutical compositions can be administered systemically or locally in any manner appropriate to the treatment of a given condition, including orally, parenterally, rectally, nasally, buccally, vaginally, topically, optically, by inhalation spray, or via an implanted reservoir. The term "parenterally" as used herein includes, but is not limited to subcutaneous, intravenous, intramuscular, intrasternal, intrasynovial, intrathecal, intrahepatic, intralesional, and intracranial administration, for example, by injection or infusion. For treatment of the central nervous system, the pharmaceutical compositions preferably readily penetrate the blood-brain barrier when peripherally administered or are administered intraventricularly.

Pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffers (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat, for example.

Tablets and capsules for oral administration can be in a form suitable for unit dose presentation and can contain conventional pharmaceutically acceptable excipients. Examples of these include binding agents such as syrup, acacia, gelatin, sorbitol, tragacanth, and polyvinylpyrrolidone; fillers such as lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; tableting lubricants, such as magnesium stearate, talc, polyethylene glycol, or silica; disintegrants, such as potato starch; and dispersing or wetting agents, such as sodium lauryl sulfate. The tablets can be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations can contain conventional additives such as suspending agents, e.g., sorbitol, syrup, methyl cellulose, glucose syrup, gelatin, hydrogenated edible fats, emulsifying agents, e.g., lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (including edible oils), e.g., almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.

Pharmaceutical compositions according to the present invention can also be administered parenterally in a sterile aqueous or oleaginous medium. The composition can be dissolved or suspended in a non-toxic parenterally-acceptable diluent or solvent, e.g., as a solution in 1,3-butanediol. Adjuvants such as local anesthetics, preservatives, and buffering agents can also be dissolved in the vehicle. Commonly used vehicles and solvents include water, physiological saline, Hank's solution, Ringer's solution, and sterile, fixed oils, including synthetic mono- or di-glycerides, etc. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or a similar alcohol.

For topical application, the drug may be made up into a solution, suspension, cream, lotion, ointment in a suitable aqueous or non-aqueous vehicle. Additives may also be included, e.g., buffers such as sodium metabisulphite or disodium edeate; preservatives such as bactericidal and fungicidal agents, including phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorhexidine, and thickening agents, such as hypromellose.

The dosage unit involved depends, for example, on the condition treated, nature of the formulation, nature of the condition, embodiment of the claimed pharmaceutical compositions, mode of administration, and condition and weight of the patient. Dosage levels on the order of about 0.1 to about 400 mg/kg per day of the active ingredient are useful in the treatment of the conditions listed above.


Claim 1 of 14 Claims

1. A method of identifying a FK506 analog that stimulates nerve cell growth, the method comprising: screening a plurality of FK506 analogs for binding to FKBP-12 and for rotamase activity; and selecting a FK506 analog of interest that binds FKBP-12 with a K.sub.d of at least 10 .mu.M without inhibition of FKBP-12 rotamase activity; thereby identifying a FK506 analog that stimulates nerve growth.

 

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