Treatment of neurodegenerative diseases

Abstract: The use of a meta-nicotine compound for the treatment of neurodegenerative diseases is described.

Description: BACKGROUND OF THE INVENTION The present invention relates to a medicament for treating patients having neurodegenerative diseases, and in particular those diseases which cause a cholinergic deficit. Senile dementia of the Alzheimer's type (SDAT) is a debilitating neurodegenerative disease, mainly afflicting the elderly; characterized by a progressive intellectual and personality decline, as well as a loss of memory, perception, reasoning, orientation and judgment. One feature of the disease is an observed decline in the function of cholinergic systems, and specifically, a severe depletion of cholinergic neurons (i.e., neurons that release acetylcholine, which is believed to be a neurotransmitter involved in learning and memory mechanisms). See, Jones, et al., Intern. J. Neurosci. , Vol. 50, p. 147 (1990); Perry, Br. Med. Bull. , Vol. 42, p. 63 (1986) and Sitaram, et al., Science , Vol. 201, p. 274 (1978). It has been observed that nicotinic acetylcholine receptors, which bind nicotine and other nicotinic agonists with high affinity, are depleted during the progression of SDAT. See, Giacobini, J. Neurosci. Res. , Vol. 27, p. 548 (1990); and Baron, Neurology , Vol. 36, p. 1490 (1986). As such, it would seem desirable to provide therapeutic compounds which either directly activate nicotinic receptors in place of acetylcholine or act to minimize the loss of those nicotinic receptors. Parkinson's disease (PD) is a debilitating neurodegenerative disease, presently of unknown etiology, characterized by tremors and muscular rigidity. A feature of the disease appears to involve the degeneration of dopaminergic neurons (i.e., which secrete dopamine). One symptom of the disease has been observed to be a concomitant loss of nicotinic receptors which are associated with such dopaminergic neurons, and which are believed to modulate the process of dopamine secretion. See, Rinne, et al., Brain Res., Vo1. 54, pp. 167-170 (1991) and Clark, et al., Br. J. Pharm., Vol. 85, pp. 827-835 (1985). Certain attempts have been made to treat SDAT. For example, nicotine has been suggested to possess an ability to activate nicotinic cholinergic receptors upon acute administration, and to elicit an increase in the number of such receptors upon chronic administration to animals. See, Rowell, Adv. Behav. Biol., Vol. 31, p. 191 (1987); and Marks, J. Pharmacol. Exp. Ther., Vol. 226, p. 817 (1983). Other studies indicate that nicotine can act directly to elicit the release of acetylcholine in brain tissue, to improve cognitive functions, and to enhance attention. See, Rowell, et al., J. Neurochem., Vo1. 43, p. 1593 (1984); Hodges, et al., Bio. of Nic., Edit. by Lippiello, et al., p. 157 (1991); Sahakian, et al., Br. J. Psych., Vol. 154, p. 797 (1989); and U.S. Patent No. 4,965,074 to Leeson. Other studies on the potential use of nicotine in the treatment of SDAT or PD include Pharmacol. Biochem. Behav.; 28(2), 1987, 299-303; Clin. Chest Med. 12(4), December 1991, 681-699; EP-A-0377520; and Nature, 336 (6196), 17 November 1988, 207-208. It would be desirable to provide a treatment for neurodegenerative diseases, such as SPAT and PD, which uses another compound. Metanicotine is a nicotine degradation product which as been known since 1861. Its biological properties were first investigated in 1895 and further studies followed in 1950 and later (see J. Pharmacol. Exp. Ther., 196 (3), March 1976, 685-696). The same article reports studies on the trans isomer, which was found to be significantly less active than nicotine in tests. Klin. Wochenschr., 63 (2), 1985, 90-92 also describes studies on metanicotine, again showing it to be less active than nicotine. SUMMARY OF THE INVENTION The present invention relates to a medicament for the treatment of a neurodegenerative disease (e.g. SDAT or PD) which includes an effective amount of a metanicotine compound, and particularly, a trans-meta-nicotine compound. The invention provides benefits to the patient in that the compounds have the potential to (i) act as a pharmacological agonist to activate nicotinic receptors, and (ii) elicit neurotransmitter secretion. In addition, the compounds are expected to have the potential to (i) increase the number of nicotinic cholinergic receptors of the brain of the patient, and (ii) exhibit neuroprotective effects. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates in particular to the use of a compound having the general formula: where n represents an integer from 1-5; m represents an integer from 0-4; R represents H or straight chain or branched C 1-5 alkyl and X represents halo (i.e., F, Cl, Br or I) or alkyl as previously defined. Preferably, each of n and m equals 1. Preferably, the 5 carbon atom of the pyridine ring includes a substitutent group other than hydrogen (e.g., a halo or alkyl substituent). Preferably, R is methyl and m is O. See, Registry No. 538-79-4 and LaForge, J.A.C.S. , Vol. 50, p. 2477 (1928). The manner in which the compounds are administered can vary. The compounds can be administered by inhalation; in the form of an aerosol either nasally or using delivery articles of the type set forth in U.S. Patent Nos. 4,922,901 to Brooks, et al. and 5,099,861 to Clearman et al.; orally (e.g., in liquid form within a solvent such as an aqueous liquid, or within a solid carrier); intravenously (e.g., within a saline solution); or transdermally (e.g., using a transdermal patch). Exemplary methods for administering such compounds will be apparent to the skilled artisan. Certain methods suitable for administering compounds useful according to the present invention are set forth in U.S. Patent No. 4,965,074 to Leeson. The administration can be intermittent, or at a gradual, continuous, constant or controlled rate to a warm-blooded animal, such as a human being or other mammal. The dose of the compound is that amount effective to treat the neurodegenerative disease from which the patient suffers. By "effective amount" or "effective dose" is meant that amount sufficient to pass across the blood-brain barrier of the patient, to bind to relevant receptor sites in the brain of the patient, and to elicit neuropharmacological effects (e.g., elicit neurotransmitter secretion, thus resulting in effective treatment of the disease). Treatment of a neurodegenerative disease involves a decrease of symptoms of the particular disease. For human patients, the effective dose of typical compounds generally does not exceed about 150 µg, often does not exceed about 100 µg, and frequently does not exceed about 50 µg, per kg patient weight. For human patients, the effective dose of typical compounds generally is at least about 5 µg, often is at least about 10 µg, and frequently is at least about 25 µg, per kg of patient weight. For human patients, the effective dose of typical compounds generally requires administering the compound in an amount of at least about 2.0, often at least about 1.0, and frequently at least about 0.1 mg/hr./patient. For human patients, the effective dose of typical compounds requires administering the compound in an amount which generally does not exceed about 10, often does not exceed about 5, and frequently does not exceed about 2.5 mg/hr./patient. The compounds useful according to the present invention have the ability to pass across the blood-brain barrier of the patient. As such, such compounds have the ability to enter the central nervous system of the patient. The log P values of typical compounds useful in carrying out the present invention generally are greater than 0, often are greater than about 0.1, and frequently are greater than about 0.5. The log P values of such typical compounds generally are less than about 3.0, often are less than about 2.5, and frequently are less than about 2.0. Log P values provide a measure of the ability of a compound to pass across a diffusion barrier, such as a biological membrane. See, Hansch, et al., J. Med. Chem. , Vol. 11, p. 1 (1968). The compounds useful according to the present invention have the ability to bind to, and hence cause activation of, nicotinic cholinergic receptors of the brain of the patient. As such, such compounds have the ability to act as nicotinic agonists. The receptor binding constants of typical compounds useful in carrying out the present invention generally exceed about 1 nM, often exceed about 200 nM, and frequently exceed about 500 nM. The receptor binding constants of such typical compounds generally are less than about 10 µM, often are less than about 7 µM, and frequently are less than about 2 µM. Receptor binding constants provide a measure of the ability of the compound to bind to half of the relevant receptor sites of certain brain cells of the patient. See, Cheng, et al., Biochem. Pharmacol. , Vol. 22, pp. 3099-3108 (1973). The compounds useful according to the present invention have the ability to demonstrate a nicotinic function by effectively eliciting neurotransmitter secretion from nerve ending preparations (i.e., synaptosomes). As such, such compounds have the ability to cause relevant neurons to release or secrete acetylcholine, dopamine, and other neurotransmitters. Generally, typical compounds useful in carrying out the present invention provide for the secretion of dopamine in amounts of at least about 5 percent, often at least about 25 percent, and frequently at least about 50 percent, of that elicited by an equal molar amount of S(-) nicotine. The following example is provided in order to further illustrate various embodiments of the invention. Unless otherwise noted, all parts and percentages are by weight. Example 1 Mice (DBA strain) were maintained on a 12 hour light/dark cycle and were allowed free access to water and food supplied by Wayne Lab Blox, Madison, WI. Animals used in the present studies were 60 to 90 days of age and weighed 20 to 25 g. Brain membrane preparations were obtained from pooled brain tissue of both males and females. Mice were killed by cervical dislocation. Brains were removed and placed on an ice-cold platform. The cerebellum was removed and the remaining tissue was placed in 10 volumes (weight:volume) of ice-cold buffer (Krebs-Ringers HEPES:NaCl, 118 mM; KCl, 4.8 mM; CaCl 2 , 2.5 mM; MgSO 4 , 1.2 mM; HEPES, 20 mM; pH to 7.5 with NaOH) and homogenized with a glass-Teflon tissue grinder. The resulting homogenate was centrifuged at 18000 x g for 20 min. and the resulting pellet was resuspended in 20 volumes of water. After 60 min. incubation at 4°C, a new pellet was collected by centrifugation at 18000 x g for 20 min. After resuspension in 10 volumes of buffer, a new final pellet was again collected by centrifugation at 18000 x g for 20 min. Prior to each centrifugation step, the suspension was incubated at 37°C for 5 min. to promote hydrolysis of endogenous acetylcholine. The final pellet was overlayered with buffer and stored at -70°C. On the day of the assay, that pellet was thawed, resuspended in buffer and centrifuged at 18000 x g for 20 min. The resulting pellet obtained was resuspended in buffer to a final concentration of approximately 5 mg protein/ml. Protein was determined by the method of Lowry, et al., J. Biol. Chem. , Vol. 193, pp. 265-275 (1951), using bovine serum albumin as the standard. The binding of L-[ 3 H]nicotine was measured using a modification of the method of Romano, et al., Science , Vol. 210, pp. 647-650 (1980) as described previously by Marks, et al., Mol. Pharmacol. , Vol. 30, pp. 427-436 (1986). The binding of L-[ 3 H]nicotine was measured using a 2 hr. incubation at 4°C. Incubations contained about 500 µg of protein and were conducted in 12 mm x 75 mm polypropylene test tubes in a final incubation volume of 250 µl. The incubation buffer was Krebs-Ringers HEPES containing 200 mM TRIS buffer, pH 7.5. The binding reaction was terminated by filtration of the protein containing bound ligand onto glass fiber filters (Micro Filtration Systems) that had been soaked in buffer containing 0.5 percent polyethyleneimine. Filtration vacuum was -50 to -100 torr. Each filter was washed five times with 3 ml of ice-cold buffer. The filtration apparatus was cooled to 2°C before use and was kept cold through the filtration process. Nonspecific binding was determined by inclusion of 10 µM nonradioactive nicotine in the incubations. The inhibition of L-[ 3 H]nicotine binding by test compounds was determined by including one of eight different concentrations of the test compound in the incubation. Inhibition profiles were measured using 10 nM L-[ 3 H]nicotine and IC 50 values were estimated as the concentration of compound that inhibited 50 percent of specific L-[ 3 H]nicotine binding. Inhibition constants (Ki values) were calculated from the IC 50 values using the method of Cheng, et al., Biochem. Pharmacol. , Vol. 22, pp. 3099-3108 (1973). The Ki values for all compounds for which an inhibition constant less than 100 µM was determined from the inhibition curves described above were also calculated independently using Dixon plots for inhibition measured using 2 nM, 8 nM and 20 nM concentrations of L-[ 3 H]nicotine. The L-[ 3 H]nicotine used in all experiments was purified chromatographically by the method of Romm, et al., Life Sci. , Vol. 46, pp. 935-943 (1990). Log P values (log octanol/water partition coefficient), which have been used to assess the relative abilities of compounds to pass across the blood-brain barrier, were calculated according to the methods described by Hansch, et al., J. Med. Chem. , Vol. 11, p. 1 (1968). Dopamine release was measured by preparing synaptosomes from the striatal area of rat brain obtained from Sprague-Dawley rats generally according to the procedures set forth by Nagy, et al., J. Neurochem. , Vol. 43, pp. 1114-1123 (1984). Striata from 4 rats were homogenized in 2 ml of 0.32M sucrose buffered with 5 mM HEPES (pH 7.5), using a glass-teflon tissue grinder. The homogenate was diluted to 5 ml with additional homogenization solution and centrifuged at 1000 x g for 10 min. This procedure was repeated on the new pellet and the resulting supernatant was centrifuged at 12,000 x g for 20 min. A 3 layer discontinuous Percoll gradient consisting of 16 percent, 10 percent and 7.5 percent Percoll in HEPES-buffered sucrose was made with the final pellet dispersed in the top layer. After centrifugation at 15,000 x g for 20 min., the synaptosomes were recovered above the 16 percent layer with a pasteur pipette, diluted with 8 ml of perfusion buffer (128 mM NaCl, 2.4 mM KCl, 3.2 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 25 mM HEPES pH 7.4, 10 mM dextrose, 1 mM ascorbate, 0.01 mM pargyline), and centrifuged at 15,000 x g for 20 min. The new pellet was collected and re-suspended in perfusion buffer. The synaptosome suspension was incubated for 10 min. at 37°C. Then [ 3 H]-dopamine (Amersham, 40-60 Ci/mmol) was added to the suspension to give a final concentration of 0.1 µM in suspension, and the suspension was incubated for another 5 min. Using this method, 30 to 90 percent of the dopamine was taken up into the synaptosomes, as determined by scintillation counting following filtration through glass fiber filters soaked with 0.5 percent polyethyleneimine. A continuous perfusion system was used to monitor release following exposure to ligand (i.e., trans-meta-nicotine). Synaptosomes were loaded onto glass fiber filters (Gelman type A/E). Perfusion buffer was dripped onto the filters (0.2-0.3 ml/min.) and pulled through the filters with a peristaltic pump. Synaptosomes were washed with perfusion buffer for a minimum of 20 min. before addition of the ligand. After the addition of a 0.2 ml of a 20 µM solution of ligand, the perfusate was collected into scintillation vials at 1 min. intervals and the dopamine released was quantified by scintillation counting. Peaks of radioactivity released above background were summed and the average basal release during that time was subtracted from the total. Release was expressed as a percentage of release obtained with an equal concentration of S(-) nicotine. Data regarding octanol-water partition coefficients, binding constants and neurotransmitter secretion capability for the ligands evaluated are set forth in Table I. Compound Ki (nM) logP Dopamine Release trans-meta-nicotine 21 ± 5 1.1 70 The data in Table I indicate that the compound has the capability of passing the blood-brain barrier, binding to high affinity nicotinic receptors, and eliciting neurotransmitter secretion. Thus, the data indicate that such compounds have the capability of being useful in treating neurodegenerative diseases.

Claims: The use in the manufacture of a medicament for treating a patient suffering from a neurodegenerative disease of a compound having the formula (1): where n represents an integer from 1-5, m represents an integer from 0-4, R represents H or C 1-5 alkyl, and X represents C 1-5 alkyl or halo. The use of claim 1 in which the neurodegenerative disease is senile dementia of the Alzheimer's type. The use of claim 1 in which the neurodegenerative disease is Parkinson's disease. The use of any preceding claim in which the compound of formula (1) is trans-meta-nicotine. The use of any of claims 1 to 3 in which the compound is such that m = 1 and n = l. The use of any preceding claim in which the medicament comprises an amount of the compound of formula (1) which provides at least 5 µg/kg patient weight, but does not exceed 150 µg/kg patient weight. The use of any of claims 1 to 5 in which the medicament comprises an amount of the compound of formula (1) which provides at least 0.10 mg/h/patient, but does not exceed about 10 mg/h/patient.

Inventors: CALDWELL WILLIAM S, LIPPIELLO PATRICK M, CALDWELL, WILLIAM S., LIPPIELLO, PATRICK M.
Applicants: REYNOLDS TOBACCO CO R, R.J. REYNOLDS TOBACCO COMPANY
Application year: 1993
Main classification: A61K31
IPC codes: A61K31, C07D213, A61P25