AU609927B2 - Compositions and methods for alleviating stress, anxiety and seizure activity - Google Patents

Description

AU-AI-28283/89

PCT

WORLD INTLLICTUAL PtopwtrY OROANIZKTION Intcrntitliul Durcau

-I

INTERNATIONAL APPLICATIONUB (51) International Patent Classification 4 A61K 33/30, C07J 1/00 frT COOPERATION TREATY (PCT) iblication Number: WO 89/ 02272 (43) International Publication Date: 23 March 1989 (23.03.89) (21) International Application Number: PCT/US88/02827 (22) International Filing Date: (31) Priority Application Number: (32) Priority Date: (33) Priority Country: 24 August 1988 (24.08.88) 089,362 25 August 1987 (25.08.87) (71)(72) Applicant and Inventor: GEE, Kelvin, W. [US/US]; 3410 Punta Del Este Drive, Hacienda Heights, CA 91745 (US).

(74)Agern: DEBRUCKY, Keith; Nilsson, Robbins, Dalgarn, Berliner, Carson Wurst, 201 N. Figueroa St., 5th Floor, Los Angeles, CA 90012-2628 (US).

(81) Designated States: AU, BE (European patent), CH (European patent), DE (European patent), DK, FR (European patent), GB (European patent), IT (European patent), JP, SE (European patent).

Published With international search report.

Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amendments.

This document contains the amendments made under Section 49 and is correct fuor pritiing.

A.O.J.P. 25 MAY 1989

AUSTRALIAN

17 APR 1989 PATENT OFFICE iL-I (54) Title: COMPOSITIONS AND METHODS FOR ALLEVIATING STRESS, ANXIETY AND SEIZURE ACTIVI-

TY

(57) Abstract A method of using 3-hydroxylated-5-reduced steroids and certain novel derivatives which act at a newly identified site on the GBR complex, to modulate brain excitability in a manner which will alleviate stress, anxiety, and seizure activi- W 89/02272 PCT/US88/02827 1 COMPOSITIONS AND METHODS FOR ALLEVIATING STRESS, ANXIETY AND SEIZURE ACTIVITY BACKGROUND OF THE INVENTION The present invention is directed to compounds and a method of use of such compounds for modulating animal brain excitability via the gamma-aminobutyric acid (GABA)/benzodiazepine (BZ) receptor-chloride ionopore complex (GBR complex).

Brain excitability is defined as the level of .arousal of an animal which is a continuum that ranges from coma to convulsions, and is regulated by various .neurotransmitters. In general, neurotransmitters are responsible for regulating the conductance of ions across neuronal membranes. At rest, the neuronal membrane possesses a potential or membrane voltage of approximately -80 mv, the interior being negative with respect to the exterior of the cell. The potential is the result of ion Na+, Cl-, organic anions) balance across the neuronal membrane, which is semipermeable. Neurotransmitters are stored in presynaptic vesicles and are released under the influence of neuronal action potentials. When released into the synaptic cleft, an excitatory chemical transmitter such as acetylcholine will cause membrane depolarization (change of potential from -80 my to -50 my). This C" vrne-~.l I 2"r_ -L WO 89/02272 PCT/US88/02827 2 effect is mediated by post-synaptic nicotinic receptors which are stimulated by acetylcholine to increase membrane permeability to Na+ ions. The reduced membrane potential stimulates neuronal excitability in the form of a post-synaptic action potential.

In the case of the GBR complex,:.the effect on brain excitability is mediated by the neurotransmitter GABA. The profound influence of GABA on overall brain excitability is related to the fact that up to 40% of the neurons in the brain utilize GABA as a neurotrans-itter. GABA regulates the excitability of individual neurons by regulating the conductance of chloride ions across the neuronal membrane. GABA interacts with its recognition site on the GBR complex to facilitate the flow of chloride ions down a concentration gradient of the GBR complex into the cell. The increase in the levels of this anion intracellularly results in the hyperpolarization of the :transmembrane potential rendering the neuron less susceptible to excitatory inputs reduced neuron excitability).

It is well-documented that the GBR complex is responsible for the mediation of anxiety, seizure activity and sedation. Thus GABA, drugs that act like GABA or facilitate the effects of GABA the therapeutically useful barbiturates and benzodiazepines (BZs) such as Valium) produce their therapeutically useful effects via their interaction with specific regulatory sites on the GBR receptor complex.

It has also been observed that a series of steroid metabolites also interact with the GBR receptor complex to alter brain excitability (Majewska, M.D. et S t 1 r- I-T ur WO 89/02272 PcTIus88102827r 3 al., "Steroid hormone metabolites area barbiturate-like modulators of the GABA receptor," Science, 232:1004- 1007, 1986; Harrison, N.L. et al., Structure-activity relationships for steroid interaction with the gammaaminobutyric acid-A receptor complex," J. Pharmacol.

Exp. Ther., 241:346-353, 1987). Prior to the present invention the potential therapeutic usefulness of these steroid metabolites was not recognized by workers in the field due to an incomplete understanding of the potency and site of action.

The ovarian hormone progesterone and its metabolites have also been demonstrated to have profound effects on brain excitability (Backstrom, T.

et al., "Ovarian steroid hormones: effects on modd, behaviour and brain excitability," Acta Obstet.

Gynecol. Scand. Suppl. 130:19-24, 1985; Pfaff, D.W..and- McEwen, "Actions of estrogens and progestins on nerve cells," Science, 219:808-814, 1983; Gyermec, et 1968. "Structure- activity relationship of some steroidal hynotic agents," J. Med. Chem. 11:117). The -levels of progesterone and its metabolites vary with the phases of the menstrual cycle. It has been welldocumented that progesterone and its metabclites decrease prior to the onset of menses. The monthly recurrence of certain physical symptoms associated with the onset of menses has also been well documented.

These symptoms which have become associated with premenstrual syndrome (PMS) include stress, anxiety, and migraine headaches (Dalton, 1984. Premenstrual Syndrome and Progesterone Therapy. 2nd edition, Chicago: Chicago Yearbook Patients with PMS have a monthly recurrence of symptoms which are present in premenses and absent in postmenses.

ctIM-1 71,M.ITC" HEE; WO 89/02272 PCT/US88/02827 4 In a similar fashion, the reduction in progesterone has also been temporally correlated with an increase in seizure frequency in female epileptics catamenial epilepsy; Laidlaw, J. "Catamenial epilepsy," Lancet, 1235-1237, 1956). A more direct correlation has been observed with the reduction in progesterone metabolites (Rosciszewska et al., "Ovarian hormones, anticonvulsant drugs and seizures during the menstrual cycle in women with epilepsy," J. Neurol.

Neurosurq. Psych., 49:47-51, 1986). In addition, for patients with primary generalized petit mal epilepsy, the temporal incidence of seizures has been correlated with the incidence of the symptoms of premenstrual syndrome (PMS) (Backstrom, T. et al. "Production of .alpha.-pregnane-3, 20-dione by human corpus luteum," Acta Endrocr. Suppl. 256:257, 1983).

A syndrome also related to low progesterone levels is postnatal depression (PND). Immediately :after birth progesterone levels decrease dramatically leading to the onset of PND. Women experiencing PND -show an increased incidence of PMS (Dalton, K. 1984.

op. cit.). The symptoms of PND range from mild depression to psychosis requiring hospitalization and is associated with severe anxiety and irritability.

The depression associated with PND is not amenable to treatment by classic antidepressants (Dalton, K. 1984, op. cit.).

Collectively, these observations imply a crucial role for progesterone in the homeostatic regulation of brain excitability which is manifested as an increase in seizure activity or symptoms associated with catamenial epilepsy, PMS, and PND. The correlation between reduced levels of progesterone and 1rbclsl~1MF WO 89/02272 PcT/us88/02827 the symptoms associated with PMS, PND and catamenial epilepsy (Backstrom, et al., 1983, op. cit.; Dalton, K.

1984, op. cit.) has prompted the use of progesterone in their treatment (Mattson, et al., 1984.

"Medroxyprogesterone therapy of catamenial epilepsy," in Advances in epileptoloqv: XVth Epilepsy International Symposium, New York: Raven Press, 279- 282 and Dalton, K. 1984, op. cit.). However, progesterone is not consistently effective in the treatment of the aforementioned syndromes. For example, no dose-response relationship exists for progesterone in the treatment of PMS (Maddocks, et al.

1986. "A double-blind placebo-controlled trial of progesterone vaginal suppositories in the treatment of premenstrual syndrome," J. Obstet. Gynecol. 154:573- 581; Dennerstein, et al., 1986. Britain Medical Journal, 290:16-17).

BRIEF DESCRIPTION OF THE DRAWINGS The present invention may be better understood and its advantages will be apparent to those skilled in the art by reference to the accompanying drawings wherein: Figures 1A and 1B are plots of the percentage of binding of [35S] t-butylbicyclophosphorothionate verse log concentration of Alphaxalone and GABA; Figures 2A and 2B are plots of the percentage of binding of 35 S] t-butylbicyclophosphorothionate verse time; SUBSTITUTE

SHEET

WO 89/02272 PCT/US88/02827 6 Figure 3 is a plot of the effect of a single dosage of pentobarbital on 5 alpha-pregnan-3 alpha-olmodulation of [3H] flunitrazepam binding in rat hippocampal homogenates; Figure 4 is a bar graph of the time to the onset of myoclonus verse different concentrations of various types of compounds of the present invention; and Figure 5 is a plot of the effect of progesterone metabolites and the progestin R5020 (promogesterone) on the binding of 3 H] R5020 to the progesterone receptor in rat cerebral cortex.

SUMMARY OF THE INVENTION The present invention is directed toward the use of 3-hydroxylated-5-reduced steroids and their :derivatives to be defined herein: acting at a newly identified site on the GBR complex, to modulate brain -excitability in a manner which will alleviate stress, anxiety, and seizure activity.

These compounds have utility as modulators of the excitability of the central nervous system as mediated by their ability to regulate chloride ion channels associated with the GABA-benzodiazepine receptor complex. Based on the unique experimental observations disclosed herein, the compounds of the invention have anti-convulsant activity similar to the actions of known anxiolytic agents such as the benzodiazepines, but act at a distinct site on the GBR complex.

WO 89/02272 PCT/US88/02827 7 The relationship of the some of the compounds of the invention, which are endogenous metabolites of progesterone, to processes associated with reproduction (estrus cycle and pregnancy) is well established (Marker, Kamm, and McGrew, R.V. [1937], "Isolation of epi-Pregnanol-3-one-20 from human pregnancy urine", J. Am. Chem. Soc. 59, 616-618).

Therefore, this invention is directed at methods, compounds and compositions of such compounds, and their prodrug derivatives, for use in the treatment of disorders such as pre-menstrual syndrome (PMS) and post-natal depression (PND).

DESCRIPTION OF THE INVENTION This invention is directed to certain compounds and new pharmaceutical applications of such compounds. In particular, the invention is directed to 3-hydroxylated-5-reduced-pregnan-20-one and .3,21-pregnandiol-20-ones and 5-reduced-3,20pregnandiols and various ester and oxime derivatives of such compounds which are known to those skilled in the art of pharmaceutical preparations as prodrugs. The expression "prodrug" denotes a derivative of a known active drug whose derivative enhances delivery characteristics and therapeutic value of the drug and is transformed into the active drug by an enzymatic or chemical process. It should be noted that some of the synthetic derivatives may not be true prodrugs by virtue of their intrinsic activity.

Our novel studies (Gee, et al. "GABAdependent modulation of the Cl- ioonophore by steroids in rat brain," European Journal of Pharmacology, 136:419-423, 1987) have demonstrated that these 3- .r 0U SUSTTUTE SHEET WO 89/02272 PCT/US88/02827 8 steroids are orders of magnitude more potent than others have reported (Majewska, M.D., et al., 1986, op. cit. and Harrison, et al., 1987, op. cit.) as modulators of the GBR complex.

Consequently, we now disclose additional experimental data from in vivg studies, which demonstrates that the high potency of these steroids will allow them to be therapeutically useful in the modulation of brain excitability via the GBR complex. The most potent steroids include major metabolites of progesterone.

These steroids can be specifically used to modulate brain excitability in stress, anxiety and seizure disorders. Furthermore, we have demonstrated that these steroids interact at a unique site on th GBR complex which is distinct from other known sites of interaction barbiturate, benzodiazepine and GABA) where therapeutically beneficial effects on stress, anxiety, sleep and seizure disorders have been previously elicited (Gee, K.W. and Yamamura, H.I., ."Benzodiazepines and Barbiturates: Drugs for the Treatment of Anxiety, Insomnia and Seizure Disorders," *in Drugs in Central Nervous System Disorders, pages 123-147, Horwell, ed. 1985]).

Exemplary of naturally occurring metabolites of progesterone for which this invention describes a new use are those having the following structural formula: FORMULA 1 ~rraar~-ra rr~f euc~T WO 89/02272 PCT/US88/02827 9 in which: R1 is a hydroxy group; R2 is acetyl group or 2-hydroxyethanone or 1hydroxyethane; and R3, is hydrogen; R4 and R5 are each a methyl group.

Exemplary of synthetic derivatives of progesterone metabolites of the invention are those having the structural formula as illustrated above (Formula 1) in which: R1 is: a pharmaceutically acceptable ester -Ywherein R6 is a C 1

-C

20 branched, straight chain, or cyclic aliphatic or aromatic radical, and wherein Y is either oxygen or sulfur. This ester can be derived from the reactions well known in the art :between the hydroxyl.group of the naturally occurring .compounds discussed above with an organic acid, acid halide, anhydride, or ester, wherein the organic acids are for example: acetic, propionic, n and i-butyric, n and i and s and t-valeric, hexanoic, heptanoic, octanoic, nonanoic, decanoic, undecanoic, dcSecanoic, cinnamic, benzylic, benzoic, maleic, fumaric, ascorbic, pamoic,' succinic, bismethylenesalicylic, methanesulfonic, ethanedisulfonic, oxalic, tartaric, salicylic, citric, gluconic, aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, and cyclohexylsulfamic; or a pharmaceutically acceptable oxime =N-O- R7 radical wherein R7 is a C 1

-C

20 branched, straight chain, or cyclic aliphatic or aromatic radical. This oxime radical may be derived from the reaction of a 3- SUBTITUTZ

SHECT

WO 89/02272 PCT/IJS88/02827 oxo derivative of progesterone by methods well know to the art with an oxyamine; or a pharmaceutically acceptable acyloxyalkyloxy--O-CH 2 radical wherein R8 is a C 1

-C

20 branched, straight chain, or cyclic aliphatic or aromatic radical. This acyloxyalkyloxy radical may be derived from the reaction of the 3-hydroxy group of the naturally occuring compounds discussed above by methods well known to the art with an organic acy)oxyalkyl halides (1 -20 carbons) or aryloxlalkyl halides,'and in particular acetyloxymethyl halide, diacetyloxymethyl halide, or amino-acetyloxymethyl halide.

R2 is: a pharmaceutically acceptable

CH

2 -O-R9; -(C=O)-CH 2 OR -(C=0)-CH 2

-O-CH

2 O-(C=O)-R11 w' 4rein R9, R10 or R11 are a C1-C20 branched, straight chain, or cyclic aliphatic or :aromatic radical, or an amide -(CH 2 )n-(C=O)-N-R16,R17 radical wherein R16 and R17 are individually a C1-C20 -branched, straight chain, or cyclic aliphatic or aromatic radical and n=l-8. An example of this type of radical wherein R10 is an amide is 5 .alpha.-pregnan-3 .alpha.-hydroxy-21-(N, These radicals may be derived from the reaction of the 21-hydroxy metabolite of progesterone by methods known in the art with an alkyl halide or organic acid, such as acetic, propionic, n and i-butyric, n and i and s and t-valeric, hexanoic, heptanoic, octanoic, nonanoic, decanoic, undecanoic, dodecanoic, cinnamic, benzylic, benzoic, maleic, fumaric, ascorbic, pamoic, succinic, bismethylenesalicylic, methanesulfonic, ethanedisulfonic, oxalic, tartaric, salicylic, citric, gluconic, aspartic, stearic, palmitic, itaconic, UBSTITUTI SHEET WO 89/02272 PCT/US88/02827 11 glycolic, p-aminobenzoic, glutamic, benzenesulfonic, and cyclohexylsulfamic acids.

a -(C-CH 3 )=N-O-R12; -C-O-CH 2 -O-(C=0)-R13; OR -(C-CH 3 )-O-R15 wherein R12, R13, R14 and R15 are a C 1

-C

20 branched, straight chain, or cyclic aliphatic or aromatic radical. These radicals may be derived from progesterone or the hydroxy metabolite of progesterone by methods known in the art with an alkyl halide or organic acids, such as acetic, propionic, n and i-butyric, n and i and s and t-valeric, hexanoic, heptanoic, octanoic, nonanoic, decanoic, undecanoic, dodecanoic, cinnamic, benzylic, benzoic, maleic, fumaric, ascorbic, pamoic, succinic, bismethylenesalicylic, methanesulfonic, ethanedisulfonic, oxalic, tartaric, salicylic, citric, gluconic, aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, and cyclohexylsulfamic acids.

a pharmaceutically acceptable thiazolidine derivative of the 20-oxo position on progesterone having the formula: S-C(R18,R19)

-(CH

3

)C

S^-NH-C(R20,R21) wherein R18 and R19 are individually a C 1

-C

20 branched, straight chain, or cyclic aliphatic or aromatic radical, and R20 and R21 are individually hydrogen or a

C

1

-C

20 branched, straight chain, or cyclic aliphatic or aromatic radical or -(C=0)-O-R22 wherein R22 is H or a C1-C 20 branched, straight chain, or cyclic aliphatic or aromatic radical.

R3 is a hydroxy, keto, alkyloxy (1 to 18 carbons), aryloxy, or amino radical.

WO 89/02272 PCTUS88/02827 12 R4 is an alkyl (2 to 18 carbons), aryl, halo (such as fluoro, chloro, bromo, or ao), or trifluroalkyl.

is an alkyl (2 to 18 carbons), aryl, halo (such as fluoro, chloro, bromo, or iodo), or trifluroalkyl.

It will be obvious to one skilled in the art that the above described compounds may be present as diastereo isomers which may be resolved into d or 1 optical isomers. Resolution of the optical isomers may be conveniently accomplished by gas or liquid chromatography or isolation from natural sources.

Unless otherwise specified herein, including the claims, reference to the compounds of the invention, as discussed above, is intended to include all isomers, whether separated or mixtures thereof.

Where isomers are separated, the desired :pharmacological activity will often predominate in one of the isomers. As disclosed herein, these compounds -display a high degree of stereospecificity. In particular, those compounds having the greatest affinity for the GABA-benzodiazepine receptor complex are those with 3 .alpha. substituted-5 .alpha.pregnane steroid skeletons. In addition, 3 .alpha.

substituted-5 .beta.-pregnane skeletons have been demonstrated to be active.

The above compounds of the invention, that being the naturally occuring metabolites of progesterone and their nontoxic pharmaceutically acceptable synthetic "prodrug" forms (synthetic derivatives of progesterone metabolites discussed N J.

0 C RcsCTSI Oueel-l WO 89/02272 PcT/us88/02827I 13 above) have novel activity in the brain at the GABAbenzodiazepine receptor complex.

The compounds of the invention may be prepared by any known technique. For example, the naturally occurring metabolites of progesterone may be extracted from various animal excretion sources, urine.

These extracted compounds may then be chemically altered to form the desired synthetic derivative, or used directly.

The pharmaceutical compositions of this invention can be prepared in conventional dosage unit forms by incorporating a compound of the invention or a mixture of such compounds, with a nontoxic pharmaceutical carrier according to accepted procedures in a nontoxic amount sufficient to produce the desired *pharmcoadynamic activity in a subject, animal or human.

Preferably, the composition will contain the active .ingredient in an active, but nontoxic amount, selected from about 50 mg to about 500 mg of active ingredient -per dosage unit. This quantity depends on the specific biological activity desired and the condition of the patient. The most desirable object of the composition and methods is in the treatment of pre-menstrual syndrome, catamenial epilepsy, and post-natal depression to ameliorate or prevent the attacks of anxiety, muscle tension, and depression common with patients suffering from these central nervous system abnormalities.

The pharmaceutical carrier employed may be, for example, either a solid, liquid, or time release (see standard reference Remington's Pharmaceutical Sciences which is incorporated herein by reference).

r I I 7.C~lTC C~rl WO 89/02272 PCT/US88/02827 14 Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stea-'ate, stearic acid, microcrystalline cellulose, polymer hydrogels and the like. Exemplary of liquid carriers are syrup, peanut oil, and olive oil and the like emulsions. Similarly, the carrier or diluent may include any time delay material well known to the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, microcapsules, microspheres, liposomes, and hydrogels.

A wide variety of pharmaceutical forms can be employed. Thus, when using a solid carrier the preparation can be tableted (however, the oral route of administration should be avoided due to first pass metabolic degradation), placed in a hard gelatin capsule in powder or pellet form, or in the form of a troche, lozenge or suppository. When using a liquid carrier the preparation can be in the form of a liquid, :such as an ampule, or as an aqueous or nonaqueous liquid suspension. Liquid dosage forms also need -pharmaceutically acceptable preservatives and the like.

In addition, because of the low doses which will be required as based on the in vitro data disclosed herein, topical administration via timed release skin .patches is also a suitable pharmaceutical form.

The method of producing anxiolyic, or anticonvulsant activity, in accordance with this invention, comprises administering internally to a subject in need of such activity a compound of the invention, usually prepared in a composition as described above with a pharmaceutical carrier, in a nontoxic amount sufficient to produce said activity as described above.

SUbSTiTUTE SHEET WO 89/02272 PCT/US88/02827 During menses the levels of excreted metabolites varies approximately fourfold (Rosciszewska, et al., op. cit.). Therefore, therapy for controlling symptoms will involve maintaining the patient at a more uniform level of progesterone metabolite. Plasma levels of active and major metabolites will be monitored during pre-menses and post-menses of the patient. The amount of the compounds, either singly or mixtures thereof, of the invention administered will reflect the physiological concentrations which naturally occur post-menses. The route of administration may be any route which effectively transports the active compound to the GABAbenzodiazepine receptors which are to be stimulated such as parenterally, rectally, intravaginally, intradermally, subligually, or nasally, the dermal route being preferred. For example, one dose in a skin patch may supply the active ingredient to the patient for a period of up to one week.

The following is a discussion of our experimental observations in support of the invention: As stated, the naturally occurring metabolites of progesterone and their derivatives interact with high affinity at a novel and specific recognition site on the GBR complex to facilitate the conductance of chloride ions across neuronal membranes sensitive to GABA (Gee, et al., 1987). This has been shown by in vitro and in vivo experimental data.

To those skilled in the art, it is known that the modulation of 35 S] t-butylbicyclophosphorothionate 35 S]TRPS) binding is a useful measure of the potency SUBSTITUTE SHEET WO 89/02272 PCT/US88/02827 16 and efficacy of drugs acting at the GBR complex which may be of potential therapeutic value in the treatment of stress, anxiety and seizure disorders (Squires, R.F.,et al., 35 S]t-Butylbicyclophophorothionate binds with high affinity to brain-specific sites coupled to a gamma aminobutyric acid-A and ion recognition site," Mol. Pharmacol. 23:326, 1983; Lawrence, et al., "Benzodiazepine anticonvulsant action: gammaaminobutyric acid-dependent modulation of the chloride ionophore," Biochem. Biophys. Res. Comm., 123:1130- 1137, 1984; Wood, et al., 1984. "In vitro characterization of benzodiazepine receptor agonists, antagonists, inverse agonists and agonist/antagonists," J. Pharmacol. Exp. Ther. 231:572-576). We performed an assay to determine the modulation of 35 S] TBPS as effected by the compounds of the invention and found that these compounds have high potency and efficacy at the GBR complex, with stringent structural requirements for such activity.

The procedures for performing this assay are *fully discussed in: Gee, et al., 1987 op. cit.; and Gee, L.J. Lawrence and H.I. Yamamura, 1986, "Modulation of the chloride ionopore by benzodiazepine receptor ligands: influence of gamma-aminobutryric acid and ligand efficacy," Molecular Pharmacology, 30, 218, both of which references are incorporated herein by reference. These procedures were performed as follows: Brains from male Sprague-Dawley rats were removed immediately following killing and the cerebral cortices dissected over ice. A P 2 homogenate was prepared as previously described (Gee, et al., 1986, op. cit.). Briefly, the cortices were gently homogenized in 0.32 M sucrose followed by U WO 89/02272 PCT/US88/02827 17 centrifugation at 1000 x g for 10 minutes. The supernatant was collected and centrifuged at 9000 x g for 20 minutes. The resultant P 2 pellet was resuspended as a 10% (original wet weight/volume) homogenate in 50mM Na/K phosphate buffer (pH 714) 200 mM NaCl.

One hundred microliter aliquots of the P 2 homogenate (0.5 milligrams (mg) protein) were incubated with 2 nanomolar (nM) [3 5 S]TBPS (70-110 curies/millimole;, New England Nuclear, Boston, MA) in the presence or absence of various concentrations of the desired compounds. Compounds were dissolved in dimethylsulfoxide (Baker Chem. Co., Phillipsbury, NJ) and added to the incubation mixture in 5 microliter aliquots. The incubation mixture was brought to a final volume of 1 millilitre (ml) with buffer. Nonspecific binding was defined as binding in the presence of 2 micromolar TBPS. The effect and specificity of :GABA (Sigma Chem. Co., St. Louis, MO) was evaluated by performing all assays in the presence of 5 micromolar GABA (+)-bicuculline (Sigma Chem. Incubations maintained at 25" C for 90 minutes (steady state conditions) were terminated by rapid filtration through glass fiber filters (No. 32, Schleicher and Schuell, Keene, NH). Filter bound radioactivity was quantitated by liquid scintillation spectrophotometry. Kinetic data and compound/[ 35 S]TBPS dose-response curves were analyzed by non-linear regression using a computerized iterative procedure to obtain rate constants and

IC

50 (concentration of compound at which half-maximal inhibition of basal 35 S]TBPS binding occurs) values.

The experimental data obtained for this assay is also published in Gee, et al., 1987 which data and SAl I 0 ^y WO 89/02272 PCT/US88/02827 18 conclusions are also incorporated herein by reference.

The data discussed in this incorporated reference is shown as plots in FIGURES 1A and lB. These plots describe the effect of (+)-bicuculline on alphaxalone (1A) and GABA (1B) modulation of 2 nanomolar 35

S]-

TBPS binding to rat cerebral cortex. In these Figures represents control without bicuculline; represents 0.5 micromoloar bicuculline; (D) represents 1.0 micromolar bicuculline; represents 2.0 micromolar bicuculline; and represents micromolar bicuculline. In this experiment the effect of (+)-bicuculline on the ability of alphaxalone or GABA to inhibit the binding of 35 S]TBPS has been determined. Bicuculline is known to be directly competitive with GABA and in Figure 1B, a classical parallel shift in the dose-response curves is observed.

In contrast, the s.teroid binding site is distinct from the GABA/bicuculline site as seen in Figure lA. The shift in dose-response curves induced by :bicuculline when the inhibition of 35 S]-TBPS binding is caused by alphaxalone is not linear. This would -definitely suggest that the GABA and steroid sites do not overlap.

We also performed this assay to determine the effect of pentobarbital on the dissociation kinetics of 35 S]TBPS in rat cerebral cortical membranes. This assay was performed in accordance with the procedures outlined above. This data indicates that the site of action of the compounds of the invention is unique and distinct from the previously known sites of action for the barbiturates and the BZs'. The results of the in vitro assay is shown in FIGURES 2A and 2B. The plots in Figures 2A or 2B describe the effect of pentobarbital, alphaxalone, 5 .alpha.-pregnan-3

C

WO 89/02272 PCT/US88/02827 19 on the dissociation kinetics for 2 nanomolar 35 S]TBPS inn cortical P2 homogenates.

Dissociation of bound 35 S]TBPS was initiated by 2 micromolar TBPS in all cases. Pentobarbital (Figure 2A) at 30 micromolar induces a biphasic dissociation mechanism which is absent for alphaxalone (300 nanomolar) and 5 .alpha.-pregnan-3 one (20 nanomolar) (Figure 2B).

A table of the kinetic rate constants and half lives obtained by this assay are seen below in Table 1.

The information presented in Table 1 indicates that the barbiturate induces a shift in the half life of dissociation and the proportion of slow and rapidly dissociating components, hallmark effects of therapeutically useful GABA agonists, barbiturates and BZs on 35 S] TBPS binding (Gee, et al., 1986; Maksay, G. Ticku, M. "Dissociation of 35 S]tbutylbicyclophophorothionate binding differentiates :convulsant and depressant drugs that modulate GABAergic transmission," J. Neurochem. 44:480-486, 1985). In contrast, the progesterone metabolite 5 .alpha.pregnan-3 .alpha.-ol-20-one, and the progestin alphaxalone do not influence the dissociation kinetics of 35 S]TBPS binding. Therefore, the steroid and barbiturate sites are distinct.

e=ll~czTE~: ~T~p 9UC WO 89/02272 PCT/US88/02827 TABLE 1 Conditions tl/ 2 (min) k_ 1 (min- 1 Total percentage of specific sites S R S R S R Control 50+4 6+1 0.145+ 0.131+ 73+2 30+2 0.0008 0.016 pM Na pentobarbital 38+3 4.4+ 0.3 0.186+ 0.158+ 0.0015 0.013 61+6* 48+6** 300nM 67+ Alphaxalone 12 4.9+1 0.01204 0.0180+ 0.0003 0.040 6.4+1 0.011+ 0.122+ 0.002 0.030 73+4 34+5 68+3 35+3 20 nM *3a-OH-DHP 76+ 11 .Significantly different from control *P<0.05 and **P<0.01 by Student's t-test. S and R represent slowly and rapidly dissociating components respectively.

Furthermore, 5 .alpha.-pregnan-3 .alpha.-oldoes not interact with pentobarbital in the enhancement of the binding of [3H] flunitrazepam to the BZ receptor in the cortical brain homogenates (FIGURE Indicating that steroids and barbiturates do not share a common site of action. The data shown in FIGURE 3 was obtained by performing an assay to determine the effect of a single concentration of pentobarbital (1.0 millimolar) on 5 .alpha.-pregnan-3 PCT/US88/02827 WO 89/02272 21 modulation of 0.25nM 3

H]

flunitrazepam ([3H]FLU) binding to the BZ receptor in rat hippocampal homogenates. 'This assay was performed in accordance with the procedures outlined above.

Each point on the plot of FIGURE 3 represents the mean SEM of 4-6 independent determinations. The data points in both curves are expressed as percent enhancements of 3 HjFLU binding, which is defined as the percentage of 3 H]FLU bound in the absence of .alpha.-pregnan-3 .alpha.-ol-20-one under the control conditions minus 100%. All assay were performed in the absence of GABA.

The above data demonstrates that the compounds of the invention interact with a novel site distinct from previously defined regulatory sites on the GBR complex.

A variety of the compounds were screened to ;determine their potential as modulators of 35

S]TBPS

binding in vitro. These assays were performed in -accordance with the above discussed procedures. Based on these assays we have established the structureactivity requirements for their specific interaction at the GBR complex and their rank order potency and efficacy (Table 2 below).

A

SUna.71TUTZ7, SWF WO 89/02272PCTU8082 PCT/US88/02827 TABLE 2

COMPOUND

CONTROL

icso (NM) +5 ~m GABA ICso (NM) MAX IMAL

INHIBITION

Sa -PREGNAN-3a ONE CNJ -PREGNAN-3c, 21- DIOL-20-ONE N?cc" co -PREGNAN-3a -CL- 1.1,20-DIONE CN3 kALPHAXALONE) 1 a~ -ANDROSTAN-3m 17a -DIOL ON 11000 15000 1500* 1000 WO 89/02272 WO 8902272PCT/US88/02827 a -PREGNAN- 21-DIOL-11, ,Z-D'IONE

CO

-ANDROSTAN-lhs OL- 3-ONE OH No9L -PREGNAN-3s (ALLOPREGNANOLONE) "s

CO

HOC6 5-PREGNEN-36 -OL-N (PRLESNENOLONE)

C

CO

1-OL3.20DIONE

CHOLESTEROL

;,10 5500 1.10, 18000 I NACT IVE I NACT IVE I NACT IVE I NACT IVE I NACT IVE I NACT IVE INACTIVE We also performed experiments to determin the physiological relevance of these interactions by WO 89/02272 PCT/US88/02827 24 measuring the ability of the compounds of the invention to modulate TBPS induced convulsions in Swiss-Webster mice. Mice were injected with various doses of the test compounds of the invention, as specified in Figure 4, 10 minutes prior to the injection of TBPS. The time to onset of myoclonus (presence of forelimb clonic activity) induced by TBPS was dete-mined by observing each mouse for a period of 45 minutes. Significant differences between the time to onset in control versus steroid treated mice were determined by Student's ttest. The relative rank order potency and efficacy of these steroids in vivo were well correlated with those observed in vitro. These observations demonstrate the therapeutic utility of these compounds as modulators of brain excitability whbi-h was predicted by their high affinity interaction .ith the GBR complex in vitro.

The correlations between reduced levels of progesterone and the symptoms associated with PMS, PND and catamenial epilepsy (Backstrom, et al. 1983 op.

cit.; Dalton, K. 1984, op. cit.) has prompted the use of progesterone in their treatment (Mattson, et al., 1984; and Dalton, 1984). However, progesterone is no+ consistently effective in the treatment of the aforementioned syndromes.. For example, no doseresponse relationship exists for progesterone in the treatment of PMS (Maddocks, et al, 1987. op. cit.).

These results are predictable when based upon our in vitro studies which demonstrate that progesterone has very low potency at the GBR complex, as seen in Table 2, compared to certain metabolites of progesterone.

The beneficial effect of progesterone is probably related to the variable conversion of progesterone to the active progesterone metabolites.

I I WO 89/02272 PCT/US88/02827 The use of specific progesterone metabolites in the treatment of the aforementioned syndromes is clearly superior to the use of progesterone based upon the high potency and efficacy of the metabolites and their derivatives (See Gee, et al., 1987 and Table 2).

We have also demonstrated that the compounds of the invention lack hormonal side effects by the lack of affinity of these compounds of the invention for the progesterone receptor (FIGURE The data plotted in FIGURE 5 was obtained by performing assays in accordance with the procedures outlined above, to determine the effect of progesterone metabolites and the progestin R5020 on the binding of 3 H]R5020 to the progesterone receptor in rat cerebral cortexes. All points on the plot of FIGURE 5 represent the mean of triplicate determinations. The following compounds are those listed in Figure 5: 5 .alpha.-pregnan-3 .alpha.- (DHP); 5 .alpha.-pregnan-3 .alpha.,21-diol- (Th-DOC); 5 beta-pregnane-3 .alpha.,20 diol

BETA).

While the preferred embodiment has been described and illustrated, various substitutions and modifications may be made thereto without departing from the scope of the invention. Accordingly, it is to be understood that the present invention has been described by way of illustration and not limitation.

SUBSiTT EE SUBSTTUTZ

SHEET

w/Is 11 SEP1989 39. An anticonvulsant composition comprising an anticonvulsant effective amount of a compound of the formula

RS

wherein R1 is selected from the group consisting of 0 0 II II -Y-C-R6, =N-0-R7, and -0-CH 2 -0-C-R8 wherein R6, R7, and R8 are individually a Ci-C2 0 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and Y is or R2 is selected from the group consisting of 0 0 0 0 0 II II II II II

-C-CH

2 -0-R9, -C-CHg-0-C-R0, -C-CH 2 -0-CH 2 -0-C-Rll,

CH

3 CH 0 CH, I I II I -C=N-0-R12, -C-0-C-R14, -C-0-R15, and H H R19 CH3 .S-C-R18 ^N-C-R21 I I H wherein R9, R10, R11, R12, R14, R15, R18, R19, R20, and R21 are individually a C 1

-C

2 0 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, with the provisos that

.M/US

W IAIi U6 rAAS 11 SEPW6 R9, R10, and R11 may also individually 0 R16 11 I be an amide -(CH 2 )n-C-N-R17 radical wherein R16 and R17 individually are a Ci-C 20 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and n=l to 8 and R20 and R21 may also individually be H or -C-0-R22 wherein R22 is H or a CI-C 20 straight chain 11 0 aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and n is an integer of 1 to 8; R3 is hydroxy, keto, C 1 alkyloxy, aryloxy, or amino; and R4 and R5 individually are C 2 -C1, alkyl, aryl, halo, or trifluoroalkyl or a compound of the formula R2' wherein R2' is selected from the group consisting of acetyl, 2-hydroxyethanone, and 1-hydroxyethane, and a pharmaceutically acceptable carrier therefor.

SUIM"M am

IPEA/US

ICT/US 88/U28 27 i I S 11SEP1989 42 A compound of the formula 4 R2

RS

wherein R1 is selected from the group consisting of 0 0 II II -Y-C-R6, =N-0-R7, and -0-CH 2 -0-C-R8 wherein R6, R7, and R8 are individually a Cl-C 20 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and Y is or R2 is selected from the group consisting of 0 0 0 0 0 II II II II II

-C-CH

2 -0-R9, -C-CH 2 -0-C-R0l, -C-CH 2 -0-CH 2 -0-C-Rll,

CH

3 CH 3 0

CH

3 -C=N-0-R12, -C-0-C-R14, -C-0-R15, and H H R19 CH, S-C-R18 N-C-R21 I I H wherein R9, R10, R11, R12, R14, R15, R18, R19, R20, and R21 are individually a C 1 -C2 0 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, with the provisos that R9, R10, and Rll may also individually 0 R16 II I be an amide -(CH 2 )n-C-N-R17 radical wherein R16 and R17 individually are a C 1

-C

20 straight chain aliphatic,

'IEA/US

PCT/US 88/02827 I IA 11SEP89 43 branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and n=l to 8 and R20 and R21 may also individually be H or -C-0-R22 wherein R22 is H or a C 1

-C

20 straight chain

II

0 aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and n is an integer of 1 to 8; R3 is hydroxy, keto, CI-C 18 alkyloxy, aryloxy, or amino; and R4 and R5 individually are C 2

-C

18 alkyl, aryl, halo, or trifluoroalkyl.

41. A method of treating the symptoms of premenstrual syndrome and postnatal depression comprising administering to a patient in need thereof a premenstrual syndrome or postnatal depression syndrome alleviating effective amount of a compound of the formula 4 R2

RS

wherein Rl is selected from the group consisting of 0 0 II II -Y-C-R6, =N-0-R7, and -0-CH 2 -0-C-R8 wherein R6, R7, and R8 are individually a C 1

-C

20 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and Y is or R2 is selected from the group consisting of 0 0 0 0 0 II II II I II

-C-CH

2 -0-R9, -C-CH 2 -0-C-Rl0, -C-CH 2 -0-CH 2 -0-C-Rll,

EA/US

PCT/US 8 8 /02 827

IP

n l11 SEP1989 44

CH

3 CH 0 CH 3 I II I -C=N-0-R12, -C-0-C-R14, -C-0-R15, and H H R19 CH3 S-C-R18 I I N-C-R21 H wherein R9, R10, R11, R12, R14, R15, R18, R19, R20, and R21 are individually a CI-C 20 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, with the provisos that R9, R10, and R11 may also individually 0 R16 II I L- an aaide -(CH 2 )n-C-N-R17 radical wherein R16 and R17 individually are a C 1

-C

20 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and n=l to 8 and R20 and R21 may also individually be H or -C-0-R22 wherein R22 is H or a C 1

-C

20 straight chain

II

0 aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and n is an integer of 1 to 8; R3 is hydroxy, keto, C 1

-C

1 alkyloxy, aryloxy, or amino; and R4 and R5 individually are C 2

-C

1 alkyl, aryl, halo, or trifluoroalkyl or a compound of the formula

IPE/US

Claims (3)

1. 42. The method of claim 41 wherein said syndrome alleviating effective amount is sufficient to maintain a level of progesterone and its metabolites prior to the onset of menses when treating premenstrual syndrome, or prior to birth when treating postnatal depression.
2. 43. A method of treating the frequency and occurrence of convulsions comprising administering to a patient in need thereof a convulsion combatting effective amount of a compound of the formula R3 R4 R2 RIRI wherein R1 is selected from the group consisting of 0 0 11 II -Y-C-R6, =N-0-R7, and -0-CH 2 -0-C-R8 wherein R6, R7, and R8 are individually a Ci-C 20 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and Y is or R2 is selected from the group consisting of 0 0 0 0 0 II II II II II -C-CH 2 -0-R9, -C-CH 2 -0-C-RlO, -C-CH 2 -0-CH-0-C-Rll, su EAr" -RTus 88/O282 7 WVfAA 11 EP 9 46 C CH 3 0 CH 3 IH1 11 1 -C=N-0-RU2, -C-0-C-R14,' -C-0-R15, arnd H H R19 S-C-Rl8 C I H wherein R9, R10, R11, R12, R14, R15, R18, Rl9, R20, and R21 are individually a C 1 -C 20 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, with the provisos that R9, R10, and R11 may also individually 0 R16 11 1 be an amide -(CH 2 ),-C-N-Rl7 radical wh~.rein R16 and R17 individually are a C 1 -C 20 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and n=1 to 8 and R20 and R2l may also individually be H or -C-0-R22 wherein R22 is H or a C 1 -C 2 straight chain 11 0 aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and n is an integer of lto 8; R3 is hydroxy, keto, C 1 -C 1 alkyloxy, aryloxy, or amino; and R4 and R5 individually are C 2 -C 18 alkyl, aryl, halo, or trifluoroalkyl or a compound of the formula R3 P4R -IM 3H IP EA/US rci/us 8d8/U 827 IPWaS 11 SEPt989 47 wherein R2' is selected from the group consisting of acetyl, 2-hydroxyethanone, and l-hydroxyethane.
3. 44. The method of claim 43 wherein said convulsion combatting effective amount is from about milligrams to about 500 milligrams. A method of modulating the excitability of neuron activity in animals comprising administering to an animal in need thereof a neuron activity modulating effective amount of a compound of the formula wherein R1 is selected from the group consisting of 0 0 II II -Y-C-R6, =N-0-R7, and -0-CH 2 -0-C-R8 wherein R6, R7, and R8 are individually a C 1 -C 20 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and Y is or R2 is selected from the group consisting of 0 0 0 0 0 II II II II II -C-CH 2 -C-CH 2 -O-C-Rl0, -C-CH 2 -O-CH-O-C-Rll, CH 3 -C=N-0-R12, CH 3 0 I II -C-0-C-R14, H H CH 3 -C-0-R15, and H -M"VINK an "'Wus P tI SEPB89 48 R19 I CH3 C-R18 C N-C-R21 I I H wherein R9, RIO, R11, R12, R14, R15, R18, R19, R20, and R21 are individually a Ci-C 20 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, with the provisos that R9, RIO, and R11 may also individually 0 R16 II I be an amide -(CH 2 ),-C-N-Rl7 radical wherein R16 and R17 individually are a C 1 -C20 straight chain aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and n=l to 8 and R20 and R21 may also individually be H or -C-0-R22 wherein R22 is H or a CI-C 20 straight chain II 0 aliphatic, branched chain aliphatic, or cyclic aliphatic radical, or aromatic radical, and n is an integer of 1 to 8; R3 is hydroxy, keto, C 1 -*C 18 alkyloxy, aryloxy, or amino; and R4 and R5 individually are C 2 -C 18 alkyl, aryl, halo, or trifluoroalkyl or a compound of the formula R'S wherein R2' is selected from the group consisting of acetyl, 2-hydroxyethanone, and l-hydroxyethane. VS I PEAUS ii.;I' MIT. N wo 89/02272 PCT/US88/02827 1/4I ANY REFERENCE TO FIGURES 1A, 1B, 2A, 2B, 3, 4 and 5 SHALL BE CONSIDERED NON-EXISTENT (See Article 14(2)). INTERNATIONAL SEARCH REPORT International Application No. PCT US88 02827 I. CLASSIFICATION OF SUBJECT MATTER (1i several classificalion symbols apply, indicate all) 6 According to Inernalional Palent Classification (IPC) or to both National Classification and IPC INT. Cl A61K 33/30, C07J 1/00 US Cl 260/397.45, 3975; 514/179, 182; 540/95 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols 260/ 7.45, 3 US 514/179, 182 540/95 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched a Ill. DOCUMENTS CONSIDERED TO BE RELEVANT 9 Category Citation of Document, it with indication, where appropriate, of the relevant passages 12 Relevant to Claim No. 1 A US,A, 4,203,981 (PIERDET ET AL) 1-16 May 1980; Col 1-4 A US,A, 4,415,554 (HORROBIN) 17-24 November 1983; Entire document A US,A, 4,605,649 (LIEHR) 17-24 12 August 1986; Entire document A US,A, 4,681,875 (AURENT ET AL) 17-16 21 July 1987; Cols 1, 8-10 Special categories of cited documents: 10 later document published alter the international filing date document deining the general state of the art which is not or priority date and not in conflict with the application but considered to be of particular relevance cited to understand the principle or theory underlying the invention earlier document but published on or alter the international document of particular relevance: the claimed Invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which is tited to establish the publication date ot another document of particular relevance: the claimed Invention citation cr other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use. exhibition or document is combined .h one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search i Date of Mailing of this International Search Report 26 February 1989 0 8 FEB 1989 International Searching Authority Sio7 re ol Au horized-efficer ISA/US tHhony M ar ane Form PCTI1SA/210 (second sheet) (Rev.1 1-87)