TUMOR NECROSIS FACTOR ALPHA (TNF- alpha ) INHIBITING PHARMACEUTICALS

Abstract: Pharmaceutical compositions are described for preventing TNF toxicity, comprising as active ingredient the stereospecific (+) enantiomer, having (3S, 4S) configuration of DELTA <6> tetrahydrocannabinol type compounds. The compositions are particulary effective in alleviating and even preventing neurotoxicity due to elevated levels of TNF, including septic shock, cachexia and trauma. They are also effective in the treatment of certain chronic degenerative diseases characterized by TNF production, including autoimmune diseases.

Description: TUMOR NECROSIS FACTOR ALPHA (TNF-Α) INHΓBΠTNG PHARMACEUTICALS FIELD OF THE INVENTION The present invention relates to the use of pharmaceutical compositions for inhibiting the production or blocking the action of Tumor Necrosis Factor α (TNF-α), and for preventing or alleviating diseases and conditions associated with this cytokine, such as septic shock or cachexia. Said pharmaceutical compositions comprise as their active ingredient the stereospecific (+) enantiomers, having (3S,4S) configuration, of Δ 6 - tetrahydrocannabinol (THC) type compounds of general formula (I), as defined hereinbelow. BACKGROUND OF THE INVENTION Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine, which has been implicated in inflammatory and immunological responses, as well as in pathogenesis of endotoxic and septic shock (reviewed by Tracey and Cerami, Ann. Rev. Med. 45, 491-503, 1994; Hauser et al. Clin. Infect Dis.. 18, suppl. 2, 205-216, 1994). TNF is one of several cytokines released mainly by mononuclear phagocytic cells in response to various stimuli. Though the role of cytokines in pathophysiological states has not been fully elucidated, it appears that TNF-α is a major mediator in the cascade of injury and morbidity. Among the serious disease states related to the production of TNF-α, a partial list includes the following: septic shock; endotoxic shock; cachexia syndromes associated with bacterial infections (e.g., tuberculosis, meningitis), viral infections (e.g., AIDS), parasite infections (e.g., malaria), and neoplastic disease; autoimmune disease, including some forms of arthritis (especially rheumatoid and degenerative forms); and adverse effects associated with treatment for the prevention of graft rejection. Septic shock is an often lethal syndrome associated with the massive release of host cytokines due to stimuli present on, or released by, invasive micro-organisms. These invasive stimuli induce polyclonal stimulation ofthe infected host immune system, and include both lipopolysaccharide (LPS), an endotoxin that stimulates B-cells and macrophages, and superantigens which are exotoxins that stimulate T-cells. Septic shock has been recognized generally as a consequence of gram-negative bacterial infection, but it is now clear that it can also result from infection with gram positive micro-organisms and probably also by fungi, viruses and parasites. The microorganism itself, its components or products trigger the host cells, especially the macrophages, to release inflammatory mediators such as TNF-α, thereby initiating a cascade of events leading to cachexia, sepsis syndrome and septic shock. TNF-α is a major mediator initiating septic shock, and therefore stands out as a potential therapeutic target (Lynn and Cohen, Clin. Infect. Dis. 20, 143-158, 1995) Despite vast improvements in intensive care and antibiotic therapy, septic shock remains associated with a very high rate of mortality (30 to 90%). The poor prognosis for this syndrome is due to the fact that this severe complication of infection results in multiple organ failure, even when the actual infection itself is- successfully treated. It is, therefore, apparent that effective therapies for this syndrome are an unmet medical need. Various therapies have been suggested for the treatment of septic shock syndrome, but as yet none of these has proven to be clinically efficacious. Antibodies against TNF-α prevent the detrimental effects of superantigen ( ethke et al., J. Exp. Med. 175, 91-98, 1992) or LPS (Beutler et al., Science 229, 869-871, 1985). The use of anti-TNF antibodies to treat septic shock is disclosed for example in WO 92 16553 (Centocor Inc.). WO 92/01472 (Celltech Ltd.) discloses a multivalent immunoglobulin used to treat diseases associated with elevated cytokine levels. Various cytokines that inhibit TNF secretion can also reduce the toxicity of LPS action (Tzung et al., Eur. J. Immunol. 22, 3097-3101, 1992; Gerard et al., J. Exp. Med. 177, 547-550, 1993). Soluble forms ofthe TNF binding protein (TBP) (Nophar et al. EMBO J., 9, 3269- 3278, 1990) may prevent the action of TNF by preventing binding to its receptors. Specific classes of compounds have been suggested for the treatment of diseases associated with elevated TNF or other inflammatory mediators, as disclosed for example in WO 95/11014 (Searle & Co.); WO 95/09627, WO 95/09624 and WO 95/09623 (Smith¬ Kline Beecham Corp.); WO 95/09619 (Wellcome Found.); WO 95/03051 (Pharmacia AB); WO 95/01980 (Pfizer Inc.); EP 629401 (Bayer AG); WO 93/14082 (SmithKline Beecham Corp.); and WO 89/05145 (Hoechst Roussel Pharm Inc.). None of these disclosures is relevant to the present invention, which deals with a class of compounds developed as non-psychotropic analogs of tetra- hydrocannabinol (THC), the active ingredient of marijuana. Some ofthe compounds of general formula (I) are disclosed in U.S. Patents Nos. 4,179,517 and 4,876,276 and 5,284,867. As disclosed in said U.S. patents, these essentially pure synthetic (+)-(3S,4S)-THC derivatives and analogues are devoid of any undesired cannabimimetic psychotropic side-effects. These known compounds have been described as having analgesic, antiemetic, antiglaucoma and neuroprotective activity. According to the present invention, it is now disclosed that the said known compounds, as well as some novel compounds, in addition to having said analgesic, antiemetic, neuroprotective and anti-glaucoma activity, are unexpectedly also effective against the diseases and conditions mentioned above, by virtue of their ability to block the production or action of TNF-α. SUMMARY OF THE INVENTION The present invention provides pharmaceutical compositions for reducing and even preventing morbidity and mortality associated with the production of TNF-α, or other cytokines. The present compositions are also effective in alleviating other cytokine induced damage including the wasting or cachexia associated with ALDS, tuberculosis, neoplasia or trauma, and may prevent or ameliorate other disease states associated with the production of cytokines, including malaria and parasitic infections. The compositions ofthe present invention are also effective in the treatment of certain chronic degenerative diseases including arthritis and other autoimmune afflictions which are characterized by production of TNF-α. In this conneαion, the compositions ofthe present invention are contemplated as therapeutically effeαive in the treatment of multiple sclerosis. The present invention relates to pharmaceutical compositions for the purposes set out above, in which the artive ingredient is a compound ofthe general formula I: having the (3S,4S) configuration and being essentially free ofthe (3R.4R) enantiomer, wherein A B indicates an optional 1 (2) or 6(1) double bond, R is (a) -Q wherein Q is a heterocyclic moiety having a labile hydrogen atom so that said moiety aαs as a carboxylic acid analogue, (b) -R"X wherein R' is C r C 5 alkyl and X is halogen, -OR" wherein R" is hydrogen, C C 5 alkyl, or -OC(O)R'" wherein R'" is hydrogen or C r C 5 alkyl, (c) -R'N(R") 2 wherein R' is C 1 -C 5 alkyl and each R", which may be the same or different, is hydrogen or C 1 -C 5 alkyl optionally containing a terminal -OR" or -OC(O)R m moiety wherein R'" is hydrogen or C r C 5 alkyl, (d) -R' wherein R' is C -C 5 alkyl, (e) -R'OR'" wherein R is C C 5 alkyl and R" is hydrogen or C C 5 alkyl, or (f) -R'-C(O)OR'", wherein R' may be absent and R' and R'" are as defined above; G is (a) halogen, (b) C C 5 alkyl, or (c) -ORi wherein Ri is (a') -R", wherein R" is hydrogen or Ci-Cj alkyl optionally containing a terminal -OR' H or -OC(0)R'" moiety wherein R" is hydrogen or C C 5 alkyl, or (b') -C(0)R'" wherein R m is as previously defined; and R 2 is (a) C1-C12 alkyl, (b) -OR"", in which R"" is a straight chain or branched C -C 9 alkyl which may be substituted at the terminal carbon atom by a phenyl group, or (c) -(CH 2 ) n OR m wherein n is an integer of 1 to 7 and R" is hydrogen or C 1 -C3 alkyl. In a currently preferred group of compounds, R 2 designates a 1,1-dimethylalkyl radical or a 1,2-dimethylalkyl radical with a total of at least 7 carbon atoms. Also preferred are precursors of such compounds. Particularly preferred compounds are those wherein R 2 is 1,1-dimethylheptyl or 1,2-dimethylheptyl. It is these embodiments of R 2 that are found in THC and its analogues. However, for the cytokine-inhibiting aαivity that charaαerizes the present invention, it is believed that any lower or mid-range alkyl substituent will be suitable at this position. The compositions ofthe present invention are particularly effeαive in alleviating and even preventing morbidity and mortality associated with the produαion of TNF-α, or other cytokines. Methods are provided for treatment of diseases associated with the production of TNF-α by administering to a patient in need thereof a composition comprising as aαive ingredient a therapeutically effeαive amount of a compound of general formula I. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the attenuating effeα of HU-211 on TNF aαivity (Fig 1 A) and in reducing TNF levels in the serum of mice (Fig. B) following the administration of endotoxin, LPS. Figure 2 shows the proteαive effeα of HU-211 in preventing mortality of mice following the administration of LPS alone, (Fig 2A) or following its administration to galaαose-amine- sensitized mice (Fig 2B). Figure 3 shows the beneficial effeα of HU-211 on blood pressure (Fig. 3 a) and on the hematocrit (Fig. 3b) of rats, following LPS administration. Figure 4 shows the beneficial effeα of HU-211 on the clinical outcome (A), on TNF levels (B) and on cerebral edema (C) in rat brain after closed head injury. The effeαs of pentoxifylline are shown for comparison (B,C). Figure 5 shows the beneficial effeα of HU-211 in collagen induced arthritis in rats. Figure 6 shows the improved clinical outcome in experimental allergic encephalomyelitis in rats treated with HU-211. DETAILED DESCRIPTION OF THE INVENTION The present invention provides pharmaceutical compositions for reducing and even preventing morbidity and mortality associated with the produαion of TNF-α, or other cytokines. The present compositions are also effeαive in alleviating other cytokine induced damage including wasting or cachexia associated with AIDS, tuberculosis, neoplasia or trauma, and may prevent or ameliorate other disease states associated with the produαion of cytokines, including parasitic infeαions. The compositions ofthe present invention may also be effeαive in the treatment of certain chronic degenerative diseases including arthritis and other autoimmune affliαions which are charaαerized by produαion of TNF-α. In this conneαion, the compositions ofthe present invention are contemplated as therapeutically effeαive in the treatment of multiple sclerosis. The present invention relates to pharmaceutical compositions for the purposes set out above, in which the aαive ingredient is a compound ofthe general formula (I): having the (3S,4S) configuration and being essentially free ofthe (3R.4R) enantiomer, wherein A B indicates an optional 1(2) or 6(1) double bond, R is (a) -Q wherein Q is a heterocyclic moiety having a labile hydrogen atom so that said moiety acts as a carboxylic acid analogue, (b) -RX wherein R * is C r C 5 alkyl and X is halogen, -OR" wherein R" is hydrogen, C r C 5 alkyl, or -OC(O)R"' wherein R" is hydrogen or Ci-Cs alkyl, (c) -R'N(R") 2 wherein R' is C C 5 alkyl and each R", which may be the same or different, is hydrogen or C 1 -C5 alkyl optionally containing a terminal -OR" or -OC(O)R'" moiety wherein R" is hydrogen or C 1 -C5 alkyl, (d) -R wherein R is C 2 -C 5 alkyl, (e) -ROR" wherein R is C1-C5 alkyl and R" is hydrogen or C C 5 alkyl, or (f) -R'-C(O)OR'" ) wherein R may be absent and R' and R'" are as defined above; G is (a) halogen, (b) C1-C5 alkyl, or (c) -ORi wherein Ri is (a') -R", wherein R" is hydrogen or C 1 -C 5 alkyl optionally containing a terminal -OR" or -OC(O)R" moiety wherein R" is hydrogen or C 1 -C5 alkyl, or (b 1 ) -C(0)R" wherein R" is as previously defined; and R 2 is (a) C r Cι 2 alkyl, (b) -OR"", in which R"" is a straight chain or branched C 2 -C 9 alkyl which may be substituted at the terminal carbon atom by a phenyl group, or (c) -(CH 2 )„OR'" wherein n is an integer of 1 to 7 and R" is hydrogen or C1-C5 alkyl. In a currently preferred group of compounds, R 2 designates a 1,1-dimethylalkyl radical or a 1,2-dimethylalkyl radical with a total of at least 7 carbon atoms. Also preferred are precursors of such compounds. Particularly prefe ed compounds are those wherein R 2 is 1,1-dimethylheptyl or 1,2-dimethylheptyl. It is these embodiments of R 2 that are found in THC and its analogues. However, for the cytokine-inhibiting aαivity that charaαerizes the present invention, it is believed that any lower or mid-range alkyl substituent will be suitable at this position. A preferred compound, with which many ofthe physiological experiments have been carried out, is the compound which may be referred to as the (+)-(3S,4S)-l,l- dimethylheptyl homologue of 7-hydroxy-Δ 6 -tetrahydrocannabinol. Said compound is designated hereinafter as HU-211, or by the trivial chemical name dexanabinol. It is stressed that all the compounds are ofthe (+)-(3S,4S) configuration, essentially free ofthe (-)-(3R,4R) enantiomer, the latter known to possess the undesired psychotropic side-effeαs. Thus, for example, the enantiomers ofthe synthetic cannabinoid 7-hydroxy-Δ 6 - tetrahydrocannabinol 1,1-dimethylheptyl homologue, have been described [Mechoulam, R., et al, Tetrahedron: Asymmetry V. 315-319, 1990; Mechoulam, R. et al., Experientia 44: 762-764, 1988]. The (-)-(3R,4R) enantiomer, herein designated HU-210, is a highly potent cannabimimetic compound (nearly 100 times more active than Δ-l -tetrahydrocannabinol, the aαive component of hashish). The (+)-(3S,4S) enantiomer, herein designated HU-211, while known to be aαive as a neuroproteαive agent, an analgesic and as an anti-emetic, is inaαive as a cannabimimetic even at doses several thousand times higher than the ED 30 of HU-210 (Mechoulam, R. et ai, Experientia 44: 762-764, 1988). Another family of stereospecific compounds that falls within the scope ofthe present invention is the family of analogs comprising (3S,4S)-delta-6- tetrahydrocannabinol-7-oic acids and derivatives thereof, in which R in formula 1 bears a carboxylic acid, as exemplified by the prototype compound HU-245. HU-245 and its analogs are disclosed in intemationai patent application WO 93/05031 which is hereby incorporated in its entirety by reference. As mentioned above, the compounds ofthe general formula (I) as defined herein are substantially devoid of cannabimimetic central nervous system activity. Pharmacology The novel compositions contain in addition to the aαive ingredient conventional pharmaceutically acceptable carriers, diluents and the like. Solid compositions for oral administration such as tablets, pills, capsules or the like may be prepared by mixing the aαive ingredient with conventional pharmaceutically acceptable ingredients such as com starch, laαose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate and gums with pharmaceutically acceptable diluents. The tablets or pills can be coated or otherwise compounded with pharmaceutically acceptable materials known in the art to provide a dosage form affording prolonged aαion or sustained release. Other solid compositions can be prepared as suppositories, for reαal administration. Liquid forms may be prepared for oral administration or for injeαion, the term including subcutaneous, transdermal, intravenous, intrathecal, and other parenteral routes of administration. The liquid compositions include aqueous solutions, with or without organic cosolvents, aqueous or oil suspensions, flavored emulsions with edible oils, as well as elixirs and similar pharmaceutical vehicles. In addition, the compositions ofthe present invention may be formed as aerosols, for intranasal and like administration. The aαive dose for humans is generally in the range of from 0.05 mg to about 50 mg per kg body weight, in a regimen of 1-4 times a day, or by constant infusion. However, administration every two days or even less frequently may also be possible, as the drug has a rather prolonged action. The preferred range of dosage is from 0.1 mg to about 25 mg per kg body weight. However, it is evident to one skilled in the art that dosages would be determined by the attending physician, according to the disease to be treated, method of administration, patient's age, weight, contraindications and the like. All the compounds defined above are inhibitors of tumor necrosis faαor alpha (TNF-α), and can be used as aαive ingredients of pharmaceutical compositions for treatment of one, or simultaneously several, symptoms ofthe disorders defined above. The effeαive dosages are essentially similar, and the more pronounced effeα is that of inhibition of TNF-α production, in addition to the known charaαeristics of these compounds. However, it is important to note that the compounds and compositions ofthe present invention may exert their beneficial effeαs by other mechanisms, and specifically may prevent the aαion of TNF-α indirectly via their induαion of and/or action on other cytokines. For example, the compositions ofthe present invention can prevent, or at least alleviate, IL-1 and IL-6 produαion, as well as poisoning caused by nitric oxide. The compounds ofthe present invention are administered for the above defined purposes in conventional pharmaceutical forms, with the required solvents, diluents, excipients, etc. to produce a physiologically acceptable formulation. They can be administered by any ofthe conventional routes of administration. The required dose for humans ranges from 0.005 mg kg to about 50 mg/kg per unit dosage form. The most preferred unit dose range is from about 0.1 mg/kg to about 20 mg kg body weight. It will be appreciated that the most appropriate administration ofthe pharmaceutical compositions ofthe present invention will depend on the type of injury or disease being treated. Thus, the treatment of septic shock will necessitate systemic administration ofthe drug as rapidly as possible after diagnosis ofthe condition. On the other hand, diminution or prophylaxis of chronic degenerative damage will necessitate a sustained dosage regimen. HU-211 conveys significant proteαion in different models of septic shock associated with baαerial infeαion or in models of traumatic shock. The invention also relates to methods of treatment ofthe various pathological conditions described above, by administering to a patient a therapeutically effective amount ofthe compositions ofthe present invention. The term administration as used herein encompasses oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, intrathecal, reαal and intranasal administration. It was shown previously that the pharmacological profile of HU-211, and some other compounds constituting the active ingredients ofthe present compositions, includes the induαion of stereotypy and locomotor hyperaαivity (Feigenbaum et al, 1989 ibid.). These effeαs were observed at doses far exceeding those contemplated for use in conneαion with repression of cytokines. Indeed Phase I clinical trials of HU-211, administered intravenously to healthy human volunteers showed no evidence of toxicity or psychoactivity of these compounds within the dose ranges contemplated for use in humans. Test systems Evaluation ofthe therapeutic effeαs of HU-211 and its analogs has now been carried out in a series of experimental systems of increasing sophistication to support the utility of these drugs as inhibitors of TNF-α. The TNF inhibitory and/or proteαive effects have been evaluated both in vitro and in vivo. These beneficial protective effeαs have been corroborated in the following systems: (a) Lowering TNF produαion in rat brain after LPS administration: The administration of LPS to experimental animals triggers the biosynthesis of large quantities of TNF. After binding to its receptor, TNF induces a wide variety of cellular responses, which are implicated in the pathogenesis of septic shock. Rats injeαed with LPS into their brain (intra-cerebral-ventricular, icv, injeαion) serve as a model for endotoxic shock, and the ability of compounds to inhibit the produαion in the brain of any ofthe mediators (e.g. TNF, interleukins) which are activated in response to LPS is a measure of their potency as anti-endotoxic agents. (h) Reduced TNF levels in the serum of mice after LPS administration: An early response to LPS administration is the elevation of serum levels of TNF, which peaks at 1.5-2 hr after LPS administration. Mice treated with LPS serve as a model for the effeα of drugs on reducing TNF levels under these conditions. (c) Reduced LPS-induced hypotension in the rat: A prominent manifestation of endotoxemia is hypotension, and the effeα of a drug on mean arterial blood pressure (MABP) indicates its potency as an anti-endotoxic shock agent. The protective effeα of a test compound is evaluated in rats treated with LPS, in which the blood pressure and other vital signs are monitored up to 4 hrs after induαion of endotoxemia . (d) Improved clinical outcome after closed head injury in rats: Closed head injury is associated with high mortality and morbidity. It induces edema formation, blood-brain-barrier (BBB) disruption, neuronal cell death and impairment of motor and cognitive funαions. Animals subjeαed to head trauma in a controlled fashion serve as models in which to test drugs of therapeutic potential. Test compounds can be evaluated both for improved clinical outcome and for reduαion of edema induced by closed head injury. The ability of compounds to reduce the severity of neurological symptoms and to reduce brain edema is considered a measure of their potency in reducing brain damage. (e Improved clinical outcome in experimental autoimmune diseases in rats: Autoimmune diseases are associated with elevated levels of TNF. The models which are most conveniently studied are experimental allergic encephalomyelitis (EAE) and experimental autoimmune arthritis in rodents. EAE is an autoimmune neurological disease elicited by sensitization ofthe animals to myelin basic protein from the central nervous system, which is also known as basic encephalitogenic protein. EAE is considered by many to represent a model ofthe human disease multiple sclerosis. Experimental autoimmune arthritis is induced in animals by immunization with collagen in complete Freund's adjuvant. The ability of compounds ofthe general formula I to prevent or attenuate the clinical symptoms of these autoimmune diseases was tested. Each of these systems represents an aspeα of endotoxin toxicity which is amenable to intervention by pharmaceutical agents. It is likely that the compounds ofthe present invention exert their demonstrated protective effeαs by virtue of their ability to prevent the produαion of TNF-α. Nevertheless, it cannot be ruled out that their aαivity is mediated by other cytokines or additional mechanisms. The prototype drug used for evaluation of prevention of release of TNF-α is the compound pentoxifylline, a known inhibitor of TNF release, and we have evaluated the similarities and differences between the biological aαivities of this compound and HU-211, as summarized in Table 1. This evaluation clearly supports the concept that HU-211 is not aαing solely by blocking the release of tumor necrosis faαor. Rather the therapeutic effeαs of HU- 11 may be attributable to additional mechanisms including suppression of TNF produαion or blockade of TNF aαion, among others. Importantly, it has been shown that the aαivity of these compounds is stereospecific and that the psycho tropic (-) compounds are of lower activity or even not active in these SUBSTITUTE SHEET {RULE 26) systems. Thus, HU-210 is not effeαive in preventing the aαion of TNF in vivo. This supports the concept that the suppression of TNF is not the known immuno-modulatory aαivity, such as has been described for a variety of psychoaαive enantiomers of cannabinoids and analogs (Lynn and Herkenham, J. Pharmacol, and Exp. Ther., 286, 1612- 1623, 1993). Even if the (-) compounds were as effeαive as the (+) compounds ofthe present invention the severe psychotropic aαivity ofthe former would make their medical use prohibitive. Compounds Experiments have shown that the (+)-(3S,4S) stereoisomer ofthe compound of formula I wherein A B designates a 6(1) double bond, R is methyl, G is OH, and R 2 is 1,1-dimethylheptyl and the compound of formula I wherein A B designates a 1(2) double bond, R is methyl, G is OH, and R 2 is 1,1-dimethylheptyl, both said compounds being essentially free ofthe (-)-(3R,4R) enantiomer, have praαically the same aαivity as that of the compound designated HU-211. The former compound is designated compound Vb Ln U.S. Patent No. 4, 179,517; the latter compound is compound Xlb therein. In addition it has been found that some novel compounds of general formula (I), wherein R designates CH 2 OR and R designates an acyl group also have the desired aαivity. These novel compounds may be prepared, for example, by esterification of compounds of general formula (I) wherein R designates CH 2 OH and G is OH, under conditions which favor the formation ofthe desired monoesters with relatively high yield. Among the novel compounds tested, monoesters including nicotinate, succinate and maleate are preferred. Most preferred compounds include the glycinate ester and N- substituted glycinate ester salts, such as the trimethyl- and triethylammonium acetate derivatives ofHU-211. These novel compounds have the added advantage of being soluble in some aqueous solutions, whereas the parent compounds are extremely hydrophobic. The highly lipophilic charaαer of HU-211 enables the compound to enter the central nervous system, as it readily passes the blood-brain barrier. However, the high lipid solubility is also associated with very poor solubility in water. This makes the development of formulations suitable for intravenous administration of HU-211 difficult, hampering its clinical application. Water-soluble derivatives of HU-211 designed to readily release the drug by hydrolysis following i.v. administration might overcome this problem and will be used as prodrugs. On the other hand, if the obtained derivatives are hydro lytically stable but possess intrinsic biological aαivity they could be used as easy-to-formulate analogs (congeners) possessing significant NMDA antagonist aαivity. The novel derivatives are obtained by the attachment of polar or permanent charge bearing groups to the allylic (C-7) or phenolic (C-3') hydroxylic funαionalities of HU-211, the two sites suitable for reversible structural modification, through a carboxylic ester or phosphate linkage. Examples TNF is released by mononuclear phagocytes and from astrocytes in response to infeαion or endotoxin. It has been implied that TNF mediates the lethal effeα of endotoxic shock. In the next examples we describe the use of LPS as a model for endotoxic shock in rats and mice and the effeα of HU-211 on a) TNF produαion in response to LPS challenge, and b) on some physiological manifestations of LPS toxicity. The following examples are intended to illustrate the present invention and these are to be construed in a non-limitative manner. Physiological Example 1: The effect of HU-211 on rat brain production of TNF after LPS administration. The biological aαivity (bioassay) of TNF in extraαs prepared from rat brains following intracerebral injection of LPS was assessed. LPS administration triggers the produαion of TNF and the effect of HU-211, and other analogs administered 15 min before LPS was evaluated 2 hr later. HU-211 and HU-245 significantly attenuated the increase in biological aαivity of TNF in homogenates prepared from brains of LPS treated rats. Experimental procedure: LPS (50 ug, dissolved in 20 ul saline) was injeαed into the cerebral lateral ventricle and within 5 min of injeαion, HU-211 7.5 mg/kg iv was administered. Two hours later, the rats were decapitated, their brains removed, and segments ofthe cortical tissue taken for cytokines extraαion in DMEM (Dulbecco's modified Eagle's medium), and analysis of biological aαivity using bioassay (Shohami et. al., J. Cereb. Blood Flow Metabol., 14, 615-619, 1994). SUBSTTTUTE SHEET (RULE 26) Results: The aαivity of TNF (expressed as S50, see Shohami et al. ibid., for details) in control (LPS) treated rats was 407±172 whereas in the HU-211- treated rats it reached only 43±21. These results clearly demonstrate that HU-211 reduced the biological aαivity of TNF in the brain homogenates. In contrast, the (-) enantiomer, which is denoted HU-210 did not affeα TNF levels when administered to rats under similar conditions (S 50 - 660±267 in LPS treated rats vs. 783±410 in the LPS + HU-210 treated rats). This establishes the stereospecificty ofthe effeα ofthe non-psychotropic (+) enantiomer. The evaluation of analogs was extended to include rats treated with HU-245 at a dose of 5 mg/kg. Upon bioassay of TNF, these animals were found to have a S5 0 of 89±36, vs. 448±146 found for the LPS control animals. The results obtained in the bioassay of TNF activity were confirmed by ELISA. Conclusion : One ofthe typical host responses to LPS is increased produαion and aαivity of TNF, which is implicated in the pathophysiology of endotoxic shock. The present results demonstrate that HU-211, but not its enantiomer HU-210, may reduce this local response (LPS was injected direαly into the brain, where the TNF production was assessed). HU-245 treated animals also had a significantly lowered level of TNF aαivity. Physiological example 2: The effect of HU-211 on serum levels and biological activity of TNF in LPS treated mice. The following experiment was designed to assess the peripheral effeα of HU-211 on serum levels of TNF in LPS treated mice. Serum levels of TNF peak within 1.5 hr following LPS administration, and at this time point, we assessed both TNF aαivity and levels in the serum. HU-211 and HU-245, when coadministered with LPS, significantly reduced both levels and aαivity of TNF in the serum of mice. Determination of TNF activity: Experimental procedure Mce (C57BIJ6) were injeαed ip with lOOμg LPS, concomitantly with HU-211, HU-210, or HU-245 (200μg mouse), and 90 min later they were bled, the sera separated and TNF was assayed for its activity (bioassay) and levels (using ELISA kit). Results: Figure 1 (A&B) depiαs the aαivity (S50) and levels (pg ml) of TNF in the sera ofthe LPS treated mice. It is apparent that HU-211 attenuated the LPS-induced produαion and action of TNF to a very pronounced extent. HU-245 also showed a pronounced effeα on TNF levels, as determined by bioassay. The S 5 o found for animals treated with HU-245 was 44±11 vs. 1233±237 found for control animals treated with LPS alone. Physiological example 3: Protective effect of HU-211 on survival of LPS treated mice The effeα ofHU-211 on the survival of mice, in response to administration of LPS was assessed. The survival was followed up to 48 hr. HU-211, administered 30 min prior to LPS, significantly reduced mortality ofthe mice. Animals and materials I BALB/c male mice 20-25 gr (Anilab, Israel); HU-211 5% in cosolvent, (Pharmos Ltd., Israel, batch no. 4T01HS); Blank cosolvent, (Pharmos batch no. c-00-502002); LPS, E.Coli 055:B5, (Difco USA, batch no. 1001007); Saline (Braun, Germany batch no. 3431A41A). Procedure: Animals are administered with either HU-211 at a dose of 10 mg/kg i.p. or vehicle alone (10 ml/kg i.p.), and 30 minutes later with LPS TV. Two dose levels of LPS were tested in the present study: 10 and 15 mg/kg. Mortality rate was recorded once a day for 2 days. RESULTS (Figure 2A) LPS 15 mg kg dose level caused 67% mortality (4/6, survival rate of 33 %) 24 hours post vehicle adπύnistration. By 48 hours post vehicle administration none of the vehicle treated mice survived. LPS 15 mg/kg dose level caused 33% mortality (2/6, 67% survival rate) 24 hours post HU-211 administration. By 48 hours post HU-211 administration none of the animals survived. The difference between HU-211 activity and vehicle was not statistically significant, at this dose level. LPS 10 mg/kg dose level caused 57% mortality (8/14, 43% survival rate) 24 hours post vehicle administration. At 48 hours after vehicle administration none of the animals survived. The same dose level caused only 9% mortality (1/12, 91 % survival rate) 24 hours post HU-211 administration. After 48 hours 4 out of the 12 tested animals survived (33%). None of the animals survived 72 hours post LPS administration. These differences were statistically significant, p < 0.02 Fisher exact test for both 24 and 48 hours. CONCLUSIONS HU-211 at a dose level of 10 mg/kg i.p. administered 30 minutes before LPS administration, induced a statistically significant protection (p< 0.02) at both 24 and 48 hrs. after LPS administration. Physiological example 4: Protection against LPS-induced hypotension The purpose of this study was to test the possible effect of HU-211 on the cardiovascular alterations induced by LPS administration. Animals and materials: Male and female Sprague Dawley rats (Harlan, Israel); HU-211 5% in cosolvent, (Pharmos Ltd., Israel, batch no. 4T01HS); Blank cosolvent, (Pharmos batch no. c-00- 502002); LPS, E.Coli 055:B5, (Difco USA, batch no. L001007); Saline (Braun, Germany batch no. 3431A41A); Halothane anesthetic (Fluthane, Zeneca, U.K.. batch no. ML040); Heparin 5000 IU/ml (Chaoy, France batch no. 1842). Procedure: Rats were individually anesthetized with halothane in 70% nitrogen, 30% oxygen. The right femoral artery was catheterized using polyethylene tubes (PE-50, Clay Adams, USA), and connected to a computerized physiograph through a pressure transducer (XTT Vigo, USA). The animals are then connected via a rectal probe to the above physiological recorder. Rectal temperature is kept throughout the study between 37°C and 38°C, using a heating lamp. Following 10-15 minutes of baseline recording, animals are intravenously administered an injection of saline, vehicle alone or HU-211 at a dose of 4 mg/kg. As soon as possible after completion of the first injection (2-5 minutes later) they are administered an intravenous injection of LPS at a dose of 15 mg/kg (in a volume of 3 ml/kg). Blood pressure and heart rate are recorded for the following 4 hrs. The hematocrit is measured and recorded just prior to drug administration, and 1, 2, and 3 hours later. At the end of the follow up period, animals are sacrificed by deep anesthesia with halothane. Results Administration of LPS at a dose of 15 mg/kg to rats caused a reduction of around 30% in mean arterial blood pressure (MABP). This drop in blood pressure was abolished by pre- administration of HU-211 (at a dose of 4 mg kg) intravenously. A transient hypotension was evident 10 minutes after LPS administration in the HU-211 treated rats. This lasted for 5-10 minutes and by 30 minutes post-LPS administration normotension was regained. The differences in MABP were statistically significant between the HU-211 treated and the control groups (Student's t-test), as shown in Fig. 3a. LPS also induced an increase of about 10% in hematocrit. This increase was prevented by administration of HU-211 at a dose of 44 mg/ml administered intravenously 2-3 minutes before LPS administration (Figure 3b). These differences were statistically significant 60 and 120 minutes post LPS administration. Physiological example 5: Protection against septic shock. The effect of HU-211 in endotoxic shock was assessed in a model of lethal toxicity by lipopolysacharide (LPS) in D-galactosamine (D-GALN)-sensitized mice (Galanos et al. PNAS 76: 5739, 1979, Lehman et al. J. Exp. Med. 165: 657, 1987). In this model 100% mortality occurs within 6-8 hrs of administration of the combined treatment. Experimental procedure: LPS (lOng/mouse) and D-GALN (18μg/mouse) were coadministered to 20 mice (C57BIJ6). Half of the mice served as controls, and the other were treated within 5 min of injection with HU-211 (7.5 mg/kg body weight), followed by 8 additional injections every hour. The survival was followed up to 24 hrs, and the results of this experiment are depicted in Table 1. Table 1. The effect of HU-211 on mice survival after endotoxic shock exp. no lethality in control group : lethality in HU-211 treated group j dead total dead/total 1 5/5 1/5 2 10/10 1/10 3 5/5 0/5 total: 20/20 (0% survival) 2/20 (90% survival) The treatment with HU-211 rescued 90% of the mice (Fig 2B), with 18 out of 20 HU- 211 treated mice surviving the shock, whereas none of the controls (20 mice) survived. Conclusion: To date, mortality from septic shock remains very high, despite advances in surgical and medical sciences. The results of our experiment are encouraging and may serve as a basis for saving lives of patients with septic shock. Physiological example 6: The effect of HU-211 on TNF production in a rat model of head trauma The effect of HU-211 on TNF production was assessed in a model of head trauma (HT) in rats. Injury was induced in anesthetized rats by a weight-drop device followed by a recovery period of up to 30 days. This type of trauma produces brain edema (i.e. increase in water content, decrease in specific gravity in the brain), breakdown of the blood brain barrier (BBB), clinical and cognitive dysfunction. HU-211 significantly reduces reduced edema and improved the clinical outcome measured 48 hours after HT (Shohami et al, J. Neurotrawna 10. 109, 1993; Brain Res. 674, 55-62, 1995) Experimental procedure: The model was described in detail by Shapira et al. , Crit. Care Med. 16: 258-265, 1988. Rats were subjected to head trauma (HT) by a weight- drop device and surviving rats were followed up for 30 days post trauma. In this experiment we compared the effects of two agents: 1) pentoxyfilline (PTX), a methylxanthine known for its ability to prevent or attenuate the LPS-induced release of TNF both in vitro and in vivo. This effect was found to be exerted at the transeriptional level (Doherty et al., 1991); and 2) HU-211, which was previously shown to have potent cerebroprotective effects in the CHI model (Shohami et al. loc. cit., Figure 4A). The levels of TNF were measured after CHI in order to determine whether the beneficial effect of HU-211 is also associated with suppression of TNF production or activity. RESULTS As shown in Figure 4, the protective effect of HU-211 is significantly superior to that of pentoxyfilline in terms of brain tissue water content (a measure of the anti-edema effect of the compounds) and in terms of the brain levels of TNF after CHI. CONCLUSION Severe head injury, or cerebral ischemia, is associated with a high mortality rate (exceeding 50%) and poor functional outcome. Despite extensive clinical and experimental research, there are no well-defined therapies for these conditions. There are very few available treatments for brain injury today and the gradual progressive biochemical changes which occur after head trauma can lead to the evolution of permanent neuronal damage. The results clearly demonstrate that the compounds of the instant invention, namely HU-211, possess protective properties in a model of closed head injury in rats. Physiological example 7: Attenuation of experimental autoimmune arthritis in mice The purpose of this study is to test the activity of HU-211 vs. arthritis, induced by collagen type 2 incorporated into complete Freund's adjuvant. MATERIALS AND METHODS CD-I male mice (27-33 gr.), five in each treatment group (supplied by Harlan Israel) were used in the present study. Each animal was administered with collagen type 2 (Sigma, batch no. 111H7180) 100 μg/ml in 0.1 ml complete Freund's adjuvant (Sigma batch no. 084H8800). The collagen was administered s.c. into the base ofthe tail. The volume of each hind paw was measured using a plathysmometer (Hugo Basill, Italy) before collagen administration and on days 1, 4, 7, 10, 13 and 16 of treatment (30 minutes after drug treatment). Five treatment groups were tested in the present study: vehicle (blank cosolvent) 10 ml kg once daily, HU-211 10 mg/kg every three days, HU-211 20 mg/kg once daily, HU-211 20 mg/kg every 3 days and diclofenac 10 mg/kg (10 ml kg) every 3 days. All treatments were administered intraperitoneally. HU-211 was used from a 5% preparation in cosolvent (Pharmos batch no. PRN-C-08623041), by diluting with saline (B. Braun batch no. 3431 A41 A) 1:25. The same procedure was performed with blank cosolvent (Pharmos batch no. 608945). Diclofenac was prepared in Pharmos, batch no. LG-121Y70 1 mg/ml. At the same time paws were measured they were clinically evaluated according to the following method (Williams R.O., Proc. Natl. Acad. Sci. USA : 89:9784-9788): 0 - normal, 1 - slight swelling and erythema, 2 - pronounced edematous swelling and 3 - joint rigidity. On day fifteen of treatment animals were euthanised with pentobarbital 100 mg/kg i.p. (Pental veterinary, C.T.S. Israel, batch no.170005). Blood samples were taken for hematocrit and blood levels of HU-211 (in heparinized serum). RESULTS a. Clinical Signs: The primary arthritis related sign that was evident in the present study was swelling ofthe paws. Animals treated with HU-211 20 mg/kg (daily or every 3 days) demonstrated the lowest number of these signs, compared to the other treatment groups. These differences were compared using a non parametric analysis (Wilcoxon Rank Sum Test), and were found to be statistical significant (p<0.01). b. Paw volume (Figure 5): All treatments (except the HU-211 20 mg kg every 3 days) reduced the collagen-induced paw edema by 30-50% compared to the vehicle treated animals (up to 30% increase in paw volume in the treated mice compared to up to 40% increase in the vehicle treated mice). This effect was statistically significant (p<0.05) with HU-211 20 mg kg (daily treatments) and with the HU-211 10 mg/kg every 3 days. The diclofenac treated animals demonstrated a smaller paw volume compared to the vehicle treated rats. But this difference did not reach statistical significance. Physiological example 8: Attentuation of experimental autoimmune encephalomyelitis in Lewis rats EAE was tested using Lewis rats in which the disease displays onset of symptoms around day 10 after induction and spontaneous recovery around 18 days after induction of the disease. The Lewis rats used were 8 week old females (from OLAC, England). The animals (5 per cage) were maintained on a 12 hour light/12 hour dark regimen, at a constant temperature of 22°C, with food and water ad libitum. EAE was induced in these animals by immunization with purified guinea pig myelin basic protein emulsified in complete Freund's adjuvant. Guinea pig myelin basic protein (mbp) was prepared from spinal cord homogenates defatted with chloroform ethanol and the isolated protein was purified using ion exchange chromatography. Each animal received 50 micrograms of the purified protein. A solution of mbp (0.5 mg/ml) was emulsified with an equal volume of Complete Freund's Adjuvant containing 4 mg/ml of mycobacterium tuberculosis, and each animal received 100 microliters (50 μl in each hind foot pad). Animals were treated with a single injection of HU-211 or vehicle control administered intravenously in a volume of 2 ml. The time of treatment was varied from day 10 to day 18 post induction of disease, with five animals per group. The results of this experiment, which are presented in Figure 6, show the diminution of mean clinical score in HU-211 treated animals. (Score of 1 indicates tail paralysis, 2 indicates paraplegia, 3 indicates quadriplegia, 4 indicates complete body paralysis and 5 indicates death) . Although the present invention has been described with respect to various specific embodiments thereof in order to illustrate it, such specifically disclosed embodiments should not be considered limiting. Many other specific embodiments will occur to those skilled in the art based upon applicants' disclosure herein, and applicants propose to be bound only by the spirit and scope of their invention as defmed in the appended claims.

Claims: THE CLAIMS 1. A method for preventing production of tumor necrosis factor by administering to a patient in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula (I): having the (3S,4S) configuration and being essentially free of the (3R,4R) enantiomer, wherein A B indicates an optional 1(2) or 6(1) double bond, R is (a) -Q wherein Q is a heterocyclic moiety having a labile hydrogen atom so that said moiety acts as a carboxylic acid analogue, (b) -R'X wherein R' is C r C 5 alkyl and X is halogen, -OR" wherein R" is hydrogen, C r C 5 alkyl, or -OC(O)R' " wherein R"' is hydrogen or -C 5 alkyl, (c) -R'N(R' ')-, wherein R' is C r C 5 alkyl and each R' ' , which may be the same or different, is hydrogen or C r C 5 alkyl optionally containing a terminal -OR' ' ' or -OC(O)R"' moiety wherein R'" is hydrogen or C 1 -C5 alkyl, (d) -R' wherein R' is C r C 5 alkyl, (e) -R'OR" ' wherein R' is C r C 5 alkyl and R' " is hydrogen or C,-C 5 alkyl, or, (f) -R'-C^-OR"' , wherein R' may be absent and R' and R"' are as defmed above; G is (a) halogen, (b) C r C 5 alkyl, or (c) -OR ! wherein R- is (a') -R" , wherein R" is hydrogen or C r C 5 alkyl optionally containing a terminal -OR' " or -OC(O)R" ' moiety wherein R'" is hydrogen or C r C 5 alkyl, or (b') -C(O)R'" wherein R'" is as previously defined; and R 2 is (a) C r C 12 alkyl ! (b) -OR"", in which R"" is a straight chain or branched C 2 -C 9 alkyl which may be substituted at the terminal carbon atom by a phenyl group, or (c) -(CH 2 ) n OR'" wherein n is an integer of 1 to 7 and R'" is hydrogen or C r C 5 alkyl. 2. The method of claim 1 wherein A — B designates a 1(2) or 6(1) double bond, R is lower alkyl of 1-3 carbon atoms or CH 2 OH, G is hydroxy or a lower acyloxy group, and R 2 is (a) a straight or branched C 6 -C 12 alkyl radical or (b) a group OR 3 in which R is a straight or branched C 5 -C 9 alkyl radical optionally substituted at the terminal carbon atom by a phenyl group. 3. The method of claim 1 wherein A — B designates a 1(2) or 6(1) double bond, R is COOR'" wherein R'" is lower alkyl of 1-5 carbon atoms or hydrogen, G is hydroxy or a lower acyloxy group, and R " is (a) a straight or branched C 6 -C 12 alkyl radical or (b) a group OR 3 in which R is a straight or branched C 5 -C 9 alkyl radical optionally substituted at the terminal carbon atom by a phenyl group. 4. The method of claim 1 wherein said compound is administered in a manner to protect against septic shock syndrome. 5. The method of claim 1 which comprises administering said compound to a patient who exhibits the symptoms associated with cachexia due to acquired immune deficiency syndrome, neoplasia or trauma. 6. The method of claim 1 which comprises administering said compound to a patient who exhibits the symptoms associated with autoimmune diseases characterized by the production of elevated levels of tumor necrosis factor. 7. A method for blocking the action of tumor necrosis factor in a patient which comprises administering to said patient, in a manner calculated to block said cytokine in a stereospeciric manner, a therapeutically effective amount of a compound of the formula (I): having the (3S,4S) configuration and being essentially free of the (3R,4R) enantiomer, wherein A B indicates an optional 1(2) or 6(1) double bond, R is (a) -Q wherein Q is a heterocyclic moiety having a labile hydrogen atom so that said moiety acts as a carboxylic acid analogue, (b) -R'X wherein R' is C r C 5 alkyl and X is halogen, -OR" wherein R" is C,-C 5 alkyl, or -OC(O)R'" wherein R" * is hydrogen or C*-C 5 alkyl, (c) -R'N(R' ') 2 wherein R' is C r C 5 alkyl and each R" ' , which may be the same or different, is hydrogen or C r C 5 alkyl optionally containing a terminal -OR"' or -OC(O)R"' moiety wherein R' " is hydrogen or C r C 5 alkyl, (d) -R' wherein R' is C r C 5 alkyl, (e) -R'OR' " wherein R' is C r C 5 alkyl and R'" is hydrogen or C r C 5 alkyl, or, (f) -R'-C(O)-OR" ', wherein R' may be absent and R' and R"' are as defined above; G is (a) halogen, (b) C r C 5 alkyl, or (c) -OR- wherein R) is (a') -R", wherein R" is hydrogen or C r C 5 alkyl optionally containing a terminal -OR' " or -OC(O)R' " moiety wherein R' " is hydrogen or C,-C 5 alkyl, or (b 1 ) -C(O)R" ' ' wherein R" ' is as previously defmed, and R 2 is (a) C r C 12 alkyl, (b) -OR"", in which R"" is a straight chain or branched C 2 -C 9 alkyl which may be substituted at the terminal carbon atom by a phenyl group, or (c) -(CrL^OR' ' ' wherein n is an integer of 1 to 7 and R" ' is hydrogen or C r C 5 alkyl. . 8. A method ~ ι Li claim 7 wherein A — B designates a 1(2) or 6(1) double bond, R designates a -CH 3 or CH 2 OH, G designates hydroxy or a lower acyloxy group and R " designates (A) a straight or branched C 6 - C 12 alkyl radical; (B) a group -O-R 3 , in which R 3 is a straight or branched C 5 - C 9 alkyl radical optionally substituted at the terminal carbon atom by a phenyl group. 9. A method as in claim 7 wherein A — B designates a 1(2) or 6(1) double bond, R designates a -COOR'.", wherein R'" is a lower alkyl group of 1-5 carbon atoms or hydrogen, G designates hydroxy or a lower acyloxy group and R designates (A) a straight or branched C 6 - C 12 alkyl radical; (B) a group -O-R , in which R 3 is a straight or branched C 5 - C 9 alkyl radical optionally substituted at the terminal carbon atom by a phenyl group. 10. The method of claim 7 wherein said compound is administered in a manner to protect against septic shock syndrome. 11. The method of claim 7 which comprises administering said compound to a patient who exhibits the symptoms of cachexia associated with acquired immune deficiency syndrome, neoplasia or trauma. 12. The method of claim 7 which comprises administering said compound to a patient who exhibits the symptoms associated with autoimmune diseases associated with the production of elevated levels of tumor necrosis factor alpha. 13. The method of claim 12 which comprises administering said compound to a patient with multiple sclerosis. 14. A method according to claim 1 or claim 7 in which said pharmaceutical composition includes a carrier or diluent. 15. The method of claim 14 which comprises selecting the carrier or diluent to be an aqueous cosolvent solution comprising a pharmaceutically acceptable cosolvent, a micellar solution prepared with natural or synthetic ionic or non-ionic surfactants, or a combination of such cosolvent and micellar solutions. 16. The method of claim 14 which comprises selecting the carrier to be a solution of ethanol, a surfactant, and water. 17. The method of claim 14 which comprises selecting the carrier to be an emulsion comprising a triglycerides, lecithin, glycerol, an emulsifier, an antioxidant, and water. 18. A method according to claim 1 or claim 7 wherein the daily dosage of said compound is between 0.1 and 25 mg/kg. 19. A method according to claim 1 or claim 7, in which said pharmaceutical composition is administered intravenously, intrathecally, orally or by inhalation.

Inventors: GALLILY RUTH, MECHOULAM RAPHAEL, SHOHAMI ESTHER, GALLILY, RUTH, MECHOULAM, RAPHAEL, SHOHAMI, ESTHER
Applicants: GALLILY RUTH, MECHOULAM RAPHAEL, SHOHAMI ESTHER, YISSUM RES DEV CO, GALLILY, RUTH, MECHOULAM, RAPHAEL, SHOHAMI, ESTHER, YISSUM RESEARCH DEVELOPMENT CO. OF THE HEBREW UNIV
Application year: 1996
Main classification: A61K
IPC codes: A61K31, A61P29, A61P37, A61P43