INHIBITORS OF CYCLOPHILIN ROTAMASE ACTIVITY

1. Field of the Invention This invention relates to the method of using neurotrophic cyclophilin inhibitor compounds having an affinity for cyclophilin immunophilins as inhibitors of the enzyme activity associated with immunophilin proteins, and particularly inhibitors of peptidyi- prolyl isomerase or rotamase enzyme activity of the cyclophilins .

2. Description of the Prior Art

The term immunophilin refers to a number of proteins that serve as receptors for the principal immunosuppressant drugs, cyclosporin A (CsA) , FK506, and rapamycin. Known classes of immunophilins are cyclophilins, and FK506 binding proteins, such as FKBP. Cyclosporin A binds to cyclophilin while FK506 and rapamycin bind to FKBP. These immunophil n-drug complexes interface with a variety of intracellular signal transduction systems, especially in the immune system and the nervous system.

Immunophilins are known to have peptidyl-prolyl isomerase (PPIase) or rotamase enzyme activity. It has been determined that rotamase activity has a role in the catalyzation of the interconversion of the cis and trans isomer of immunophilin proteins.

Immunophilins were originally discovered and studied in immune tissue. It was initially postulated

by those skilled m the art that inhibition of the immunophilins rotamase activity leads to the inhibition of T-cell proliferation, thereby causing the immunosuppressive action exhibited by immunosuppressive drugs such as cyclosporin A, FK506, and rapamycin. Further study has shown tnat the inhibition of rotamase activity, m and of itself, is not sufficient for lmmunosuppressant activity Instead immunosuppression appears to stem from the formulation of a complex of lmmunosuppressant drugs and immunophilins. It has been shown that the lmmunophxlm-drug complexes interact with ternary protein targets as their mode of action In the case of FKBP-FK506 and FKBP-CsA, the drug-immunophilin complexes bind to the enzyme calcineurin, inhibiting T-cell receptor signalling leading to T-cell proliferation Similarly, the complex of rapamycin and FKBP interacts with the RAFTl/FRAP protein ana inhibits signalling from the IL-2 receptor Immunophilins have been found to be present at high concentrations in the central nervous system Immunophilins are enriched 10-50 times more m the central nervous system than m the immune system. Within neural tissues, immunophilins appear to influence nitric oxide synthesis, neurotransmitter release, and neuronal process extension

Surprisingly, it has been found that picomolar concentrations of an lmmunosuppressant such as

cyclospoπn A stimulates neurite out growth ιr PC ₁ 2 cells and sensory neurons, namely dorsal root ganglion cells (DRGs; More particularly, it has been found that drugs with a high affinity for cyclophilin are potent rotamase inhibitors and exhibit excellent neurotrophic effects Snyder et al , "Immunophilins and the Nervous System", Na ture Medi cine , Volume 1, No. 1, January 1995, 32-37 These findings suggest the use of inhibitors of cyclophilin rotamase activity m treating various peripheral neuropathies and enhancing neuronal regrowth in the central nervous system (CNS) Studies have demonstrated that neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease may occur due to the loss, or decreased availability, of a neurotrophic substance specific for a particular population of neurons affected m the disorder

Several neurotrophic factors effecting specific neuronal populations m the central nervous system have been identified. For example, it has been hypothesized that Alzheimer's disease results from a decrease or loss of nerve growth factor (NGF) . It has thus been proposed to treat SDAT patients with exogenous nerve growth factor or other neurotrophic proteins such as brain derived growth factor, glial derived growth factor, ciliary neurotrophic factor, and neurotropm-3 to increase the survival of degenerating neuronal populations.

Clinical application of these proteins m various neurological disease states is hampered by difficulties m the delivery and bioavailability of large proteins to nervous system targets By contrast, lmmunosuppressant drugs with neurotrophic activity are relatively small and display excellent bioavailability and specificity However, when administered chronically, lmmunosuppressants exhioit a number of potentnally serious side effects including nephrotoxicity, such as impairment of glomerular filtration and irreversible interstitial fibrosis (Kopp et al , 1991, J Am . Soc . Nephrol 1 162) ; neurological deficits, such as involuntary tremors, or non-specific cerebral angina such as non- localized headaches (De Groen et al . , 1987, N. Engl . J Med . 317:861) ; and vascular hypertension with complications resulting therefrom (Kahan et al . , 1989 N Engl . J. Med 321- 1725)

The present invention provides both immunosuppressive and non-immunosuppressive cyclophilin inhibitor compounds containing small molecule cyclophilin rotamase inhibitors which are extremely potent in augmenting neurite outgrowth, and for promoting and stimulating neuronal growth and regeneration m various neuropathological situations where neuronal repair can be facilitated. These situations include augmenting neurite outgrowth and promoting neuronal growth and regeneration for nerve

damage by physical injury or disease state such as diabetes, including peripheral nerve damage, damage to motor neurons, damage to the central nervous system (spinal cord and brain) including damage to spinal neurons and neurons in the brain, brain damage associated with stroke, and for the treatment of neurological disorders relating to neurodegeneration, including Parkinson's disease and Alzheimer's disease .

SU-4MARY OF THE INVENTION This invention relates to the method of using neurotrophic cyclophilin inhibitor compounds having an affinity for cyclophilin-type immunophilins as inhibitors of the enzyme activity associated with immunophilin proteins, and particularly inhibitors of peptidyl-prolyl isomerase or rotamase enzyme activity

A preferred embodiment of this invention is a method of treating a neurological activity m an animal, comprising: administering to an animal an effective amount of an immunosuppressive cyclosporin represented by formula I and pharmaceutically acceptable salts thereof, wherein: R ¹ is MeBmt or [3-DesoxyMeBmt] or represented by formula III, wherein positions 2, 3, and 4 have the configurations S, R and R, respectively;

wherein -X-Y- is connected by a double bond or a single bond; wherein:

R ² is independently selected from the group consisting of Abu, Ala, Thr, Val, and norVal ; R ³ is Sar; R ⁴ is MeLeu; R ⁵ is Val; R° is MeLeu; R" is

Ala; R ⁸ is D-Ala; R ⁹ is MeLeu; R ¹⁰ is MeLeu; and, R ¹¹ is Val or [D-MeVal] .

Another preferred embodiment of this invention is a method of treating a neurological activity in an animal, comprising: administering to an animal an effective amount of a non-immunosuppressive cyclosporin of formula IV: wherein:

R ¹ is MeBmt or dihydro MeBmt or represented by formula III; wherein positions 2, 3, and 4 have the configurations S, R and R, respectively; wherein -X-Y- is connected by a double bond or a single bond; wherein: R ² is Abu or a fluoπnated analog thereof; R ³ is Sar, D-MeAla, or a fluormated analog thereof; R ⁴ is an N-methylated ammo acid residue with a (C1-C9) straight or branched chain alkyl or alkenyl group; these straight or branched alkyl or alkenyl groups may be substituted by cycloalkyl (C3-C8) ; R _x may also be (C3-C8) cycloalkyl or (C5-C7) cycioalkenyl; the above alkyl, alkenyl, cycloalkyl or cycioalkenyl groups may be substituted with (Cl- C4) alkyl or (C1-C4) alkenyl, or hydroxy,

especially where R ⁴ is MeLeu, MeVal, Me homo-Ala, or [Me- (α-methyl) Thr] ; R ⁵ is Val or a fluorinated analog thereof; R ⁶ is MeAla, MeAbu, or a fluorinated analog thereof; R ⁷ is Ala or a fluorinated derivative thereof; R ⁸ is (a) D-Ala or a fluorinated analog thereof; or (b) O-acyl-D-Ser or O-acyl-D-Thr wherein the acyl group is defined as R ¹² -CO- where R ¹² represents hydrogen, C ₁ . ₆ alkyl, phenyl or substituted phenyl of formula V wherein X ¹ and X ² independently are (a) C _M alkyl ,-

(b) C^alkanoyl; (c) CH ₂ OH; (d) halo; (e) C,_ ₆ alkoxy; (f) -NH ₂ ; (g) -N0 ₂ ; (h) -COOH; (i) -COOC _: _ ₆ alkyl; or (j) -H; R ⁹ and R ¹⁰ are independently MeLeu or a fluorinated analog thereof; and, R ¹¹ is MeVal or a fluorinated analog thereof.

Another preferred embodiment of this invention is a method of treating a neurological disorder in an animal, comprising: administering to an animal an effective amount of a cyclophilin inhibitor having an affinity for cyclophilin-type immunophilins to stimulate growth of damaged peripheral nerves or to promote neuronal regeneration, wherein the cyclophilin-type immunophilin exhibits rotamase activity and the cyclosporin derivative inhibits said rotamase activity of the immunophilin.

Another preferred embodiment of this invention is a me nod of treating a neurological disorder in an animal, comprising administering to an animal an effective amount of a cyclophilin inhibitor having an affinity for cyclophilin-type immunophilins in combination with an effective amount of a neurotrophic factor selected from the group consisting of neurotrophic growth factor, brain derived growth factor, glial derived growth factor, cilial neurotrophic factor, and neurotropιn-3 to stimulate growth of damaged peripheral nerves or to promote neuronal regeneration, wherein the cyclophilin-type immunophilin exhibits rotamase activity and the cyclosporin derivative inhibits said rotamase activity of the immunophilin

Another preferred embodiment of this invention is a method of stimulating growth of damaged peripheral nerves, comprising; administering to damaged peripheral nerves an effective amount of a cyclophilin inhibitor compound having an affinity for cyclophilin-type immunophilins to stimulate or promote growth of the damaged peripheral nerves, wherein the cyclophilin-type immunophilins exhibit rotamase activity and the cyclosporin derivative inhibits said rotamase activity of the immunophilin.

Another preferred embodiment of this invention is a method of stimulating growth of damaged peripheral nerves, comprising administering to damaged peripneral nerves an effective amount of an FKBP inhibitor compound having an affinity for cyclophilin-type immunophilins to stimulate growth of damaged peripheral nerves, wherein the cyclophilin-type lmmunopnilin exhibit rotamase activity and the cyclosporin derivative inhibits said rotamase activity of the immunophilin

Another preferred embodiment of this invention is a method for promoting neuronal regeneration and growth m animals, comprising administering to an animal an effective amount of a cyclophilin inhibitor compound having an affinity for cyclophilin-type immunophilins to promote neuronal regeneration, wherein the cyclophilin-type immunophilins exhibit rotamase activity and the cyclosporin derivative inhibits said rotamase activity of the immunophilin

Yet another preferred embodiment of this invention is a method for preventing neurodegeneration in an animal, comprising, administering to an animal an effective amount of a cyclophilin inhibitor having an affinity for cyclophilin-type immunophilins to prevent neurodegeneration, wherein the cyclophilm-type immunophilin exhibits rotamase activity and the

cyclosporin derivative inhibits said rotamase activity of the immunophilin.

BRIEF DESCRIPTION OF THE DRAWINGS FIG 1 is dose-response curve for Cyclosporin A.

FIG 1 shows a typical dose at which ED _5Q for CsA, the dose at which 50% of the maximal response was elicited, is calculated to be 50 nM.

DETAILED DESCRIPTION OF THE INVENTION

The novel neurotrophic cyclophilin inhibitor compounds f this invention have an affinity for the cyclosporin binding protein, cyclophilin When the neurotrophic compounds of the invention are bound to cyclophilin, they have been found to inhibit the prolyl-peptidyl cis-trans isomerase activity, or rotamase activity of the binding protein and unexpectedly stimulate neurite growth.

The compounds of the present invention can be used m the form of salts derived from inorganic or organic acids and bases Included among such acid salts are the following: acetate, adipate, algmate, aspartate, benzoate, benzenesulfonate, bisulfate butyrate, citrate, camphorate, camphorsulfonate, cyclopentaneprop onate, digluconate, dodecylsulfate, ethanesulfonate , fumarate, glucoheptanoate , glycerophosphate, hemissulfate heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-

hydroxyethanesul fonate , lactate, maleate, methanesulfonate, 2-naphthalensulfonate, nicotmate, oxalate, pamoate, pectinate, propionate, succmate, tartrate, thiocyanate, tosylate and undecanoate . Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salt with organic bases such as dicyclohexyiamme salts, N- methyl-D-glucamme, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained. The neurotrophic compounds of this invention can be periodically administered to a patient undergoing treatment for neurological disorders or for other reasons in which it is desirable to stimulate neuronal regeneration and growth, such as in various peripheral neuropathic and neurological disorders relating to neurodegeneration. The compounds of this invention can also be administered to mammals other than humans

for treatment of various mammalian neurological disorders .

The novel compounds of the present invention are potent inhibitors of rotamase activity and possess an excellent degree of neurotrophic activity. This activity is useful the stimulation of damaged neurons, the promotion of neuronal regeneration, the prevention of neurodegeneration, the stimulation of growth of damaged peripheral nerves, the stimulation of growth of damaged neurons the spinal cord, the stimulation of growth of motor nerves, and m the treatment of several neurological disorders known to be associated with neuronal degeneration and peripheral neuropathies . The neurological disorders that may be treated include but are not limited to: tπgeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis, muscular dystrophy, progressive muscular atrophy, progressive bulbar inherited muscular atrophy, herniated, ruptured or prolapsed invertabrae disk syndromes, cervical spondylosis, plexus disorders, thoracic outlet destruction syndromes, peripheral neuropathic such as those caused by lead, dapsone, ticks, porphyria, or Gullain-Barre syndrome, Alzheimer's disease, and Parkinson's disease.

For these purposes the compounds of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally,

buccally, vagmally or via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, mtrapeπtoneally, mtrathecally, mtraventricularly, trasternal and lntracramal iniection or infusion techniques .

To be effective therapeutically as central nervous system targets the immunophilin-drug complex should readily penetrate the blood-bram barrier when peripherally administered. Compounds of this invention which cannot penetrate the blood-bra barrier can be effectively administered by an mtraventricular route.

The pharmaceutical compositions may be m the form of a sterile mjectable preparation, for example as a sterile mjectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques know in the art using suitable dispersing or wetting agents and suspending agents. The sterile mjectable preparation may also be a sterile mjectable solution or suspension m a non- toxic parenterally-acceptable diluent or solvent, for example as a solution 1, 3-butanediol . Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils

are conventionally employed as a solvent or suspending medium For this purpose any bland fixed oil may £>e employed including synthetic mono- or digiycerides Fatty acids such as oleic acid and its gl/ceride derivatives find use m the preparation of mjectables, olive oil or castor oil, especially m their polyoxyethylated versions Tnese oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant The compounds may be administered orally in the form of capsules or tablets, for example, or as an aqueous suspension or solution. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch Lubricating agents, such as magnesium stearate, are also typically added

For oral administration m a capsule form, useful diluents include lactose and dried corn starch When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents If desired, certain sweetening and/or flavoring and/or coloring agents may be added

The compounds of this invention may also be administered in the form of suppositories for rectal administration of the drug These compositions can be prepared by mixing the drug with a suitable non- lrritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt m the rectum to release the drug

Ξuch materials include cocoa butter, beeswax ana polyethylene glycols.

The compounds of this invention may also be administered optically, especially when the conditions addressed for treatment involve areas or organs readily accessible by topical application, including neurological disorders of the eye, the sκιn, or the lower intestinal tract Suitable topical formulations are readily prepared for each of these areas For ophthalmic use, the compounds can be formulated as micronized suspensions m lsotonic, pH adjusted sterile saline, or, preferably, as solutions is isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkomum chloride. Alternatively for the ophthalmic uses the compounds may be formulated an ointment such as petrolatum

For application topically to the skm, the compounds can be formulated in a suitable ointment containing the compound suspended or dissolved , for example, a mixture with one or more of the following mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water Alternatively, the compounds can be formulated m a suitable lotion or cream containing the active compound suspended or dissolved m, for example, a mixture of one or more of the following mineral oil, sorbitan monostearate,

polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol , benzyl alcohol and water.

Topical application for the lower intestinal tract an be effected m a rectal suppository formulation (see above) or in a suitable enema formulation

Dosage levels on the order of about 0 lmg to about 10,000mg of the active ingredient compound are useful m the treatment of the above conditions, with preferred levels of about 0 lmg to about 1000 mg The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It is understood, however, that a specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated and form of administration.

The compounds can be administered with other neurotrophic agents such as neurotrophic growth factor (NGF) , glial derived growth factor, brain derived growth factor, ciliary neurotrophic factor, and neurotropm-3. The dosage level of other neurotrophic drugs will depend upon the factors previously stated

and the neurotropnic effectiveness of the drug combination.

Methods and Procedures Nerve Extension Elicited in Chick Dorsal Root Ganglia by Cyclophilin Ligands

In the present study we have employed explants of chick dorsal root ganglia m order to demonstrate the powerful neurotrophic effects of the cyclophilin binding compounds The maximal increase the number of processes, their length and branching is quite similar at maximally effective contractions of the cyclophilin ligands and of NGF (100 ng/ml) With progressively increasing concentrations of the various drugs, one observes a larger number of processes, more extensive branching and a greater length of individual processes .

We evaluated the potencies of drugs m binding to cyclophilin A by examining inhibition of peptidyl prolyl-isomerase activity (Table 1) There is a striking parallel between their potencies m stimulating neurite outgrowth and inhibiting rotamase activity.

The very close correlation between the potencies of drugs m binding to immunophilins, inhibiting their rotamase activity and stimulating neurite outgrowth implies that inhibition of rotamase activity is responsible for neurotrophic effects of the drugs The extraordinarily high potency of the drugs m

stimulatmg neurite outgrowth and m binding to cyclophilin makes it most unlikely that any other target could account for the neurotrophic effects.

Because of the extraordinary potency of the drugs and the close correlation between rotamase inhibition and neurotrophic actions, we conclude that rotamase inhibition of cyclophilin is likely involved neurotrophic effects .

The neurotrophic actions of the drugs studied here are exerted at extremely low concentrations indicating potencies comparable to those of neurotrophic protems such as bram derived growth factor, neurotropm-3 and neurotrophic growth factor.

The following examples are illustrative of preferred embodiments of the invention and are not to be construed as limiting the invention thereto. All polymer molecular weights are mean average molecular weights All percentages are based on the percent by weight of the final delivery system or formulation prepared unless otherwise indicated and all totals equal 100% by weight.

Illustrative cyclophilin inhibitor compounds which can be used for the purposes of this invention include, but are not limited to, the following. Cyclosproins of general structural Formula I, comprising cyclic peptides which contain 11 ammo acids :

.10 ι11

-— R

Formula I More Particularly, naturally occurring cyclosporms have the general structure shown Formula II and embodied in the present invention:

10 11 3 MeLeu MeVal MaC — A Sar

9

MeLeu

(D)AIe Ate MeLeu Val MeLeu 4 a 7 S 5

Formula II wherein "MeC ₉ " represents the so-called "C9-ammo acid" residue of Formula III (also known as "MeBmt" = (4R) -4- [ (E) -2-butenyl] -4-methyl-L-threonine) :

X

I Y N

C. _^

HO \ ³ -CH OH 4 ^' CH _^

3

Formula III

in which positions 2, 3, and 4 have the configurations S, R and R, respectively, -X-Y- is CH=CH- (trans; where

Me = methyl

Abu = a-aminobutyric acid Val = valine norVal = norvaline Ala = alanine

MeLeu = N-methyl-leucine MeVal = N-methyl-valine Sar = sarcosine

[3-DesoxyMeB t] = MeBmt wherein the oxygen at position 3 is removed.

Unless specified, the amino acid configuration is L; and A is, for cyclosporin A, -Abu-; for cyclosporin B, -Ala-; for cyclosporin C, -Thr-; for cyclosporin D, -Val-,- and for cyclosporin G, -norVal-, all of which are incorporated herein by reference. Additional known naturally ocurring cyclosporins are related to cyclosporin A, above, by replacement of an ammo acid; thus for cyclosporin E, position 11 is Val; for cyclosporin F, position 1 is [3- DesoxyMeBmt] ; and for cyclosporin H, position 11 is [D-MeVal] , all of which are incorporated herein by reference.

In the following discussion, all references to numbered positions in the cyclosporin type structure are with reference to formula II. As is common in the field, a specific cyclosporin analog may be named using the above numbering system, using a shorthand notation to identify how the cyclosporin analog differs from cyclosporin A (CsA) . Thus, [MeVal] ⁴ -CsA denotes the cyclosporin in which MeLeu in cyclosporin A (Formula II) has been replaced by MeVal, and so forth.

In the case of the dihydrocyclosporms, -X-Y- of formula III is -CH--CH-- Thus, dihydrocyclosporin A, dihydrocyclosporin C and ail other dihydro analogues of naturally occurring as well as semi- synthetic and synthetic cyclospor s are to be understood as falling within the spirit and scope of the invention

In addition to these known cyclosporms and their dihydro derivatives, a number of non-natural cyclosporms have been prepared by synthetic means, either by total synthesis or synthetic modification of naturally ocurring materials, or by modified culture techniques. These synthetic and semi- synthetic cyclosporin analogues and derivatives have been shown to potently inhibit the rotamase activity of cyclophilin, and are thus to be understood as specifically falling with the scope and spirit of the present invention Such modified cyclosporms include, but are not in any way limited to, the following- cyclosporms with modified "C9 ammo acids"; such modified cyclosporms are disclosed by Witzel (U.S. Patent 4,885,276 and U.S. Patent 4,798,823) , and are incorporated herein by reference; cyclospor s with modified residues at position

8, such as [dehydro-Ala] 8-CsA and cyclosporms containing a sulfur-containing ammo acid at posιtιon-8, as disclosed by Patchett et al (U S

Patents 5,122,511 and 5,214,130) and incorpora _t ed herein by reference; cyclospor s wherem the ammo acid at the 8- position is a (D) -acyloxy-a-ammo acid residue, as disclosed by Wenger (U.S. Patent 4,764,503) and incorporated herein by reference; cyclosporms having an a-hydroxycarboxylic acid at posιtιon-8, as described by Dreyfuss et al . (U.S. Patent 5,116,816) and incorporated herein by reference; cyclospor s containing hydroxy-substituted serme residue at posιtιon-8, such as [0-(2- hydroxyethyl) (D) Ser] ⁸ -CsA, are disclosed by Eberle m U.S. Patent 5,284,826 and mcoprporated herein by reference; cyclosporins containing one or more fluorinated amino acids, as for example described by Durette et al . U.S. Patent 5,227,467 and incorporated nerem by reference; cyclosporins which contain at the 3-position an optically active, N-methylated a-amino acid residue of the (D) configuration, as disclosed by Seebach n U.S. Patent 4,703,033 and mcoprporated herein by reference; cyclosporms which contain an allyl-Gly residue in the 2-position and/or a - (D) -Ser residue in the 8-posιtιon, as disclosed by Bollinger et al . (U.S.

Patent 4,384,996) and incorporated herein by reference

In addition to the above cyclospor s and cyclosporin analogs which possess cyclophilin inhibitory and immunosuppressive activity, it is also an intention of the present invention to provide a method for the use of non- lmmunosuppressive cyclosporin analogs as neurotrophic agents Such non-immunosuppressive cyclosporin analogs bind to cyclophilin and mnibit its rotamase activity, while not eliciting lmmunosuppression, and are potent neurotrophic agents

The following structural Formula IV is illustrative of such embodiment, and is not meant to be construed as limiting the scope of the mvention m any way

Formula IV where R ¹ is MeBmt or dihydro MeBmt,

R ^z is Abu or a fluorinated analog thereof,

R ³ is Sar, D-MeAla, or a fluorinated analog thereof ,-

R ⁴ is an N-methylated ammo acid residue with a (C1-C9) straight or branched chain alkyl or alkenyl group; these straight or branched alkyl or alkenyl groups may be substituted by cycloalkyl (C3-C8) ; R _x may also be (C3-C8) cycloalkyl or (C5-C7) cycioalkenyl; the above alkyl, alkenyl, cycloalkyl or cycioalkenyl groups may be substituted with (C1-C4) alkyl or

(C1-C4) alkenyl, or hydroxy; R ⁹ and R ¹⁰ are independently MeLeu or a fluorinated analog thereof ,-

R ⁵ is Val or a fluorinated analog thereof; R ^δ is MeAla, MeAbu, or a fluorinated analog thereof ,-

R ⁷ is Ala or a fluorinated derivative thereof; R ^a is

(a) D-Ala or a fluorinated analog thereof; or

(b) O-acyl-D-Ser or O-acyl-D-Thr wherein the acyl group is defined as R ¹² -CO- where R ¹² represents hydrogen, C _M alkyl, phenyl or substituted phenyl of formula V:

wherem X ¹ and X ² independently are

(a) C _M alkyl;

(b) C-_ ₆ alkanoyl;

(c) CH ₂ OH; (d) halo;

(e) C-. ₆ alkoxy;

(f) -NH ₂ ;

(g) -N0 ₂ ; (h) -COOH; (i) -COOC- _e alkyl; or

( -H,

Particularly preferred are cyclosporin analogs wherem R ¹ is MeBmt or dihydro MeBmt;

R ² is Abu;

R ³ is Sar or D- eAla;

R ⁴ is MeLeu, MeVal, Me homo-Ala, or [Me- (a- methyl) Thr] ; R ⁹ and R ¹⁰ are independently MeLeu or a fluorinated analog thereof;

R ⁵ is Val or a fluorinated analog thereof;

R ^δ is MeAla, MeAbu, or a fluorinated analog thereof;

R ⁸ is D-Ala; and

R ¹¹ is MeVal or a fluorinated analog thereof .

Fluormated analogs represent the amino acid residues where one or more of the various C-K bonds the side chain are replaced with C-F bonds For example, fluorinated analog of Abu represents

—NH CH CO—

I x ₂ —C X _!

X C X ₂ χ ₅

wherem X. to X ₅ independently are H or F with the proviso that at least one of X-, X ₂ , X- , X., and X ₅ is F.

Known derivatives and analogs of cyclosporms, which are known to be potent inhibitors of immunophilin rotamase activity and lack lmmunosuppressant effects include modified 6- position analogs, described by Dumont et al . (U.S. Patent 4,914,188) and Durette (U.S. Patent 5,236,899) and incorporated herein by reference. Other known non-immunosuppressive cyclosporin analogs include N-alkylated [Val] ⁴ derivatives, such as [MeVal] ⁴ -CsA, disclosed by Fliri et al . (Annal . N.Y. Acad. Sci 696, 47, 1993) , and incorporated herein by reference, and other derivatives with various side chain on the 4-posιtιon residue, as described by Papageorgiou et al . (BioMed. Chem. Lett. 1994, 2, 267-272) and incorporated herein by reference .

K._ Test Procedure

Inhibition of the peptidyl-prolyl isomerase (rotamase) activity of cyclophilin by the inventive compounds can be evaluated by known methods described the literature (Harrison and Stem, Biochemistry, 1990, 29, 3813-3816) . These values are obtained as apparent K. ' s and are presented for some of Examples 1-30 m Table I. The cis- rans lsomerization of an alanme-prolme bond m a model substrate, N-succmyl-Ala-Ala-Pro-Phe-p- nitroamlide, is monitored spectrophotometrically m a chymotrypsin-coupled assay, which releases para- nitroanilide from the trans form of the substrate. The inhibition of this reaction caused by the addition of different concentrations of inhibitor is determined, and the data is analyzed as a change m first-order rate constant as a function of inhibitor concentration to yield the apparent K. values . The absorbance at 390 nm versus time is monitored for 90 sec using a spectrophotometer and the rate constants are determined from the absorbance versus time data files .

The data for these experiments is presented m Table I.

TABLE I

Example Cyclosporin Ki , cyclophilin inhibition

1 CsA

2 dihydro-CsA

3 [dehydro-Ala] ⁸ -CsA 4 [MeVal] ⁴ -CsA

5 [Me-homoAla] ⁴ -CsA

6 [Me- (a-methyl) Thr] ⁴ -CsA 18 nM

Chick Dorsal Root Ganglion Cultures and Neurite Outgrowth

Dorsal root ganglia were dissected from chick embryos of ten day gestation. Whole ganglion explants were cultured on thin layer Matrigel-coated 12 well plates with Liebovitz L15 plus high glucose media supplemented with 2mM glutamine and 10% fetal calf serum, and also containing 10 μM cytosine -3-D arab ofuranoside (Ara C) at 37°C in an environment containing 5% C0 ₂ . Twenty-four hours later, the DRGs were treated with various concentrations of cyclosporin drugs. Forty-eight hours after drug treatment, the ganglia were visualized under phase contrast or Hoffman Modulation contrast with a Zeiss Axiovert inverted microscope. Photomicrographs of the explants were made, and neurite outgrowth was quantitated. Neurites longer than the DRG diameter were counted as positive, with total number of neurites quantitated per each experimental condition. Three to four DRGs are cultured per well, and each treatment was performed in duplicate.

The data for these experiments are presented in Table II. Referring to FIG. 1 of the drawings, the ED ₃₀ for cyclosporin A, the dose at which 50% of the maximal response was elicited, was obtained from dose-response curves (Fig. 1 is a typical example) and calculated to be 50 nM. The relative potencies of the other compounds m Table II are given relative to cyclosporin A, the number of "+" marks denoting the relative potency.

Table II

Neurite Outgrowth in Chick DRG

The above data demonstrate that compounds which are inhibitors of cyclophilin' s rotamase activity, whether immunosuppressive or non-immunosuppressive, are capable of promoting neurite outgrowth in cultured neurons, and are capable of achieving maximal effects comparable to nerve growth factor itself.

The invention being thus described, it will be obvious that the same may be varied in many ways . Such variations are not to be regarded as a departure from the spirit and scope of the invention and all such modifications are intended to be included within the scope of the following claims.