(12) United States Patent (10) Patent No.: US 7,737,180 B2 Mak0vec Et Al

USOO773718OB2

(12) United States Patent (10) Patent No.: US 7,737,180 B2 Mak0Vec et al. (45) Date of Patent: Jun. 15, 2010

(54) USE OF NEBOGLAMINE (CR 2249). ASAN WO WOO3O45913 5, 2003 ANTIPSYCHOTIC AND NEUROPROTECTIVE OTHER PUBLICATIONS Flint et al. A qualitative analysis of the negative symptoms of schizo (75) Inventors: Francesco Makovec, Lesmo (IT); Lucio phrenia interfering with academic and Social Success, and the Claudio Rovati, Monza (IT) exacerbators and diminishers of those symptoms. Dissertation Abstracts International, 2003 vol. 64, No. 12A. p. 4420. Abstract.* (73) Assignee: Rottapharm S.p.A., Milan (IT) Garofalo et al. CR 2249: a New putative memory enhnacer. Behavioural studies on learning and memory in rats and mice. J. (*) Notice: Subject to any disclaimer, the term of this Pharm. Pharmacol. 1996, 48, pp. 1290-1297.* patent is extended or adjusted under 35 Victor Peralta, Are There MoreThanTwo Syndromes in Schizophre U.S.C. 154(b) by 476 days. nia? A Critique of the Positive-Negative Dichotomy, British Journal of Psychiatry (1992), 161, 335-343. (21) Appl. No.: 11/569,545 R.D. Porsolt, Behavioural Despair in Mice: A Primary ScreeningTest for Antidepressants, Arch. int. Pharmacodyn. 229,327-336 (1977). N. R. Swerdlow, Effects of Spiperone, Raclopride, SCH 23390 and (22) PCT Filed: May 23, 2005 Clozapine on Apomorphine Inhibition of Sensorimotor Gating of the Startle Response in the Rat, The Journal of Pharmacology and (86). PCT No.: PCT/EP2005/052340 Experimental Therapeutics, vol. 256, No. 2, U.S. J. David Jentsch. The Neuropsychopharmacology of Phencyclidine: S371 (c)(1), From NMDA Receptor Hypofunction to the Dopamine Hypothesis (2), (4) Date: Nov. 22, 2006 of Schizophrenia, Neurospychopharmacology, 1999-vol. 20, No. 3. Paolo Garofalo, CR 2249: a New Putative Memory Enhancer. (87) PCT Pub. No.: WO2005/115373 Behavioural Studies on Learning and Memory in Rats and Mice, J. Pharm. Pharmacol. 1996, 48: 1290-1297, Feb. 27, 1996. PCT Pub. Date: Dec. 8, 2005 M. Lanza, Characteriztion of a Novel Putative Cognition Enhancer Mediting Facilition of Glycine Effect on Strychnine-Resistant Sites (65) Prior Publication Data Coupled to NMDA Receptor Complex, Neuropharmacology, vol. 26. No. 8, pp. 1057-1064, 1997. US 2007/O2497.15A1 Oct. 25, 2007 Yousry Sayed, The Dopamine Hypothesis of Schizophrenia and the Antagonistic Action of Neuoleptic Drugs—A Review; The British (30) Foreign Application Priority Data Library, vol. 19, No. 2, 1983. Nancy C. Andreasen, Methods for Assessing Positive and Negative May 24, 2004 (IT) ...... TO2OO4AO343 Symptoms, Department of Psychiatry, University of Iowa College of (51) Int. Cl. Medicine, 1990, vo. 24, pp. 73-88. A 6LX 3L/95 (2006.01) * cited by examiner (52) U.S. Cl...... 514/561 Primary Examiner Jennifer M Kim (58) Field of Classification Search ...... 514/561 (74) Attorney, Agent, or Firm—Roylance, Abrams, Berdo & See application file for complete search history. Goodman, L.L.P. (56) References Cited (57) ABSTRACT U.S. PATENT DOCUMENTS Use of neboglamine, (S)-4-amino-N-(4.4-dimethylcyclo 3,665,037 A * 5, 1972 Murakami et al...... 564,300 hexyl)glutamic acid (CR 2249) (CAS Registry Number 163000-63-3), of the racemate thereof or of a pharmaceuti cally acceptable salt thereof for the preparation of a medica FOREIGN PATENT DOCUMENTS ment for the treatment of schizophrenia. WO WO942O454 9, 1994 9 Claims, No Drawings US 7,737,180 B2 1. 2 USE OF NEBOGLAMINE (CR 2249). ASAN glycine site coupled to the NMDA receptor complex Lanza ANTIPSYCHOTIC AND NEUROPROTECTIVE et al., Neuropharmacology 36, 1057-64 (1997) together with interesting properties promoting memory and learning in FIELD OF THE INVENTION various animal models Garofalo et al. J. Pharm. Pharmacol. 48, 1290-97 (1996). The present invention relates to a novel therapeutic use for The facilitatory activity exerted by neboglamine at the (S)-4-amino-N-(4,4-dimethylcyclohexyl)glutamic acid (CR level of the NMDA receptor complex should be of therapeutic 2249-neboglamine) (CAS Registry Number 163000-63-3), utility under conditions involving glutamatergic hypofunc of the pharmaceutically acceptable salts thereof and of the tionality, which, as explained above, may contribute to the main metabolite thereof, 4.4-dimethylcyclohexylamine (CR 10 negative symptomatological picture of Schizophrenia. 2863), in the treatment of schizophrenia with predominant negative symptomatology, of type II bipolar disorders (hypo SUMMARY OF THE INVENTION mania with major depression) and in cyclothymic disorders (numerous episodes of hypomania and minor depression). Phenylcyclidine (PCP) induced psychosis is one model 15 which better reflects the pathophysiology of the disease. In BACKGROUND OF THE INVENTION fact, both in experimental animals and in humans, PCP induces behavioural effects which exhibit considerable simi Schizophrenia is a psychiatric condition characterised by larities with the symptoms of schizophrenia Jentsch et al., positive symptoms, for example hallucinations, delusions, Neuropsychopharmacology 20, 201-225 (1999). Accord disturbances of thought, paranoia Andreson, Mod. Probl. ingly, in humans, chronic administration of PCP (as may Pharmacopsychiatry 24, 73-88 (1990); Peralta et al., Br. J. occur, for example, in drug addicts) brings about persistent Psychiatry 161, 335-343 (1992), negative symptoms, for neurobiological changes which are manifested with both example apathy which primarily manifests as emotional and positive and negative typologies, and mimic those present in motivational deficits and deficits in Social interactions, cog Schizophrenic patients. nitive deficit together with depression. 25 Thus, neboglamine and the main metabolite thereof CR This complex scheme of symptoms which is observed in 2863 have been evaluated in experimental animal models in Schizophrenic patients suggests the existence of dysfunctions which PCP was used and which are recognised as being at various levels in the brain of these individuals and it is thus predictive for the evaluation of antipsychotic drugs. Such difficult to hypothesise that malfunctioning of a single neu models in fact reproduce both glutamatergic hypofunctional rotransmitter system could explain the complex pathological 30 ity and hyperactivity of the dopaminergic system (hyperac picture of the disease. tivity and stereotype behaviour) with a psychotomimetic State The until recently accepted hypothesis suggested hyperac which greatly resembles schizophrenia, unlike amphetamine tivity of the dopaminergic system as the primary cause of the induced models which only bring about positive type psycho clinical manifestations of schizophrenia. This hypothesis was SCS. largely derived from the observation that amphetamine brings 35 The first model used was the study of PCP-induced inhi about positive symptoms which resemble those present in bition of the acoustic prestimulus (PPI) in the rat. paranoid patients; this symptomatology is brought about by Principle of the Method increased dopaminergic neurotransmission in the central ner The extent of the startle reflex response to an acoustic vous system (CNS) (Sayed et al., Psychopharmacol. Bull. 19, stimulus is reduced if it is preceded by a weak stimulus which, 283-288 (1983). 40 by itself, does not bring about a significant startle response. However, many schizophrenic patients and in particular This phenomenon is known as “pre-pulse inhibition’ (PPI). those exhibiting predominantly negative symptoms do not This PPI response is present both in many animal species and respond adequately to treatment with dopamine antagonist in humans, while it has on the contrary been reported that drugs, so demonstrating that the dopaminergic model can schizophrenic patients generally exhibit a PPI deficit. Such a only provide a partial explanation of the complexity of the 45 deficit may be reproduced experimentally with drugs which disease. induce psychotic phenomena, Such dopamine agonists and Functional anatomical Studies have revealed morphomet NMDA antagonists. ric changes in the cerebral cortex of Schizophrenic individu The method is based on that reported by Swerdlow et al. J. als, these changes probably arising from modified cortical Pharmacol. Exp. Ther. 256530-536 (1991) with slight modi development. Glutamate is the primary neurotransmitter at 50 fications. An acrylic cylinder is used which is equipped with this level and it is thus probable that dysfunction of the a piezoelectric sensor mounted under the cylinder which glutamatergic pathways may well play an important part in detects and transduces movements of the cylinder. A back Schizophrenia as it does in bipolar disorders. The dopamin ground noise of 60 dB is provided and subsequently two ergic and glutamatergic hypotheses are not mutually exclu acoustic stimuli separated by 500 ms, the first of which is 20 sive because there are major functional interactions between 55 dB above the background noise, while the second (startle these two neurotransmitter systems. stimulus) is 120 dB for a duration of 40 ms. The session of 30 One limitation of preclinical studies into novel molecules trials at 30 second intervals with an overall duration of with potential antipsychotic activity is the limited availability approx. 15 minutes involved subjecting the animal randomly of animal models capable of meaningfully reproducing a to a startle stimulus either isolated or preceded by the low complex pathological picture Such as that presented by 60 intensity stimulus. PPI was calculated as a percentage reduc Schizophrenia. Despite this, there are models which can Suf tion in the level of startle in the presence of the prestimulus ficiently effectively reproduce the symptoms of such disease compared to that without the acoustic prestimulus. Rats with the aim of assessing the pharmacological activity of the weighing approx. 250 g were used, which were treated intra potential candidate drug in specific functional changes. peritoneally (i.p.) with the products under investigation 15 On the basis of published preclinical data, the compound 65 minutes prior to Subcutaneous (s.c.) administration of 3 neboglamine (CR 2249) has shown that it possesses consid mg/kg of PCP and 30 minutes before the beginning of the erable modulating properties for the (Strychnine-insensitive) experiment. The results obtained are shown in Table 1 below. US 7,737,180 B2 3 4 ity of the PCP-treated group in fact increased by approx. 3 TABLE 1. times relative to the untreated control group In the dose range from 3-10 mg/kg, neboglamine Successfully inhibits this Reversion of PCP-induced inhibition of the effect, which becomes statistically significant at a dose of 10 acoustic prestimulus (PPI) in the rat mg/kg, with virtually complete inhibition (87.8%) of the PPI (% inhibition) % inhibition by PCP’ depressive effect. Neboglamine's ability to reverse the effect of PCP at a Control 559, PCP 20% neuronal level was also evaluated in an in vitro test carried out PCP+N (1) 279 2O.O on slices of rat frontal cortex. PCP+N (3): 40% 57.1 10 The method involved preparing 0.4 mm thick cortical PCP+N (10) 48* 80.0 slices, incubating them for 20 minutes with H-dopamine in PCP + CR2863 (10) 38: 51.4 physiological Solution in the presence of 0.1 uM 6-nitroqui Note N = neboglamine pazine and 0.1 uM nisoxetine (selective serotonin and nora (): The results relate to 10 animals per group: * P< 0.05 (ANOVA vs drenalinereuptake inhibitors), aerated with 95% O+5% CO, PCP only group) 15 at 37° C., which, after appropriate washing with artificial ’: Perdcentage inhibition iss calculated with the formula: cerebrospinal fluid (aCSF), were superperfused for 30 min CONTROLPCp* 100 utes with aGFS at a rate of 1 ml/minute to equilibrate the system. Then, PCP and neboglamine were added at the stated : The numbers in brackets are the doses of drug in mg/kg i.p. concentrations to the perfusion liquid for the entire duration of the experiment, while, after 45 minutes, NMDA (100 uM) Analysis of the PPI data of the animals pretreated with the stimulation was provided for 5 minutes. Another four 5 drugs under investigation shows how neboglamine dose-de minute fractions of eluate were then collected and their radio pendently inhibits the blocking of PPI by PCP. This inhibition activity determined. is already significant at a dose of 3 mg/kg (i.p.). The metabo The effect of the drugs was evaluated by calculating the lite CR 2863 proves to be approx. 3 times less active than the 25 ratio between the percentage radioactivity present in the “parent in this experimental model. effluent fraction corresponding to the maximum effect and An animal model of depression (which may be considered that in the first effluent fraction collected. The results obtained as a negative symptom of Schizophrenia) was then used to are shown in Table 3 below. study the activity of neboglamine on the PCP-induced depres sive effects in the mouse using the Swimming test according 30 TABLE 3 to Porsoltet al. Arch. Int. Pharmadyn. 229,327-336 (1977). Effect of neboglamine in inhibiting PCP-induced Method blocking on the release of tritiated dopamine The test involves inducing a state of depression in the brought about by NMDA in slices off rat prefrontal animal, which is forced to Swim in a glass cylinder from cortex 35 which it cannot escape. The state of depression was accentu % increase in ated by 14 days’ pre-treatment with a daily 10 mg/kg (s.c.) NMDA-evoked % inhibitory injection of PCP. On the 15th day, the animals were addition 3H-dopamine effect of ally pretreated (i.p.) with physiological Solution or GroupSS release PCP(3) neboglamine 30 minutes before the beginning of the test. The I: Control (NMDA) 120 duration of immobility (sign of depression) in seconds was 40 (100)(1) II: NMDA + PCP (0.1) 64 evaluated during the experiment of an overall duration of 360 III: NMDA + PCP + neboglamine 842 35.7 seconds. The results obtained in this manner, calculated (10) between the 3rd and 6th minute of the experiment, i.e. for a IV: NMDA + PCP+ neboglamine 110° 82.1 (30) period of 240 seconds, are shown in Table 2. 45 ''': All concentrations are M: TABLE 2 (): P < 0.05 (ANOVA) (n = 9x group) : Percentage inhibition was calculated with the formula: Reversion of PCP-induced depression in the mouse Swinning test III(IV) - II 100 50 I-II) × Control group Immobility in seconds % effect vs. PCP Control 48 It may be inferred from the data shown in Table 3 how PCP 130 neboglamine powerfully and dose-dependently prevents Neboglamine (3) + 84 56.0 PCP PCP-induced blocking of NMDA-evoked dopamine release. Neboglamine (N) (10) + 5891) 87.8 55 This inhibition was deemed significant from a dose of as low PCP as 10 M. It should furthermore be noted that, at both the ''': P < 0.01 vs PCP only group (n = 10 animals/group) concentrations tested, neboglamine has no effect on basal (): Percentage inhibition iss calculated with the formula: dopamine release. PCP- (N+PCP) Neuroprotection Experiments -(PCP-CONTROL)* - X 100 60 It was decided to investigate whether neboglamine and the main metabolite thereof (CR 2863) could additionally exhibit neuroprotective activity: Such properties could in fact prove It may be inferred from the data shown in Table 2 how 2 to be extremely useful because a considerable sub-population weeks’ chronic treatment with PCP at a dose of 3 mg/kg of Schizophrenic patients Suffers from progressive structural brings about a behavioural change in the animals in an experi 65 degeneration of the brain (neurodegeneration), the prevention ment which is considered as a possible model of the negative of which is essential if cognitive function is to be maintained symptomatology of schizophrenia. The duration of immobil or restored. US 7,737,180 B2 5 6 It was accordingly decided to investigate the possible neu desired therapeutic effect, for example solid formulations for roprotective effects of neboglamine and CR 2863 on a model oral use with delayed action which allows slow release of the of hippocampal ischaemia brought about in the gerbil by active ingredient over time. bilateral occlusion of the carotid arteries. Substances commonly used in the pharmaceutical sector In brief, halothane-anaesthetised animals are subjected to Such as excipients, binders, disintegrants, Substances capable bilateral occlusion of the carotid arteries for 5 minutes. After of promoting transdermal absorption may be used together 7 days, the animals are sacrificed, the brain is removed, deep with the active ingredient in the pharmaceutical formulation. frozen and 10 um sections from the hippocampal area are Neboglamine compounds and the compound thereof. CR prepared which are stained with cresyl Violet. A quantitative 2863, may accordingly be used as such or as pharmaceuti determination (in mm) of the area occupied by CHI pyrami 10 cally acceptable salts. In the case of neboglamine, the Sodium dal neurons is then performed by means of an image analyser. or potassium salt or the hydrochloride is preferred, while the The type of hippocampal neurodegeneration arising from this hydrochloride is preferred in the case of CR 2863. model of ischaemia involves a significant reduction in the The effective therapeutic quantity of neboglamine to be area of the neuronal nuclei. The drugs were administered used for the treatment of schizophrenia should be between 10 intraperitoneally (i.p.) 1 h before the ischaemia. The results 15 and 600mg of active ingredient per day, preferably from 30 to obtained in this manner are shown in Tables 4 and 5 below. 300 mg. depending on the specific condition of the treated patient, on the individual response to treatment, the age and TABLE 4 weight of the patient. The following Examples are intended to illustrate better the Ischaemia induced in the gerbil by bilateral occlusion of present invention in a purely exemplary, non-limiting man the carotid arteries: protective effect of neboglamine . Dose % % Substances mg/kg area (mm) protection mortality EXAMPLE 1. Controls 25.222.13 O Composition of Neboglamine Capsules (Sham) 25 Ischaemia 9. OS 5.988 50 CR 2249 12.5 8.42 - 4.46* O 50 CR 2249 25 16.47 S.S4 45.9 25 CR 2249 50 20.48 S.3 70.7 O Snap Fit hard gelatin capsules, Size 1 The data are means - SD. 30 P<0.05 ws sham animals. Neboglamine 100 mg P< 0.05 vs. ischaemic animals Pregelatinised maize starch 228 mg USP talcum 2 mg TABLE 5 35 Ischaemia induced in the gerbil by bilateral occlusion EXAMPLE 2 of the carotid arteries: protective effect of CR 2863 Dose % % Composition of an Oral Solution (Syrup) of Substances mg/kg area (mm2) protection mortality Neboglamine (Per 100 ml of Syrup) 40 Controls 22.39 - 1.15 O (Sham) Ischaemia 5.55.3.1: 28.57 CR 2863 4 9.74 O.9* 24.88 11.11 Neboglamine 1 g CR 2863 8 12.165.49* 39.25 5.88 Sodium hydroxide pellets 0.15 g CR 2863 16 15.235.49* 57.82 O 45 Essential oil 0.05 g The data are means - SD. Sorbitol, 70% 65 g P<0.05 ws sham animals. Ethyl alcohol, 95% 5 g "Ps 0.05 vs. ischaemic animals Distilled water to make up to 100 ml

It can be seen from the data shown in Tables 4 and 5 how 50 neboglamine provides dose-dependent protection from neu EXAMPLE 3 ronal degeneration. Indeed, while ischaemia produces a mor tality of 50% in the control animals, neboglamine at the Composition of a Sterile Vial of Neboglamine for higher dose completely eliminates any mortality. Further Parenteral Use more, in this model, the metabolite CR 2863 proves to be still 55 more active than the “parent', since complete protection against mortality is achieved at a dose of as low as 16 mg. These results would suggest that the neuroprotective effect Neboglamine 50 mg exhibited by neboglamine is principally due to its main Sodium hydroxide pellets 7.5 mg metabolite CR 2863. 60 Pyrogen-free physiological Solution 3 ml Pharmaceutical formulations for the use of the compounds to make up to according to the invention may be prepared using conven tional methods. The formulations include those suitable for oral use such as capsules, tablets, Suspensions, emulsions, The invention claimed is: Solutions; sterile Solutions for parenteral use (including Sub 65 1. A method for treating schizophrenia in a patient by cutaneous, intramuscular, intravenous), or preparations for administering to said patient in need thereof an effective topical or rectal use or other forms suitable for achieving the amount of an active ingredient selected from the group con US 7,737,180 B2 7 8 sisting of (S)-4-amino-N-(4.4-dimethylcyclohexyl)glutamic 6. The method of claim 1, wherein said active ingredient acid; a racemate containing (S)-4-amino-N-(4.4-dimethylcy comprises 4,4-dimethylcyclohexylamine (CR 2863) or of a clohexyl)glutamic acid; 4.4-dimethylcyclohexylamine; and pharmaceutically acceptable salt thereof and where said a pharmaceutically acceptable salt thereof. method comprises administering said active ingredient in an 2. The method of claim 1, further comprising administer ing to said patient having symptoms of type II bipolar (manic amount effective for the treatment of schizophrenia. depressive) disorders the active ingredient in an effective 7. A method for treatment of symptoms of type II bipolar amount for the therapeutic treatment of type II bipolar (manic-depressive) disorder in a patient Suffering from (manic-depressive) disorders. Schizophrenia comprising the step of administering to said 3. The method of claim 1, further comprising administer 10 patient in need thereof a 4.4-dimethylcyclohexylamine or a ing to said patient having symptoms of cyclothymic disorders pharmaceutically acceptable salt thereof in an amount effec the active ingredient in an effective amount for the therapeutic tive to treat type II bipolar (manic-depressive) disorder. treatment of cyclothymic disorders. 8. The method of claim 7, comprising administering to said 4. The method of claim 1, further comprising administer patient having symptoms of progressive structural degenera ing to said patient having symptoms of progressive structural 15 tion of the brain said 4.4-dimethylcyclohexylamine in an degeneration of the brain the active ingredient in an effective amount effective for therapeutic neuroprotective treatment of amount for the therapeutic neuroprotective treatment of pathological conditions characterized by progressive struc pathological conditions characterized by progressive struc tural degeneration of the brain. tural degeneration of the brain. 5. The method of claim 1, wherein said active ingredient 9. A method for treating negative symptoms of schizophre comprises 4,4-dimethylcyclohexylamine (CR 2863) or of a nia in a patient by administering to said patient an effective pharmaceutically acceptable salt thereof and where said amount of 4.4-dimethylcyclohexylamine and a pharmaceuti method comprises administering said active ingredient in an cally acceptable salt thereof. amount effective for the treatment of symptoms of depression by said patient.