PH26630A - Method for preventing or treating anxiety employing an ace inhibitor - Google Patents

Description

WALA OF N° TTNTS TRACEPRARY '

ANG TE O6Y TRANSFER

METHOD FOR PREVENTING OR TREATING .

ANXIETY EMPLOYING AN ACE iNHIRITOW AG26 P2:13 "

REFERENCE TO OTHER APPLICATION ecoived By: Lo

This is a continuation-in-part of rp TIER Serr

No. 188,827 filed May 2, 1988, now ahandoned and . application Ser. No. 132,457, filed Dec. “14% PORT HALE ioe abandoned.

FIELD OF THE INVENTION

The present invention relates ta a method for preventing or treating anxiety by administering an ACE inhibitor, such as captopril, SQ 29,852, zofenopril, foginopril or enalapril, alone or in combination with a calcium channel blocker, such as diltiazem, nifedipine or verapamil. 1.8. Pat. Nos. 4,046,889 and 4,105,776 to Ondetti : et al discloses proline derivatives, including captopril, which are angiotensin converting enzyme (ACE) inhibitors useful for treating hypertension.

U.S. Pat. No. 4,337,201 to Petrilo discloses phosphinylalkanoyl enhstituted prolines, including fosinepril, which are ACE inhibitors useful for treating hypertension.

U,S. Pat. No. 4,374,829 discloses carhoxyvalkyl dipeptide derivatives, including enalapril, which are

ACE inhibitors useful for treating hypertensioen.

U.S. Pat. No. 4,452,790 to Karanewsky et al. iG discloses phosphonate eubstituted amino or imino acids and Salts thereof and covers (s)-1-[h-amino- ye 2-[ [hydroxy (4-phenylbutyl)-phosphinyllexy]-1-oxohexyl]-

L-proline (SQ 29,8852). These compounds are ACE inhibitors useful in treating hypertension. . r

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U.S. Pat, No. 4,216,960 to Ondetti et al, } discloses ether and thioether mercaptoacyl prolines which are ACE inhibitors useful in treating hypertension.

This Ondetti et al patent covers zofenopril.

DESCRIPTION OF THE INVENTION

In accordance with the present invention, a method is provided for Preventing or treating anxiety in mammalian species wherein an antianxiety effective . 10 amount of an angiotensin converting enzyme inhibitor alone or in combination with a calcium channel blocker is systemically, such as orally or parenterally, administered,

The method of the invention is useful in treating or preventing anxiety including chronic and acute anxiety disorders (or anxiety and phohic neuroses) including panic disorder with or without agoraphobia, ' agoraphobia, social phobia, simple phobia, ohsessive compulsive disorder (or ohsessive compulsive neurosis), post-traumatic stress disorder, generalized anxiety disorder, anxiety disorder not otherwise specified, and mixed anxiety-depression,

In addition, the method of the invention is useful in treating or preventing anxiety associated with ” 25 withdrawal from drugs of dependency and/or addiction.

Thus, the method of the invention is useful in reducing : anxiety and thus facilitates withdrawal from aleohal . dependency, nicotine dependency, cocaine dependency and henzodiazepine dependency as well as withdrawal from other drug dependency.

Where a combination of ACE inhibitor and calcium

BAD ORIGINAL 9 2 ee ‘

channel hlocker are to be used, the ACE inhibitor will he emploved in a weight ratio to the calcium channel hlocker of within the range of from ahout 0.1:1 to ahout 10:1 and preferahly from ahout 0.4:1 to about 2,5:1.

The angiotensin converting enzyme inhibitor which may he employed herein includes substituted proline derivatives, such as any of those disclosed in U.S. Pat,

No. 4,046,889 or 4,106,776 to Ondetti et al mentioned ahove, with captopril, that is, 1-{(28)-3-mercapto-2- methylpropionyl]-L-proline, heing preferred, carboxyalkyl dipeptide derivatives, such as any of those disclosed in U.S, Pat. No, 4,374,829 mentioned ahove, with N-(1-ethoxycarhoenyl-3-phenylpropyl)-L-alanyl-L- 3 proline, that is, enalapril, being preferred.

Other examples of angiotensin converting enzyme inhibitors suitable for use herein include any of the rhosphonate substituted amino or imino acids or salts disclosed in U.S, Pat. No. 4,452,790 with (S)-1-[6- amino-2-[ [hydroxy-(4-phenylbutyl)phosphinyl]oxyl-1- axnhexyl]l-L-proline (SQ 29,852) heing preferred, phosphinylalkanoyl prolines disclosed n U,S. Pat. No. 4,168,267 mentioned ahove with fogsinapril being preferred, mercaptoacyl derivatives of substituted prolines, disclosed in U.S. Pat. No. 4,316,906 with zofenopril heing preferred, any of the phosphinylalkanoyl substituted prolines disclosed in

U.S. Pat. No. 4,337,201 discussed above; and the phosphonamidates disclosed in U.S. Pat. No. 4,432,971 discussed above, i} d a0 Other examples of ACE inhibitors that may be : : - employed herein include Reecham’s BRL 36,378 as {fi Pe “a \ heen 3 ~~ BAD ORIGINAL 2

} disclosed in European Pat. Nog. 80822 and 60668;

Chugai’s MC-838 disclosed in CA. 102:72588v and Jap. J.

Pharmacol. 40:373 (198A); Ciba-Geigy's CGS 14824 (3- ([1-ethoxycarbonyl-3-phenvl-(1S)-propyllaminn)-2,3,4,5- tetrahydro-2-oxo0-1-(3S8)-henzazepine-1 acetic acid HCl) disclosed in U.K. Patent No. 2103614 and CGS 16,617 (3(S)-[[(1S)-5-aminn-1-carboxypentyllamino]l-2,3,4,5- tetrahydro-2-oxno-1H-1l-henzazepine-l-ethanoic acid) disclnsed in U.S. Pat. No. 4,473,575; cetapril (alacepril, Dainippon) disclosed in Eur. Therap. Reg, 39:671 (1986); 40:543 (1986); ramipril (Hoechst) disclosed in Eur. Patent No. 79-022 and Curr. Ther. Res. 40:74 (1986); Ru 44570 (Hoechst) disclosed in

Arzneimittelforschung 35:1254 (1985), cilazapril (Hof fman-LaRoche ) disclosed in J. Cardiovasc. Pharmacol. 9:39 (1987); R, 31-2201 (Hof fman-LaRoche) disclosed in

FEBS Lett. 165:201 (1984); lisinopril (Merck) disclosed in Curr. Therap. Res. 37:342 (1985) and Eur. Pat. appl.

No. 12-401, indalapril (delapril) disclesed in U.S. Pat.

No. 4,385,081; rentiapril (fentiapril, Santen) disclosed in Clin. Exp. Pharmacol. Physiol. 10:131 (1983); indolapril (Schering) disclosed in J. Cardiovasc.

Pharmacanl. 5:643,A55 (1983); spirapril (Schering) disclosed in Acta, Pharmacol, Toxienl., B59 (Supp. 5): 173 (19868); perindopril (Servier) disclosed in Eur. J.

Clin. Pharmacol. 231:5819 (1987); quinapril (Warner-

Lambert) disclosed in U.S. Pat. No, 4,344,949 and CI 925 (Warner-Lambert) ([3S-[2[R(*)R(*)]]J3R(*)]-2-[2-[[1~- (ethoxycarbonyl)-3-phenylpropyl Jamino[-1-oxopropyl]- an 1,2,3,4-tetrahydro-6,7-dimethoxyv-3- isoquinolinecarboxylie acid HCl) disclosed in

Pharmacnlogist 26:243,266 (1984), WY-44221 (Wyeth) ] disclosed in J. Med. Chem. 26:394 (1983),

Preferred are those ACE inhibitors which are proline or substituted proline derivatives.

The abnve-mentioned U.S. patents are incorporated herein by reference.

The calcium antagonist which will he used herein may be diltiazem which is disclesed in U.S. Pat. No. 3,862,257 and which has the chemical name 3-(arcetyloxy)-

F-[2-{dimethylamino)ethyl-2,3-dihydro-2-(4- methoxyphenyvl)-1,5-henzothiazepin-4-(5H)-one and the structure s 2 Hoon

H

HE

OOoCC

N” So 5

CH,CH,N(CHy), ’ 4-Phenyl-1,4-dihydropyridine calcium antagonists may be employed which will have the structure

H

HCN _ CH,

R,00 COCR,

CD h wherein Ry and Rg may he the same or different and are lower alkyl or lower alkoxy (lower alkyl) where lower alkyl and lower alkoxy contain 1 to 4 carbons.

The above compounds and methods for preparing same are disclosed in U.S. Pat. Nos, 3,644,627, 23,485,847, 2,488,359, 3,574,843, 13,799,924, 3,932,645 and 4,154,839 which are incorporated herein by reference.

The dihydropyridine calcium antagonist present in the composition of the invention will preferably he nifedipine, that is, the compound of formula C wherein

Ry is CH,, Ro is CHgq and NO, is at the 2-pasition, namely,

H

HC NN cH

H3COO COOCH; 0, : which is disclosed in U.S. Pat. Nos. 3,644,627 and 3,485,847.

Other preferred 4-phenyl-1,4-dihydropyridine calcium antagonists suitable for use herein include niludipine, that is, the compound of formula C wherein 0C3H7 and Nd, 1s nt le ARI Rion her in U.S,

E 20 Pat. Nos, 3,488,259 and 3,574,843); nimodipine, that is the campound of the formula C wherein rl is -(CHy)9OCH4, oo ] —

BAD ORIGINAL P)

Ro is -CH(CH3) 9 and NO, is at the 3-position (disclosed in U.S. Pat, Nos. 31,799,934 and 3,932,645); nitrendipine, that is, the compound of the formula C wherein Ry is -CH,CH,, Ry is -CHgq and NO, is at the 3- position (disclosed in U.S. Pat. Nos. 3,799,934 and 3,932,645); and nisoldipine, that is, the compound of the formula C wherein Ry is -CH;, Rg is -CH,CH(CHy) 5 and

NO, js at the 2-position (disclosed in U.S. Pat. Nos. 3,799,934, 3,832,645 and 4,154,839). Verapamil may also he employed.

In addition, verapamil may he employed.

The disclosure of the ahove-mentioned U.S, Patents are incorporated herein by reference.

In carrying out the method of the present invention, the angiotensin converting enzyme inhihitor alone or in comhination with the calcium channel blocker may he administered to mammalian species, such as monkeys, dogs cats, rats and humans, and as such may be incorporated in a conventional systemic dosage form, such as a tahlet, capsule, elixir or injectable. The ahove dosage forms will ‘also include the necessary carrier material, excipient, lubricant, buffer, antibacteria, bulking agent (such as mannitol), anti- oxidants (ascorbic acid of sodium bisulfite) ar the like. Oral dosage forms are preferred, although parenteral forms such as intramuscular, intraperitoneal, or intravenous are quite satisfactory as well.

The dose administered must he carefully adjusted ' 0 according to age, weight and condition of the patient, as well as the route of administration, dosage form and

- TT TTT— -—— — EN i SE regimen and the desired result.

Thus, for aral administration, a satisfactory result may be obtained employing the ACE inhibitor in an amount within the range of from ahout 0.01 mg/kg to about 100 mg/kg and preferahly from ahout 0.1 mg/kg to ahout 25 mg/kg alone or in combination with the calcium channel hlocker in an amount within the range of from ahout 0,01 mg/kg to ahout 100 mg/kg and preferably from about 0.1 mg/kg to ahonut 25 mg/kg with the ACE inhibitor and calcium channel hlocker being employed together in the game oral dosage form or in separate oral dosage forms taken at the same time.

A preferred oral dosage form, such as tablets or capsules, will contain the ACE inhibitor in an amount of from about 0.1 ta abhaut 500 mg, preferably from about 5 to about 200 mg, and more preferably from about 25 to about 150 mg, alone or with the calcium channel blocker in an amount of frem about 1 to about 350 mg, preferaby oo from ahout 2 ta about 200 mg, and more preferably from about 30 to about 150 mg.

For parenteral administration, the ACE inhibitor will he employed in an amount within the range of from about 0.005 mg/kg to about 10 mg/kg and preferably from about 0.01 mg/kg tn about 1 mg/kg, alone or with the ’ 25 calcium channel blocker in an amount within the range of from ahout 0.005 mg/kg to ahout 20 mg/kg and preferahy from ahout 0.01 mg/kg ta about 2 mg/kg.

The composition described ahove may he administered in the dosage forms as described ahove in . 30 single or divided doses of one te four times daily. It may be advisable to start a patient on a low dose ]

camhination and work up gradually to a high dose combination.

Tablets of various sizes can be prepared, e.g. of about 50 to 700 mg in total weight, containing one or hath of the active substances in the ranges descrihed ahnove, with the remainder being a physiologically acceptahle carrier of other materials according to accepted pharmaceutical practice. These tablets can, of course, be scored to provide for fractional doses.

Gelatin capsules can he similarly formulated.

Liquid formulations can alsa he prepared hy dissolving or suspending one ar the combination of active substances in a conventional liquid vehicle acceptable for pharmaceutical administration so as tao provide the desired dosage in one to four teaspoonfuls.

Such dosage forms can he administered to the patient on a regimen of one to four doses per day.

According to another modification, in nrder to more finely regulate the dosage schedule, the active . 20 substances may he administered separately in individual dosage units at the same time or carefully coordinated - times. Since hlood levels are built up and maintained hy a regulated schedule of administration, the same result is achieved by the simultaneous presence of the twn substances. The respective substances can he jndividually formulated in separate unit dosage forms in a manner similar to that descrihed ahove.

Fixed combinations of ACE inhibitor and calcium : channel hlocker are more convenient and are preferred, : ao especially in tablet or capsule form for oral administration. : . § Co J

In formulating the compositions, the active substances, in the amounts descrihed above, are compounded according to accepted pharmaceutical practice with a physiologically acceptable vehicle, carrier, excipient, hinder, preservative, stahilizer, flavor, ete., in the particular type of unit dosage form.

Illustrative of the adjuvants which may be incorporated in tahlets are the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate or cellulose; a disintegrating agent such as corn starch, potato starch, alginic acid or the like; a lubricant such as stearic acid or magnesium stearate; a sweetening agent such as sucrose, lactose or saccharine; a flavoring agent such as orange, peppermint, oil of wintergreen or cherry.

When the dosage unit form is a capsule, it may contain . in addition to materials of the above type a liquid carrier such as a fatty oil. Various other materials may he present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tahlets or capsules may be cnated with shellac, sugar or both.

A syrup of elixir may contain the active compound, water, alcohol or the like as the carrier, glycerol as : soluhilizer, sucrose as sweetening agent, methyl and propyl parabens as preservatives, a dye and a flavoring such as cherry or orange. . Many of the active substances descrihed above form ] commonly known, pharmaceutically acceptable salts such : as alkali metal and other common basic salts or acid addition salts ete, References to the base substances are therefore intended to include those common salts 10 3 Le _—

known to he substantially equivalent tao the parent compound.

The formulations as described ahove will be administered for a prolonged period, that is, for as long as the potential for onset of anxiety remains or . the symptoms of anxiety continue. Sustained release forms of such formulations which may provide such amounts hiweekly, weekly, monthly and the like may also he employed. A dosing period of at least one to two weeks are required to achieve minimal henefit.

BRIEF DESCRIPTION OF FIGURES

All of the accompanying Figures are graphs or charts of test data obtained as described in the working g

Examples,

FIG. 1 shows the action of diazepam on mouse hehavior in a hlack:white test box, n¥5. S.E.M.s shown less than 12.1% *¥pP<0,001.0=sedation;

FIG. 2 shows the action of captopril on mouse hehavier in the black:white test hox.n =5. S.E.M.s shown less than 12.6%. *pP<0.001;

FIG. 3 shows the action of SQ 29,852 on mouse hehavior in the black:white test box.n =5. S.E.M.s shown less than 12.5%. *P<0.001;

FIG. 4 shows the action of fosinopril on mouse hehavior in the black:white test bhox.n=5.S.E.M.s shown less than 12.9%. [*])P<0.05, ¥P<0.01-P<0.001;

FIG. 5 shows the action of zofenopril on mouse hehavior in the black:white test box.n=5.S.E.M.s shown less than 13.1%. [*]P<0.08, *¥P<0,01-P<0.001; an FIGS. 6 and 7 show anxiolytic potentials of captopril, zofenopril, SQ 29,R52 and fosinopril following oral administration on the mouse. In FIG. 6,

S.E.M.s shown less than 11.3% and in FIG. 7, S.E.M.s shown less than 12.2%. In FIGS. 6 and 7, n=5. *P<0,01-~

P<0,001;

FIg. 8 shows action of epicaptopril on mouse hehavior in the black:white test bhox.n=56. S.E.M.s shown less than 12.2%;

FIGS. 9 and 10 show action of enalapril on mouse behaviar in the mouse black:white text box.n =5, For

FIG, 9, S.E.M.s shown less than 11.6%. *P<0,001, For

FIG. 10, S.E.M.s shown less than 10.3%. *P<0,001; ~ FIGS, 11 and 12 show the action of lisinopril on mouse hehavior in the black:white test hox.n=5.

For FIG. 11, S.E.M.s shown less than 10.9%. *¥P<0,001,

For FIG, 12, S.E.M.s shown less than 11.2%. *P<0.001;

FIGS. 13 and 14 show anxiolytic ‘potential of ACE inhibitors on subchronic treatment assessed in the mouse

Lo hlack:white test hox. n=5, [*]P<0.05. *P<0.01-P<0.01-

P<0.001, S.E.M. shown less than 12.0% in FIG. 13 and less than 11.6% in FIG. 14. 0=Sedation.;

FIGS. 15 and 16 show anxiolytic potential of the " ACE inhibitors on subchronic treatment and effects of " : abrupt withdrawal assessed in the mouse hlack:white test ) 25 hox. Treatment time 6 days, b.d, withdrawn 24 hours. : In FIG. 15, S,E.M.s shown less than 12.0% and in FIG. 16, S.E.M.s shown less than 12.7%. In both FIGS. 15 and : } 16, n=5, ¥*P<0,05-P<0.001(anxiolysis). +P<0.,058-~ ) P<0.001(anxiogenesis); : 30 FIGS. 17, 18, 19, 20 and 21 show anxieolytic action : ; of nicotine during intake, anxiogenesis of withdrawal : Co 12 : i | | en and antagonism of withdrawal anxiogenesis by diazepam. n=5. S.E.M.s shown less than 12.9%. [*]P<0.05, *P<0.01 -P<0.001 (comparison with C) +P<0.001 (reversal withdrawal anxiogenesis); diazepam FIG. 17, captopril

FIG. 18, SQ 29,852 FIG. 19, fosineopril FIG. 20, and zopennpril FIG. 21.

FIGS. 22 and 23 show anxieclytie action of alcohol during intake, anxiogenesis of withdrawal and antagonism of withdrawal anxiogenesis hy SQ 29,852, zofenopril and diazepam.

FIGS. 24 and 25 show anxiolytic action of diazepam given subchronically, anxiogenesis of withdrawal and antagonism of anxiogenesis by the ACE inhibitors;

FIGS. 26, 27 and 28 show ability of ACE inhibitors to antagonize the anxiogenesis caused by FGT7142 in the mouse hlack:white test.

FIG. 29 shows anxiolytie patential of ACE inhibitors seen on the rat social interaction test; and

FIGS. 30, 31 and 32 show anxinlytie potential detected in a marmoset human threat test. .

The following Examples represent preferred embodiments of the present invention.

EXAMPLE 1

A captopril formulation suitahle for oral administration in inhibiting onset of or treating . anxiety is set out helow. 1000 tablets each containing 100 mg of 1-[(2S)-3- mercapto-2-methylpropionyl]-L-preline were praduced from the following ingredients, }

1-[(2S)-3-Mercapto-2-methylpropionyl}- 100 ¢

L-proline (captopril)

Corn starch 50 g

Gelatin 7.5 g

Aviecel (microcrystalline cellulose) 25 g

Magnesium stearate 2.5 g

The captopril and corn starch are admixed with an aqueous solution of the gelatin. The mixture is dried and ground to a fine powder. The Avicel and then the magnesium stearate are admixed with the granulation,

This is then compressed in a tablet to form 1000 tablets ’ each containing 100 mg of active ingredient which is used for inhibiting onset of or treating anxiety.

EXAMPLE 2

By substituting 100 g of 1-(3-mercapto-2-D-methyl propanoyvl)-L-proline for the captopril in Example 1, , 1000 tablets each containing 100 mg of the 1-(3- mercapto-2-D-methylpropanoyl)-L-preoline are produced which is useful in inhihiting onset of or treating anxiety,

EXAMPLE 3 1000 tablets each containing 200 mg of captopril are produced from the following ingredients:

Captopril 200 g

Lactose 100 g

Avicel 150 ¢

Corn starch 50 g

Magnesium stearate 5 g

The captopril, lactose and Avicel are admixed, then blended with the corn starch. Magnesium stearate 4 is added. The dry mixture is compressed in a tablet h ; press to form 1000 505 mg tahlets each containing 200 mg

C14 1 ne of active ingredient. The tahlets are coated with a solution of Methocel E 15 {methyl cellulose) including ’ as a color a lake containing yellow #6. The resulting tablets are useful in inhibiting onset of or treating anxiety. : EXAMPLE 4

Twn piece #1 gelatin capsules each containing 250 mg of captopril are filed with a mixture of the following ingredients: ee —

Captopril 250 mg

Magnesium stearate 7 mg

USP lactose 193 mg ee

The resulting capsules are useful in inhibiting onset of or treating anxiety, , 1s EXAMPLE 5

Co An injectable solution for use in inhibiting onset - of or treating anxiety is produced as follows: oo

Captopril . 500 mg

Methyl paraben 5 mg

Propyl paraben 1 mg

Sodium chloride 25 mg

Water for injection qs. 5 1.

The captopril, preservatives and sodium chloride are dissolved in 3 liters of water for injection and then the volume is brought up to 5 liters. The solution : is filtered through a sterile filter and aseptically filled into presterilized vials which are then closed with presterilized rubber closures. Each vial contains ' 5 ml of solution in a concentration of 100 mg of active a0 ingredient per ml of solutien for injection. ' 15 o~

EXAMPLE 6

Tablets for use in inhibiting onset of or treating anxiety are prepared as described in Example 1 except that N-(l-ethoxycarhonyl-3-phenylpreopyl)L-alanyl-L- proline (enalapril) is used in place of captopril.

EXAMPLE 7 : An injectable for use in inhibiting onset of or treating anxiety is prepared as described in Example 5 except that N-(l-ethoxycarbonyl-3-phenylpropyl)-L- alanyl-L-proline (enalapril) is employed in place of captopril.

EXAMPLE 8

A zofenopril formulation suitable for oral administration in inhihiting onset of or treating anxiety igs set out helow. 1000 tablets each containing 100 mg of zofenopril . are produced from the following ingredients. [(1(S),4(S)-1-3-(henzoylthio)-2- 100 g methyl-1-oxopropyl-4-(phenylthio)-

L-proline (zofenopril)

Corn starch 50 ¢g

Gelatin 7.5 g

Aviecel (microcrystalline cellulose) 25 ¢

Magnesium stearate 2.5 g . ) 25 : The zofenopril and corn starch are admixed with an aqueous solution of the gelatin. The mixture is dried and ground to a fine powder. The Avicel and then the magnesium stearate are admixed with the granulation.

This is then compressed in a tahlet to form 1000 tablets 4 30 each containing 100 mg of active ingredient which is

Cy used for inhibiting onset of or treating anxiety.

EXAMPLE 9

By substituting 100 g of fosinopril for the zofenopril in Example 8, 1000 tablets each containing 100 mg of the fosinopril are produced which is useful in inhihiting onset of or treating anxiety.

EXAMPLE 10 1000 tablets each containing 200 mg of fosinopril are produced from the following ingredients:

Ce —————————ee

Fosinopril 200 g

Lactose 100 g

Avicel 150 ¢

Corn starch 50 ¢

Magnesium stearate 5g

EE

The fosinnpril lactose and Avicel are admixed, then blended with the corn starch. Magnesium stearate is added. The dry mixture is compressed in a tablet press to form 1000 508 mg tahlets each containing 200 mg nf active ingredient, The tablets are coated with a enlution of Methocel E 15 (methyl cellulose) including as a color a lake containing yellow #6. The resulting tahlets are useful in inhibiting onset of or treating anxiety.

EXAMPLE 11

Tahlets for use in inhibiting onset of or treating anxiety are prepared as described in Example 1 except that 1-[N-[hydroxy(4-phenylbutyl)phosphinyl}-L-alanyl-L- proline, disodium salt (prepared as described in U.S, } Pat. No. 4,422,971) is used in place of captopril.

EXAMPLE 12 20 An injectable for use in inhibiting onset of or treating anxiety is prepared as described in Example 5§ except that 1-{N-[hydroxy(4-phenylbutyl )phosphinyvl]-L- alanyl]-L-proline, disodium salt (prepared as descrihed in U.S. Pat, No. 4,432,971) is used in place of captopril. : EXAMPLE 13

A captopril-diltiazem formulation suitable for or an administration in the treatment of anxiety is set out helow. 1000 tablets each containing 100 mg of 1-[(28)-23- mercapto-2-methylpropionyl]-L-proline and 100 mg of diltiazem produced from the following ingredients: ) 1-{(2S)-3-mercapto-2-methylpropionyvl]-L- 100 g rroline (captopril)

Diltiazem 100 g

Corn starch 50 g

Gelatin 7.5 ¢

Avicel (microcrystalline cellulose) © 25 g

Magnesium stearate 2.5 g -_—

The captopril diltiazem and corn starch are admixed with an aqueous solution of the gelatin. The mixture is dried and ground to a fine powder. The

Avicel and then the magnesium stearate are admixed with the granulation. This is then compressed in a tablet tan : 25 form 1000 tablets each containing 200 mg of active ingredients which is used for preventing or treating anxiety. -

EXAMPLE 14

By substituting 100 g of 1-(3-mercapto-2-D- methylpropannoyl)-L-proline for the captopril in Example 13, 1000 tablets each containing 100 mg of the 1- : (3-mercapto-2-D-methylpropanoyl)-L-proline and 100 me

Co 18

Ailtiazem are produced which is useful in preventing or treating anxiety.

EXAMPLE 15 1000 tablets each containing 200 mg of captopril and 200 mg nifedipine are produced from the following ingredients: ee

Captopril 200 g

Nifedipine 200 g

Lactose 100 g

Avicel 150 g

Corn starch 50 g

Magnesium stearate 5 ¢

Co ———

The captopril, nifedipine, lactose and Avicel are ' admixed, then blended with the corn starch. Magnesium stearate is added. The dry mixture is compressed in a tahlet press to form 1000 505 mg tablets each containing 200 mg of each active ingredient. The tahlets are coated with a solution of Methocel E 15 {methyl cellulase) including as a color a lake containing yellow £6, The resulting tablets are useful in preventing or treating anxiety.

EXAMPLE 16

Two piece #1 gelatin capsules each containing 250 mg of enalapril and 150 mg of nitrendipine are filled with a mixture of the following ingredients:

Co ————————————— " Enalapril 250 mg

Nitrendipine 150 mg

Magnesium stearate 7 mg

USP lactose 193 mg an The resulting capsules are useful in preventing or treating anxiety.

EXAMPLE 17

An injectable solution for use in treating or preventing anxiety is produced as follows: -_—

Captopril 500 mg

Diltiazem 300 mg

Methyl parahen 5 g

Propyl paraben 1g

Sodium chloride 25 g

Water for injection qs. 5 L. _—

The captopril, diltiazem, preservatives and sodium chloride are dissolved in 3 liters of water for injection and then the volume is hrought up to 5 liters, - The solution is filtered through a sterile filter and ' aseptically filled into presterilized vials which are then closed with presterilized rubber closures. Each vial contains 5 ml of solution in a concentration of 100 : mg of active ingredient per ml of solution for injection.

EXAMPLE 18

Tablets for use in preventing or treating anxiety : are prepared as described in Example 13 except that N- (1-ethoxycarbonyl-3-phenyvlpropyl)-L-alanyvl-L-proline (enalapril) is used in place of captopril and nifedipine is used in place of diltiazem.

EXAMPLE 19

Tablets for use in treating or preventing anxiety are prepared following the procedure of Example 123 except that zofenopril is employed in place of captopril and nisoldipine is used in place of diltiazem.

EXAMPLE 20

Tahlets for use in treating or preventing anxiety are prepared following the procedure of Example 13 except that fosinopril is employed in place of captopril.

EXAMPLE 21

Tahlets for use in treating or preventing anxiety are prepared following the procedure of Example 13 except that alacepril is employed in place of captopril.

EXAMPLE 22

Tablets for use in treating or preventing anxiety are prepared following the procedure of Example 13 except that (S)-1-[h-amino-2-[[hydroxy-(4- phenylbutyl)phosphinyl]oxy}-1-oxohexyl]-L-proline or lisinopril is employed in place of captopril.

EXAMPLE 23

Captopril, SQ 29,852, fosinopril and zofenapril, and diazepam (as a positive control) were tested for their anti-anxiety effect in the mouse hlack:white test hox employing the following test procedure.

The mouse black:white test hox (test of anxiolytic potential using mouse aversion tn a brightly 1it, white environment)

The studies used naive male alhino BKW mice, 25-30 g. Ten mice were normally housed in each cage with free access to food and water. The mice were kept on a 12 hours light-dark cycle with lights off at 10:00 hour. - For assessment of anxiety responding animals were taken in a dark container from a dark holding room to the dimly lit testing room where the experiments were conducted between 13:00 and 18:00 hours, The apparatus used for the detection of changes in anxiety consisted of an open-topped box (45x27x27 ecm high) having a smaller portion painted black (40% of area) and illuminated under a dim red light (1x60W) and partitioned from the remainder of the hox which was painted white and brightly illuminated with a 60W white light sourece located 17 cm ahove the hox. The floor area was lined into 9 cm squares, Access hetween these areas was enahled hy means of a 7.5x7.5 cm opening : located at floor level in the center of the partition.

Animals that had received drug or vehicle injections were placed individually inte the center of the white area and their behavior ohserved over a 5 minute period hy remote video recording. An increased exploratory activity (rearings, line crnssings) in the brightly-1lit environment was taken as an index of anxiolytic action when a dark environment was simultaneously available,

Anxinlytic activity was also associated with a delayed latency to move from the white to the hlack environment and a reduced % of time spent in the black environment.

Thus, four hehavioral parameters were noted every minute, the number of exploratory rearings in the white and hlack areas, the number of line crossings in the white and black areas, the latency to mnve from the white to the black area, and the % of time spent in the hlack area, Experimenters remained blind to drug treatment throughout, the code only being broken after . : : analysis was complete, = a0 Animals were used on a single occasion only, in : experimental groups of 5 (n=5). Vehicle treated controls were run on each day of testing. Testing was carried out heath after intraperitoneal (i.p.) and oral (p.o.) dosing. Where time courses of drug action were assessed, fresh groups of animals were used on each test occasion. To determine whether drug effects were maintained on subchronic treatment mice were dosed twice daily (b.d.) for 6 days and then tested 45 or 60 minutes after administration of the last dose (see helnw). To determine whether anxiogenesis or other adverse effects developed on withdrawal of subchronic treated twice daily with drug for 6 days then assessed in the hlack:white box 24 hours after drug withdrawal {withdrawal effects, or lack of, were confirmed by testing further groups of mice 48 hours and 96 hours - 15 after withdrawal of drug). a oo wr

Throughout the studies diazepam (Roche) was used : as the positive control. Preparation was in minimum PEG : made up to volume with distilled water (route i.p., pretreatment 60 minutes). Captopril (route i.p., pretreatment 45 minutes), fosinepril (route i.p., pretreatment 60 minuteg), SQ 29,852 (route i.p., pretreatment 60 minutes), enalapril (route p.o., pretreatment 2 hours), lisinopril (route p.o., pretreatment 2 hours) and epicateopril (route p.o., pretreatment 2 hours) were prepared in distilled water,

Zafenopril (route i.p., 60 minutes pretreatment) was prepared in phosphate huffer, pH 6.0). Hydergine {commercial preparation, route p.o., pretreatment 60 minutes) was prepared as Aa suspension in 0.1% carhoxymethylcellulose.

Results

Diazepam caused changes in mouse responding consistent with an anxiolytic potential at doses of 0.125-1 mg/kg i.p. Sedation developed at 10 mg/kg. The anxiolytic action was characterized hy increased i exploratory rearings and line crossings in the white section of the test box, with corresponding reductions in the white, reduced % of time spent in the black, and delayed latency to move from the white to the black compartment (FIG. 1).

Captopril caused a similar change in hehavior a recorded for diazepam but at somewhat higher doses (1-50 mg/kg i.p.) and without sedation at the highest dose (FIG. 2). Similarly, treatment with SQ 29,8582 lead to increased rearings and line crossings in the white, ) normally averse, environment, with corresponding ' reductions in the black, and reduced % of time spent in the black, and delayed latency to move to the black. : The latter parameter changed significantly at doses of 1 and 10 mg/kg, hut other parameters significantly changed in the dose range 0.01-10 mg/kg (FIG. 3). The - characteristic redistribution of exploratory rearings and line crossings was seen at 0.01-1 mg/kg fosinopril. ” These characteristics of anxiolytic potential were also . 25 reflected in reduced % time spent in the hlack environment although, surprisingly, the latency to move from the bhlack to the white environment was never changed by fosinopril treatment (FIG. 4). Also, as the dose of fosinopril was increased so the anxiolytic potential decreased (FIG. 4).

Zofenopril altered exploratory rearings and line

IR

. 24 | os BAD ORIGINA- J

Ne ee TT crossings in a manner characteristics of anxielytic agents at doses of 0.1-1.0 mg/kg (problems in drug preparation precluded the use nf higher doses) but % time in hlack remained unchanged and latency to move from the white to the hlack compartment was delayed only hy the highest dose of 1 mg/kg i.p. zofenopril (FIG. 5).

The anxiolytic actions of captopril, zofenopril,

SQ 29,852 and fosinopril were maintained on oral treatment, Thus, aversion to the white, hrightly lit environment was reduced by captopril (1-100 mg/kg), zofenopril (0.01-1 mg/kg) SQ 29,852 (0.1-10 mg/kg) and fosinopril (0.1-10 mg/kg) given orally (FIGS. 6 and 7).

The failure of zofenopril to influence latency to move from the white to the hlack area on systemic injection was again seen on oral administration (FIG. 7). : In contrast to captopril, SQ 29,852, zofenopril : and fosinopril, epicaptopril failed th modify behavior of mice in the black:white test hox. In this test, and this species, therefore, epicaptopril would appear to he devoid of anxiolytic potential (FIG. 2).

Enalapril was given orally and different groups of mice assessed 1 hour and 2 hours after treatment, A maximal anxiolytic potential was apparent at both times (FIG. 9). The onset of action of enalapril is therefore less than 1 hour, hut maximal activity is maintained at 2 hours and the full dose-response curve for enalapril was therefore carried out at 2 hours. The dose-response cenirve for enalapril was steep, with maximal anxielytic oo action apparent at doses of 0.01-1 mg/kg p.o. (seen as ] ’ 30 increased rears in the white, increased line crossings : in the white, reduced % time iri the hlack and markedly po

- DE mm —————— - = I delayed latencies to move from the white) (FIG. 10).

The threshold dose for enalapril was Approximately 0.005 mg/kg and this was given twice daily in the hahituation test (see later), Using this treatment regime over 7 days an anxiolytic potential was seen in only 1-2 animals after longer treatment. ; In contrast ta enalapril, the onset of action of lisinopril was delaved for 2 hours: at this time the anxiolytic action was maximum. A dose-response analysis : 10 carried out after 2 hour pretreatments showed lisinopril "to be approximately equipotent to enalapril. Again, in subsequent habituation tests the threshold dose was ] i found to he 0,005 mg/kg and, again, there was some accumulation of effect in 1 to 2 animals where an anxiolytic potential developed after 5-7 days of treatment with 0.005 mg/kg p.o. h.d, '

In subsequent studies captopril, SQ 29,852, fosinopril and zofenopril were given bh.,d. for 6 days, and comparisons of anxiolytic potential made with diazepam. The anxiolytic actions of captopril and SQ 29,852 were maintained on sub-chronic treatment whilst those of fosinopril and zofenopril waned (FIGS. 13 and 14), Testing was done 45 minutes after last dose . (captopril) or AO minutes (other ACE inhibitors and diazepam). On withdrawal from subchronic treatment with ) : diazepam (10 mg/kg i.p. bh.d., 6 days) an anxiogenesis ' develops (characterized by increased exploratory rears in the hlack, increased line crossings in the black, increased % time in the hlack, and reduced latency to \ 30 move from the white to the hlack compartment, FIGS. 15 and 168), In contrast, the mice withdrawn from captopril ‘ C26 Ce resumed normal control responding (indistinguishahle from that of vehicle control animals) and the anxieclytic activity of SQ 29,852 was etill present at 24 hours.

This activity of SQ 29,852 waned to normal control values after 48 hours and subsequent -measures showed } there was no rehound anxiogenesis. The anxiolytic activity of fosinopril was lest on long-term treatment and behavior remained at control levels even on drug withdrawal. In contrast, the anxiolytic potential of zofenopril, lost on sub-chronic treatment, was regained on withdrawal, This anxiolytic activity waned over the 96 hours following withdrawal and anxiogenesis was never ohserved (animal hehavior followed for up to 10 days after withdrawal) (FIGS. 15 and 16).

The ahove test results clearly show that ACE inhibitors have anti-anxiety activity and they are ngeful in reducing anxiety. ‘

EXAMPLE 24

Antagonism of Anxiogenesis of Withdrawal from

Subchronic treatment with Nicotine

The same test procedure as deseribed in Example 23 } was used in a dependency study wherein an ACE inhibitor was administered to a test animal to reduce anxiety and thus facilitate withdrawal from nicotine dependency.

Methods

The studies used mice and the hlack:white test hox system described in Example 23. Anxionlysis was measured as reduced aversion for the white, hrightly-1lit compartment (increased rearings and line crossings in the white, with corresponding reductions in the black,

delayed latency to move out of the white and reduced % time spent in the black) and anxingenesis as increased aversion for the white, brightly-1lit area (decreased rearings and line crossings in the white, markedly increased in the hlack, rapid movement out of the white environment and increased time in the Thlack environment).

Nicotine was given at a dose of 0.1 mg/kg i.p. h.d., for 7 davs (a dose carefully selected from extensive preliminary studies) (tested day 5 of treatment) and withdrawn for 24 hours (time of maximum ’ anxiogenesis, again established from extensive preliminary studies). In FIG. 17, diazepam, 1.0 mg/kg i.p. bh.d. (3 doses) was used as the positive control.

In FIG. 18, captopril, 10 mg/kg i.p. b.d. (3 doses) was used as the positive control, In FIG. 19, SQ 29,852 1 mg/kg i.p. b.d. (3 doses) was used as the positive control. In FIG. 20, fosinopril, 0.5 mg/kg i.p. h.d. (3 doses) was used as the positive control. In FIG, 21, zofenopril, 1.0 mg/kg i.p. h.d, (3 doses) was nsed as the positive control.

Results

Mice exhihit an anxiolysis during treatment with nicotine and an anxiogenesis on withdrawal. The ’ ’ 25 anxiogenesis of withdrawal can be antagonized by diazepam. 1.0 mg/kg i.p. given at the time of nicotine } withdrawal and then h.d. (3 doses, last dose given 45

Co minutes hefore test) (FIG. 17). The same profile of behavioral change was seen in experiments using 20 captopril 910 mg/kg given at time of nicotine withdrawal and then h.d. (3 doses, last dose 60 minutes hefore test)), SQ 29,R52 (1 mg/kg given at a time of nicotine withdrawal and then b.d. (3 doses, last dose 60 minutes before test)), fosinopril (0.5 mg/kg given at time of nicotine withdrawal and then b.d. (3 doses, last dose 60 minutes hefore test)) and zofenopril (1 mg/kg given at time of nicotine withdrawal and then b.d. (3 doses, last dose B60 minutes before test)). Thus, in each experiment anxiolysis was seen during treatment with nicotine, anxiogenesis followed withdrawal from the subchronic nicotine treatment, and the withdrawal anxiogenesis was antagonized by captopril (10 mg/kg h.d,), SQ 29,R52 1,0 mg/kg h.d.), fosinopril (0.5 mg/kg h.d.) and zofenopril 1.0 mg/kg hb.d.): in each situation the anxiogenegsis nf withdrawal was converted to an anxinlysis. This marked change in response from anxingenesis to anxiolysis was also seen for diazepam (see FIGS. 17, 18, 19, 20 and 21). n=5 in all Figures.

In FIG. 17, S.E.M.'s shown less than 12.9%, (¥]P<0.05, *P<0,.01-P<0.001 (comparison with € (Contrel)) +P<0,001 (reversal withdrawal anxiogenesis),

In FIG. 18, S.E.M.’s shown less than 12.2%. ¥P<0,001 (comparison with C) +P<0.001 (reversal withdrawal anxiogenesis).

In FIG. 19, S.E.M.'s shown less than 11.7%. ¥*P<0,001 (comparison with C}. +P<0.001 (reversal withdrawal),

In FIG, 20, S,.E.M.’s shown leas than 12.8%. © %¥P<0.01-P<0,001 (comparison with €C) +P<0.001 (reversal withdrawal anxiogenesiaz), a0 In FIG. 21, S.E.M.’s shown less than 12.2%, *P<0,001 (comparison with C) +P<0.,001 (reversal withdrawal anxiogenesis). 4 EXAMPLE 25 . Antagonism of Anxiogenesis of Withdrawal from

Suhchronic Treatment with Alcohol Methods

The same test procedure as described in Example 23 was used in a dependency study wherein an ACE inhibitor was administered to a test animal to reduce anxiety and thus facilitate withdrawal from aleohol dependency,

The studies used mice and the hlack:white test hox ; 10 system described in Example 23, Anxinlysis was measured as reduced aversion for the white, brightly-1lit ; Co compartment (increased rearings and line crossings in - the white, with corresponding reductions in the black, . delayed latency to move aut of the white and reduced % time spent in the black) and anxiogenesis as increased aversion for the white, brightly lit area (decreased rearings and line crossings in the white, markedly incresed in the black, rapid movement nut of the white environment and increased time in the hlack environment),

Alcohol was presented in the drinking water (no choice) at a concentration of 8% w/v for 7 days (tested " day 5 of treatment, also 24 hours after withdrawal),

N Extensive studies showed this approach to he acceptahle (free choice situation and mice take 2% w/v alcohol), to cause anxiolysis during treatment and anxiogenesis within 24 hours of withdrawal, Antagonists were given i at time of withdrawal and then h.d. (3 doses), : Captopril (10 mg/kg i.p. bh.d.), SQ 29,852 (1.0 mg/kg a0 i.p. b.d.), fosinopril (0.5 mg/kg i.p. h.d.), zafenopril

(1.0 mg/kg i.p. h.d.) and diazepam (1.0 mg/kg i.p. b.d.) were used as positive controls.

Results

Mire exhibited anxielysis during intake of alcohol {measured on day 5) and a marked anxiogenesis 24 hours after withdrawal of 7 davs alcohol intake. The anxiogenesis nf alcohol withdrawal was not antagonized by captopril (10 mg/kg i.p- h.d.) or hy fosinepril (0.5 mg/kg i.p. b.d.) but was antagonized hy SQ 29,852 1.0 mg/kg i.p. b.d.), zofenopril (1.0 mg/kg i.p. b.d.) and hy diazepam 1.0 mg/kg i.p. b.d.) (FIGS. 22 and 23). In

FIGS. 22 and 23 n=5. In FIG. 22 S.E.M.’S shown less than 11.6%. ¥P<0.001 (comparison with Cc) +P<0.001-

P<0.001 (reversal withdrawal). In FIG. 23, S.E.M.’'s shown less than 12%. ¥P<0,005-P<0,001 (comparison with ©) +P<0.,01-P<0.001 (reversal withdrawal).

EXAMPLE 26

Antagonism of Anxiongenesis of Withdrawal from

Subechronic Treatment with Diazepam Method

The same test procedure as descrihed in Example 23 was used in a dependency study wherein an ACE inhibitor was administered to a test animal to reduce anxiety and thus facilitate withdrawal from diazepam dependency.

The studies used mice and the hlack:white test box system descrihed in Example 23. Anxiolysis was measured as reduced aversion for the white, brightly-1lit compartment (increased rearings and line crossings” in the white, with corresponding reductions in the black, delayed latency to move out of the white and reduced % time spent in the black) and anxiogenesis as increased

. TTT Ee = aversion for the white, brightly it area {decreased rearings and line crossings in the white, markedly increased in the black, rapid movement nut of the white ) environment and increased time in the black environment),

Diazepam was administered at a dose of 10 mg/kg i.p. b.d. for 7 days. Tolerance to sedation developed within 3 days and a full anxiolytic response was measured by day 5 (FIGS. 24 and 25). Within 8 hours of ceasing treatment with diazepam an anxiogenesis was developed, and this was marked 24 hours after withdrawal (FIGS. 24 and 25). The effects of potential antagonists were assessed 24 hours after withdrawal of diazepam treatment, Antagonists were given at time of withdrawal and then b.,d. (3 doses). Captopril (10 mg/kg i.p. h.d.), SQ 29,852 (1 mg/kg i.p. h.d.), fosinopril (0.5 mg/kg i.p. b.d.), zofenopril (1 mg/kg i.p. bh,d,) and diazepam (10 mg/kg i.p. h.d.) were used as positive controls.

Results

The anxiogenesis of withdrawal from diazepam was antagonized by captopril (10 mg/kg i.p. bh.d.), SQ 29,852 (1.0 mg/kg i.p. b.d.), fosinopril (0.5 mg/kg i.p. bh.d.) " and zofenopril (1.0 mg/kg i.p. b.d.). Indeed, in each . | 25 situation the anxiogenesis was converted to a full anxiolysis (FIGS. 24 and 25). In FIGS. 24 and 25, n=5

S.E.M.’s shown are less than 9.6% (FIG, 24) and less : than 10.1% (FIG. 25) *P<0.001 (comparison with C) +P<0.,00]1 (reversal withdrawal). * ' 32: Co Ce : )

EXAMPLE 27

The same test procedure as described in Example 23 was used in a dependency study wherein an ACE inhibitor was administered to a test animal to reduce anxiety and thus facilitate withdrawal from f-carbhnline dependency.

Vehicle treated controls were run on each day of testing. All agents were given hy the intraperitoneal routes using 20-60 minutes pretreatment times as defined in the figure legends. Anxiogenesis was induced by the f-carboline FG7142 at 1 mg/kg i.p. 30 minutes. The following agents were administered as positive controls and to determine their potential to antagonized the anxiogenesis caused by FG7142:diazepam, captopril, epicaptopril, SQ 29,852, fosinopril and zofenopril,

Lo 15 Results oo

Treatment with y mg/kg i.p. 20 minutes FGT142 caused an anxiogenesis as ~haracterized in the hlack:white test hox as decreased rearings in the white with increases in the hlack (FIG. 26), decreased line crossings in the white with rorresponding increases in the black (FIG. 27), increased % time in the black (FIG. 28) and reduced latency to move from the white to the black compartment of the tect hox (FIG. 28).

These anxiogenic effects of FGT142 were . 25 antagonized by diazepam (1 mg/kg i.p. 30 minutes), : captopril (10 mg/kg i.p. 45 minutes), SQ 29,852 (1 mg/kg i.p. 60 minutes), fosinopril (0.5 mg/kg i.p. 60 minutes), enalapril (0.1 mg/kg i.p. 60 minutes) and lisinopril (0.1 mg/kg i.p. 60 minutes and zofenopril (1 * 30 mg/kg i.p. 60 minutes (FIGS. 26, 27 and 28). .

Epicaptopril (10 mg/kg i.p. 60 minutes) and Control

(vehicle 1 ml/100 g i.p. 30 minutes) failed to influence : the anxiogenesis caused by FG7142 (FIGS. 26, 27 and 28%,

The experiments carried out and shown on FIGS. 268, 27 and 28 were carried out on 3 separate occasions and therefore 3 sets of comparative control (vehicle) and

FG7142 data are given.

Conclusion

The ACE inhibitors, like diazepam, are ahle to antagonize the anxingenesis caused hy the B-carboline

FG7142, Epicaptopril was without effect to confirm the specificity of the ACE inhibitor response, n=5 in each of FIGS. 26, 27 and 28, S.E.M.’s shown are less than 7.6-11.8% (FIG. 26), less than 10.2-12.1% (FIG. 27) and less than 9.4-12.7% (FIG. 28), °P<0.1-P<0.001 anxiogenesis), *P<0,001 (anxiolysis), +P<0.001 {antagonism of FG7142), ‘

EXAMPLE 28

Test of Anxiolytic Potential Using the Rat Social

Interaction Test Methods

Male Sprague-Dawley rats, 225-275 g, were normally ’ housed in groups of 5 and kept on a 12 hour light/dark cycle with lights on at 08.00 hour. Tests were conducted between 13,00-1R.00 hours in an illuminated ; room. The apparatus used for the detection of changes in rat social interaction and exploratory behavior consisted of an opaque white Perspex open-topped box (45x32 ecm and 20 cm high) with 15 x 16 em areas marked on the floor. Two naive rats, from separate housing - cages, were placed inte the hox (with a 100 W bright white illumination 17 ecm above) and their hehavior ahserved over a 10 minute perind by remote video recording. Two hehaviors were noted, (a) social interaction hetween the animals was determined by timing (sec), sniffing of partner, crawling under or climhing aver partner, genital investigation of partner, following partner and (b) exploratory locomotion was measured as the number of crossings of the lines marked on the floor of the test hox. Values for time spent in sncial interaction and moving around the nhservation cage were determined for individual animals, Naive animals were used in drug treated pairs in treatment groups of 6. As with the mouse studies, data obtained ) wags analyzed using single-factor Analysis of Variance followed hy Dunnett's test. Drugs were prepared as : described for the mouse experiments. Pretreatment was for 45-60 minutes,

Results

The anxiolytic action of diazepam was seen in the rat as increased social interaction (0.125-1 mg/kg i.p.). At doses above 1 mg/kg (2, 5 and 10 mg / kg i.p.) locomotor activity was suppressed. The marked increase in rat social interaction seen after diazepam treatment was also seen after administration of captopril (1-50 } mg/kg i.p.) and SQ 29,852 (0.01-10 mg/kg i.p.). The activity of both of these compounds was maintained on oral administration (1-50 mg/kg p.o. captopril, 0.1-10 mg/kg p.o. SQ 29,852). Increased social interaction could also he seen following treatment with fosincpril (0.01 mg/kg, hut loss of effect at 1 mg/kg) or ’ zofenopril (0.1 mg/kg) although the intensity of 3b oo ~ .

response was less marked than for diazepam, captopril or

SQ 29,252 (FIG. 29). n=5 S.E.M.’s shown less than 13,6% {*]pP<n.05. *P<0.,01-P<0.001, .

EXAMPLE 29

Assessment of Anxiolytiec Potential Using the

Marmoset Human Threat Test

Male and female lahoratory bred common marmosets {(Callithrix jacchus) weighing hetween 2350-400 g, were housed in single sex pairs. Holding rooms were maintained at 25% + 1° C. at a humidity of 55% and on a . 12 hour light/dark cycle (with simulated dawn and : . twilight periods, red illumination) with lights on at 07.00 hours. Tests were conducted between 13.30-15.30 hours in the normal holding room (to avoid unwanted disruption of hehavioer by movement to a novel room or cage). The holding cages measured 75 com high, 50 cm wide and 60 cm deep. A hehavioral change characterized hy retreat from, and posturing towards a human threat (a behavior sensitive to known anxiolytic agents) was initiated by a human observer standing in close proximity in front of the holding cage. Changed hehavior was recorded over a 2 minute period by the ohserver, The behavioral measures selected for the present study were, (a) the % of time spent on the cage front in direct confrontation with the human threat and (bh) the number of hody postures, primarily shown as raising of the tail to expose the genital region with varying degrees of hody piloerection, anal scent marking and slit stare with flattened ear tufts, 20 12 marmosets were used at 7 day intervals : throughout the study and were subject to a random cross- 36. Co : ce 9 | a | | — Co over of treatments. They were separated according to their hasal anxiety responding. Statistical analysis utilized a one-way analysis of variance followed hy

Dunnett’s test, Drugs were prepared for the marmoset studies as described for the mouse (except that normal saline was used instead of distilled water). In the marmoset the route of administration was always suhecutaneous (s.c.).

Results

Reduced anxiety is exhibited hy marmosets as reduced numher of postures exhibited in a fixed period

N and increased time on the cage front in direct confrontation with the human threat, This is clearly seen for diazepam in FIG. 30 with =a dose-dependent effect hetween 10 and 25 ug/kg s.c. . The first group of marmosets used to assess the potential anxiolytic actions of the ACE inhibitors had relatively low haseline anxiety responses. Nevertheless, the . | anxiolytic actions of diazepam (0.25 mg/kg s.c.}, : | 20 . captopril (1.0 mg/kg s.c.) and SQ 29,852 (0.1 mg/kg s.c.) were clearly demonstrated as reduced posturing ) and, for diazepam and captopril, as increased time spent an the cage front (FIG. 31).

In a second group of marmosets the hasal anxiety : 25 responding was higher (FIG. a2). In these animals diazepam (0.1 mg/kg s.c.), captopril (1.0 mg/kg s.c.) and zofenopril (0.1 mg/kg s.c.) were all shown to exert marked anxiolytic action, seen both as reduced posturing and (with the exception of zofenopril) increased time on : 30 the cage front, Of these compounds zofenopril was the a7 5 i - Ca ee ——————————— i } least effective anxinlytic agent, However, in contrast to diazepam and the ACE inhibitors tested, epicatopril was shown in the marmoset, as in the mouse, to he devoid of anxiolytic potential (FIG, 32).

In FIGS. 30, 31 and 22, n=4. S.E.M.’s shown less than 10.9% *P<0.01-P<0.001 in FIG. 30, less than 11.7% ¥*P<0.001 in FIG. 31 and less than 10.3% ¥pP<0.,05, ¥*¥P<0.,001 in FIG, 32. i } : KE]

Claims (1)

1. A method for inhibiting onset of or treating anxiety in Aa mammalian specie, which comprises administering to a mammalian specie in need of such treatment an anxiolytic effective amount of an angiotensin converting enzyme inhibitor, alone or in combination with a calcium channel blocker.

2. The method as defined in claim 1 wherein the angiotensin converting enzyme inhibitor is a phosphonate suhstituted amino or imino acid or salt thereof, =a proline derivative, a substituted proline derivative, Aa carhoxyalkyl dipeptide derivative, a phosphinylalkanoyl proline derivative or a phosphonamidate derivative.

7. The method as defined in claim 1 wherein said angintensin converting enzyme inhibitor is a proline derivative or a substituted proline derivative.

4. The method as defined in claim 1 wherein said angiotensin converting enzyme inhihiter is a carboxyalkyl dipeptide derivative. :

5. The method as defined in claim 1 wherein =aid angiotensin ronverting enzyme inhibitor is A phosphinylalkaneyl proline derivative, a phosphoramidate derivative, or a phosphonate substituted amino or imine arid or salt thereof.

6. The method as defined in claim 1 wherein said angiotensin converting enzyme inhibitor js captopril.

7. The method as defined in claim 1 wherein said angiotensin converting enzyme inhibitor is enalapril.

: 8. The method as defined in claim 1 wherein said a 30 angintensin converting enzyme inhibitor is lisinopril.

: 9. The method as defined in claim 1 wherein said angiotensin converting enzyme inhibitor is zofenopril.

10. The method as defined in claim 1 wherein said angiotensin converting enzyme inhibitor is fosinopril.

11. The method as defined in claim 1 wherein said angiotensin converting enzyme inhihiteor is (S)-1-[6- amino-2-[[hydroxy(4-phenylbutyl)phosphinyl Joxy}-1- oxohexyll}-L-proline.

12. The method as defined in claim 1 wherein said angiotensin converting enzyme inhihitar is administered in single or divided doses of from about 0.1 to about 500 mg/one to four times daily and where present the calcium channel blocker is administered in single or divided doses of from about 1 to about 300 mg/1 to 4 times daily.

13. The method as defined in claim 1 wherein the angiotensin converting enzyme inhibitor is administered with a calcium channel blacker. ‘ } 14. The method as defined in claim 13 wherein the y calcium channel hlocker is diltiazem, a 4-phenyl-1,4- : 20 dihydropyridine or verapamil. : 15. The method as defined in claim 14 wherein the 4-phenyl-1,4-dihydropyridine is nifedipine or ! nitrendipine. . " 16. The method as defined in claim 13 wherein the . 25 angiotensin converting enzyme inhibitor is employed in a weight ratio to the calcium channel blocker of within the range of from ahout 0.1:1 to ahout 10:1.

17. A method for inhibiting onset of or treating anxiety in a mammalian specie associated with withdrawal from drugs of dependency and/or addiction, which comprises administering to a mammalian specie in need of such treatment an anxiolytic effective amount of an angiotensin converting enzyme inhibitor, alone or in combination with a calcium channel hlocker.

18. The method as defined in claim 17 for reducing anxiety associated with nicotine withdrawal, alcohol withdrawal, diazepam withdrawal or cocaine withdrawal.

19. The method as defined in claim 17 for reducing anxiety and thus facilitating withdrawal from nicotine, alcohol, diazepam or cocaine.

20. The method as defined in claim 17 for reducing anxiety assnciated with nicotine withdrawal wherein the angiotensin converting enzyme inhibitor administered is captopril, SQ 29,852, fosinopril, zofenopril, enalapril or lisinopril.

21. The method as defined in claim 17 for reducing anxiety associated with alcohol withdrawal wherein the angiotensin converting enzyme inhibitor administered is SQ 29,852, zofenopril, enalapril or lisinopril.

22. The method as defined in claim 17 for reducing anxiety associated with diazepam withdrawal wherein the angiotensin converting enzyme inhihitor administered is captopril, SQ 29,852, fosinopril, znfenopril, enalapril or lisinopril.

23. A method for inhibiting onset of or treating anxiety in a mammalian specie, which comprises administering to a mammalian specie in need of such treatment an anxiolytic effective amount of an a0 angiotensin converting enzyme inhibitor.

24. The method as defined in claim 23 wherein the

= ——— eee SE , angiotensin converting enzyme inhibitor is a phosphonate substituted amino or imino acid or salt thereof, a proline derivative, a substituted proline derivative, a carboxyalkyl dipeptide derivative, a rhosphinylalkanayl proline derivative or a phosphonamidate derivative.

25. The method as defined in claim 223 wherein said angiotensin converting enzyme inhibitor is captopril. ‘ 26. The method as defined in claim 23 wherein said angiotensin converting enzyme inhibitor is : ’ enalapril. . 27. The method as defined in claim 23 wherein said angiotensin converting enzyme inhibitor is lisinopril.

28. The method as defined in claim 23 wherein said angiotensin converting enzyme inhibitor is : zoafinopril. :

29. The method as defined in claim 23 wherein said angiotensin converting enzyme inhibitor is fosinopril,

30. The method as defined in claim 23 wherein said angiotensin converting enzyme inhibitor is (S)-1-(6-amino-2-[ [hydroxy (4-phenylbutyl )phosphinyl Joxy] -l1-oxohexyl]-L-proline.

31. A method for inhibiting onset of or treating anxiety in a mammalian specie associated with withdrawal from drugs of dependency and/or addiction, which comprises administering to a mammalian specie in need : of such treatment an anxiolytic effective amount of an ; 30 angiotensin converting enzyme inhibitor. . 32. The method as defined in claim 21 for i} 42 i ——

reducing anxiety associated with nicotine withdrawal, alcohol withdrawal, diazepam withdrawal or cocaine withdrawal.

13. The method as defined in claim 31 for reducing anxiety and thus facilitating withdrawal from nicotine, alcohol, diazepam or cocaine.

34. The method as defined in claim 31 for reducing anxiety associated with nicotine withdrawal wherein the angiotensin converting enzyme inhibitor administered is captopril, SQ 29,8582, fosinopril, zofenopril, enalapril or lisinopril. as, The method as defined in claim 31 for reducing anxiety associated with alcohol withdrawal wherein the angiotensin converting enzyme inhibitor administered is SQ 29,852, zofenopril, enalapril or lisinopril. «

36. The method as defined in claim 21 for reducing anxiety associated with diazepam withdrawal wherein the angiotensin converting enzyme inhibitor administered is captopril, SQ 29,852, fosinopril, zafenopril, enalapril or lisinopril. - Inventors - ABRAHAM SUDILOVSKY ZOLA P. HOROVITZ