Patent Application
20050137191
This invention relates to nitrate esters and use thereof in mitigating cellular damage. Particularly, this invention relates to selected organic nitrates, most particularly those bearing a sulfur or phosphorus atom β or γ to the nitrate group, having therapeutic utility as agents that protect tissues from oxidative injury.
The nitrate ester glyceryl trinitrate (GTN) or nitroglycerin, has been used as a vasodilator in the treatment of angina pectoris for over a hundred years, and the dominant contemporary belief is that GTN exerts its therapeutic effect through in vivo release of nitric oxide (NO). Other organic nitrates, such as isosorbide dinitrate, have also been identified as effective and clinically important vasodilators. NO itself has been identified as Endothelium Derived Relaxing Factor (EDRF) and several classes of compounds, for example nitrosothiols, in addition to organic nitrates, have been proposed as NO donors or NO prodrugs. Endogenous stimulation or exogenous administration of NO have been shown to inhibit production of reactive oxygen species (ROS) and expression of oxidant-mediated molecular or tissue injury. Well-known examples of these classes of compounds and one nitrate, GTN itself, have been suggested to demonstrate neurotoxic or neuroprotective effects by dint of interactions with the redox modulatory site of the N-methyl-D-aspartate (NMDA) excitatory amino acid receptor. Thus GTN is firstly a potent vasodilator and secondly possesses potential neuroprotective properties. In addition, GTN has been found to suppress renal oxidant damage caused by potassium bromate (Rahman et al., Redox Rep. 4: 263-9, 1999). Several attempts have been made to increase the efficacy or potency of alternative organic nitrates as vasodilators relative to GTN, for example, by incorporation of propanolamine or cysteine functionalities. However, no attempt has been made to separately regulate the vasodilatory and cytoprotective effects of GTN. Indeed, postural hypotension and weakness are signs of cerebral ischemia, and are adverse effects associated with the vasodilatory effects of GTN. Observed in treatment, these effects are highly contraindicative of GTN itself, and by extrapolation GTN derivatives (1,2,3-trinitratopropane derivatives), as clinically useful protective therapeutic agents.
In as much as the potent vasodilatory effects of organic nitrates may prove (a) deleterious to, or alternatively (b) synergistic with the protective effects of GTN, it is postulated herein that regulation of these two effects is required for development of new and useful protective therapeutic agents. Further, it is postulated that such regulation may be achieved through use of an appropriate organic nitrate, such as, for example, nitrate esters incorporating sulfur-containing or phosphorus-containing functionalities into the structure of the nitrate esters.
Accordingly, a first aspect the invention features nitrate esters having the formula:
In accordance with the present invention, one skilled in the recognizes that one of m, n, or p must be equal to at least one.
In one embodiment, the compound of the invention is:
wherein G1 is Me, OMe, Cl, NO2, Br, or H; G2 is CO2Et, CO2H, CO2Me, CONH2, or CO(CH2)2NEt2; and G3 is Cl, OMe, or CONH2.
In another embodiment, the compound is:
In another embodiment, the compound is:
In another embodiment, the compound is:
In another embodiment, the compound is:
In yet another embodiment, the compound is:
In a second aspect, the invention features a compound having the formula:
In one embodiment of the second aspect, the compound is:
In another embodiment, the compound is:
In another embodiment, the compound is:
In another embodiment the compound is:
In another embodiment, the compound is:
In another embodiment, the compound is:
In yet another embodiment of the second aspect, the compound is:
In a third aspect, the invention features a compound having the formula:
In a fourth aspect, the invention features a composition that includes a pharmaceutically acceptable carrier and a compound having the general formula:
In a fifth aspect, the invention features a composition that includes a pharmaceutically acceptable carrier and one of the following compounds:
In a sixth aspect, the invention features a method for preventing or mitigating tissue and/or cellular damage in a subject by modulating intercellular and/or intracellular free radical concentration in the subject by administering to the subject an effective amount of a compound containing at least one aliphatic nitrate group and at least one sulfur atom in proximity to the nitrate group. By “sulfur atom in proximity” or “proximal functional group” is meant a sulfur atom or functional group that is connected through bonds in a β, γ, or δ relationship to a nitrate ester group (i.e., the atom connectivity is 1,2, or 1,3, or 1,4). The functional group may also be referred to as “proximally located” or “situated in proximity.” Proximal functional groups also include those groups that have a through-space intramolecular juxtaposition with a nitrate group that is within 3 Å.
In one embodiment, the tissue and/or cellular damage can be associated with aging, septic shock, ischemia/reperfusion injury, ulcers, gastritis, ulcerative colitis, Crohn's disease, diabetes, rheumatoid arthritis, asthma, cirrhosis of the liver, allograft rejection, encephalomyelitis, meningitis, pancreatitis, peritonitis, vasculitis, lymphocytic choriomeningitis, glomerulonephritis, uveitis, glaucoma, blepharitis, chalazion, allergic eye disease, corneal ulcer, keratitis, cataracts, age-related macular degeneration, optic neuritis, ileitis, hemorrhagic shock, anaphylactic shock, bacterial infection, viral infection, fungal infection, parasitic infection, hemodialysis, chronic fatigue syndrome, stroke, toxic shock syndrome, adult respiratory distress syndrome, cachexia, myocarditis, eczema, psoriasis, dermatitis, urticaria, cerebral ischemia, systemic lupus erythematosis, chronic neurodegenerative disease, priapism, cystic fibrosis, schizophrenia, depression, premenstrual syndrome, anxiety, addiction, migraine, gastrointestinal motility disorders, obesity, hyperphagia, hematologic cancers, myelofibrosis, graft-versus-host disease, CNS trauma, hepatitis, renal failure, chronic hepatitis C, drug-induced lung injury (e.g., paraquat), bacterial translocation, circulatory shock, traumatic shock, vascular aneurysm, metastatic cancer, or myocardial infarction.
In another embodiment, the tissue and/or cellular damage can associated with neurological diseases such as, for example, Parkinson's disease; Alzheimer's disease; Huntington's disease; multiple sclerosis; amylotrophic lateral sclerosis; AIDS-induced dementia; epilepsy; alcoholism; alcohol withdrawal; drug-induced seizures; viral/bacterial/fever-induced seizures; trauma to the head; hypoglycemia; hypoxia due to myocardial infarction; cerebral vascular occlusion; cerebral vascular hemorrhage; hemorrhage; or environmental excitotoxins of plant, animal, or marine origin.
In yet another embodiment, the tissue and/or cellular damage can be associated with cytokine therapy, wherein a nitrate ester of the invention is administered to the subject before, during, and/or after the administration of the therapeutic cytokine.
In another embodiment of the sixth aspect, the compound has the formula:
In another embodiment, the compound contains at least 2 nitrate groups. In another embodiment, the nitrate is beta or gamma to said sulfur atom.
In another embodiment of the sixth aspect, the compound can be any of the compounds that were cited individually as an embodiment of either the first or second aspect of the invention.
In another embodiment, the compound is:
In yet another embodiment of the sixth aspect, the compound is:
As used herein, the terms “alkyl” and the prefix “alk-” are inclusive of both straight chain and branched chain saturated or unsaturated groups, and of cyclic groups, i.e., cycloalkyl and cycloalkenyl groups. Unless otherwise specified, acyclic alkyl groups are from 1 to 6 carbons and contain at least one C—H bond. The number of carbons in an alkyl group refers to the total number of carbons contained in the group. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 8 ring carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclopentyl, cyclohexyl, and adamantyl groups. Unless otherwise indicated, alkyl groups may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halogen, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
By “aryl” is meant a carbocyclic aromatic ring or ring system. Unless otherwise specified, aryl groups are from 6 to 18 carbons. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl, and indenyl groups.
By “heterocycle” is meant an aromatic or non-aromatic ring system that contains at least one ring heteroatom (e.g., O, S, N). The term “heteroaryl” refers to an aromatic heterocyclic ring or ring system that contains at least one ring heteroatom (e.g., O, S, N). Unless otherwise specified, heteroaryl rings contain from 1 to 9 carbons. Exemplary heteroaryl groups include furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, oxatriazolyl, pyridyl, pyridazyl, pyrimidyl, pyrazyl, triazyl, benzofuranyl, isobenzofuranyl, benzothienyl, indole, indazolyl, indolizinyl, benzisoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, naphtyridinyl, phthalazinyl, phenanthrolinyl, purinyl, and carbazolyl rings or ring systems.
Unless otherwise specified, non-aromatic heterocyclic groups are from 2 to 9 carbons and can include, for example, dihydropyrrolyl, tetrahydropyrrolyl, piperazinyl, pyranyl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophene, tetrahydrothiophene, and morpholinyl groups. As defined in the present application, the definition of heterocycle specifically excludes β-lactam rings.
Unless otherwise specified, aryl, heteroaryl, or heterocyclic groups may be unsubstituted or substituted by one or more substituents selected from the group consisting of C1-6 alkyl, hydroxy, halo, nitro, C1-6 alkoxy, C1-6 alkylthio, trifluoromethyl, C1-6 acyl, arylcarbonyl, heteroarylcarbonyl, nitrile, C1-6 alkoxycarbonyl, arylalkyl (wherein the alkyl group has from 1 to 6 carbon atoms) and heteroarylalkyl (wherein the alkyl group has from 1 to 6 carbon atoms).
By “halide” or “halogen” or “halo” is meant bromine, chlorine, iodine, or fluorine. As used herein, the terms “alkyl” and the prefix “alk-” are inclusive of both straight chain and branched chain saturated or unsaturated groups, and of cyclic groups, i.e., cycloalkyl and cycloalkenyl groups. Unless otherwise specified, acyclic alkyl groups are from 1 to 6 carbons. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 8 ring carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclopentyl, cyclohexyl, and adamantyl groups. Alkyl groups may be substituted or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halogen, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
By “alkoxy” is meant a chemical substituent of the formula —OR, where R is an alkyl group. By “aryloxy” is meant a chemical substituent of the formula —OR′, where R′ is an aryl group. By “alkaryl” is meant a chemical substituent of the formula —RR′, where R is an alkyl group and R′ is an aryl group, By “alkheteraryl” is meant a chemical substituent of the formula RR″, where R is an alkyl group and R″ is a heteroaryl group.
What is meant by “aliphatic nitrate” is a nitrate group that is connected to a non-aromatic carbon.
What is meant by “antioxidant” is a compound that attenuates or prevents oxidation of a target biomolecule through the inhibition of the initiation or propagation steps that constitute oxidative and peroxidative processes.
“Mitigating neurodegeneration” as used herein involves affecting neuroprotection, inhibiting or preventing neurodegeneration, and/or ameliorating the manifestations or impact of neurodegeneration. Such amelioration includes effecting cognition enhancement, as is quantified by tests known in the art (e.g., Venault et al., 1992, incorporated herein by reference). “Modulating” a biological process as used herein (for example, modulating activity of the non-glutamate neuroreceptors), encompasses both increasing (positively modulating) and decreasing (negatively modulating) such activity, and thus inhibition, potentiation, agonism, and antagonism of the biological process.
It is an object of the present invention to provide provide novel organic nitrates, including aliphatic nitrate esters bearing a sulfur or phosphorus moiety β or γ to a nitrate group, or congeners thereof. Another object of the present invention is to provide methods for making such novel organic nitrates. Another object of the invention is to provide methods for affecting neuroprotection, mitigating neurodegeneration, affecting cognition enhancement, and/or protecting tissues from oxidative injury employing selected organic nitrates. Another object of the present invention is to provide novel drugs as neuroprotective agents. Another another object of the present invention is to provide novel drugs for use in cognition enhancement. Another object of the invention is to provide novel drugs for use in protecting tissues from oxidative injury.
It will be understood by those skilled in the art, that when organic radicals, such as Rn, X, or Y, are represented as “does not exist,” that the valency of the carbon bound to the radical is adjusted accordingly (i.e., by a point of unsaturation).
Compounds may be constructed according to formulas of the invention in which an S atom is appropriately proximally placed with respect to a nitrate functional group, but in which a carbonyl group intervenes to form a thioester linkage. Such compounds do not form part of the invention if cleavage of this thioester bond produces two entirely separate molecules, one containing the nitrate functionality, and another the S-functionality. An example of such a compound is one containing the motif, O2NOCH2C(CH3)2C(O)S-(organic radical), which would liberate on thioester cleavage an aliphatic nitrate that does not contain a S-functionality. It is understood by one skilled in the art that facile cleavage of a thioester in an aqueous biological milieu will render an aliphatic nitrate that does not contain a proximal S, and therefore, such a thioester does not comprise part of the current invention.
This invention pertains to methods and compositions useful for treating neurodegeneration or preventing or mitigating tissue and/or cellular damage by administering to a subject a therapeutic nitrate ester. Neuroprotection and/or cognition enhancement can be affected, for example, by modulating an interaction with guanylyl cyclase (GCase, the enzyme responsible for cGMP production in various areas of the brain), modulating a glutamate or non-glutamate neuroreceptor or attenuating free radical damage. The attenuation of free radical concentration by a nitrate ester of the invention can also be useful for preventing or mitigating tissue and/or cellular damage.
According to certain aspects of the invention, neurodegeneration is mitigated by stimulating cerebral GCase. One of the major targets for the novel organic nitrates of the invention is GCase activation, resulting in the production of cGMP. Experimental evidence obtained in a number of in vitro model systems supports the notion that elevated levels of cGMP help to prevent apoptotic (programmed) cell death. Thus, a cGMP-dependent mechanism significantly increases the survival of trophic factor-deprived PC12 cells and rat sympathetic neurons (Farinelli et al., 1996), and of primary cultures of rat embryonic motor neurons (Estevez et al., 1998). The mechanism of action for selected organic nitrates in preventing apoptotic cell death may be inhibition of caspase-3 activation indirectly through elevations in cGMP levels or directly via protein S-nitrosylation of the enzyme by an NO-intermediate (Kim et al., 1997). Caspase-3 is a member of the cysteine protease family of enzymes that are essential for the execution step in apoptosis (Cohen, 1997; Nicholson and Thornberry, 1997). Activation of caspase-3 is required for apoptotic cell death in trophic factor-deprived PC12 cells (Haviv et al., 1997) and in glutamate-mediated apoptotic cell death of cultured cerebellar granule neurons (Du et al., 1997). In animal models of cerebral ischemia, caspase-3 activity is induced and may be responsible for the apoptotic component of delayed neuronal cell death (Chen et al., 1998; Namura et al., 1998; Ni et al., 1998). Inhibitors of caspase-3 significantly decrease the apoptotic component of delayed neuronal cell death in response to ischemic injury both in vitro (Gottron et al., 1997) and in vivo (Endres et al., 1998). A secreted region of the Alzheimer's disease β-amyloid precursor protein lowers intracellular calcium levels and provides neuroprotective effects on target cells through increases in cGMP levels and activation of protein kinase G (Barger et al., 1995; Furukawa et al., 1996). In preferred embodiments of the methods of the invention, nitrated molecules that have the capacity to activate GCase directly or via release of an NO-containing intermediate are used to modulate GCase activity.
According to certain other aspects of the invention, cognition enhancement (e.g., improved memory performance) is achieved by stimulating cerebral GCase. Several lines of experimental evidence support the notion that GCase and cGMP are involved in the formation and retention of new information. cGMP has been directly implicated in both long-term potentiation (LTP) and long-term depression (LTD), which are proposed cellular models for learning and memory (Arancio et al., 1995; Wu et al., 1998). In animal models, elevation of hippocampal cGMP levels leading to increased protein kinase G activity has been shown to be important for retention and consolidation of new learning (Bernabeu et al., 1996, 1997). Thus, stimulation of cerebral GCase activity is expected to improve learning and memory performance in individuals in whom cognitive abilities are impaired by injury, disease, or aging.
We have shown that novel organic nitrate esters have differential effects to activate soluble GCase and to cause cGMP accumulation in vascular and brain tissue. There is a clear dissociation between the vascular relaxation effects of organic nitrate esters and ability to affect neuroprotection. Activation of GCase and accumulation of cGMP have been shown to be important in the neuroprotection of hippocampal brain slices subjected to a period of in vitro ischemia.
Cerebral ischemia results in marked increases in the release of the excitatory amino acid glutamate in the affected brain region (Bullock et al., 1998; Huang et al., 1998; Yang et al., 1998). In both humans (Bullock et al., 1998) and experimental animals (Huang et al., 1998; Goda et al., 1998; Yang et al., 1998), the amount of glutamate released during ischemia is positively correlated with the extent of brain injury. In experimental animal models of cerebral ischemia, decreased release of glutamate during ischemia (Goda et al., 1998) or blockade of glutamate receptors with antagonists (Ibarrola et al., 1998; O'Neill et al., 1998; Umemura et al., 1997) significantly reduces the extent of brain injury. However, these interventions are only effective when given prior to or during the ischemic insult. To be broadly useful, a therapeutic intervention is preferably effective when administered after the period of ischemia. We have designed a class of novel organic nitrate esters having high efficacy in effecting neuroprotection in vivo in models of transient global and focal cerebral ischemia when given after the ischemic insult. It will be appreciated, therefore, that these selected organic nitrates can be used for treatment of conditions including but not limited to: stroke; Parkinson's disease; Alzheimer's disease; Huntington's disease; multiple sclerosis; amylotrophic lateral sclerosis; AIDS-induced dementia; epilepsy; alcoholism; alcohol withdrawal; drug-induced seizures; viral/bacterial/fever-induced seizures; trauma to the head; hypoglycemia; hypoxia; myocardial infarction; cerebral vascular occlusion; cerebral vascular hemorrhage; hemorrhage; environmental excitotoxins of plant, animal, or marine origin; and the like.
The direct effects of selected organic nitrates on amino acid neurotransmitter receptors has been tested using the Xenopus oocyte expression system and two-electrode voltage-clamp recording methods. Selected organic nitrates were found to have direct, modulatory effects on GABAA receptor function (see Working Examples below). These allosteric modulatory effects of selected organic nitrates were not shared by direct NO-generating compounds, indicating a novel mechanism of action for selected organic nitrates to interact with GABAA receptors. In behavioural models of learning and memory, drugs which decrease GABAA receptor function improve performance on learning and memory tasks (Venault et al., 1992). Thus, the behavioural effect of selected organic nitrates, developed to act as modulators of GABAA receptor function, will be to improve memory performance and cognition in patient populations. It will be appreciated, therefore, that these selected organic nitrates can be used for treatment of conditions including but not limited to: stroke; dementias of all type; trauma; drug-induced brain damage; and aging.
According to certain aspects of the invention, neurodegeneration is mitigated by inhibition of free radical damage. Reoxygenation and reperfusion after a period of ischemia contributes significantly to the development of brain injury. Oxygen radicals, especially superoxide and peroxynitrite, formed in the period after an ischemic event may initiate processes such as breakdown of membrane lipids (lipid peroxidation), leading to loss of cell membrane integrity and inhibition of mitochondrial function (Macdonald and Stoodley, 1998; Gaetani et al, 1998). Oxidative stress is also believed to be one factor involved in initiation of apoptotic neuronal cell death (Tagami et al., 1998). In experimental animal models of ischemic brain injury, free radical scavengers and enhanced activity of superoxide dismutase have been found to reduce the extent of neuronal injury and cell death (Chan et al., 1998; Mizuno et al., 1998; Tagami et al., 1998).
It has been shown that 2,3-dinitrooxy-(2,3-bis-nitrooxy-propyldisulfanyl)-propane, compound Va, improves task acquisition in cognitively impaired animals (see Smith et al., NeuroReport 11: 3883, 2000), suggesting to us that novel nitrates administered to an intact animal can have direct effects on the brain. These observations also lead us to postulate that the nitrates of the present invention could protect tissues against oxidative injury. Accordingly, we tested the effects of compound Va in the 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. 6-OHDA is a neurotoxin selectively taken up into dopaminergic neurons, resulting in a selective killing of these neurons, via a mechanism involving oxidative stress that is evident by 4 days after injection of the toxin. Previous studies have demonstrated that the monoamine oxidase type B (MAO-B) inhibitor, deprenyl, can prevent 6-OHDA-induced killing of dopaminergic neurons. We therefore employed deprenyl as a positive control in this study. Loss of dopaminergic innervation to the striatum results in an upregulation of postsynaptic dopamine receptors, and the development of dopamine receptor supersensitivity 2-3 weeks after 6-OHDA lesioning of the substantia nigra. This supersensitivity to dopamine can be unmasked by challenging the animal with a dopamine receptor agonist, such as apomorphine. Since the substantia nigra pars compacta is a bilateral structure, unilateral destruction of the substantia nigra will induce a dopamine receptor imbalance, which can manifest behaviourally as apomorphine-induced turning in the direction contralateral to the lesion. This behavioural manifestation of 6-OHDA toxicity provides a convenient, quantifiable index of neuronal injury and neuroprotection. Immunocytochemical analysis confirmed that compound Va preserved TH-positive neurons in the substantia nigra of 6-OHDA-injected rats (see Example 3 and
Accordingly, in certain aspects and embodiments of the invention, the invention features nitrated molecules which have the capacity to inhibit production of free radicals and/or which act as free radical scavengers.
Free radical overproduction is associated with a wide range of disease states and/or indications, such as, for example, aging, septic shock, ischemia, overexpression of cytokines, ulcers, inflammatory bowel disease (e.g., gastritis, ulcerative colitis or Crohn's disease), diabetes, arthritis (e.g., rheumatoid arthritis), asthma, cirrhosis, allograft rejection (e.g., transplant rejection), encephalomyelitis, meningitis, pancreatitis, peritonitis, vasculitis, lymphocytic choriomeningitis, glomerulonephritis, ophthalmologic diseases (e.g., uveitis, glaucoma, blepharitis, chalazion, allergic eye disease, corneal ulcer, keratitis, cataract, retinal disorders, age-related macular degeneration, optic neuritis, and the like), ileitis, inflammation induced by overproduction of inflammatory cytokines (e.g., liver inflammation, renal inflammation, airway inflammation, and the like), hemorrhagic shock, anaphylactic shock, burn, infection leading to the overproduction of inflammatory cytokines (including bacterial (e.g., E. coli infection), viral (e.g., HIV), fungal (e.g., Candidiosis and histoplasmosis) and parasitic (e.g., Leishmaniasis and Schistosomiasis) infections), hemodialysis, chronic fatigue syndrome, stroke, cancers, including metastatic cancers (e.g., breast cancer, bladder cancer, lung cancer, colon cancer, or cancer of the other organs, or skin or other noncutaneous portions of the body), cardiovascular diseases associated with overproduction of inflammatory cytokines (e.g., heart disease, cardiopulmonary bypass, ischemic/reperfusion injury, and the like), ischemic/reperfusion associated with overproduction of inflammatory cytokines, toxic shock syndrome, adult respiratory distress syndrome, cachexia, myocarditis, autoimmune disorders, eczema, psoriasis, heart failure, dermatitis, urticaria, cerebral ischemia, systemic lupus erythematosis, AIDS, neurodegenerative disorders (e.g., chronic neurodegenerative disease), chronic pain, priapism, cystic fibrosis, schizophrenia, depression, premenstrual syndrome, anxiety, addiction, migraine, gastrointestinal motility disorders, obesity, hyperphagia, solid tumors (e.g., neuroblastoma), malaria, hematologic cancers, myelofibrosis, lung injury, graft-versus-host disease, head injury, CNS trauma, hepatitis, renal failure, liver disease (e.g., chronic hepatitis C), drug-induced lung injury (e.g., paraquat), transplant rejection and preservation, fertility enhancement, bacterial translocation, circulatory shock, traumatic shock, and vascular aneurysm (e.g., aortic aneurysm), ileus, or myocardial infarction.
In addition, the compounds or methods of the present invention may find use in cytokine therapy (with consequent induction of free radical overproduction) which, for example, is commonly used in the treatment of cancers, including metastatic cancers (e.g., breast cancer, bladder cancer, lung cancer, colon cancer, or cancer of the other organs, or skin or other noncutaneous portions of the body), autoimmune disease, and in AIDS patients. Systemic hypotension due to the induction of free radical overproduction is a dose-limiting side effect of cytokine therapy. Thus, a large patient population exists which will benefit from the invention methods.
It will also be appreciated by those skilled in the art, that any organic nitrate in which vasodilatory potency is reduced and neuroprotective potency increased, represents a new and useful therapeutic agent for use in neuroprotection, particularly in treatment of conditions including but not limited to: stroke; Parkinson's disease; Alzheimer's disease; Huntington's disease; multiple sclerosis; amylotrophic lateral sclerosis; AIDS-induced dementia; epilepsy; alcoholism; alcohol withdrawal; drug-induced seizures; viral/bacterial/fever-induced seizures; trauma to the head; hypoglycemia; hypoxia; myocardial infarction; cerebral vascular occlusion; cerebral vascular hemorrhage; hemorrhage; environmental excitotoxins of plant, animal, or marine origin. GTN itself, proposed as a neuroprotective agent, has no clinical utility as a neuroprotective agent in therapy owing to its extraordinarily high vasodilatory potency. Similarly, by extrapolation, 1,2,3-trinitratopropane (GTN) derivatives are not expected to have clinical utility as neuroprotective agents in therapy owing to their especially high vasodilatory potency.
It will additionally be appreciated by those skilled in the art, that the use in therapy of any organic nitrate in cognition enhancment, represents a new and useful treatment for cognition enhancement, particularly in treatment of conditions including but not limited to: stroke; dementias of all type, trauma, drug-induced brain damage, and aging.
In particular, the therapeutic compounds of the invention comprise at least one nitrate group. The nitrate groups(s) can optionally be covalently bound to a carrier moiety or molecule (e.g., an aromatic group, an aliphatic group, peptide, steroid, nucleoside, peptidomimetic, steroidomimetic, or nucleoside analogue, or the like). In addition to functioning as a carrier for the nitrate functionality, the carrier moiety or molecule can enable the compound to traverse biological membranes and to be biodistributed preferentially, without excessive or premature metabolism. Further, in addition to functioning as a carrier for the nitrate functionality, the carrier moiety or molecule can enable the compound to exert amplified neuroprotective effects and/or cognition enhancement through synergism with the nitrate functionality.
In one aspect, the invention provides a method of treating a neurological condition and/or preventing an undesirable mental condition (e.g., memory loss) including the step of administering to a subject an effective amount of a therapeutic compound capable of mitigating neurodegeneration which has at least one nitrate group. In one embodiment, the therapeutic compound is capable of effecting neuroprotection. In another embodiment of the invention, the therapeutic compound is capable of effecting cognition enhancement. The therapeutic compound has the formula (Formula I):
In another aspect, the invention provides a pharmaceutical composition including a physiologically acceptable carrier and a compound having the formula (Formula I):
In another aspect, therapeutic compounds of the invention that effect neuroprotection and/or effect cognition enhancement in a subject to which the therapeutic compound is administered have the formula (Formula II):
Pharmaceutical compostions comprising a compound of Formula II in admixture with a pharmaceutically acceptable carrier therefor are provided by the invention. The invention further provides methods of mitigating neurodegeneration, effecting neuroprotection and/or effecting cognition enhancement in a subject comprising the step of administering a compound of Formula II to a subject such that said mitigation and/or said neuroprotection an/or cognition enhancement occurs.
According to this aspect of the invention, preferred therapeutic compounds for effecting neuroprotection and/or cognition enhancement in a subject to which the compound is administered include compounds in which R19 is X—Y. In some preferred embodiments: R19 is X—Y and R5, R6, R8, R9, R12, R13, R14, R15, R16 are the same or different alkyl groups containing 1-24 carbon atoms which may contain 1-4 ONO2 substituents, or C1 or C2 connections to R1-R3 in cyclic derivatives; R1 and R3 are the same or different and selected from H, C1-C4, alkyl chains, which may include one O, linking R1 and R3 to form pentosyl, hexosyl, cyclopentyl, or cycohexyl rings, which rings optionally bear hydroxyl substituents; R2 and R4, are the same or different and selected from H, a nitrate group, C1-C4 alkyl optionally bearing 1-3 nitrate group, and acyl groups (—C(O)R5); and R7, R11 are the same or different C1-C8, alkyl or acyl.
In certain embodiments in which R19 is X—Y, m, p=1, and n=0. In other embodiments in which R19 is X—Y, X is selected from: CH2, O, NH, NMe, CN, NHOH, N2H3, N2H2R13, N2HR13R14, N3, S, SCN, SC(═NH)N(R15)2, SC(═NH)NHR15, SC(O)N(R15)2, SC(O)NHR15, SO3M, SH, SR7, SO2M, S(O)R8, S(O)2R9, S(O)OR8, S(O)2OR9, PO3HM, PO3M2, P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(R15)(OR8), P(O)(OM)R15, CO2M, CO2H, CO2R11, C(O), C(O)R12, C(O)(OR13) PO2M, P(O)(OR14), P(O)(R13), SO, SO2, C(O)(SR13), SSR4. In certain other embodiments in which R19 is X—Y, Y is selected from CN, N2H2R13, N2HR13R14, N3, SCN, SC(═NH)N(R15)2, SC(O)N(R15)2, SC(O)NHR15, SO3M, SR4, SO2M, PO3HM, PO3M2, P(O)(OR15)(OR16), P(O)(OR16)(OM), P(O)(R15)(OR8), P(O)(OM)R15, CO2M, CO2H, CO2R11, C(O)R12, C(O)(SR13), SR5, SSR5, or does not exist. In some embodiments, X and/or Y contains a sulfur-containing functional group. In certain embodiments, the compound of the invention comprises a heterocyclic functionality, more preferably, a nucleoside or nucleobase. In other embodiments, the compound of the invention comprises a carbocyclic functionality, more preferably, a steroidal or carbohydrate moiety.
In another aspect of the invention, a therapeutic compound of the invention is represented by the formula (Formula III):
Examples and preferred embodiments of this aspect include:
In another aspect of the invention, a therapeutic compound of the invention is represented by the formula III in which the compound contains from 1 to 3 nitrate groups and an S atom in proximity to a nitrate group, where each of m and n is, independently, an integer from 0 to 10; R1 is a hydrogen or A; each of R2, R5, and R18 is, independently, hydrogen or A; each of R3, R4, and R17, is independently, a hydrogen, a nitrate group, or A; each of R6, R7, R8, R9, R11, R12, R13, R14, R15, and R16 is, independently, A, a hydrogen, a nitrate group, or a C1-C24 alkyl or acyl group, optionally containing 1-4 ONO2 substituents or a C1-C6 linkage to R1, R2, R3, or R4 in cyclic derivatives; each of R7 and R11 is, independently, a substituted or unsubstituted C1-C8 alkyl or acyl group;
Examples and preferred embodiments of this aspect include:
Pharmaceutical compostions comprising a compound of Formula III in admixture with a pharmaceutically acceptable carrier therefor are provided by the invention. The invention further provides methods of mitigating neurodegeneration, effecting neuroprotection and/or effecting cognition enhancement in a subject comprising the step of administering a compound of Formula III to a subject such that said mitigation and/or said neuroprotection and/or cognition enhancement occurs.
In another aspect of the invention, a therapeutic compound of the invention can be represented by the formula (Formula IV):
Pharmaceutical compositions comprising a compound of Formula IV in admixture with a pharmaceutically acceptable carrier therefor are provided by the invention.
The invention further provides methods of mitigating neurodegeneration, effecting neuroprotection and/or effecting cognition enhancement in a subject comprising the step of administering a compound of Formula IV to a subject such that said mitigation and/or said neuroprotection and/or cognition enhancement occurs.
Examples and preferred embodiments of compounds of the invention according to Formula IV are as follows:
In yet another aspect of the invention, a compound of the invention can be represented by the formula (Formula V):
Pharmaceutical compositions comprising a compound of Formula V in admixture with a pharmaceutically acceptable carrier therefor are provided by the invention.
The invention further provides methods of mitigating neurodegeneration, effecting neuroprotection and/or effecting cognition enhancement in a subject comprising the step of administering a compound of Formula V to a subject such that said mitigation and/or said neuroprotection and/or cognition enhancement occurs.
Examples and preferred embodiments of compounds of the invention according to formula V (Formulae Va-c) are as follows:
In another aspect, the invention features one of the following nitrate esters:
In another aspect, the invention features a method for preventing or mitigating tissue and/or cellular damage in a subject by modulating intercellular and/or intracellular free radical concentration in the subject. The method includes administering to the subject an effective amount of a compound containing at least one aliphatic nitrate group and at least one sulfur atom in proximity to said nitrate, such as, for example, a compound of formula I. In one embodiment, the nitrate of this method contains at least 2 nitrate groups. In another embodiment, the nitrate is beta or gamma to a sulfur atom.
Yet another embodiment features compounds of formula III for preventing or mitigating tissue and/or cellular damage where m is an integer from 0 to 10; n is an integer from 0 to 10; each of R3,4,17 is, independently, hydrogen, a nitrate group, or A; R1 is hydrogen or A, with A is selected from: a substituted or unsubstituted C1-C24 alkyl group, optionally containing 1 to 4 O, S, NR6, and/or unsaturations in the chain, optionally bearing from 1 to 4 hydroxy, nitrate, Cl, F, amino or unsubstituted or substituted aryl, or unsubstituted or substituted heterocyclic groups; an unsubstituted or substituted cyclic moiety having from 3 to 7 carbon atoms in the ring, which optionally contains O, S, NR6, and/or unsaturations in the ring, optionally bearing from 1 to 4 hydroxy, nitrate, Cl, F, amino or unsubstituted or substituted aryl, or unsubstituted or substituted heterocyclic groups; an unsubstituted or substituted moiety constituting a linkage from 0 to 5 carbons, to or between any of R1, R3, R17 and R4, which optionally contains O, S, NR6, and/or unsaturations in the linkage, and optionally bearing from 1 to 4 hydroxy, nitrate, Cl, F, amino or unsubstituted or substituted aryl, or unsubstituted or substituted heterocyclic groups; a substituted or unsubstituted C1-C24 alkyl group, containing 1-4 linkages selected from C═O, C═S, and C═NOH, which optionally contains O, S, NR6, and/or unsaturations in the chain, optionally bearing from 1 to 4 hydroxy, nitrate, Cl, F, amino or unsubstituted or substituted aryl, or unsubstituted or substituted heterocyclic groups; a substituted or unsubstituted aryl group; a substituted or unsubstituted heterocyclic group; an amino group selected from alkylamino, dialkylamino, cyclic amino, cyclic diamino, cyclic triamino, arylamino, diarylamino, and alkyarylamino; a hydroxy group; an alkoxy group; and a substituted or unsubstituted aryloxy group;
Preferred compounds include any of the compounds of formulas II, III, IV, and V that have been individually described herein. Other preferred compounds include:
Particularly preferred compounds for preventing or mitigating tissue and/or cellular damage are:
It will be noted that the structure of some of the compounds of this invention includes asymmetric carbon atoms. It is to be understood accordingly that the isomers (e.g., enantiomers, diastereomers) arising from such asymmetry are included within the scope of this invention. Such isomers can be obtained in substantially pure form by classical separation techniques and by asymmetric synthesis. For the purposes of this application, unless expressly noted to the contrary, a compound shall be construed to include both the R and S stereoisomers at each stereogenic center.
In certain embodiments, a therapeutic compound of the invention comprises a cation (i.e., in certain embodiments, one of X or Y includes a cation, e.g., in the compound of formula IVd). If the cationic group is a proton, then the compound is considered an acid. If the proton is replaced by a metal ion or its equivalent, the compound is a salt. Pharmaceutically acceptable salts of the therapeutic compound are within the scope of the invention. For example, M can be a pharmaceutically acceptable alkali metal (e.g. Li, Na, K), ammonium, alkaline earth metal (e.g. Ca, Ba, Mg), higher valency cation, or polycationic counter ion (e.g., polyammonium cation) (see e.g., Berge et al., 1977). It will be appreciated that the stoichiometry of an anionic portion of the compound to a salt-forming cation will vary depending on the charge of the anionic portion of the compound and the charge of the counterion. Preferred pharmaceutically acceptable salts include a sodium, potassium, or calcium salt, but other salts are also contemplated within their pharmaceutically acceptable range.
A therapeutic compound of the invention can be administered in a pharmaceutically acceptable vehicle. As used herein “pharmaceutically acceptable vehicle” includes any and all solvents, excipients, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like which are compatible with the activity of the compound and are physiologically acceptable to the subject. An example of a pharmaceutically acceptable vehicle is buffered normal saline (0.15 M NaCl). The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the therapeutic compound, use thereof in the compositions suitable for pharmaceutical administration is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Carrier or substituent moieties useful in the present invention may also include moieties which allow the therapeutic compound to be selectively delivered to a target organ. For example, delivery of the therapeutic compound to the brain may be enhanced by a carrier moiety using either active or passive transport (a “targeting moiety”). Illustratively, the carrier molecule may be a redox moiety, as described in, for example, U.S. Pat. Nos. 4,540,654 and 5,389,623, both to Bodor. These patents disclose drugs linked to dihydropyridine moieties which can enter the brain, where they are oxidized to a charged pyridinium species which is trapped in the brain. Thus drugs accumulate in the brain. Other carrier moieties include compounds, such as amino acids or thyroxine, which can be passively or actively transported in vivo. Such a carrier moiety can be metabolically removed in vivo, or can remain intact as part of an active compound. Structural mimics of amino acids (and other actively transported moieties) including peptidomimetics, are also useful in the invention. As used herein, the term “peptidomimetic” is intended to include peptide analogues which serve as appropriate substitutes for peptides in interactions with, for example, receptors and enzymes. The peptodomimetic must possess not only affinity, but also efficacy and substrate function. That is, a peptidomimetic exhibits functions of a peptide, without restriction of structure to amino acid constituents. Peptidomimetics and methods for their preparation and use are described in Morgan et al. (1989), the contents of which are incorporated herein by reference. Many targeting moieties are known, and include, for example, asialoglycoproteins (see e.g., Wu, U.S. Pat. No. 5,166,320) and other ligands which are transported into cells via receptor-mediated endocytosis (see below for further examples of targeting moieties which may be covalently or non-covalently bound to a target molecule).
In the methods of the invention, neurodegeneration in a subject is mitigated, and/or neuroprotection and/or cognition enhancement is effected, by administering a therapeutic compound of the invention to the subject. The invention also features methods for preventing or mitigating tissue and/or cellular damage in a subject by administering a therapeutic compound of the invention to the subject, thereby modulating intercellular and/or intracellular free radical concentration. The term “subject” is intended to include living organisms in which the particular neurological condition to be treated can occur. Examples of subjects include humans, apes, monkeys, cows, sheep, goats, dogs, cats, mice, rats, and transgenic species thereof. As would be apparent to a person of skill in the art, the animal subjects employed in the working examples set forth below are reasonable models for human subjects with respect to the tissues and biochemical pathways in question, and consequently the methods, therapeutic compounds and pharmaceutical compositions directed to same. As evidenced by Mordenti (1986) and similar articles, dosage forms for animals such as, for example, rats can be and are widely used directly to establish dosage levels in therapeutic applications in higher mammals, including humans.
In particular, the biochemical cascade initiated by cerebral ischemia is generally accepted to be identical in mammalian species (Mattson and Scheff, 1994; Higashi et al., 1995). In light of this, pharmacological agents that are neuroprotective in animal models such as those described herein are believed to be predictive of clinical efficacy in humans, after appropriate adjustment of dosage. Specifically, there are comparable memory-deficit patterns between brain-damaged rats and humans, which indicates that the rat can serve as an excellent animal model to evaluate the efficacy of pharmacological treatments or brain damage upon memory (Kesner, 1990). The only approved drug for the clinical treatment of occlusive stroke in humans is tissue plasminogen activator, which is administered at a dose of 0.9 mg/kg by intravenous injection (Wittkowsky, 1998). This drug is also effective in protecting the rat brain subjected to cerebral ischemia by occlusion of the middle cerebral artery, when administered at a dose of 10 mg/kg intravenously (Jiang et al., 1998). Thus, the rat model of focal cerebral ischemia used in the development of the novel organic nitrate esters described herein has been shown to be shown to be predictive of clinical efficacy with at least one other class of pharmacological agents.
As would also be apparent to a person skilled in the art, the invention further encompasses methods of the invention employed ex vivo or in vitro. For example, the Examples describe studies utilizing tissue homogenates according to the invention. Furthermore, diagnostic tests or studies of efficacy of selected compounds may conveniently be performed ex vivo or in vitro, including in animal models. Such tests, studies and assays are within the scope of the invention.
The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). Administration of the compositions of the present invention to a subject to be treated can be carried out using known procedures, at dosages and for periods of time effective to mitigate neurodegeneration, to effect neuroprotection, to effect cognition enhancement, and/or to prevent or mitigate tissue and/or cellular damage in the subject. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the amount of neurodegeneration that has already occurred at the clinical site in the subject, the age, sex, and weight of the subject, and the ability of the therapeutic compound to mitigate neurodegeneration, to effect neuroprotection, to effect cognition enhancement, and/or to prevent or mitigate tissue and/or cellular damage in the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the invention (e.g., Va) is between 0.5 and 500 mg/kg of body weight per day. In an aqueous composition, preferred concentrations for the active compound (i.e., the therapeutic compound that can mitigate neurodegeneration, effect neuroprotection, effect cognition enhancement, and/or prevent or mitigate tissue and/or cellular damage) are between 5 and 500 mM, more preferably between 10 and 100 mM, and still more preferably between 20 and 50 mM.
The therapeutic compounds of the invention can be effective when administered orally. Accordingly, a preferred route of administration is oral administration. Alternatively, the active compound may be administered by other suitable routes such as transdermal, subcutaneous, intraocular, intravenous, intramuscular or intraperitoneal administration, and the like (e.g., by injection). Depending on the route of administration, the active compound may be coated in a material to protect the compound from the action of acids, enzymes and other natural conditions which may inactivate the compound.
The compounds of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB, they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs (“targeting moieties”), thus providing targeted drug delivery (see, e.g., Ranade et al., 1989). Exemplary targeting moieties include folate and biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., 1988); antibodies (Bloeman et al., 1995; Owais et al., 1995); and surfactant protein A receptor (Briscoe et al., 1995). In a preferred embodiment, the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety.
Delivery and in vivo distribution can also be affected by alteration of an anionic group of compounds of the invention. For example, anionic groups such as phosphonate or carboxylate can be esterified to provide compounds with desirable pharmocokinetic, pharmacodynamic, biodistributive, or other properties. Exemplary compounds include IVl and pharmaceutically acceptable salts or esters thereof.
To administer the therapeutic compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the therapeutic compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al., 1984).
The therapeutic compound may also be administered parenterally (e.g., intramuscularly, intravenously, intraperitoneally, intraspinally, or intracerebrally). Dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
Sterile injectable solutions can be prepared by incorporating the therapeutic compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the therapeutic compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient (i.e., the therapeutic compound) optionally plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The therapeutic compound can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The therapeutic compound and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the therapeutic compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the therapeutic compound in the compositions and preparations may, of course, be varied. The amount of the therapeutic compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a therapeutic compound for the treatment of neurological conditions in subjects.
Therapeutic compositions can be administered in time-release or depot form, to obtain sustained release of the therapeutic compounds over time. The therapeutic compounds of the invention can also be administered transdermally (e.g., by providing the therapeutic compound, with a suitable carrier, in patch form).
Active compounds are administered at a therapeutically effective dosage sufficient to mitigate neurodegeneration, to effect neuroprotection, to effect cognition enhancement, and/or to prevent or mitigate tissue and/or cellular damage in a subject. A “therapeutically effective dosage” preferably mitigates neurodegeneration by about 20%, more preferably by about 40%, even more preferably by about 60%, and still more preferably by about 80% relative to untreated subjects. The ability of a compound to mitigate neurodegeneration can be evaluated in model systems that may be predictive of efficacy in mitigating neurodegeneration in human diseases, such as animal model systems known in the art (including, e.g., the method of transient middle cerebral artery occlusion in the rat) or by in vitro methods, (including, e.g., the assays described herein).
It will be appreciated that the ability of a compound of the invention to mitigate neurodegeneration will, in certain embodiments, be evaluated by observation of one or more symptoms or signs associated with neurodegeneration in vivo. Thus, for example, the ability of a compound to mitigate neurodegeneration may be associated with an observable improvement in a clinical manifestation of the underlying neurodegeneration-related disease state or condition, or a slowing or delay in progression of symptoms of the condition. Thus, monitoring of clinical manifestations of disease can be useful in evaluating the neurodegeneration-mitigating efficacy of a compound of the invention.
The method of the invention is useful for treating neurodegeneration associated with any disease in which neurodegeneration occurs. Clinically, neurodegeneration can be associated with conditions including but not limited to: stroke; Parkinson's disease; Alzheimer's disease; Huntington's disease; multiple sclerosis; amylotrophic lateral sclerosis; AIDS-induced dementia; epilepsy; alcoholism; alcohol withdrawal; drug-induced seizures; viral/bacterial/fever-induced seizures; trauma to the head; hypoglycemia; hypoxia; myocardial infarction; cerebral vascular occlusion; cerebral vascular hemorrhage; hemorrhage; environmental excitotoxins of plant; animal, or marine origin; dementias of all type; trauma; drug-induced brain damage; and aging; or result from surgical procedures such as cardiac bypass.
Novel compounds according to the invention can be synthesized by methods set forth herein or in U.S. Pat. Nos. 5,807,847; 5,883,122; 6,310,052; and 6,365,579, for example. Various compounds for use in the methods of the invention are commercially available and/or can be synthesized by standard techniques. In general, nitrate esters can be prepared from the corresponding alcohol, oxirane or alkene by standard methods, that include: nitration of alcohols and oxiranes, mixed aqueous/organic solvents using mixtures of nitric and sulfuric acid and/or their salts, with temperature control (see Yang et al., 1996); nitration of alcohols and oxiranes in acetic anhydride using nitric acid or its salts with or without added acid catalyst, with temperature control (see, e.g., Louw et al., 1976); nitration of an alcohol with a nitronium salt, e.g. a tetrafluoroborate; nitration of an alkene with thallium nitrate in an appropriate solvent (see Ouellette et al., 1976).
The following Examples further illustrate the present invention and are not intended to be limiting in any respect. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
Synthesis of Compound IVr
As shown in
Synthesis of Compound Va, Vbb, and Vbc
As shown in
Synthesis of Compounds Ve, Vf, Vg, Vh, Vi, Vj, and Vu
Alkyl bromides or alkyl mercaptans were obtained commercially or by adaptation of literature procedures. Bunte salts were obtained from the appropriate alkyl bromide by reaction with sodium thiosulfate, as described above for compound IVd. Bunte salts (9.67 mmoles) were dissolved in distilled water (10 mL). To this solution, a solution of mercaptan (6.46 mmoles) in 1M NaOH (7 mL) was added dropwise. The resulting emulsion was stirred for 1 to 15 minutes and then extracted with dichlorometane or ethylacetate. The combined organic extracts were washed with H2O, dried over MgSO4 and concentrated under vacuum. The residue was purified by flash column chromatography on silica gel to give the following: Compound Vj (53% yield), 13C NMR (75 Mhz, CDCl3) 36.9, 69.8, 77.6, 128.6, 129.5, 129.8, 136.0; Compound Vi (43% yield), 13C NMR (75 Mhz, CDCl3) 36.9, 69.8, 77.4, 122.7, 130.9, 132.9, 135.1; Compound Vh (9% yield), 13C NMR (75 Mhz, CDCl3) 36.9, 69.6, 77.6, 124.5, 126.9, 144.7, 147.0; Compound Vg (81% yield), 13C NMR (75 Mhz, CDCl3) 36.8, 69.8, 77.5, 129.9, 130.8, 134.5, 134.8; Compound Vf (56% yield), 13C NMR (75 Mhz, CDCl3) 36.5, 55.8, 69.7, 77.8, 115.4, 126.7, 133.7, 160.9; Compound Ve (52% yield), 13C NMR (75 Mhz, CDCl3) 21.5, 36.8, 69.8, 77.7, 130.5, 130.6, 132.5, 139.2; and Compound Vuj (61% yield), 13C NMR (75 Mhz, CDCl3) 36.7, 69.9, 77.6, 116.8-117.1(d), 131.3-131.3(d), 132.5-132.6(d), 161.6-164.9(d).
Synthesis of Compound Vb
1,4-dibromo-2,3-dinitrobutanediol (8.88 mmol) and Na2S2O3.5H2O (2.81 g; 18 mmol) were dissolved in a mixture of 100 mL of MeOH and 45 mL of H2O. The resulting solution was heated during 4 days at 40-45°. After this time the reaction mixture was partially evaporated to reduce the volume of solvents. The resulting mixture was extracted with 4×50 mL of Et2O. The extracts were combined, washed (H2O), dried (MgSO4) and concentrated. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc 85:15), yielding the title compound (10%) 13C(CDCl3): δ3.9, 31.7.
Tables 1 and 2 list characterization data determined for other compounds of the invention.
1H NMR
13C NMR
Characterization of Cardioprotection in Isolated, Perfused Heart
In order to test for potential cardioprotective properties, the effects of Va and compound IIIam were tested in an in vitro model of cardiac ischemia, in which isolated, perfused rat hearts were subjected to transient left coronary artery occlusion (LCAO) followed by reperfusion. Drug treatments [DMSO (drug vehicle), GTN, compound Va or compound IIIam were initiated at two distinct time points: (i) prior to and throughout the 45 minute period of LCAO (protection) or (ii) prior to and throughout the 90 minute reperfusion period (salvage). Drug-induced reduction of lactate dehydrogenase (LDH) release and reduction of infarct size were assessed as measures of cardioprotection. Rat hearts were excised and mounted for retrograde aortic perfusion at a constant flow rate of 6-8 mL/min/g heart weight. The coronary perfusion pressure was monitored by a pressure transducer connected to the perfusion line. To induce regional ischemia, the left coronary artery was occluded for 45 minutes, after which the occlusion was released and the heart reperfused for 90 minutes. At the end of the reperfusion period, the LCA was re-occluded and 0.5 mL of 1% Evan's Blue dye was slowly infused into the heart, via the aotic cannula, to stain the area of myocardium perfused by the patent right coronary artery. Thus the area-at-risk (AAR) for infarction was determined by negative staining. Acute ischemic damage was assessed by measuring the release of the cytosolic enzyme, LDH, into the perfusate, and by quantitation of infarct size by staining for viable tissue using 2,3,5-triphenyltetrazolium chloride (TTC) followed by computerized planimmetry. Infarct size was expressed as infarct area (negative staining after TTC staining) as a percent of the area-at-risk (negative staining after Evan's Blue dye). Left coronary artery occlusion was associated with a 5-10 fold increase in LDH release (
Neuroprotection Against 6-hydroxydopamine-induced Killing of Dopaminergic Neurons in the Rat Substantia Nigra Pars Compacta
Male Long-Evans rats were anesthetized with sodium pentobarbital, and received stereotaxic, unilateral injections of 6-OHDA (6 μg in 2 μl) into the right substantia nigra pars compacta. Vehicle (dimethylsulfoxide, DMSO) or compound Va were given by subcutaneous injection every hour for 6 hours, beginning 30 minutes before 6-OHDA. Each dose of compound Va was 200 μmol/kg. Two weeks after the administration of 6-OHDA or vehicle, the rats received a single injection of apomorphine (1 mg/kg, s.c.), and contralateral rotations were counted at 15 minute intervals for 60 minutes. In some animals, the brains were fixed, and frozen sections cut for immunocytochemical analysis of tyrosine hydroxylase (TH).
In vehicle-treated animals, apomorphine induced rotations contralateral to the lesion that persisted for the entire 60 minute observation period. In contrast, animals that received compound Va exhibited essentially no rotations over 60 minutes in response to apomorphine injection, suggesting virtually complete protection against the neurotoxic effects of 6-OHDA (Table 3). Immunocytochemical analysis confirmed that compound Va preserved TH-positive neurons in the substantia nigra of 6-OHDA-injected rats (
*p < 0.001 compared to vehicle control, One-way analysis of variance with Bonferroni's Multiple Comparison's test.
Synaptosome Assay The methodology for measurement of thiobarbituric acid reactive substances (TBARS) from synaptosomes was adapted from that of Keller et al. Neuroscience 80: 685-696, 1997. Adult Sprague-Dawley rats (250-300 g) were anesthetized with halothane for 20 sec. and decapitated. The brain was removed and the cerebral cortex separated from white matter. The cerebral cortex was homogenized in a solution containing sucrose 0.32 M, EDTA 2 mM and TRIS.HCl 10 mM, pH 7.2, using a Teflon pestle. The tissue was 5% w/v in the homogenizing buffer. The homogenate was centrifuged for 10 minutes at 310 g at 4° C. The supernatant was then centrifuged for 10 minutes at 20,000 g at 4° C. The pellet was collected, resuspended in Locke's buffer (154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2.2H2O, 1.0 mM MgCl2.6H2O, 3.6 mM NaHCO3, 5 mM glucose, 5 mM HEPES, pH 7.2) and centrifuged for 10 minutes at 20,000 g at 4° C. The procedure of washing the pellet was repeated 2-3 times in order to reduce transition metal ion concentrations. Finally, the pellet was resuspended in Locke's buffer for use in the lipid peroxidation assay. All assays were performed in triplicate and on three separate synaptosome preparations from different animals.
Potential antioxidants and prooxidants were freshly prepared: in Locke's buffer [FeSO4; ascorbic acid (AA); Trolox; cysteine]; in 10 mM NaOH [NONOates]; or in organic solvent, such as methanol or DMSO, [α-tocopherol (αTH); nitrates; nitrites; PhSH; lipoic acid (LA); dihydrolipoic acid (LAH2)]; final dilution ≦2.5% (v/v) organic component). Pro/antioxidants (or solvent vehicle in control experiments) were added to the synaptosome preparation, followed immediately by freshly prepared aqueous FeSO4 (or buffer in control experiments) and incubated for 30 minutes at 37° C. (air equilibrated). After incubation, TBA reagent (TBA 0.375% w/v, Cl3CCO2H 15% w/v, HCl 1M 25% v/v) was added to the homogenate (homogenate: TBA reagent, 1:2 v/v), and the sealed samples were boiled for 15 minutes at approx. 96-100° C. The cooled samples were then centrifuged for 10 minutes at 9,000 g at room temperature. The pink supernatant was transferred into microplates and the absorbance was measured at 530 nm on a Dynex MRX microplate reader. TBA reagent solutions were freshly made and calibrated using solutions of authentic malondialdehyde.
Lipid peroxidation: synaptosome experimental results. Preliminary lipid peroxidation experiments explored the time course of synaptosome lipid peroxidation, incubating homogenate with FeSO4 (10 μM-150 μM) in Locke's buffer, for time intervals from 15 minutes to 180 minutes (data not shown). Under these experimental conditions, the level of peroxidation, as measured by TBARS, was seen to be below saturation at 30 minutes using 50 μM FeSO4. The ability of this system to provide concentration dependent lipid peroxidation data was demonstrated using the antioxidant α-tocopherol (αTH), and ascorbic acid (AH), which is known to act as a prooxidant in Fe(II)-induced lipid peroxidation systems (see
The Fe/synaptosome/TBARS system was designed to provide concentration-response curves for inhibition of lipid peroxidation, which might be quantified by EC50 values. Absolute EC50 values measured in such systems are highly dependent on experimental conditions, and therefore must be benchmarked against well-studied antioxidants, such as Trolox, a water soluble chroman carboxylate derivative of αTH (see
Thiols can display mixed pro- and antioxidant activity towards lipid peroxidation. In particular, in the presence of transition metals, either added to lipid preparations, or adventitious metal ions present in tissue homogenates, thiols may act as prooxidants. The vic-dithiol, dihydrolipoic acid (LAH2, ▪, solid line in
The antioxidant trolox yielded a potent concentration dependent reduction in TBARS products with EC50=6.8×10−5 M (
GTN alone had no effect on Fe-induced lipid peroxidation (data not shown), nor did varied concentrations of GTN have any significant effect in the presence of added LAH2 (1 mM) (
Addition of PhSH (1 mM) yielded a concentration dependent inhibition curve for lipid peroxidation: EC50=1.4×10−5 M. At high millimolar concentrations, compound Va showed some prooxidant activity in the presence of cysteine.
Data for inhibition of iron-induced lipid peroxidation by compound IVr can be fit to a curve leading to 100% efficacy with an EC50 of 1.2×104M (or fitted without constraint on efficacy to EC50=2.7×10−4 M; efficacy=78%). Moreover, compound Va, in the absence of thiol, showed a modest inhibition of lipid peroxidation, more pronounced at lower concentrations (see
Concentration response curves were derived from TBARS data for the NO donor NONOate, Sper/NO (spermine NONOate, dashed line), and for DEA/NO (diethylamine NONOate, solid line) (see
All publications and patents cited in this specification are hereby incorporated by reference herein as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
This application is a continuation-in-part of application Ser. No. 10/147,808, filed May 20, 2002, which is a division of application Ser. No. 09/267,379, filed Mar. 15, 1999 and issued on Oct. 30, 2001 as U.S. Pat. No. 6,310,052, which is in turn a continuation-in-part of application Ser. No. 08/867,856, filed Jun. 3, 1997 and issued Mar. 16, 1999 as U.S. Pat. No. 5,883,122, which is in turn a continuation-in-part of application Ser. No. 08/658,145, filed Jun. 4, 1996 and issued Sep. 15, 1998 as U.S. Pat. No. 5,807,847. This application also claims the benefit of application Ser. No. 09/473,713, filed Dec. 29, 1999. Each of above applications is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09267379 | Mar 1999 | US |
| Child | 10147808 | May 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10147808 | May 2002 | US |
| Child | 10943264 | Sep 2004 | US |
| Parent | 08867856 | Jun 1997 | US |
| Child | 09267379 | Mar 1999 | US |
| Parent | 08658145 | Jun 1996 | US |
| Child | 08867856 | Jun 1997 | US |
| Parent | 09473713 | Dec 1999 | US |
| Child | 10943264 | Sep 2004 | US |
