`
`
`
`
`
`
`
`
`US005447952A
`[19J
`5,447,952
`
`[11]Patent Number:
`
`United States Patent
`[45]Date of Patent:
`Sep. 5, 1995
`Wulfert et al.
`
`macological Profile In Animals" [Published erratum
`
`
`
`[54]TREATMENT OF ANXIETY WITH IBE AID
`
`
`appears in Eur J Pharmacol 1993 Jan. 19; 230(3):389].
`OF
`(S)-(-)-a-ETHYL-2-0X0-
`1-PYR
`
`
`Loscher et al., Eur. J. Pharmacol (Netherlands), Mar. 2,
`1993, vol. 232, No. 2�3, pp. 147-158.
`ROLIDINEACETAMIDE
`
`
`"Profile of UCB L059, A Novel Anticonvulsant Drug,
`
`[75]Inventors: Ernst Wulfert; Jean Gobert,'both of
`
`
`
`
`In Models of Partial and Generalized Epilepsy In Mice
`
`Brussels; Alma Gower,
`and Rats" (1993).
`
`
`Braine-1' Alleud; Eric Cossement,
`Wulfert et al., Psychopharmacol Bull (United States),
`
`
`
`Brussels, all of Belgium
`1989, vol. 25, No. 3, pp. 498-502.
`"Facilitation of Calcium-Dependent Cholinergic Func
`
`
`
`[73]Assignee:
`
`U C B S.A., Brussels, Belgium
`
`tion By UCB L059, a New 'Second Generation' Noo
`[21]Appl. No.: 309,186
`
`tropic Agent" (1992).
`File et al., N. Affect Disord (Netherlands), Dec. 1979,
`
`[22]Filed:Sep. 20, 1994
`
`
`vol. 1, No. 4, pp. 227-235 "Evidence That Piracetam
`
`Has An anxiolytic Action".
`[30] Foreign Application Priority Data
`
`
`
`Henley, III
`Sep. 24, 1993 [GB] United
`
`Kingdom ................. 9319732
`Primary Examiner-Raymond
`R. A. Jarvis
`
`Assistant Examiner-William
`[51] Int. Q.6 ..............................................
`A61K 31/40
`
`
`
`Attorney, Agent, or Firm-Wenderoth, Lind & Ponack
`514/424
`[52]U.S. a .
`[57]
`
`[58]Field of Search ......................................... 514/424
`ABSTRACT
`
`
`
`A method is disclosed for the treatment of anxiety in a
`References
`Cited
`
`
`
`patient in need thereof, by administering to said patient
`U.S. PATENT DOCUMENTS
`
`
`an effective amount of (S)-(-)-a-ethyl-2-oxo-1-pyr
`
`rolidineacetamide of the formula
`
`4,696,943 9/1987 Gobert et al . .
`
`
`4,837,223 6/1989 Gobert et al . .
`4,943,639 7/1990 Gobert et al . .
`
`
`FOREIGN PATENT DOCUMENTS
`
`
`
`0162036 11/1985 European Pat. Off . .
`OTHER PUBLICATIONS
`
`
`Gower et al., Eur. J.Pharmacol. (Netherlands), No. 10,
`1992, vol. 222, No. 2-3, pp. 193-203.
`
`"UCB L059, A Novel Anti-Convulsant Drug: Phar-
`
`6 Oaims, No Drawings
`
`(I)
`
`....................................................
`
`[56]
`
`ARGENTUM Exhibit 1096
` Argentum Pharmaceuticals LLC v. Research Corporation Technologies, Inc.
`IPR2016-00204
`
`Page 00001
`
`
`
`1
`
`5,447,952
`
`TREATMENT OF ANXIETY WITH THE AID OF
`(S)-(—)-a-ETHYL-2-OXO-l-PYRROLIDINEACETA
`MIDE
`
`5
`
`The present invention relates to the use of (S)-(—)-a
`ethyl-Z-oxo-l-pyrrolidineacetamide for the treatment of
`anxiety.
`_
`The use of levorotatory (S)-a-ethyl-2-oxo-l-pyr
`rolidineacetamide as a protective agent for the treat
`ment and the prevention of hypoxic and ischemic type
`aggressions of the central nervous system is disclosed in
`US. Pat. Nos. 4,696,943, 4,837,223 and 4,943,639, all
`three assigned to the assignee of the present invention.
`This compound is also effective in the treatment of
`epilepsy, a therapeutic indication for which it has been
`demonstrated that its dextrorotatory enantiomer
`(R)-(+)-a-ethyl-2-oxo-l-pyrrolidineacetamide
`com
`pletely lacks activity (A. J. GOWER et al., Eur. J.
`Pharmacol., 222, (1992), 193-203). No disclosure of the
`use
`of
`levorotatory
`(S)-a-ethyl-2-oxo-l-pyr
`rolidineacetamide for the treatment of anxiety is known,
`however.
`In the above-mentioned U.S. Pat. Nos. 4,696,943,
`4,837,223 and 4,943,639, processes for preparing (S)
`(—)-a-ethy1-2-oxo-l-pyrrolidineacetamide are also de
`scribed. These processes involve the synthesis of a start
`ing reactant obtained by resolution of the correspond
`ing racemate. In British Patent No. 2,225,322, also as
`signed to the assignee of the present invention, a process
`for the preparation of this compound is described,
`which offers the advantage of using a naturally occur
`ring amino acid with the desired stereochemical config
`uration as the starting material. This process thus avoids
`tedious separation of the enantiomers.
`35
`Continuing research work in this ?eld, we have now
`found that (S)-(—)-a-ethyl-2-oxo-l-pyrrolidineaceta
`mide possesses anxiolytic properties of considerable
`therapeutic interest.
`Moreover, this anxiolytic activity could not be found
`for the dextrorotatory enantiomer, (R)-(+)-a-ethyl-2
`oxo-l-pyrrolidineacetamide.
`Thus, the present invention relates to a new and use
`ful method for the treatment of anxiety in a patient in
`need thereof, which comprises administering to said
`patient an effective amount of (S)-(—)-a-ethyl-2-oxo
`lpyrrolidineacetamide of the formula
`
`45
`
`[3. N
`CHQCHZ '%\
`
`H CONH;
`
`O
`
`(I)
`
`55
`
`The anxiolytic activity of (S)-(—)-a-ethyl-2-oxo-l
`pyrrolidineacetamide is particularly pronounced in
`pharmacological situations in which the initial emo
`tional state has been exacerbated, for example by choos
`ing particularly sensitive or aged animals, or by using
`60
`experimental conditions that involve the anticipation of
`an aversive stimulus.
`The relation between anxiolytic activity of the com
`pound of the formula I and the intensity of the initial
`emotional state, suggests that the therapeutic applica
`tion of this compound will preferably be directed
`towards the treatment of pathological anxiety states.
`This selectivity clearly distinguishes the compound
`
`65
`
`2
`used according to the invention from known anxiolytic
`medicaments of the benzodiazepine type, and offers an
`important advantage over these other classes of prod
`ucts, which act without distinguishing between an anx
`ious animal and a normal animal. In the latter case, the
`anxiolytic activity is accompanied by disinhibition of
`the normal general behavior, thereby inducing an inade
`quate adaptative response in healthy subjects, that
`should be avoided. In this context, (S)-(—)-a-ethyl-2
`oxo-l-pyrrolidineacetamide facilitates recovery and
`return to a more normal situation from pathological
`anxiety states caused by disorders of a neuroendocrine
`origin produced by stress in aged subjects, as opposed
`to the benzodiazepines which do not favor this recov
`ery. Contrary to benzodiazepines, for which amnesia
`and neuromotor disturbances such as ataxia, muscular
`relaxation and sedation are well known and undesirable
`side-effects (J. H. WOODS et al., Pharmacol. Rev.,39
`(1987), 251-419), therapeutic doses of (S)-(—)-a-ethyl
`2-oxo-l-pyrrolidineacetamide do not produce the
`slightest harmful effect on memory and do not cause
`awkward neuromotor effects. Indeed, there is a large
`safety margin between the anxiolytic doses and the
`neurotoxic or sedative doses in animals (A. J. GOWER
`et al., loc.cit.).
`(S)-(—)-a-ethyl-2-oxo-1-pyr
`Furthermore,
`rolidineacetamide also reduces the anxiety induced by
`the withdrawal from chronic administration of ben
`zodiazepines.
`The result of this unexpected range of properties is
`that
`the
`use
`of
`(S)-(-)-a-ethyl-2-oxo-l-pyr
`rolidineacetamide is particularly suited in the treatment
`of anxiety states, such as general anxiety, panic, agora
`phobia, social phobia, obsessive-compulsive disorders,
`anxiety due to acute posttraumatic stress, feelings of
`impending danger, absence of tonus, fear and tension,
`which are sometimes accompanied by physiological
`symptoms such as tachycardia, dyspnea, sweating,
`trembling, weakness and fatigue (International Statisti
`cal Classi?cation of Diseases and Related Health Prob
`lems-Tenth Revision, Vol. 1, World Health Organiza
`tion, Geneva, 1992).
`The present invention requires administration of a
`dose of the compound of the formula I effective to treat
`anxiety. The dose required according to the present
`invention should be suf?ciently high to permit the relief
`of anxiety. Pharmaceutical compositions containing the
`compound of the formula I may be administered, for
`example, orally or parenterally, i.e. intravenously, intra
`muscularly and subcutaneously.
`The pharmaceutical compositions which can be used
`for oral administration may be solid or liquid, for exam
`ple, in the form of tablets, pills, dragees, gelatine cap
`sules, solutions, syrups, and the like.
`For this purpose, the active compound can be mixed
`with an inert diluent or a pharmaceutically acceptable
`non-toxic carrier, such as for example starch or lactose.
`Optionally, these pharmaceutical compositions can also
`contain a binder such as microcrystalline cellulose, gum
`tragacanth or gelatine, a disintegrating agent such as
`alginic acid, a lubricant such as magnesium stearate, a
`glidant such as colloidal silicon dioxide, a sweetening
`agent such as sucrose or saccharin, a coloring agent or
`a flavoring agent such as peppermint or methyl salicyl
`ate. These compositions also include compositions that
`allow controlled release of the active substance.
`
`Page 00002
`
`
`
`5,447,952
`3
`4
`The pharmaceutical compositions which can be used
`tests performed on animals, using standard tests, which
`for parenteral administration are the pharmaceutical
`are recognized for their capacity to demonstrate the
`anxiolytic activity of new compounds.
`forms known for this mode of administration, for exam
`Anxiety can exist in different physiological and path
`ple, aqueous or oily solutions or suspensions generally
`contained in ampules, disposable syringes, vials made of
`ological forms. The heterogeneous nature of anxiety
`disorders is clinically accepted in the same way that it is
`glass or plastic, or infusion containers.
`accepted that various anxiety tests, performed on ani
`Besides the active compound, these solutions or sus
`pensions can optionally also contain a sterile diluent
`mals and based on behavioral changes are sensitive to
`different types of anxiety (S. E. FILE, “Animal models
`such as water for injection, physiologic saline solution,
`oils, polyethylene glycols, glycerine, propylene glycol
`of anxiety” in “Biological Psychiatry”, Vol. 2, G. Raca
`gui et al. (eds), Excerpta Medica, Amsterdam, (1991), p.
`or other synthetic solvents, antibacterial agents such as
`596-599).
`benzyl alcohol, antioxidants such as ascorbic acid or
`sodium bisul?te, chelating agents such as ethylenedi
`However, to be sure that a given behavioral test actu
`ally permits to detect anxiolytic activity, the latter must
`aminetetraacetic acid, buffers such as acetates, citrates
`or phosphates and agents for adjusting the osmolality
`also be con?rmed by clinical tests. A test in man has
`enabled to con?rm the therapeutic activity of (S)- (—)~
`such as sodium chloride or dextrose.
`These pharmaceutical forms can be prepared accord
`a-ethyl-2-oxo-l-pyrrolidineacetamide for the treatment
`of various types of anxiety.
`ing to conventional methods used by pharmacists.
`1. Hole-board Test (Exploratory Activity)
`The percentage of active compound in the pharma
`The hole-board test offers a simple method for study
`ceutical compositions can vary within very wide con
`ing the behavior of a rodent and for measuring the
`centration limits and depends on a variety of factors
`such as the sex, age, weight and medical condition of
`response of an animal to an unfamiliar environment.
`This reaction, known as the “exploratory behavior”,
`the patient, as well as the method of administration.
`Thus the amount of active compound in compositions
`relates both to the curiosity of the animal and to its
`natural ?ght-or-?ight reaction and is in?uenced by psy
`intended for oral administration, is at least 0.5% by
`25
`choactive drugs. In particular, this method has proved
`weight, and can reach 80% by weight with respect to
`useful in predicting the potential anxiolytic activity of
`the weight of the composition. In the preferred oral
`compositions, the dosage unit is between 50 mg and
`benzodiazepines (N. A. NOLAN and M. W. PARKES,
`Psychopharmacologia (Berl) 29, (1973), 277-288).
`1000 mg of active compound.
`In compositions intended for parenteral administra
`Using the methodology of J. R. BOISSIER and P.
`SIMON (Arch. Int. Pharmacodyn. 147, (1964),
`tion, the amount of active compound present is at least
`0.5% by weight and can reach 33% by weight of the
`372-387), the test consists of placing a mouse in the
`composition. In the preferred parenteral compositions,
`center of a square board, perforated by 16 regularly
`spaced holes, and counting the number of times the
`the dosage unit is between 1 mg and 200 mg of active
`compound.
`animal dips its head into a hole during a ?ve-minute
`exploration period. Three genetically distinct strains of
`As regards the daily dosage, this can vary within a
`wide range of dosage units and is preferably between 5
`mice are used in this test; a normal strain of NMRI mice
`and 70 mg/kg. An average dose of 250 mg, twice a day,
`and two strains which are emotionally more sensitive,
`has proved to be effective in relief of anxiety in man. It
`one being prone to audiogenic seizure (Dilute Brown
`Agouti-derived (DBA-derived)), and the other having
`is to be understood, however, that the speci?c doses can
`the normal ?ght-or-flight reaction blocked by fear (C57
`be adapted for particular cases, depending on individual
`need, at the discretion of the responsible physician. The
`Black Mice strain).
`above-mentioned dosages are given exemplary only and
`of (S)-(—)-a-ethyl-2-oxo-l-pyr
`The activities
`rolidineacetamide (compound I), of its dextrorotatory
`by no means limit the scope of practice of the invention.
`As non-limiting examples of compositions containing
`enantiorner (R)-(+)-cL-ethyl-2-oxo-l-pyrrolidineaceta
`(S)-(—)-a-ethyl-2-oxo-l-pyrrolidineacetamide, which
`mide (compound II) and of diazepam are compared on
`can be administered orally, four compositions are given
`these three strains.
`hereinafter for white and opaque gelatine capsules:
`The compounds are administered to the animals by
`intraperitoneal injection (10 ml/kg of mouse) 30 min
`utes before exposure to the board. Animals in the con
`trol group receive the carrier only.
`Table I shows the mean of the number of holes ex
`plored per group of 16 animals (Xi-SEM) for each of
`the strains (control and treated animals) at the doses
`indicated (SEM: Standard Deviation from the Mean). It
`also gives the percentage change of the score with re
`spect to the score of the control groups.
`
`5
`
`20
`
`35
`
`45
`
`50
`
`55
`
`Number of the capsules
`1
`1
`
`1
`
`0
`
`Compound I
`Lactose
`Magnesium stearate
`
`62.5 mg 125 mg 250 mg
`362.5 mg 264 mg 89 mg
`1 mg
`1 mg
`1 mg
`
`500 mg
`50 mg
`2 mg
`
`of (S)-(-)-a-ethyl-2-oxo-l-pyr
`The ef?cacy
`rolidineacetamide for the treatment of anxiety is demon
`strated by its activity in the following pharmacological
`
`TABLE I
`3%
`NMRI strain
`DEA-derived strain
`C57 Black Mice strain
`Dose
`(mg/kg) X i SEM % Change X i SEM % Change X i SEM % Change
`9.5
`16.1 i 3.1
`80.9
`22.5 i 3.1
`47.1
`NT
`17.0
`21.5 1': 3.4‘
`141.6
`21.0 i- 2.7
`37.3
`NT
`30.6
`17.1 i 2.1‘
`92.1
`24.9 i 3.3‘
`62.7
`29.4 i 2.8
`Control
`8.9 i 1.4
`—
`15.3 i 2.6
`—
`32.7 i 2.5
`9.5
`9.9 i 1.4
`0
`17.0
`7.9 i 2.0
`—20.2
`
`NT
`
`NT
`
`—l0.l
`—
`
`Compound
`Compound]
`
`Compound II
`
`Page 00003
`
`
`
`5
`
`5,447,952
`
`6
`
`TABLE I-continued
`Hole-board test
`NMRI strain
`DBA-den'ved strain
`C57 Black Mice strain
`Dose
`(mg/kg) X i SEM % Change X i SEM % Change X it SEM % Change
`30.6
`10.1 i 2.7
`2.0
`Control
`9.9 i 2.4
`-
`0.5
`19.7 i 2.7‘
`45.9
`1.0
`14.9 i 3.0
`10.4
`Control 13.5 i 1.9
`—
`
`49.8 i 3.8‘
`51.1 i 2.9‘
`39.7 i 2.8
`
`25.4
`28.7
`-—
`
`33.7 i 3.0‘
`42.1 i 3.4‘
`21.0 i 3.3
`
`60.5
`100.5
`—
`
`Compound
`
`Diazepam
`
`NT: not treated
`(‘)z signi?cant increase in activity compared with the control group: P § 0.05; Mann-Whitney U-test
`
`The results show that the three strains of mice tested
`differ from one another by their baseline levels (control
`tests), which are much higher for the normal NMRI
`mice than for the other two strains. It is known that the
`number of holes explored is considerably reduced under
`the in?uence of anxiogenic agents, such as caffeine and
`yohimbine (R. LISTER, Pharmacol. Then, 46, (1990),
`321-340). Consequently, the low level observed for the
`control groups of unhealthy DBA-derived and C57
`Black Mice strains correctly re?ects the increased level
`of intrinsic anxiety in these two strains.
`As with diazepam, (S)-(-)-a-ethyl-2-oxo-1-pyr
`rolidineacetamide considerably increases the score
`25
`achieved by the DEA-derived and C57 Black Mice
`strains, but unlike diazepam, compound I has practically
`no anxiolytic effect on the normal NMRI strain. Diaze»
`pam is also very active on the latter strain and the in
`crease in score re?ects very well the disinhibition effect
`which is speci?c to benzodiazepines. The dependence
`of the anxiolytic effect of compound I on the nature of
`the strain, and the pronounced selectivity of this effect
`for strains which exhibit unhealthy anxiety, suggests
`that compound I is particularly useful in the treatment
`
`Compound
`CompoundI
`
`chlordiazepoxide
`
`counted (unpunished crossings). In a second stage, the
`animal is punished by an electric shock to the paws,
`each time it crosses right over the surface (punished
`crossings), which induces on the animal an immobiliz
`ing reaction. Under conditions with punishment (pun
`ished crossings), the number of crossings over the sur
`face to explore the environment is strongly reduced. On
`the other hand, the reduction in the number of crossings
`under conditions with punishment is inhibited in ani
`mals which have been previously treated with an anxi
`olytic agent.
`The compounds tested, (S)-(-)-a-ethyl-2-oxo-l-pyr
`rolidineacetamide (compound I) and chlordiazepoxide,
`are administered to the animals by intraperitoneal injec
`tion (10 ml/kg of mouse) 30 minutes before the begin
`ning of the test. The animals in the control groups re
`ceive the carrier only. Table II gives the mean
`(XiSEM) of the number of crossings made under pun
`ished conditions and under unpunished conditions for
`groups of 30 NMRI mice, over a period of 1 minute and
`at the doses indicated. .
`It also gives the percentage change of the score with
`respect to the score of the control groups.
`TABLE II
`Four-plates test
`Number of Crossings
`Punished crossings
`Unpunished crossing
`Dose
`(mg/kg) X i SEM % Change X i SEM % Change
`30.6
`7.8 -_P 0.6
`16.4
`14.7 t 0.7
`3.5
`54.0
`8.9 i 0.6‘
`32.8
`12.7 i 0.7
`—10.6
`95.2
`8.5 i 0.5‘
`26.9
`13.7 i 0.6
`-—3.5
`Control
`6.7 i 0.4
`-—
`14.2 i 0.8
`—
`2.0
`10.9 i 0.5
`18.5
`17.4 :L 1.0
`13.7
`4.0
`10.9 i 0.5‘
`18.5
`16.1 :L 0.7
`5.2
`8.0
`13.2 -_t- 0.6’
`43.5
`' 22.7 i 0.8‘
`48.4
`16.0
`13.7 i 0.7‘
`48.9
`22.8 i 1.0‘
`49.0
`Control
`9.2 i 0.5
`—
`15.3 i 0.8
`—
`
`('): signi?cant increase in activity with respect to the control group: P § 0.05; Mann-Whitney U-test
`
`of exacerbated emotional states, independently of any
`disinhibiting effect on the behavior. The (R)-(+)-a
`ethyl-2-oxo-l-pyrrolidineacetamide enantiomer (com
`pound II) is inactive on these strains.
`2. Four-Plates Test (Punished Behavior)
`The general approach used in this type of experiment
`consists of inducing the inhibition of a speci?c response
`by applying a stimulus which produces aversion at the
`time of that speci?c response. The “four-plates” test is
`an easy method described for the ?rst time by J. R.
`BOISSIER et al., in Eur. J. Pharmacol, 4, (1968),
`145-151, for evaluating the potential anxiolytic activity
`of new compounds in laboratory animals. In a ?rst
`stage, it involves placing a mouse in an unfamiliar envi
`ronment, which consists of a surface covered by four
`metal plates which can be electri?ed, and during a spe
`ci?c period of time, the number of times that the animal
`crosses the surface, passing from one plate to another is
`
`55
`
`60
`
`65
`
`The results show that, under punished conditions, the
`number of crossings is lower than under unpunished
`conditions, both for the control groups and for the
`treated groups.
`Compound I and chlordiazepoxide increase the num
`ber of crossings under punished conditions but, unlike
`compound I, chlordiazepoxide also has the same effect
`under unpunished conditions by disinhibiting normal
`behavior.
`These results show that (S)-(—)-a-ethyl-2-oxo-l-pyr
`rolidineacetamide (compound I) offers an advantage
`over existing drugs because it only exhibits its anxiolytic
`activity under conditions which induce severe anxiety
`and not under normal conditions.
`This selectivity of compound I enables to distinguish
`an anxiolytic effect from an effect which would result in
`an increase of activity per se.
`
`Page 00004
`
`
`
`10
`
`5,447,952
`8
`light stimulus that has been previously paired with an
`electric shock to the paws of the animals. In this case,
`the anxiety induced by the combined sound and light
`aggressions (potentiated startle) originates from the
`premonition of a painful and unpleasant event (M. DA
`VIS, Psychopharmacology, 62, (1979), l-7).
`Anxiolytic compounds, such as benzodiazepines or
`buspirone, reduce the amplitude of the potentiated star
`tle response proportionally to the dose used (S.
`GREEN et a1, “Animal Models of Anxiety” in “Behavi
`oural Models in Psychopharrnacology”, P. Willner
`(ed), Cambridge Univ. Press, 21-49, 1991; M. DAVIS,
`Trends Pharmacol. Sci., 13, (1992), 35-41). The tech
`nique used in this test is based upon that proposed by M.
`DAVIS (loc. cit.) and comprises essentially two stages:
`1st stage: Training—the animals are trained to react
`to a light stimulus accompanied by an electric
`shock to the paws;
`2nd stage: Main test—the amplitude of the startle
`response of the animals to a sound aggression ac
`companied by a light stimulus without electric
`shock is measured—20 tests—(potentiated startle
`response or PSR), and the amplitude of the startle
`response of the animals to sound aggression, nei
`ther accompanied by a light stimulus, nor by an
`electric shock, is also measured—20 tests-(acous
`tic startle response or ASR).
`Groups of 10 male untrained Sprague-Dawley rats
`are used. The compounds tested are (S)-(—)-a-ethyl-2
`oxo-l-pyrrolidineacetamide (compound I) and chlordi
`azepoxide. They are administered by intraperitoneal
`injection (1 ml/kg of rat) 60 minutes before the main
`test. Animals in the control group receive only the
`saline solution (0.9% NaCl). At the time of the test, each
`animal is placed in a cage connected to accelerometers
`which record automatically the startles of the animals
`and express the amplitude of the response in arbitrary
`units.
`Table IV gives the mean of the amplitudes obtained
`for ASR and PSR responses at the doses indicated.
`
`20
`
`30
`
`7
`3. Elevated Plus-Maze Test
`This test is a simple and rapid method, widely used
`for detecting the anxiolytic activity of new compounds.
`Unlike most anxiety tests which use nociceptive stimuli,
`for example an electric shock, this test relies solely on
`measuring the spontaneous activity of an animal con
`fronted with a natural anxiogenic situation, which
`causes a con?ict between two opposed tendencies: the
`desire to explore a novel environment and the desire to
`?ee from an open elevated area. Relative exploration of
`the open or closed arms of a maze is a re?ection of an
`anxiety state of the animal, exploration of the open arms
`being strongly reduced in animals that exhibit a high
`anxiety state (S. PELLOW et al., J. Neurosci. Methods,
`14, (1985), 149-167).
`An anxiolytic compound increases the number of
`visits to the open arms and the time spent to explore
`them, whereas with benzodiazepines, part of this in
`crease has been attributed to induction of stereotypy by
`the drug at the doses used (U. FALTER et al., Behav.
`Processes, 29, (1993), l28—l29). The technique used in
`this test is that described by S. PELLOW (loc. cit)
`modi?ed by R. J. RODGERS et al. (Psychopharmacol
`ogy, 106 (1992), 102-110). This modi?cation consists of
`enhancing the anxiety state of the animals, by previ
`25
`ously placing them on an elevated maze (pre-test) of
`which the four arms are open, which has the effect of
`reducing the number of explorations into the open arms
`(baseline) when the animal is placed, 24 hours later, on
`a conventional maze having 2 open and 2 closed arms.
`The activities
`of (S)-(—-)-a-ethyl-2-oxo-l-pyr
`rolidineacetamide (compound I), of the dextrorotatory
`enantiomer (compound II) and of chlordiazepoxide
`have been examined using this test. The compounds are
`administered to Sprague-Dawley rats by intraperitoneal
`injection (10 ml/kg of rat) 60 minutes before the begin
`ning of the test. The animals in the control group re
`ceived a saline solution (0.9% NaCl). Table III gives the
`mean values for the total number of entries into the arms
`of the maze, the percentage of entries into the open
`40
`arms with respect to the total number of entries, and the
`.
`.
`time spent exploring these open arms, for groups of 15
`rats (control groups and groups treated with the doses
`indicated), each in the mazgflg?énpliptes.
`
`'
`
`'
`
`'
`
`“
`
`TABLE IV
`_
`D-——-—-—P°t‘_mmt°d wisest
`
`tug/kg
`x i SEM
`
`056 in
`
`ASR
`X It SEM
`
`Compound
`
`Elevated plus-maze test
`OEn arms
`Total number
`% entries
`Time (sec)
`of entries
`Dose
`x : SEM
`x i SEM
`(mg/kg) x i SEM
`10.3 i 3.0
`5.2 i 2.0
`8.6 i 1.0
`20.2 i 3.0'
`16.1 i 3.1*
`11.9 i 0.7‘
`17.0
`10.9 i 3.1
`5.7 i 2.2
`8.3 i 0.9
`17.0
`11.8 i 2.7
`6.8 i 1.6
`9.2 i 0.7
`54.0
`27.1 i 2.7‘
`27.1 i 3.7’
`15.0 i 1.3t
`5.0
`Chlordiazepoxide
`(‘) signi?cant increase in activity compared with the control group: P § 0.05; Mann-Whitney U-test.
`
`Compounds
`Control
`Compound I
`Compound II
`
`The results show that, for the control group, the
`number of entries into the open arms only represents a
`very low percentage of the total number of entries.
`Control
`Treatment of animals with compound I or with chlor-
`60 Compound]
`iaze xi 6 i i?cant
`increa es th number
`of
`Chlordiazepoxide
`d
`p0 d s gn
`1y
`8
`e
`entries into the open arms as well as the time spent to
`explore them. On the other hand, compound II has no
`activity.
`These results con?rm the value of compound I in the
`treatment of pathological anxiety states.
`4. Potentiated Startle Test
`The startle response to a loud sound (sound aggres
`sion) is potentiated by simultaneous presentation of a
`
`22965 i- 4760"
`14817 i 3056“
`10393 i 2079"
`
`17326 i 3126
`14388 i 2214
`8759 i 2352"“
`
`17.0
`5.0
`
`(') Signi?cant difference between PSR and ASR: P § 0.05, paired t-test
`(') Signi?cant difference between treated and control groups: P § 0.05; Student's
`[-[CSK
`
`65
`
`The results for the animals in the control group show
`that the PSR response is, as expected, more intense than
`the ASR response. Compound I strongly reduces the
`amplitude of the potentiated startle response (PSR) but
`
`Page 00005
`
`
`
`5,447,952
`
`9
`only has a small effect on the acoustic startle response
`(ASR).
`chlordiazepoxide, on the other hand, attenuates the
`amplitude of the two responses in a signi?cant manner.
`The in?uence of benzodiazepines on ASR illustrates an
`inherent disadvantage with this class of compounds,
`which is attributable to their sedative effect.
`A compound such as (S)-(-)-a-ethyl-2-oxo-l-pyr
`rolidineacetamide, which acts speci?cally on the PSR
`response, may be predicted as having greater speci?city
`for the treatment of pathological anxiety states and,
`hence offers an advantage over existing therapies.
`5. Neuroendocrine Response to Stress
`Adaptive responses to stress, involving negative
`feedback control mechanisms in the hypothalamic
`pituitary-adrenergic system, are profoundly impaired in
`the aged rat. These age-related impairments can be
`demonstrated by measuring the basal plasma cortico
`sterone level, which is much higher in aged subjects
`than in the young (R. M. SAPOLSKY, Neurobiol.
`Aging, 13, (1991), 171-174).
`A stress situation rapidly increases the plasma corti
`costerone level both in young and in aged animals but,
`for the latter, recovery from stress occurs much more
`slowly and less completely (R. M. SAPOLSKY et. al,
`Exp. Gerontol., 18, (1983), 55-64; A. M. ISSA, et al, J.
`Neurosci, 10, (1990), 3247-3254).
`The consequences of these age-related effects are
`important because, while it is true that glucocorticoids
`are essential for survival and adaptation to various
`stressors, prolonged exposure to excessive levels of
`glucocorticoids may be pathogenic for the system.
`The test used here is the immobilization test described
`by R. M. SAPOLSKY (1983, 10c. cit.). An animal,
`placed for 5 minutes in a restrainer adjusted so as to
`fully immobilize it (immobilization stress), develops an
`anxiety response which results in a rapid increase in the
`corticosterone level.
`In this experiment, groups of 4 to 8 young (2 months
`of age) and aged (20 to 21 months of age) Sprague-Daw
`ley rats are used. The treated groups receive 17 mg/kg
`of compound I or 5 mg/kg of chlordiazepoxide per os,
`60 minutes before the immobilization test. The control
`groups only receive the saline solution (0.9% NaCl).
`The level of plasma corticosterone is measured before
`stress, at the end of stress and 30 minutes later, in order
`to determine the state of recovery of the animals.
`Table V gives the mean of the plasma level of corti
`costerone for each group of animals, measured under
`these conditions.
`
`TABLE V
`ResEnse to an immobilization stress
`Plasma corticosterone level (ng/ml)
`at the
`end of stress
`X -_t— SEM
`
`after 30 min
`X i SEM
`
`basal
`Animals/Treatment X 1 SEM
`
`50
`
`55
`
`10
`but 30 minutes after the end of the stress period, this
`level still remains very high in the aged/control ani
`mals. Thus, recovery in these animals is slower than in
`young rats.
`In the groups of aged animals which have been
`treated, either with compound I or with chlordiazepox
`ide, the basal level of corticosterone is lower than in the
`group of aged control animals, while being greater than
`the level in the young control animals.
`Stress also increases considerably the level of plasma
`corticosterone in aged animals, however, the aged
`group treated with compound I recovers much more
`rapidly than that treated by chlordiazepoxide, as indi
`cated by the level of corticosterone measured after 30
`minutes. This result shows that, contrary to chlordiaz
`epoxide, (S)-(—)-a-ethyl-2-oxo-l-pyrrolidineacetamide
`has a bene?cial action on aged stressed subjects by
`facilitating physiological adaptation to a stress situation.
`6. Syndrome of Withdrawal from Benzodiazepines
`The abrupt discontinuation from prolonged therapeu
`tic treatment with benzodiazepines is frequently accom
`panied by a withdrawal syndrome characterized by a
`wide range of symptoms due to a physical dependence
`on the drug, such as trembling, nausea, dizziness or
`hypertension to quote but a few, as well as usual anxiety
`symptoms which are intense in man (J. H. WOODS et
`al., Pharmacol. Rev., 39, (1987), 251-419; M. H.
`LADER, “Abuse Potential, Tolerance and Depen
`dence on Chronic Anxiolytic Treatment” in “Target
`Receptors for Anxiolytics and Hypnotics: From Molec
`ular Pharmacology to Therapeutics”, J. Mendlewicz
`and G. Racagni (eds), Karger, Basel, Vol. 3, (1992), p
`46-54).
`Based on the observation that withdrawal is associ
`ated with anxiogenic stimuli, the use of animal anxiety
`models for detecting signs of withdrawal in animals has
`been suggested (M. W. EMMETT-OGLESBY et al.,
`Psychopharmacology, 101, (1990), 292-309).
`The anxiogenic response to withdrawal from diaze
`pam has in particular been the subject of studies in the
`rat, using the elevated plus-maze anxiety test, and is
`expressed by a signi?cant reduction in the exploration
`of the open arms (S. E. FILE et al., Psychopharmacol
`ogy 105, (1991), 578-582).
`The same model has been used to study the anxio
`genic effect of withdrawal from chlordiazepoxide in
`mice and to demonstrate the anxiolytic activity of (S)
`(-)-a-ethyl-2-oxo-l-pyrrolidineacetamide (compound
`I) on this syndrome.
`Groups of 17 mice (NMRI) were used, split up as
`follows:
`group 1: animals are treated intraperitoneally, twice a
`day, for 21 days, with increasing doses of chlordi
`azepoxide ranging from 10 to 40 mg/kg; the last
`injection is performed 60 minutes before the test;
`group 2: withdrawal from chlordiazepoxide takes
`place 24 hours before the test and the withdrawn
`animals receive a physiological salt solution;
`group 3: the withdrawn animals receive compound I
`intraperitoneally at a dose of 17 mg/kg;
`group 4: the withdrawn animals receive compound I
`intraperitoneally at a dose of 54 mg/kg.
`The control group only receives 10 ml/kg of a physi
`ologic solution. Table VI g