WO1993025203A1

WO1993025203A1 - Method of treating and protecting against central nervous system ischemia, hypoxia, degeneration, and trauma with a 5-aminocarbonyl-sh-dibenzo[a,d]cyclohepten-5,10-imine

Info

Publication number: WO1993025203A1
Application number: PCT/US1993/005845
Authority: WO
Inventors: Michael A. Rogawski
Original assignee: The United States Of America, Represented By The Secretary, Department Of Health And Human Services
Priority date: 1992-06-17
Filing date: 1993-06-16
Publication date: 1993-12-23
Also published as: AU4540193A

Abstract

A method of treating and protecting against central nervous system ischemia, hypoxia, degeneration, and trauma, particularly glutamate-mediated excitotoxicity, in mammals by administering a therapeutically effective amount of a 5-aminocarbonyl-5H-dibenzo[a,d]cyclohepten-5,10-imine or pharmaceutically acceptable salt thereof of formula (I), especially 5-aminocarbonyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine.

Description

METHOD OF TREATING AND PROTECTING AGAINST CENTRAL NERVOUS SYSTEM ISCHEMIA HYPOXIA, DEGENERATION, AND TRAUMA WITH A 5-AMINOCARBONYL-SH-DIBENZO (A.D) CYCL0HEPTEN-5. 10-IMINE.

TECHNICAL FIELD OF THE INVENTION

The present invention concerns a method of treating and protecting against central nervous system disorders, particularly central nervous system ischemia, hypoxia, degeneration, and trauma. In particular, the present invention concerns the use of 5-aminocarbonyl-5H- dibenzo[a,d]cyclohepten-5,10-imines to treat and protect against glutamate-mediated excitotoxicity in mammals.

BACKGROUND OF THE INVENTION Many compounds have been developed for the treatment of neuronal loss caused by excessive activation of glutamate receptors such as may occur in central nervous system ischemia, hypoxia, trauma, and neurodegenerative disorders such as Alzheimer's disease, 0 Down's syndrome, olivopontocerebellar atrophy, Korsa off's syndrome, or similar conditions. For example, U.S. Patent 4,399,141 discloses 5-methyl-10,ll- dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) and derivatives thereof. The principal clinical utility 5 of MK-801 has been shown to be anticonvulsant. MK-801 has also been reported to be an N-methyl-D-aspartate (NMDA) antagonist useful in the treatment of neurodegenerative diseases.

Similarly, U.S. Patent 5,011,834 discloses 10,5- 0 (iminomethano)-10,ll-dihydro-5H-dibenzo[a,d]cycloheptene (IDDC) and analogs thereof for treating or preventing neuronal loss in ischemia, hypoxia, hypoglycemia, brain and spinal cord trauma, as well as for the treatment of Alzheimer's disease, amyotrophic lateral sclerosis, 5 Huntington's disease, and Down's syndrome. Both MK-801 and IDDC bind with high affinity to the ion channel of the NMDA receptor (also known as the PCP receptor) in mammalian neurons. Although they are effective in protecting against glutamate-induced neuronal destruction, these high affinity compounds also cause a variety of unacceptable side effects at therapeutic doses including locomotor activation, impairment of motor performance, disruption of learning and memory, signs of sympathetic nervous system stimulation such as hypertension and tachycardia, stereotypies, and catalepsy. Derivatives of MK-801 have been disclosed which also purport to be anticonvulsants and NMDA antagonists useful in the prevention and treatment of neurodegeneration in a variety of pathological conditions. U.S. Patent 4,870,079 discloses hydroxy- and fluoro-derivatives of MK-801 in which the substituents are on non-benzenoid carbons. One of the derivatives is reportedly a major mammalian metabolite of MK-801. U.S. Patent 4,996,211 discloses other derivatives of MK-801 with a hydrocarbon or a hydrocarbyloxycarbonyl group of the formula -C0₂R in which R represents a hydrocarbon group. Other compounds related to MK-801 and IDDC for use in treating a variety of central nervous system disorders are disclosed in numerous other references, including U.S. Patents 3,509,158, 3,542,787, 3,597,433, 3,641,038, 3,716,541, 3,717,641, 3,892,756, 4,009,273, 4,052,508, 4,064,139, 4,232,158, 4,252,810, 4,374,838, 4,414,154, and 4,940,789.

None of these compounds, however, have been demonstrated to protect against glutamate-induced neuronal degeneration and also to have a favorable toxicity profile. Many of these compounds are not as therapeutically effective as is desired, and others are quite toxic or produce a variety of unacceptable side effects, including impairment of motor performance, disruption of learning and memory, stereotypies, and, at high doses, catalepsy. Moreover, and quite significantly, glutamate- induced neuronal degeneration can occur as a result of glutamate acting on non-NMDA receptors, that is o-amino- 3-hydroxy-5-methyl-4-isoxazole (AMPA) and kainate receptors, as well as NMDA receptors. Acceptable protection from glutamate-induced neuronal degeneration, therefore, may be achieved in many circumstances only by preventing the action of glutamate on both NMDA and non- NMDA receptors. Both MK-801 and IDDC, as well as their derivatives, have not been demonstrated to prevent the action of glutamate on non-NMDA receptors.

Since the existing compounds are of limited usefulness due to their lack of therapeutic effectiveness and/or unacceptable toxicity, there remains a need for a method of treating central nervous system disorders, particularly central nervous system ischemia, hypoxia, degeneration, and trauma, with compounds which are therapeutically effective in such treatments and which have acceptable levels of toxicity. It is an object of the present invention to provide such a method for the treatment of central nervous system ischemia, hypoxia, degeneration, and trauma. It is a further object of the present invention to provide a method for the treatment or prevention of glutamate- mediated excitotoxicity resulting from glutamate action on NMDA and non-NMDA receptors.

The present invention overcomes the problems associated with the existing compounds inasmuch as it is effective in protecting against glutamate-mediated cell damage, but does not cause side effects at therapeutic doses. The present invention, therefore, represents a significant therapeutic advance. The present invention concerns the use of a compound which (i) binds with low affinity to the PCP receptor and (ii) prevents glutamate-mediated excitotoxicity resulting from glutamate action on both NMDA and non-NMDA receptors. In these two respects, the compound used in the context of the present invention differs significantly in its biochemical actions from the aforementioned high affinity compounds such as MK-801. The present invention's biochemical actions and its highly efficacious neuroprotective activity is unexpected, but the fortuitous discovery provides for a practical treatment that is superior to previously disclosed treatments.

These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION The present invention encompasses a method of treating and protecting against central nervous system ischemia, hypoxia, degeneration, and trauma, particularly glutamate-mediated excitotoxicity, in mammals by administering a therapeutically effective amount of a 5-aminocarbonyl-5H-dibenzo[a,d]cyclohepten- 5,10-imine, especially5-aminocarbonyl-10,ll-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 depicts the results of an experiment determining cultured cortical neuron LDH release (U/ml) , which is an indication of neuronal disintegration, at various ADCI concentrations (μM) . The figure illustrates the protective effects of ADCI on cultured cortical neurons from glutamate-induced excitotoxicity.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated on the discovery that 5-aminocarbonyl-5H-dibenzo[a,d]cyclo- hepten-5,10-imines and pharmaceutically acceptable salts thereof, particularly 5-aminocarbonyl-10,ll-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine, can be used to effectively treat and protect cells against glutamate- mediated excitotoxicity. Such compounds are useful in the treatment and prevention of central nervous system ischemia, hypoxia, degeneration, and trauma in mammals with minimal toxicity and adverse side effects.

The active compound of the present inventive method is a compound from the class of 5-aminocarbonyl-5H- dibenzo[a,d]cyclohepten-5,10-iminesandpharmaceutically acceptable salts thereof. The 5-aminocarbonyl-5H- dibenzo[a,d]cyclohepten-5,10-imines have the formula:

wherein R_x, R₂, R₃, R₄, and R₅ are suitable substituent groups.

Preferably, each of R_x and R₂ is independently selected from hydrogen, alkyl groups of from 1 to 20 carbon atoms, alkenyl groups of from 2 to 20 carbon atoms, alkynyl groups of from 2 to 20 carbon atoms, cycloalkyl groups of from 3 to 8 carbon atoms, and cycloalkenyl groups of from 3 to 8 carbon atoms, although R_λ and R₂ may be taken together with the adjoining nitrogen in -ΝR;-^ to form an Ν-containing cyclic structure having 2 to 8 carbon atoms, each of R₃ and R₄ is independently selected from hydrogen, halogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms, alkynyl groups of from 2 to 10 carbon atoms, hydroxyl, amino, alkoxy, cyano, nitro, and mercapto, and R₅ is selected from hydrogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms, alkynyl groups of from 2 to 10 carbon atoms, hydroxyl, alkoxy, and phenyl, and R₁ and R₅ may be taken together to form a cyclic structure containing two nitrogen atoms and from 2 to 6 carbon atoms. Any of these R groups can be substituted with one or more suitable substituents, such as those selected from the group consisting of alkyl, alkenyl, oxo, thio, alkoxy, hydroxy, amino, phenyl, halogen, cyano, mercapto, thio, and combinations thereof. Any of the R groups may be also linear or branched.

More preferably, each of R± and R₂ is independently selected from the group consisting of hydrogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms, and alkylamino groups of from 2 to 10 carbon atoms, each of R₃ and R is independently selected from the group consisting of hydrogen and halogen, and R₅ is selected from the group consisting of hydrogen and alkyl groups of from 1 to 5 carbon atoms. Most preferably, each of R_λ and R₂ is independently selected from the group consisting of hydrogen, alkyl groups of from 1 to 5 carbon atoms, and alkylamino groups of from 2 to 5 carbons atoms, each of R₃ and R₄ is independently selected from the group consisting of hydrogen, chlorine, and fluorine, and R₅ is selected from the group consisting of hydrogen and methyl.

The family of compounds of Formula I include the tautomeric forms of the described compounds, isomeric forms such as diastereomers, and the pharmaceutically acceptable salts thereof. Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid and organic acids as maleic acid, succinic acid, and citric acid.

The most preferred compound for use in the context of the present invention is 5-aminocarbonyl-10,ll- dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine or ADCI. This compound has the formula:

Other species within the family of 5-aminocarbonyl- 5H-dibenzo[a,d]cyclohepten-5,10-imines are disclosed in U.S. Patent 5,196,415 and in U.S. patent application Serial No. 07/697,395 (filed on May 9, 1991). The preparation of 5-aminocarbonyl-5H-dibenzo[a,d]cyclo¬ hepten-5,10-imines, including 5-aminocarbonyl-10,11- dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine, is described in Monn et al., J. Med. Chem.. 33. 1069-1076 (1990) .

ADCI and its analogs have been shown to have relatively low toxicity and a minimum of side effects (Rogawski et al., J. Pharmacol. Exp. Ther.. 259(1) , 30-

37 (1991); Rogawski et al., Epilepsia. 31. 620 (1990)).

It has now been discovered that ADCI and its analogs, e.g., other 5-aminocarbonyl-5H-dibenzo[a,d]- cyclohepten-5,10-imines and pharmaceutically acceptable salts thereof, are useful in the treatment of central nervous system ischemia, hypoxia, degeneration, and trauma. In particular, ADCI and its analogs are useful in the treatment of, as well as the protection from, neuronal loss caused by excessive activation of glutamate receptors such as may occur in central nervous system ischemia, hypoxia, trauma, and neurodegenerative disorders. For example, 5-aminocarbonyl-5H- dibenzo[a,d]cyclohepten-5,10-imines are useful in the treatment of neurodegeneration in pathological conditions such as stroke, transient cerebral ischemic attack, cerebral ischaemia during cardiac pulmonary surgery or cardiac arrest, Alzheimer's disease, Down's syndrome, olivopontocerebellar atrophy, Korsakoff's syndrome, dementia associated with HIV infection, brain or spinal cord injury associated with trauma, and similar conditions. In particular, 5-aminocarbonyl-5H- dibenzo[a,d]cyclohepten-5,10-imines are effective in the treatment and prevention of glutamate-mediated excitotoxicity in mammals.

In this respect, it has been found that the low affinity NMDA antagonists 5-aminocarbonyl-5H-dibenzo- ta,d]cyclohepten-5,10-imines prevent the action of glutamate on both NMDA and non-NMDA receptors and are, therefore, superior in their ability to treat and prevent glutamate-mediated excitotoxicity in comparison to high affinity selective NMDA antagonists. Specifically, ADCI and its analogs have been found to be highly effective in protecting against seizures induced by 4-aminopyridine (Yamaguchi et al., Epilepsy Res.. 11. 9-16 (1992)) and dendrotoxin (Coleman et al., Brain Res. _f 575. 138-142 (1992)). Such seizures are caused by the massive release of glutamate acting on non-NMDA receptors. While ADCI and its analogs have been demonstrated to protect against the seizures resulting from glutamate acting on non-NMDA receptors, such a functionality of MK-801 and other selective NMDA antagonists has not been demonstrated. Indeed, MK-801 and other selective NMDA antagonists are completely ineffective in protecting against such seizures resulting from glutamate acting on non-NMDA receptors, thereby demonstrating the unique nature of the present inventive method.

The precise mechanism of ADCI and its analogs is not known with certainty. Since ADCI is not a non-NMDA antagonist, it is currently believed that the superior properties of ADCI and its analogs in treating and preventing glutamate-mediated excitotoxicity may be due to the ability of ADCI and its analogs to prevent glutamate release from presynaptic nerve terminals, thereby preventing activation of non-NMDA receptors. The present inventive method includes the administration to an animal, particularly a human, of a therapeutically effective amount of ADCI and its analogs. The use of such a compound in treating animals, particularly humans, circumvents the disadvantages, particularly the toxicity, resulting from the use of other active agents for the effective treatment and prophylaxis of central nervous system ischemia, hypoxia, degeneration, and trauma. ADCI and its analogs may be administered in accordance with the present inventive method in any suitable manner, preferably with pharmaceutically acceptable carriers. One skilled in the art will appreciate that suitable methods of administering such a compound in the context of the present invention to an animal are available, and, although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective reaction than another route. Pharmaceutically acceptable carriers are also well-known to those who are skilled in the art. The choice of carrier will be determined in part by the particular compound, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention.

Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water or saline, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to *the active ingredient, such carriers as are known in the art.

The active ingredient, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

Suitable formulations for rectal administration include, for example, suppositories, which consist of the active ingredient with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the active ingredient with a base, such as, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.

Formulations suitable for parenteral administration, such as, for example, by intravenous, intramuscular, intradermal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidantε, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the animal over a reasonable time frame. The dose will be determined by the strength of the particular compound employed and the condition of the animal, as well as the body weight of the animal to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound in a particular animal. In determining the effective amount of the active ingredient to be administered in the treatment or prophylaxis of central nervous system ischemia, hypoxia, degeneration, and trauma, the physician need only evaluate the effects of the active ingredient in the animal being treated by incrementally increasing the dosage in increments ranging from about 0.1 to about 20 mg/kg body weight to achieve as high a cumulative level of the active ingredient in the animal as possible without adverse side-effects being manifested. The active ingredient will typically be administered to the animal being treated for a time period ranging from a day to a few weeks, consistent with the clinical condition of the treated animal. This dosage regimen will usually be within the range of about 0.1 to about 500 mg/kg body weight per day, although higher dosage amounts may be required in some situations. ADCI and its analogs will be generally administered to a mammal, such as a human, in an amount of about 0.5 mg/kg to about 100 mg/kg of body weight per day, more typically in an amount of about 1 mg/kg to about 50 mg/kg of body weight per day. A suitable dose can be administered in suitable subdoses per day, particularly in a prophylactic regimen. The precise treatment level will be dependent upon the response of the animal, e.g., the human patient, being treated. The following examples further illustrates the present invention but, of course, should not be construed as in any way limiting its scope.

Example 1 This example demonstrates that ADCI protects against glutamate-mediated excitotoxicity in cerebellar granule cells. Glutamate is considered a mediator of neuronal death in numerous pathologic conditions, including central nervous system ischemia, hypoxia, degeneration, and trauma. —-"

The procedure which was followed was similar to that set forth in Novelli et al., Brain Res.. 451_f 205-

212 (1988), and Lysko et al.. Brain Res.. 499. 258-266

(1989) . Specifically, primary cultures of rat cerebellar granule cells were prepared from 8-day old rats. The cultures were grown with cytosine arabinoside on poly-L-lysine coated petri dishes at 37°C in 5% C0₂ at 100% humidity. The cells were incubated in glucose- free buffer for 40 minutes and then with 100 μM glutamate for 30 minutes. In some of the cultures, ADCI was added at various concentrations 20 minutes prior to the glutamate addition.

Neurotoxicity was estimated at the end of the glutamate exposure by staining with fluorescein diacetate (5 μg/ml) for 5 minutes. Viability was determined as the number of cells retaining fluorescein. In cultures not exposed to ADCI, less than 5% of the cells were viable. ADCI produced a concentration- dependent increase in viability with a 50% inhibition of cell death at a concentration of 45 μM. At sufficiently high concentrations, there was complete protection from cell death.

Example 2 This example further demonstrates that ADCI protects against glutamate-mediated excitotoxicity. A sample of ADCI was provided to a research facility unaffiliated with the inventor to confirm the results set out in Example 1.

Since excessive exposure to glutamate causes a large influx of calcium and sodium and since increased levels of intracellular free calcium have been implicated as being directly responsible for starting a destructive cascade of events which results in cell death (Choi, Cerebrovascular and Brain Metabolism Reviews, 2., 105-47 (1990)), a lactate dehydrogenase (LDH) efflux assay was used as a biochemical method for measuring glutamate-induced neuronal cell death and to evaluate the effect of ADCI in protecting against such cell death. The leakage of this enzyme from a variety of cultured cell types, including neurons exposed to glutamate, has been used to quantitate cell injury (Koh et al., J. Neuroscience Methods. 20. 83-90 (1987); Frandensen et al., Neuroscience Int'l. 10. 583-91 (1987); Patel et al., J. Neurochem.. 54. 349-54 (1990); Choi et al., J. Neuroscience. ϋ(l) , 185-96 (1988)). The amount of LDH efflux correlates in a linear fashion with the number of neurons damaged by glutamate exposure (Koh et al., J. Neuroscience Methods. 20. 83-90 (1987)).

In conducting this example, mixed neuronal cultures, containing both neuronal and glial elements, were prepared by removing the cerebral cortices from Charles River rat embryos on the eighteenth day of gestation. The cortices were halved and incubated at room temperature in 0.1% trypsin in Hank's Balanced Salt Solution (HBSS) lacking Ca²⁺ and Mg⁺ (Sigma H2387) but supplemented with 20 mM N-[2-hydroxyethyl] piperazine- N'-[2-ethanesulfonic acid] (HEPES) for 15 minutes. The tissue was then rinsed three times with minimum essential medium (MEM), with Earles's salts, and L- glutamine (Gibco 410-1100EB) supplemented with glucose (30 mM) and sodium bicarbonate (26 mM) . Cells were dissociated by trituration. The resulting suspension was then diluted in a plating medium consisting of 10% fetal calf serum and 10% horse serum (Sigma) in MEM. Cells were plated at approximately 5,000,000 cells per well on 12-well plates that had been previously coated with poly-1-lysine and that contained 0.5 mL of plating media. Cells were maintained at 37°C in a humidified 5% C0₂ atmosphere.

One day after plating, the culture media were replaced with plating media lacking fetal serum. On the fourth day after plating, nonneuronal cell division was reduced by exposure to 15 μg/mL 5-fluoro-2-doxyuridine and 35 μg/mL uridine in medium lacking fetal serum. Subsequent feedings, beginning on day 10 or 11, involved removing approximately half of the medium from the cells and replacing it with medium lacking fetal serum. Additional feedings usually occurred every other day until day 17 or 18. Only mature (15 to 20 days in vitro) cortical cultures were selected for study; whenever possible, comparison of treatments were made between matched sister cultures from a single plating.

On day 17 or 18, exposure to glutamate at room temperature was in an exposure solution (substituted for culture medium by triple exchange) with the following composition: 120 mM NaCl, 5.4 mM KC1, 0.8 mM MgCl₂, 1.8 mM CaCl₂, 20 mm HEPES (pH 7.4 at 25°C) , 15 mM glucose, and 500 μM glutamate. After 5 minutes, the exposure solution was thoroughly replaced with culture medium (lacking serum) by triple exchange, and the plates were returned to the incubator for LDH measurements the following day.

ADCI was added to the glutamate exposure medium at concentrations of 0 (control), 1, 3, 10, 30 and 100 μM and incubated with the culture along with the added 500 μM glutamate for 5 minutes. The exposure medium was then replaced with culture medium (lacking serum) by triple exchange. Overall neuronal injury was quantitatively assessed by measurement of LDH released into the culture medium one day after glutamate exposure. This method has been previously described (Koh et al., J. Neuroscience Methods. 20. 83-90 (1987); Klingman et al., J. Neuroscience Methods. 31 47-51 (1989); Wroblowski et al., Proc. Soc. Exp. Biological Medicine. 90, 210-213 (1955)). By using a minor modification of this method, a kinetic enzyme assay was carried out for the purpose of LDH measurement. Twenty- five μL medium samples from each well were placed into a clean 96-well microtiter plate and mixed with 225 μL 0.1 M KP0₄ buffer (pH 7.5 at 25°C) containing 30 μg NADH (reduced α-nicotinamide adenine dinucleotide) . Following a 10 minute incubation at 37°C, the reaction was initiated by the addition of 30 μL 2.4 mM sodium pyruvate to each well. The absorbance of each well at 340 nM, an index of NADH concentration, was measured repetitively with a Multiskan MCC/340 plate reader with an interval time of 5 seconds (sufficient to complete a single reading of 96 samples) , for a total of 2 minutes. LDH activity (conventional units per mL) was then calculated from the absorbance readings by applying a correction factor for temperature and light path. Accuracy of the assay was verified by periodic checks with a standard LDH enzyme solution. A small amount of LDH was present in the media of cultures carried through the exposure protocol but not exposed to glutamate. No significant LDH efflux occurred when cultures of cortical glia were similarly exposed to glutamate.

The results of this example are set out in Figure 1, which depicts the effect of ADCI concentration (μM) on LDH release (U/ml) as an indication of glutamate- induced excitotoxicity of the cultured cortical neurons. The mean background level of LDH release in the absence of added glutamate was determined and is depicted in Figure 1. The control for this example involved the addition of 500 μM glutamate with no ADCI and resulted in a mean LDH release which is also depicted in Figure 1. Without the addition of ADCI, widespread neuronal disintegration was visually observed 18 to 24 hours after the addition of the glutamate. The addition of 1, 3, 10, 30, and 100 μM ADCI with the 500 μM glutamate resulted in a concentration-dependent decrease in LDH release and a corresponding decrease in neuronal disintegration. The mean values of LDH release for the various ADCI concentrations are set out in Figure 1. In general, as the concentration of ADCI was increased, LDH release decreased, with the exception of an anomalously large decrease in LDH release at an ADCI concentration of 1 μM which is believed to have been an experimental aberration and is not set out in Figure 1. A concentration of 30 μM ADCI was shown to be nearly entirely effective in counteracting the adverse effect of the added glutamate, while a concentration of 100 μM ADCI was shown to be effective in counteracting the adverse effect of the added glutamate as well as any glutamate which otherwise existed in the cultured samples, e.g., which was produced by the cultured cells in situ.

All of the references cited herein, including articles, patents, and patent applications, are hereby incorporated in their entireties by reference. While this invention has been described with an emphasis upon a preferred embodiment, it will be obvious to those of ordinary skill in the art that variations in the preferred composition and method may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating and protecting against central nervous system ischemia, hypoxia, degeneration, and trauma in mammals which comprises administering to a mammal in need thereof a therapeutically effective amount of a 5-aminocarbonyl-5H-dibenzo[a,d]cyclohepten- 5,10-imine or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein said 5-amino- carbonyl-5H-dibenzo[a,d]cyclohepten-5,10-imine has the formula

wherein each of R-L and R₂ is independently selected from hydrogen, alkyl groups of from 1 to 20 carbon atoms, alkenyl groups of from 2 to 20 carbon atoms, alkynyl groups of from 2 to 20 carbon atoms, cycloalkyl groups of from 3 to 8 carbon atoms, and cycloalkenyl groups of from 3 to 8 carbon atoms, although R₁ and R₂ may be taken together with the adjoining nitrogen in -NR^ to form an N-containing cyclic structure having 2 to 8 carbon atoms, each of R₃ and R₄ is independently selected from hydrogen, halogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms, alkynyl groups of from 2 to 10 carbon atoms, hydroxyl, amino, alkoxy, cyano, nitro, and mercapto, and R₅ is selected from hydrogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms, alkynyl groups of from 2 to 10 carbon atoms, hydroxyl, alkoxy, and phenyl, and R₁ and R₅ may be taken together to form a cyclic structure containing two nitrogen atoms and from 2 to 6 carbon atoms, or a pharmaceutically acceptable salt thereof.

3. The method of claim 2, wherein each of R_x and R₂ is independently selected from the group consisting of hydrogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms, and alkylamino groups of from 2 to 10 carbon atoms, each of R₃ and R is independently selected from the group consisting of hydrogen and halogen, and R₅ is selected from the group consisting of hydrogen and alkyl groups of from 1 to 5 carbon atoms.

4. The method of claim 2, wherein each of R₁ and R is independently selected from the group consisting of hydrogen, alkyl groups of from 1 to 5 carbon atoms, and alkylamino groups of from 2 to 5 carbons atoms, each of R₃ and R₄ is independently selected from the group consisting of hydrogen, chlorine, and fluorine, and R₅ is selected from the group consisting of hydrogen and methyl.

5. The method of claim 1, wherein said compound is 5-aminocarbony1-10,ll-dihydro-5H-dibenzo[a,d]cyclo¬ hepten-5,10-imine.

6. The method of claim l, wherein said compound is administered in an amount of about 0.5 mg/kg to about 100 mg/kg of body weight per day.

7. The method of claim 6, wherein said compound is administered in an amount of about 1 mg/kg to about 50 mg/kg of body weight per day.

8. The method of claim 6, wherein said mammal is a human.

9. The method of claim 8, wherein said compound is 5-aminocarbonyl-10,ll-dihydro-5H-dibenzo[a,d]cyclo¬ hepten-5,10-imine..

10. The method of claim 7, wherein said mammal is a human and said compound is 5-aminocarbonyl-10,ll- dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine.

11. The method of claim 1, wherein said central nervous system ischemia, hypoxia, degeneration, and trauma is the result of glutamate-mediated excitotoxicity.

12. The method of claim 11, wherein said 5-amino- carbonyl-5H-dibenzo[a,d]cyclohepten-5,10-imine has the formula

wherein each of R₁ and R₂ is independently selected from hydrogen, alkyl groups of from 1 to 20 carbon atoms, alkenyl groups of from 2 to 20 carbon atoms, alkynyl groups of from 2 to 20 carbon atoms, cycloalkyl groups of from 3 to 8 carbon atoms, and cycloalkenyl groups of from 3 to 8 carbon atoms, although R_x and R₂ may be taken together with the adjoining nitrogen in -NR--^ to form an N-containing cyclic structure having 2 to 8 carbon atoms, each of R₃ and R₄ is independently selected from hydrogen, halogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms, alkynyl groups of from 2 to 10 carbon atoms, hydroxyl, amino, alkoxy, cyano, nitro, and mercapto, and R₅ is selected from hydrogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms, alkynyl groups of from 2 to 10 carbon atoms, hydroxyl, alkoxy, and phenyl, and R_x and R₅ may be taken together to form a cyclic structure containing two nitrogen atoms and from 2 to 6 carbon atoms, or a pharmaceutically acceptable salt thereof.

13. The method of claim 12, wherein each of R_λ and R₂ is independently selected from the group consisting of hydrogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms, and alkylamino groups of from 2 to 10 carbon atoms, each of R₃ and R₄ is independently selected from the group consisting of hydrogen and halogen, and R₅ is selected from the group consisting of hydrogen and alkyl groups of from 1 to 5 carbon atoms.

14. The method of claim 12, wherein each of R_λ and R₂ is independently selected from the group consisting of hydrogen, alkyl groups of from 1 to 5 carbon atoms, and alkylamino groups of from 2 to 5 carbons atoms, each of R₃ and R₄ is independently selected from the group consisting of hydrogen, chlorine, and fluorine, and R₅ is selected from the group consisting of hydrogen and methyl.

15. The method of claim 11, wherein said compound is 5-aminocarbonyl-10,ll-dihydro-5H-dibenzo[a,d]cyclo¬ hepten-5,10-imine.

16. The method of claim 11, wherein said compound is administered in an amount of about 0.5 mg/kg to about 100 mg/kg of body weight per day.

17. The method of claim 16, wherein said compound is administered in an amount of about 1 mg/kg to about 50 mg/kg of body weight per day.

18. The method of claim 16, wherein said mammal is a human.

19. The method of claim 18, wherein said compound is 5-aminocarbonyl-10,ll-dihydro-5H-dibenzo[a,d]cyclo¬ hepten-5,10-imine.

20. The method of claim 17, wherein said mammal is a human and said compound is 5-aminocarbonyl-10,ll- dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine.

21. The method of claim 2, wherein each of R_λ and R is independently selected from hydrogen, alkyl groups of from 1 to 20 carbon atoms, alkenyl groups of from 2 to 20 carbon atoms, alkynyl groups of from 2 to 20 carbon atoms, cycloalkyl groups of from 3 to 8 carbon atoms, and cycloalkenyl groups of from 3 to 8 carbon atoms, each of R₃ and R₄ is independently selected from hydrogen, halogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms-;"^" alkynyl groups of from 2 to 10 carbon atoms, hydroxyl, amino, alkoxy, cyano, nitro, and mercapto, and R₅ is selected from hydrogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms, alkynyl groups of from 2 to 10 carbon atoms, hydroxyl, alkoxy, and phenyl.

22. The method of claim 21, wherein each of R₃ and R₄ is hydrogen.

23. The method of claim 22, wherein each of _λ and R₂ is independently selected from the group consisting of hydrogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms, and alkylamino groups of from 2 to 10 carbon atoms, and R₅ is selected from the group consisting of hydrogen and alkyl groups of from 1 to 5 carbon atoms.

24. The method of claim 22, wherein each of R_x and R₂ is independently selected from the group consisting of hydrogen, alkyl groups of from 1 to 5 carbon atoms, and alkylamino groups of from 2 to 5 carbons atoms, and R₅ is selected from the group consisting of hydrogen and methyl.

25. The method of claim 12, wherein each of R± and R₂ is independently selected from hydrogen, alkyl groups of from 1 to 20 carbon atoms, alkenyl groups of from 2 to 20 carbon atoms, alkynyl groups of from 2 to 20 carbon atoms, cycloalkyl groups of from 3 to 8 carbon atoms, and cycloalkenyl groups of from 3 to 8 carbon atoms, each of R₃ and R₄ is independently selected from hydrogen, halogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms, alkynyl groups of from 2 to 10 carbon atoms, hydroxyl, amino, alkoxy, cyano, nitro, and mercapto, and R₅ is selected from hydrogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbo atoms, alkynyl groups of from 2 to 10 carbon atoms, hydroxyl, alkoxy, and phenyl.

26. The method of claim 25, wherein each of R₃ and R₄ is hydrogen.

27. The method of claim 26, wherein each of R_λ and R₂ is independently selected from the group consisting of hydrogen, alkyl groups of from 1 to 10 carbon atoms, alkenyl groups of from 2 to 10 carbon atoms, and alkylamino groups of from 2 to 10 carbon atoms, and R₅ is selected from the group consisting of hydrogen and alkyl groups of from 1 to 5 carbon atoms.

28. The method of claim 26, wherein each of R_λ and R₂ is independently selected from the group consisting of hydrogen, alkyl groups of from 1 to 5 carbon atoms, and alkylamino groups of from 2 to 5 carbons atoms, and R₅ is selected from the group consisting of hydrogen and methyl.