Use of Tofisopam as a PDElOA inhibitor

The present invention relates to a novel use of the compound Tofisopam. The invention provides a method of producing a composition comprising Tofisopam useful for treating psychosis and schizophrenia. In a preferred embodiment, a composition is made for treatment of the positive symptoms and cognitive deficits seen in schizophrenic patients.

Background of the Invention

The subject of the invention relates to the treatment of disorders of the central nervous system. More particularly, the invention relates to treatment of neurologic and psychiatric disorders, for example psychosis and disorders comprising deficient cognition as a symptom. This invention relates to PDElOA inhibition.

Cyclic nucleotides cAMP and cGMP are second messengers that regulate many functions in various tissues. Intracellular cAMP and cGMP are generated by adenyl and guanyl cyclases, and are degraded by cyclic nucleotide phosphodiesterases (PDEs). Both steps are controllable by intracellular signaling, and particularly stimulation/repression of adenyl and guanyl cyclases in response to GPCR stimulation is critical and well characterized way of controlling cyclic nucleotide concentrations (Antoni et al 2000).

There are 21 phosphodiesterase genes that can be divided into 11 gene families. Some PDEs degrade cAMP, some cGMP and some both. Most PDEs have a relatively widespread expression and have roles in many tissues, while some are more spatially restricted. Phosphodieasterase 10a (PDElOA) is a dual specificity phosphodiesterase that can convert both cAMP to AMP and cGMP to GMP (Loughney et al 1999, Fujishige et al 1999, Soderling et al 1999). It is primarily and strongly expressed in neurons in the striatum, nucleus accumbens and in the olfactory tubercle (Kotera et al 1999; Seeger et al 2003)

Dopamine D2 receptor antagonism is well established in treatment of schizophrenia. Since the 1950' s, dopamine D2 receptor antagonism has been the mainstay in psychosis treatment and all effective antipsychotic drugs antagonise D2 receptors. The effects of D2 are likely to be mediated primarily through neurons in the striatum, nucleus accumbens and olfactory tubercle, since these areas receives the densest dopaminergic projections and have the strongest expression of D2 receptors (reviewed in Konradi and Heckers 2001. D2 receptor agonism leads to decrease in cAMP levels in the cells where it is expressed through adenylate cyclase inhibition and this is a critical

component of D2 signalling (Stoof and Kebabian 1981; Neve et al 2004). Conversely, antagonism effectively increases cAMP levels, and this effect could be mimicked by inhibition of cAMP degrading phosphodiesterases.

Most of the 21 phosphodiesterase genes are widely expressed, wherefore inhibition are likely to have side effects, but PDElOA has the in this context desired expression profile with high and relatively specific expression in neurons in striatum, nucleus accumbens and olfactory tubercle. Thus, PDElOA inhibition is likely to have effects similar to D2 receptor antagonism and therefore have antipsychotic effects.

While PDElOA inhibition is expected to mimmick D2 receptor antagonism in part, it will be expected to have a somewhat different profile. D2 receptor has other signalling components than cAMP (Neve at al 2004), wherefore interference with cAMP through PDElOA inhibition is likely to negatively modulate rather than directly antagonise Dopamine signalling through D2 receptors. This may reduce the risk of the extrapyrimidal side effects that are seen with strong D2 antagonism. Conversely, PDElOA inhibition will have some effects not seen with D2 receptor antagonism. PDElOA is also expressed in Dl receptor expressing striatal neurons (Seeger et al 2003). Since Dl receptor agonism leads to stimulation of adenylate cyclase and resulting increase in cAMP levels, PDElOA inhibition is likely to also have effects that mimic Dl receptor agonism. Finally, PDElOA inhibition will not only increase cAMP in cells, but will also be expectd to lead to an increase in cGMP, since it is a dual specificity phosphodiesterase. cGMP activates a number of target protein in cells like cAMP and also interacts with the cAMP signalling pathways (ref), but the overall impact on Psychotic symptoms is difficult to predict. In conclusion, PDElOA inhibition is likely to mimic D2 receptor antagonism in part and therefore have antipsychotic effect, but the profile is likely to differ from that of classical D2 receptor antagonists.

The PDElOA inhibitor papaverin has been shown to be active in several antipsychotic models. Papaverin potentiated the cataleptic effect of the D2 receptor antagonist haloperidol in rats, but did not cause catalepsy on its own (WO 03/093499_A2). Papaverin reduced hyperactivity in rats induced by PCP, while reduction of amphetamine induced hyperactivity was insignificant (WO 03/093499 A2). These models suggest that PDElOA inhibition indeed has the classic antipsychotic potential that would be expected from theoretical considerations. Finally, PDElOA inhibition reverses subchronic PCP-induced deficits in attentional set-shifting in rats (Rodefer et al. 2005). This model suggests that PDElOA inhibition might also alleviate some of the cognitive deficits associated with schizophrenia.

The molecular structure and conformational properties of tofisopam have been determined by NMR,

CD and x-ray crystallographic methods. Visy, J. and Simongi, M., Chirality 1:271-275 (1989). The 2,3 diazepine ring exists in two kinds of boat conformation. In the case of the major conformers (+)R and (-) S the ethyl group attached to the center of asymmetry C-5 has quasi-equatorial orientation while in the minor conformer (-)R and (+)S this group is positioned quasi-axially. As a result, racemic tofisopam in solution contain four molecular species, i.e., two enantiomers each of which exists in two chiral conformations. The sign of optical rotation is reversed upon inversion of the diazepine-ring. In crystal form tofisopam exists only as the major conformations, with levorotatory-tofisopam being of the (S) absolute configuration. Toth, G. et al.,J. Heterocyclic chem. 20:709-713,1983. Fogassy, E. et al., In: Bio-Organic Heterocycles, Van der Plas, H. C, Otvos, L, Simongi, M., eds. Budapest Amsterdam: Akademia; Kiado-Elsevier, 1984, 229:233.

Tofisopam has previeosly been indicated to inhibit PDE2 and PDE4 (WO2005113517), but the in vivo target of tofisopam and related compounds are unknown. PDE4 inhibition has previously been linked to causing emesis and other side effects, such as those observed with the selective PDE4 inhibitor Rolipram (Martin 2001 IDrugs vol 4 No 3, p312-338).

References:

Kotera J, Fujishige K, Yuasa K, Omori K. Biochem Biophys Res Commun. 1999 Aug ll;261(3):551-7.

Seeger TF, Bartlett B, Coskran TM, CuIp JS, James LC, Krull DL, Lanfear J, Ryan AM, Schmidt CJ, Strick CA, Varghese AH, Williams RD, Wylie PG, Menniti FS. Brain Res. 2003 Sep 26;985(2): 113-26.

Konradi C, Heckers S. Antipsychotic drugs and neuroplasticity: insights into the treatment and neurobiology of schizophrenia. Biol Psychiatry. 2001 Nov 15;50(10):729-42.

Lee KW, Hong JH, Choi IY, Che Y, Lee JK, Yang SD, Song CW, Kang HS, Lee JH, Noh JS, Shin HS, Han PL. Impaired D2 dopamine receptor function in mice lacking type 5 adenylyl cyclase. J Neurosci. 2002 Sep 15;22(18):7931-40.

Stoof JC, Kebabian JW. Opposing roles for D-I and D-2 dopamine receptors in efflux of cyclic AMP from rat neostriatum. Nature 1981; 294:366-368.

Neve KA, Seamans JK, Trantham-Davidson H. Dopamine receptor signaling.

J Recept Signal Transduct Res. 2004 Aug;24(3):165-205. Review.

Rodefer JS, Murphy ER, Baxter MG. PDElOA inhibition reverses subchronic PCP-induced deficits in attentional set-shifting in rats. Eur J Neurosci. 2005 Feb;21(4):1070-6.

Horvath EJ, Salamon C, Bakonyi A, Fekete MI, Palkovits M. [(3)H]-girisopam, a novel selective benzodiazepine for the 2, 3 -benzodiazepine binding site. Brain Res Brain Res Protoc. 1999 Jul;4(2):230-5.

Antoni FA. Molecular diversity of cyclic AMP signalling. Front Neuroendocrinol. 2000 Apr;21 (2): 103-32. Review.

Loughney K, Snyder PB, Uher L, Rosman GJ, Ferguson K, Florio VA. Isolation and characterization of PDElOA, a novel human 3', 5'-cyclic nucleotide phosphodiesterase. Gene. 1999 Jun 24;234(1): 109-17.

Fujishige K, Kotera J, Michibata H, Yuasa K, Takebayashi S, Okumura K, Omori K. Cloning and characterization of a novel human phosphodiesterase that hydrolyzes both cAMP and cGMP (PDElOA). J Biol Chem. 1999 Jun 25;274(26):18438-45.

Soderling SH, Bayuga SJ, Beavo JA. Isolation and characterization of a dual-substrate phosphodiesterase gene family: PDElOA. Proc Natl Acad Sci U S A. 1999 Jun 8;96(12):7071-6.

Nemoz G, Sette C, Conti M. Selective activation of rolipram- sensitive, cAMP-specific phosphodiesterase isoforms by phosphatidic acid. MoI Pharmacol. 1997 Feb;51(2):242-9.

Summary of the Invention

It has been found by the inventors of the present invention, that Tofisopam and both of its enantiomers are inhibitors of PDElOA, which indicate that these compounds will be useful for treatment of Psychosis and schizophrenia, especially the positive symptoms of schizophrenia and the cognitive deficits of schizophrenia.

The object of the present invention is therefore to provide pharmaceutical compositions useful for treatment of schizophrenia and psychosis. The compositions are in a preferred embodiment especially useful for treatment of cognitive deficits and the positive symptoms of schizophrenia. The present invention is based on the novel finding of the inventors, that Tofisopam is an inhibitor of PDElOA. The inventors have observed that both Tofisopam racemate, and each of the enantiomers (R and S) have the ability to inhibit PDElOA, with approximately the same IC50 value. This finding open new possibilities for the use of Tofisopam racemate, as well as each enantiomer as active compounds in compositions made for the treatment of psychosis and schizophrenia, especially for the treatment of the cognitive deficits and positive symptoms of schizophrenia.

Further, the inventors has observed that although both racemic Tofisopam and the S form of Tofisopam exhibit inhibition of PDE4D, this is not the case for R-Tofisopam, indicating that this enantiomer is less likely than the racemate or the S-enantiomer to cause emesis and other side effects.

The term "Tofisopam" as used throughout the description is intended to include any form of the compound, such as any isoform, any conformer, and any mixture thereof, pharmaceutically acceptable salts, e.g. pharmaceutically acceptable acid addition salts, hydrates or solvates of the base or salt, as well as anhydrates and also amorphous, or crystalline forms.

The present invention provides a method of treating an anxiety or psychotic disorder in a mammal, including a human, comprising administering to said mammal a therapeutically effective amount of a Tofisopam effective in treating said anxiety or psychotic disorder.

The invention also provides a method of treating an anxiety or psychotic disorder in a mammal, including a human, which comprises administering to said mammal a therapeutically effective amount of Tofisopam effective in inhibiting PDElOA.

Examples of psychotic disorders that can be treated according to the present invention include, but are not limited to, schizophrenia, for example of the paranoid, disorganized, catatonic, undifferentiated, or residual type; schizophreniform disorder; schizoaffective disorder, for example of the delusional type or the depressive type; delusional disorder; substance-induced psychotic disorder, for example psychosis induced by alcohol, amphetamine, cannabis, cocaine, hallucinogens, inhalants, opioids, or phencyclidine ; personality disorder of the paranoid type; and personality disorder of the schizoid type.

Examples of anxiety disorders that can be treated according to the present invention include, but are not limited to, panic disorder; agoraphobia; a specific phobia; social phobia; obsessive-compulsive disorder; post-traumatic stress disorder; acute stress disorder; and generalized anxiety disorder.

This invention also provides a method of treating a movement disorder selected from Huntington's disease and dyskinesia associated with dopamine agonist therapy in a mammal, including a human, which method comprises administering to said mammal an amount of Tofisopam effective in treating said disorder.

This invention also provides a method of treating a movement disorder selected from Huntington's disease and dyskinesia associated with dopamine agonist therapy in a mammal, including a human, which method comprises administering to said mammal an amount of Tofisopam effective in inhibiting PDElOA.

This invention further provides a method of treating a movement disorder selected from Parkinson's disease, restless leg syndrome, and essential tremor in a mammal, including a human, comprising administering to said mammal an amount of Tofisopam effective in treating said disorder.

This invention also provides a method of treating a movement disorder selected from Parkinson's disease, restless leg syndrome, and essential tremor in a mammal, including a human, comprising administering to said mammal an amount of Tofisopam effective in inhibiting PDElOA.

This invention also provides a method of treating a disorder selected from obsessive/compulsive disorders, Tourette's syndrome and other tic disorders in a mammal, including a human, which method comprises administering to said mammal an amount of Tofisopam effective in treating said disorder.

This invention also provides a method of treating obsessive/compulsive disorder, Tourette's syndrome and other tic disorders in a mammal, including a human, which method comprises administering to said mammal an amount of Tofisopam effective in inhibiting PDElOA.

This invention further provides a method of treating a drug addiction, for example an alcohol, amphetamine, cocaine, or opiate addiction, in a mammal, including a human, which method comprises administering to said mammal an amount of Tofisopam effective in treating drug addiction.

This invention also provides a method of treating a drug addiction, for example an alcohol, amphetamine, cocaine, or opiate addiction, in a mammal, including a human, which method comprises administering to said mammal an amount of Tofisopam effective in inhibiting PDElOA.

A "drug addiction", as used herein, means an abnormal desire for a drug and is generally characterized by motivational disturbances such a compulsion to take the desired drug and episodes of intense drug craving.

This invention further provides a method of treating a disorder comprising as a symptom a deficiency in attention and/or cognition in a mammal, including a human, which method comprises administering to said mammal an amount of Tofisopam effective in treating a deficiency in attention and/or cognition.

This invention also provides a method of treating a disorder comprising as a symptom a deficiency in attention and/or cognition in a mammal, including a human, which method comprises administering to said mammal an amount of Tofisopam effective in inhibiting PDElOA.

The phrase "deficiency in attention and/or cognition" as used herein in "disorder comprising as a symptom a deficiency in attention and/or cognition" refers to a subnormal functioning in one or more cognitive aspects such as memory, intellect, or learning and logic ability, in a particular individual relative to other individuals within the same general age population. "Deficiency in attention and/or cognition" also refers to a reduction in any particular individual's functioning in one or more cognitive aspects, for example as occurs in age-related cognitive decline.

Examples of disorders that comprise as a symptom a deficiency in attention and/or cognition that can be treated according to the present invention are dementia, for example Alzheimer's disease, multi-infarct dementia, alcoholic dementia or other drug-related dementia, dementia associated with intracranial tumors or cerebral trauma, dementia associated with Huntington's disease or Parkinson's disease, or AIDS-related dementia; delirium; amnestic disorder; post-traumatic stress disorder; mental retardation; a learning disorder, for example reading disorder, mathematics disorder, or a disorder of written expression; attention-deficit/hyperactivity disorder; and age-related cognitive decline.

This invention also provides a method of treating a mood disorder or mood episode in a mammal, including a human, comprising administering to said mammal an amount of Tofisopam effective in treating said disorder or episode.

This invention also provides a method of treating a mood disorder or mood episode in a mammal, including a human, comprising administering to said mammal an amount of Tofisopam effective in inhibiting PDElOA.

Examples of mood disorders and mood episodes that can be treated according to the present invention include, but are not limited to, major depressive episode of the mild, moderate or severe type, a manic or mixed mood episode, a hypomanic mood episode; a depressive episode with atypical features; a depressive episode with melancholic features; a depressive episode with catatonic features; a mood episode with postpartum onset; post- stroke depression; major depressive disorder; dysthymic disorder; minor depressive disorder; premenstrual dysphoric disorder; post- psychotic depressive disorder of schizophrenia; a major depressive disorder superimposed on a psychotic disorder such as delusional disorder or schizophrenia; a bipolar disorder, for example bipolar I disorder, bipolar 11 disorder, and cyclothymic disorder.

This invention further provides a method of treating a neurodegenerative disorder or condition in a mammal, including a human, which method comprises administering to said mammal an amount of Tofisopam effective in treating said disorder or condition.

This invention further provides a method of treating a neurodegenerative disorder or condition in a mammal, including a human, which method comprises administering to said mammal an amount of Tofisopam effective in inhibiting PDElOA.

As used herein, and unless otherwise indicated, a "neurodegenerative disorder or condition" refers to a disorder or condition that is caused by the dysfunction and/or death of neurons in the central nervous system. The treatment of these disorders and conditions can be facilitated by administration of an agent which prevents the dysfunction or death of neurons at risk in these disorders or conditions and/or enhances the function of damaged or healthy neurons in such a way as to compensate for the loss of function caused by the dysfunction or death of at-risk neurons.

The term "neurotrophic agent" as used herein refers to a substance or agent that has some or all of these properties. Examples of neurodegenerative disorders and conditions that can be treated according to the present invention include, but are not limited to, Parkinson's disease; Huntington's disease; dementia, for example Alzheimer's disease, multi-infarct dementia, AIDS-related dementia, and Fronto temperal Dementia; neurodegeneration associated with cerebral trauma; neurodegeneration associated with stroke, neurodegeneration associated with cerebral infarct;

hypoglycemia-induced neurodegeneration; neurodegeneration associated with epileptic seizure; neurodegeneration associated with neurotoxin poisoning; and multi-system atrophy. In one embodiment of the present invention, the neurodegenerative disorder or condition comprises neurodegeneration of striatal medium spiny neurons in a mammal, including a human. In a further embodiment of the present invention, the neurodegenerative disorder or condition is Huntington's disease.

"Neurotoxin poisoning" refers to poisoning caused by a neurotoxin. A neurotoxin is any chemical or substance that can cause neural death and thus neurological damage. An example of a neurotoxin is alcohol, which, when abused by a pregnant female, can result in alcohol poisoning and neurological damage known as Fetal Alcohol Syndrome in a newborn. Other examples of neurotoxins include, but are not limited to, kainic acid, domoic acid, and acromelic acid; certain pesticides, such as DDT; certain insecticides, such as organophosphates ; volatile organic solvents such as hexacarbons (e. g. toluene); heavy metals (e. g. lead, mercury, arsenic, and phosphorous); aluminum ; certain chemicals used as weapons, such as Agent Orange and Nerve Gas; and neurotoxic antineoplastic agents.

Further embodiments of the present invention encompass the use of Tofisopam, for the preparation of pharmaceutical compositions made for treatment of each of the above listed diseases.

In one embodiment, the compositions of the present invention are made for administration simultaneously with treatment with another drug made for the treatment of psychosis or schizophrenia.

Thus, the preferred embodiment of the present invention relates to the use of Tofisopam for preparation of a pharmaceutical composition for the treatment of psychosis or schizophrenia, preferably the positive symptoms and/or the cognitive deficits in schizophrenic patients.

In one embodiment, the present invention relates to the use of Tofisopam or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition as above, which is adapted for simultaneous administration of the active ingredients. In particular, such pharmaceutical compositions may contain the active ingredients within the same unit dosage form, e.g. in the same tablet or capsule. Such unit dosage forms may contain the active ingredients as a homogenous mixture or in separate compartments of the unit dosage form.

In another embodiment, the present invention relates to the use of Tofisopam or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition or kit as above, which is

adapted for sequential administration of the active ingredients. In particular, such pharmaceutical compositions may contain the active ingredients in discrete unit dosage forms, e.g. discrete tablets or capsules containing either of the active ingredients. These discrete unit dosage forms may be contained in the same container or package, e.g. a blister pack.

As used herein the term kit means a pharmaceutical composition containing each of the active ingredients, but in discrete unit dosage forms.

The invention also relates to a pharmaceutical composition or kit comprising Tofisopam or a pharmaceutically acceptable salt thereof and a compound, which is a serotonin reuptake inhibitor, or any other compound, which causes an elevation in extracellular 5-HT and optionally pharmaceutically acceptable carriers or diluents.

The pharmaceutical composition or kit of the invention may be adapted for simultaneous administration of the active ingredients or for sequential administration of the active ingredients, as described above.

Detailed Description of the Invention

The present invention relates to the use of Tofisopam or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition to be used either alone or in combination with an antipsychotic compound for the treatment of psychosis or schizophrenia.

In particular, the present invention relates to the use of Tofisopam or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical composition useful for the treatment of the cognitive deficits of schizophrenia and/or of the positive symptoms of schizophrenia.

In a further embodiment, the invention relates to the use of Tofisopam or a pharmaceutically acceptable salt thereof and a compound, which is antipsychotic or a compound which is known to be effective for the treatment of any of the diseases listed in EP1250923 as responsive for treatment with an inhibitor of PDElOA, for the preparation of a pharmaceutical composition for the treatment of diseases or disorders responsive to the therapeutic effect of a PDElOA inhibitor.

In a further embodiment, the invention relates to the use of Tofisopam or a pharmaceutically acceptable salt thereof and a compound, which is effective in the treatment of any of the diseases listed in EP1250923 as being sensitive for inhibition of PDElOA, for the preparation of a kit-of-parts

(kit) for the treatment of diseases or disorders responsive to the therapeutic effect of a PDElOA inhibitor.

The magnitude of a prophylactic or therapeutic dose of the active ingredient (e. g. Tofisopam, in a preferred embodiment R-Tofisopam is used) in the treatment or prevention of psychosis and schizophrenia will vary with the severity of the patient's affliction and the route of administration. A therapeutically effective dose and dose frequency will also vary according to the age, weight and response of the individual patient.

In general, the recommended daily dose range for the conditions described herein lies within the range of from about 10 mg to about 1200 mg per day, generally divided equally into doses given one to four times a day. Preferably, a daily dose range should be between 50 mg and 600 mg per day, usually divided equally into a two to four times a day dosing. Most preferably, a daily dose range should be between 100 mg and 400 mg per day, usually divided equally into a two to four times a day dosing. It may be necessary to use dosages outside these ranges in some cases, and the treating physician will know how to increase, decrease or interrupt treatment based upon patient response.

The various terms described above such as "therapeutically effective amount" are encompassed by the above- described dosage amounts and dose frequency schedule.

For use in treating or preventing anxiety or anxiety disorders, the physician will generally prescribe the period of treatment and frequency of dose Tofisopam on a patient-by-patient basis. In general, however, treatment or prevention of psychosis and schizophrenia with Tofisopam may be carried out for as long a period as necessary, either in a single, uninterrupted session or in discrete sessions. Most preferably, Tofisopam therapy may be carried out for a period of 4 to 18 weeks. Tofisopam may be administered before, along with, or after other psychoactive compounds, particularly those with antidepressant or antipsychotic activity. Such compounds include tricyclic antidepressants such as amitriptyline, clomipramine, doxepin, imipramine, trimipramine, amoxapine, desipramine, maprotiline, nortriptyline, and protryptiline; serotonin-reuptake inhibitors such as racemic fluoxetine and enantiomers, fluvoxamine, paroxetine, citalopram, escitalopram, sertraline, and ()-venlafaxine; atypical antidepressants such as bupropion, nefazodone, and trazodone; and other monoamine oxidase inhibitors, such as phenelzine, tranylcypromine, and (-)-selgiline, either singly or in combination. In particular, the present invention encompasses the use of tofisopam in conjunction with other known psychoactive medications.

Tofisopam may also be administered before, along with, or after traditional psychotherapy. Thus,

Tofisopam may be utilized in accordance with the present invention as an adjunct to conventional behavioral therapy or psychotherapy.

Any suitable route of administration may be employed for providing the patient with an effective dosage of Tofisopam. For example, oral, rectal, parenteral, transdermal, subcutaneous, sublingual, intranasal, intramuscular, intrathecal and the like may be employed as appropriate. Dosage forms include tablets, coated tablets, caplets, capsules (e. g. hard gelatin capsules), troches, dragees, dispersions, suspensions, solutions, patches and the like, including sustained release formulations well known in the art. See, e. c. Introduction to Pharmaceutical Dosaae Forms, 1985, Ansel, H. C, Lea and Febiger, Philadelphia, PA; Remington's Pharmaceutical Sciences, 1995, Mack Publ. Co., Easton, PA.

The pharmaceutical compositions of the present invention comprise Tofisopam as the active ingredient or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The term "pharmaceutically acceptable salt" refers to salts prepared from pharmaceutically acceptable non-toxic acids including inorganic acids and organic acids.

Since the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable nontoxic acids including inorganic and organic acids. Such acids include malic, acetic, benzene-sulfonic (besylate), benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic; nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like.

The acid addition salts of the compounds of the invention are pharmaceutically acceptable salts formed with non-toxic acids. Exemplary of such organic salts are those with maleic, fumaric, benzoic, ascorbic, succinic, oxalic, bis-methylenesalicylic, methanesulfonic, ethanedisulfonic, acetic, propionic, tartaric, salicylic, citric, gluconic, lactic, malic, mandelic, cinnamic, citraconic, aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic and theophylline acetic acids, as well as the 8-halotheophyllines, for example 8-bromotheophylline. Exemplary of such inorganic salts are those with hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric and nitric acids.

Particularly preferred are hydrobromic, hydrochloric, maleic, phosphoric, and sulfuric acids.

The compositions include compositions suitable for oral, rectal, transdermal, sublingual, and parenteral administration (including subcutaneous, intramuscular, intrathecal and intravenous), although the most suitable route in any given case will depend on the nature and severity of the condition being treated. The most preferred route of administration of the present invention is the oral route. The composition may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

In the case where an oral composition is employed, a suitable dosage range for use is, e. g., from about 10 mg to about 1200 mg per day, generally divided equally into a one to four times a day dosing, preferably from about 50 mg to about 600 mg per day, generally divided equally into a two to four times a day dosing and most preferably from about 100 mg to about 400 mg per day, generally divided equally into a two to four times a day dosing. Patients may be upward titrated from below to within this dose range to achieve satisfactory control or prevention of symptoms as appropriate.

In practical use, Tofisopam can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e. g., oral or parenteral (including intravenous injections or infusions). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, for example, suspensions, elixirs and solutions; or aerosols; or carriers such as starches, sugars, microcrystalline cellulose, stabilizers, diluents, granulating agents, lubricants, binders, fillers, disintegrating agents and the like in the case of oral solid preparations such as, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. The preferred solid oral preparation is tablets. The most preferred solid oral preparation is coated tablets. Because of their ease of administration tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.

The pharmaceutical compositions of the present invention may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydropropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes and/or microspheres.

In general, a controlled-release preparation is a pharmaceutical composition capable of releasing the active ingredient at the required rate to maintain constant pharmacological activity for a desirable period of time. Such dosage forms provide a supply of a drug to the body during a predetermined period of time and thus maintain drug levels in the therapeutic range for longer periods of time than conventional non- controlled formulations.

U. S. Patent No. 5,674,533 discloses controlled-release pharmaceutical compositions in liquid dosage forms for the administration of moguisteine, a potent peripheral antitussive.

U. S. Patent No. 5,059,595 describes the controlled-release of active agents by the use of a gastro- resistant tablet for the therapy of organic mental disturbances.

U. S. Patent No. 5,591,767 describes a liquid reservoir transdermal patch for the controlled administration of ketorolac, a non-steroidal anti- inflammatory agent with potent analgesic properties.

U. S. Patent No. 5,120,548 discloses a controlled-release drug delivery device comprised of swellable polymers.

U. S. Patent No. 5,073,543 describes controlled-release formulations containing a trophic factor entrapped by a ganglioside-liposome vehicle.

U. S. Patent No. 5,639,476 discloses a stable solid controlled-release formulation having a coating derived from an aqueous dispersion of a hydrophobic acrylic polymer. Biodegradable microparticles are known for use in controlled-release formulations.

U. S. Patent No. 5,354,566 discloses a controlled-release powder that contains the active ingredient.

U. S. Patent No. 5,733,566, describes the use of polymeric microparticles that release antiparasitic compositions. The use of R-tofisopam in a controlled- release formulation has not been previously disclosed.

The controlled-release of the active ingredient may be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds. Various mechanisms of drug release exist. For example, in one embodiment, the controlled-release component may swell and form porous openings large enough to release the active ingredient after administration to a patient.

The term "controlled-release component" in the context of the present invention is defined herein as a compound or compounds, such as polymers, polymer matrices, gels, permeable membranes, liposomes and/or microspheres, that facilitate the controlled-release of the active ingredient (i. e., R- tofisopam) in the pharmaceutical composition. In another embodiment, the controlled-release component is biodegradable, induced by exposure to the aqueous environment, pH, temperature, or

enzymes in the body. In another embodiment, sol-gels may be used, wherein the active ingredient is incorporated into a sol-gel matrix that is a solid at room temperature. This matrix is implanted into a patient, preferably a mammal, having a body temperature high enough to induce gel formation of the sol-gel matrix, thereby releasing the active ingredient into the patient.

Pharmaceutical stabilizers may also be used to stabilize compositions containing tofisopam or salts thereof; acceptable stabilizers include but are not limited to L-cysteine hydrochloride, glycine hydrochloride, malic acid, sodium metabisulfite, citric acid, tartaric acid and L-cysteine dihydrochloride.

Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, or tablets or aerosol sprays, each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in- water emulsion, or a water-in-oil liquid emulsion.

Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free- flowing form such as powder or granules, optionally mixed with one or more of a binder, filler, stabilizer, lubricant, inert diluent, and/or surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

Desirably, each tablet contains from about 10 mg to about 100 mg of the active ingredient, and each cachet or capsule contains from about 10 mg to about 200 mg of the active ingredient. In a preferred embodiment, the tablet, cachet or capsule contains one of four dosages: about 10 mg, about 50 mg, about 100 mg, and about 150 mg of active ingredient. For example, tablets containing 20 mg of active agent (e. g., optically pure S- or R-tofisopam may be prepared in a manner known in the art. The composition of one such tablet is as follows: Tablets Formula Quantity per Tablet in mg. Active ingredient 20.0 Magnesium stearate 1.0 Stearin 1.0 Talc 2.0 Gelatin 3.5 Maize starch 20.5 Lactose 122.0 Microcrvstalline cellulose 10.0 Several animal models are suitable for measuring the

relative activities of the optically pure R and S enantiomers of tofisopam, as well as racemic tofisopam, in treating anxiety in humans. Evaluation of the compounds is based on their relative potencies in a test designed to measure the anxiety generated by a novel situation. In the "Elevated Plus-maze" Test, anxiety is generated by placing the animals on the elevated open arm of an apparatus in the shape of a cross (or "plus") with two open and two enclosed arms.

Experimental Section

Example 1

The compounds of the invention may be prepared as follows:

Tofisopam Enantiomers

The molecular structure and conformational properties of tofisopam have been determined by NMR, CD and x-ray crystallographic methods. Visy, J. and Simongi, M., Chirality 1:271-275 (1989). The 2,3 diazepine ring exists in two kinds of boat conformation. In the case of the major conformers (+)- R and (-)S the ethyl group attached to the center of asymmetry C-5 has quasi-equatorial orientation while in the minor conformer (-)R and (+)S this group is positioned quasi-axially. As a result, racemic tofisopam in solution contain four molecular species, i.e., two enantiomers each of which exists in two chiral conformations. The sign of optical rotation is reversed upon inversion of the diazepine-ring. In crystal form tofisopam exists only as the major conformations, with levorotatory- tofisopam being of the (S) absolute configuration. Toth, G. et al.,J. Heterocyclic chem. 20:709- 713,1983. Fogassy, E. et al, In: Bio-Organic Heterocycles, Van der Plas, H. C, Otvos, L, Simongi, M., eds. Budapest Amsterdam: Akademia; Kiado-Elsevier, 1984, 229:233.

The chemical synthesis of the racemic mixture of tofisopam can be performed by the method described in U.S. Pat. No. 3,736,315. Racemic Tofisopam (Cas nr 22345-47-7) is also commercially available from these two sources: Sigma; catalog nr T8200 and Wako; catalog nr 203-15121. The chemical synthesis of the (R)-tofisopam can be performed by the method described in U.S. Pat. No. 6,080,736. The chemical synthesis of the (S)-tofisopam can be performed by the method described in U.S. Pat. No. 2005/10288277 Al. The separation of the tofisopam stereosiomers have also been described in J. Liq. Chrom. & ReI. TechnoL, 22(5), 713-719 (1999).

Furthermore, the (R)-tofisopam and (S)-tofisopam can be prepared from racemic tofisopam by resolution using chiral Super Critical Fluid (SFC) chromatography as described here:

Racemic tofisopam was subjected to resolution by chiral SFC using a Gilson SF3 supercritical fluid chromatography system equipped with chiralpak AD-H columns (4.6 mm x 25 cm for analytical runs). The particle size in the columns was 5 μm. A lmg/ml solution of racemic tofisopam in methanol was injected in 15 μL portions on the column. The column was eluted with carbondioxide - modifier (60:40). The modifier was methanol containing 0.1% diethylamine. The flow was 3 mL/min at a pressure of 200 bar. UV-detection was done by (230 nm and 250 nm). The R- enantiomer was isolated from two peaks (the two conformers of the R-isomer) with retention times 1.55 min and (peak 1) and 4.6 min and (peak 4) and, correspondingly, the S-isomer isolated from two peaks (the two conformers of the S-isomer) with retention times 1.7 min and (peak 2) and 3.5 min and (peak 3).

Preparative separations were done using the Berger Multigram®II prep-SFC system equipped with chiralpak AD-H columns (21 mm x 25 cm). The particle size in the columns was 5 μm. A lOOmg/lOml solution of racemic tofisopam in methanol was injected in 200 μL portions on the column. The column was eluted with carbondioxide - modifier (70:30). The modifier was methanol containing 0.1% diethylamine. The flow was 70 mL/min at a pressure of 100 bar. Fraction collection was triggered by UV-detection (230 nm). The fractions containing the separate products were pooled and evaporated in vacuo which gave the two enantiomers The R-enantiomer was isolated from two peaks (the two conformers of the R-isomer) with retention times 1.55 min and (peak 1) and 4.6 min and (peak 4) and, correspondingly, the S-isomer isolated from two peaks (the two conformers of the S-isomer) with retention times 1.7 min and (peak 2) and 3.5 min and (peak 3).

The absolute configuration of the enantiomers was determined by CD-spectroscopy and compared to the literature J. Liq. Chrom. & ReI. Technol., 22(5), 713-719 (1999).

Example 2

Testing for PDE4 and PDElOA inhibition

The compounds of the invention were tested in well recognised and reliable tests. The tests were as follows:

We tested tofisopam racemate and its enantiomers for inhibition of PDElOA and Pde4D. PDElOA was inhibited by tofisopam racemate with an IC50 of 305nM, while Pde4D was inhibited with an IC50 of 220nM/1067nM. Thus, tofisopam and its enantiomers are potent inhibitors of PDElOA and it will therefore be expected to have antipsychotic potential.

Active Pde4D and PdelOA enzyme can be and have been prepared in a number of ways for PDE assays (for example Nemoz et al 1997, Loughney et al 1999, Fujishige et al 1999, Soderling et al 1999). They can be expressed as full length proteins or as truncated proteins expressing as long as they express the catalytic domain. They can be prepared in different cell types for example insect cells or E. coli.

An example of a method to obtain catalytically active Pde4D and PdelOA is described below: The catalytic domain of human PDE4D (amino acids 252-579 from the sequence with accession number NP_006194) and human PDElOA (amino acids 440-779 from the sequence with accession number NP_006652) was amplified from total human brain total RNA by standard RT-PCR and cloned into the BamHl and Xhol sites of the pET28a vector (Novagen). Expression in coli was performed according to standard protocols. Briefly, the expression plasmids were transformed into the BL21(DE3) E. coli strain, and 50ml cultures inoculated with the cells being allowed to grow to an OD600 of 0.4-0.6 before protein expression was induced with 0.5mM IPTG using. Following induction, the cells were incubated overnight at room temperature, after which cells were collected by centrifugation. Cells expressing Pde4D were resuspended in 12 ml [50 mM NaHPO4-pH8.0, 300 mM NaCl, 10 mM imidazole and protease inhibitors], while cells expressing PdelOA were resuspended in 12 ml [50 mM TRIS-HCl-pH8.0, 1 mM MgC12 and protease inhibitors]. The cells were lysed by sonication, and after all cells were lysed, TritonXlOO was added according to Novagen protocols. PDE4D was purified by standard Nickel affinity chromatography, eluted with 25OmM imidazole and subsequently further purified by ion exchange on Q sepharose with pooling of the most active fractions. PDElOA was partially purified on Q sepharose with pooling of the most active fractions.

Both Pde4D and PdelOA assays have been described previously (Nemoz et al 1997, Loughney et al 1999, Fujishige et al 1999, Soderling et al 1999). They could for example be performed as described below:

PDE assays were performed in 60ul samples containing a fixed amount of the relevant PDE enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer [5OmM HEPES7.6; 1OmM MgC12], 0.1mg/ml BSA, 225 pCi of 3H-labelled cyclic nucleotide substrate, cold cyclic nucleotide substrate to a final concentration of 20OnM and varying amounts of tofisopam. PDE4D assays were performed with cAMP as a substrate, while PDElOA assays were performed with cGMP as a substrate Reactions were initiated by addition of cyclic nucleotide substrate, and reactions were allowed to proceed for one hour at room temperature before being terminated through mixing with 15ul 8 mg/ml yttrium silicate SPA beads (Amersham). Beads were allowed to settle for one hour in the

dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signal can be converted to activity relative to an uninhibited control (100%) and IC50 values can be calculated using the Xlfit extension to EXCEL.

Inhibition of the binding of Tofisopam and its enantiomers to the PDElOA and PDE4....

Table 1: Binding Data (% inhibition of binding at .. nM)

The results in Table 1 clearly show that Tofisopam racemate and both R- and S-Tofisopam exhibit a similar IC50 value with regard to inhibition of PDElOA. Further, it is very clear from Table 1, that while Tofisopam racemate and S-Tofisopam are relatively potent in inhibiting PDE4, the R-form of Tofisopam is approximately 10 times as weak an inhibitor of PDE4.

Thus, the compounds of the invention are therefore considered useful in the treatment of diseases responsive to inhibition of PDElOA. Further, the compounds of the invention are useful for the preparation of pharmaceutical compositions made for treatment of diseases responsive to inhibition of PDElOA (listed above). In a particular preferred embodiment, the R-enantiomer is used.

Formulation

The pharmaceutical formulations of the invention may be prepared by conventional methods in the art.

For example tablets may be prepared by mixing the active ingredient with ordinary adjuvants and/or diluents and subsequently compressing the mixture in a conventional tabletting machine. Examples of adjuvants or diluents comprise: corn starch, potato starch, talcum, magnesium stearate, gelatine, lactose, gums, and the like. Any other adjuvants or additives usually used for such purposes such as colourings, flavourings, preservatives etc. may be used provided that they are compatible with the active ingredients.

Solutions for injections may be prepared by dissolving the active ingredient and possible additives in a part of the solvent for injection, preferably sterile water, adjusting the solution to desired volume,

sterilising the solution, and filling it in suitable ampules or vials. Any suitable additive conventionally used in the art may be added, such as tonicity agents, preservatives, antioxidants, etc.