BG63150B1 - Method for the treatment of traumatic cerebral impairment - Google Patents

Abstract

The invention relates to a method for the treatment of traumatic cerebral impairment where 5-(2-ethyl-2H-terazol-yl)-1,2,3,6-tetrahydro-1-methylyridine with the formula The compound improves the identification manifestations and decreases the reduction of the cholinergic neurons in traumatically impaired cerebral models. 3 claims

Description

The invention relates to the use of 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine for the treatment of traumatic brain injury (TMU).

BACKGROUND OF THE INVENTION

EP-A1 0 296 721 describes a class of piperidine or 1,2,3,6-tetrahydropyridine compounds substituted at the 5-position with a 15 5-membered heterocyclic group comprising a subclass of optionally substituted 5-tetrazolyl 1,2,3 , 6-tetrahydro-pyridine compounds. According to the description, the compounds have high affinity for central cholinergic receptors, in particular high affinity for central muscarinic M _t receptors, and as such are useful in the treatment of Alzheimer's disease, senile dementia, and difficulty in learning and remembering. 25

The linkage structure-activity of this subclass is described by Moltzen et al., J.Med Chem., 1994, 37, 4085-4099. The compound (2etil-2H-tetrazol-5-yl) -1,3,6-tetrahydro-1-methylpyridine is selective for muscarinic re- 30 acceptor with a several fold higher affinity for M than for M2 and _M3 receptors (Subtypes of Muscarinic Receptors, The Sixth International Symposium, Nov. 9-12, 1994, Fort Lauderdale). It is disclosed that functional το 35 behaves as a partial agonist at M ₁ receptors and an antagonist at _M2 and M3 receptors. The only reported pronounced effect in vivo is the effect on spatial memory acquisition in young and 40 adult rat, respectively.

The treatment of traumatic brain injury, caused by a physical or neurological condition or by various diseases, is of increasing importance to humans and 45 is in dire need of an effective and safe remedy for the treatment of such disorders and their consequences.

It is surprisingly found that the compound 5- (2-ethyl-2H-tetrazal-5-yl) -1,2,3,6-tetrahydro 1-50 methylpyridine has a beneficial treatment effect (TMU) and its effects.

SUMMARY OF THE INVENTION

The invention relates to the use of 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine or an acid addition salt thereof

for the treatment of traumatic brain injury or its consequences.

The term traumatic brain injury (TMU) in this description means all conditions related to brain or spinal cord injury, such as caused by physical forces on the skull or spine, ischemia, stroke, paused breathing, cardiac arrest, cerebral thrombosis or embolism , neurological problems caused by AIDS, cerebral hemorrhage, encephalomyelitis, hydrocephalus, post-operative complications, cerebral infections, concussions, or increased internal pressure.

The pharmaceutically acceptable acid addition salts of the compounds used in the invention are formed with non-toxic organic or inorganic acids. Examples of such organic salts are those with maleic, fumaric, benzoic, ascorbic, pam, succinic, wine, salicylic, citric, gluconic, milky, almond, cinnamon, citric, aspartic, stearic, palmitic, itaconic, glucosalic, glucosalic glutamic, benzenesulfonic and theophylline acetic acid, as well as 8-halogenteophilins, for example 8-bromoethophylline. Examples of inorganic salts are those of hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric and nitric acids.

The compound of the invention has been found to be useful in the treatment of TMU. It improves cognitive performance after moderate traumatic brain damage and reduces damage-induced reduction of cholinergic neurons. Moreover, it was found not to cause adverse cardiac or other adverse effects at doses considered clinically relevant.

According to another aspect, the invention provides a method for the prevention or treatment of TMU in humans, comprising administering a medicamentally effective amount of 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1 -methylpyridine or an acid addition salvage thereof in a patient in need of such treatment.

The use of the compound of the invention and its pharmaceutically acceptable acid addition salts may be effected in any suitable manner, for example, orally or parenterally, and the compounds may be in any suitable form for such administration, for example in the form of tablets, capsules, powders, syrups or solutions or as dispersions for injection.

An effective daily dose of the compound of the invention or a pharmaceutically acceptable salt thereof is from 10 pg / kg to 10 mg / kg body weight, preferably 25 mkg / day / kg body weight to 1.0 mg / day / kg body weight. A suitable daily dose is 500 pg to 600 pg / day, preferably 1.0 mg to 100 mg.

The compound of the invention can be prepared as described in EP-A1 0 296 721 and its acid addition salts by methods well known to those skilled in the art.

Pharmacology

The compound used according to the invention is tested according to the following accepted and reliable method.

Cognitive function after TMU

Rats were subjected to central fluid percussion by traumatic brain injury, as described by Dixon, CEet al., J. Neurosurgery, 67 (1987) 110-119. Injured animals were treated subcutaneously daily for 1 to 15 days, such as start 24 hours after injury with saline or 5 (2-ethyl-2H-tetrazal-5-yl) -1,2,3,6-tetrahydro-1 methylpyridine, 3.6 pmol / kg or 15 pmol / kg.

Recovery of suppression of reflex after TMU was determined according to the method of Dioxon, C.E.et al. 1987 (see above).

Body weight reading is performed before injury and on days 1-5 after injury and rotarod expression is determined from 1 to 5 days after injury by the method of Hamm, RJ, et al., J. Neurotrauma, 11 (1994) 187 -196. The Rotarod test is used to measure motor performance after TMU.

Finally, the average latency (+ S.E.M) for finding the target platform at Morris water confusion is measured from 11 to 15 days after injury. The results were analyzed by ANOVA. During the water confusion tests, all animals were injected 10 minutes before the evaluation in this test.

False-injured animals (animals prepared for damage but not water-rippled) are included as a comparison.

Quantification of ChAT neurosis after TBI

TMU and treated rats were subjected to central liquid percussion as described above. They are injected subcutaneously with saline (n = 5) or with the test compound 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine, 15 pmol / kg (n = 5). False-damaged rats were injected subcutaneously with saline (n = 4) or test compound 15 pm/kg (n = 4).

Reflex suppression is performed as described above.

Possible loss of cholinergic neurons after TMU is quantified by choline acetyltransferase (C11AT) immunoreactive neurons in the basal forebrain. On the 15th day after injury (2-4 h after the last injection), all animals were anesthetized with phenobarbital (90 mg / kg intraperitoneally) and perfused transaortically with 200-250 ml isotonic saline and then with 500 ml 4.0% paraformaldepide / 0.2% picric acid in 0.1 M phosphate buffer at 500 ml / 30 min at room temperature. After perfusion, the brains were stacked in two blocks and left in the same solution at 10 ° C for 24 h. Coronal 40 pc sections were collected on vibrate through the nucleus of the forebrain and every fifth section was processed for ChAT immunoreactivity. Parallel sections are stained for Nissi cresyl violet substance for quantification for possible cholinergic and non-cholinergic neuronal loss in the basal forebrain.

Free flooded cerebral sections were incubated at a final concentration of ChAT antibodies (1:50) of 1.0 pg / tnl in 0.01 M phosphate buffered saline (PBS) containing 0.1% Triton X-100. The forebrain sections were sorted and incubated with the original antibody for 24 hours at room temperature in culture plates (4 sections / 300 μl / well).

After 4 washes in PBS, sections were incubated in the secondary antibody (equine anti-mouse IgG; Vector Laboratories, Burlingame, CA) for 1 h at 37 ° C. After 3 washes in PBS, the sections were incubated in a mouse avidin3 Biotin Peroxidase Complex (ABC) (Vector) technique (Hsu et al., J.Histochem. Cytochem., 29, 1981, 577-580) at 37 ° C. . After three washes with PBS and one c 0.1 M Tris-Buffered Saline (TBS), the free-floating sections were treated by the glucose oxidazadiaminobenzidine-nickel method described by Shu et al., Neurosci. Lett. 85 1988, 169-171. The reaction is terminated by transferring the sections to TBS. The sections were plated on gelatin-coated glass tiles and allowed to dry overnight at room temperature, dried with decreasing concentrations of ethanol and xylene, and then overlapped with Permount.

ChAT immunoreactive neurons in the middle septal nucleus (MSN), diagonal coupling vertical end nucleus (VDB) and basal magnocellulitis (NML) were counted using a microcomputer imaging system (MCID) (Ontario, Canada, imaging Research inc. ). The boundary between MSN and VDB is defined as the front link. NMB is defined as immunolabeled neurons located in the dlobulus pallidus and the adjacent inner hull. Celle counting is done for each animal obtained at 0.2 mm intervals through the forebrain nucleus. The number of cells was counted as the mean for the 10,000 gm ^{2 group} for each forebrain nucleus.

Neurons from MSN, VDB and NMB of parallel sections stained with cresyl violet are also counted on MCID. Due to the large number of total cholinergic and non-cholinergic neurons stained by cresyl violet in these regions, a bilateral beuron sample is recorded in each nucleus. A network with predetermined cell count sections for each nucleus is used. For NSN and VDB cores, three 2,000 gm ² sections on each side are counted (so a total area of 12,000 gm ² is checked for each core in each of the four sections counted). For NMB, one 12,000 gm ² plot on each side is counted (total area ”24,000 gm ² for each of the four sections counted). Only large cells (> 20 gm in diameter) with visible somatic Nissi bodies and with clear neural type nuclei are quantified.

Results

There is no significant difference between the individual injured animals in the evaluation of reflex suppression after TBI. This means that in each experiment, the injured groups receive an equivalent severity of injury. False-injured animals recovered significantly faster than any of the FAs (p <0.0001 for each comparison). The suppression of the recovery of the false-injured group is due to the gas anesthesia used before the injury.

There is no significant difference between the individual injured groups in terms of weight loss after TMU, which again shows an equivalent in severity of injury between the individual injured groups.

There were no significant differences in the rotarod expression from 1 to 5 days after injury in the individual injured groups. Because the test compounds are administered 1 to 15 days after injury, these trials also show that the drug has no effect on body weight or rotarod expression after injury.

ANOVA shows that the injured animals injected daily subcutaneously with the test compound exhibited better waterborne Morris confusion than the animals treated with saline. Injured animals,? treated with the test compound 15 gmol / kg 7 improved their expression with pCO.

TMU caused a significant reduction in the number of ChAT-IR neurons in VDB and NMB in saline-treated rats and the test compound, respectively. However, the compound of the invention significantly reduced this reduction in the CIAT-IRb damage score (32% reduction compared to the saline group relative to VDB and 51% in the NBM). Parallel cresyl violet sections did not show a decrease in the number of cells in MSN, VDB or NMB, indicating that the loss of ChAT-IR neurons was not due to cell mortality.

Formulation examples

The pharmaceutical formulations according to the invention can be prepared by commonly used methods.

Tablets may be prepared by mixing the active ingredient with conventional additives and / or diluents and then compressing the mixture into a conventional tabletting machine. Examples of additives or diluents are corn starch, lactose, talc, magnesium stearate, gelatin, lactose, resins and the like. Additional substances such as coloring agents, preservatives, flavors, etc. may also be used. provided that they are compatible with the active ingredients.

Injectable solutions can be prepared by dissolving the active ingredient and possible additives in a portion of the carrier, preferably sterile water, adjusting the solution to the desired volume, sterilizing the solution and filling it in suitable ampoules or vials. Other commonly used suitable additives, such as isotonizing agents, preservatives, antioxidants, etc., may be added.

Typical Formulation Examples of Form10 Propyl Paraben 0.1 mg Ethanol 0.005 ml Flavor 0.05 mg Saccharin Sodium 0.5 mg Water to 1 ml

4) Solution:

5- (2-ethyl-2H-tetrazol-5-yl) 1,2,3,6-tetrahydro-1 methylpyridine 1.0 mg sorbitol 5.1 mg acetic acid 0.08 mg water for injections up to 1 ml

the snippets of the invention are as follows (which, active substance is a free base): 1) Tablets: 5- (2-ethyl-2H-tetrazol-5-yl) - 1,2,3,6-tetrahydro-1 - calculated methylpyridine 20 mg lactose 60 mg cornstarch 30 mg hydroxypropyl cellulose 2.4 mg microcrystalline cellulose 19.2 mg croscarmellose sodium A2.4 mg magnesium stearate 0.84 mg 2. Tablets: 5- (2-ethyl-2H-tetrazol-5-yl) - 1,2,3,6-tetrahydro-1 - methylpyridine 10 mg lactose 46.9 mg cornstarch 23.5 mg povidone 1.8 mg microcrystalline cellulose 14.4 mg croscarmellose sodium type A 1.8 mg magnesium stearate 3) Syrup: 5- (2-ethyl-2H-tetrazol-5-yl) - 1,2,3,6-tetrahydro-1- 0.63 mg methylpyridine 5.0 mg sorbitol 500 mg hydroxypropyl cellulose 15 mg glycerol 50 mg methyl paraben 1 mg

Claims

1. Use of 5- (2-ethyl-2H-tetrazole-

Claims (3)

1. Use of 5- (2-ethyl-2H-tetrazole- 5-yl) -1,2,3,6-tetrahydro-1 methylpyridine and its pharmaceutically acceptable acid addition salts for the treatment of 25 traumatic brain injuries. The use according to claim 1, wherein 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine or a pharmaceutically acceptable acid addition salt thereof 50 salts are in the amount of 500 pg / day, preferably 1.0 mg to 100 mg / day. Use according to claims 1 to 3 for the treatment of traumatic brain injury caused by physical forces on the skull or 55 spine, ischemia, stroke, paused breathing, cardiac arrest, cerebral thrombosis or embolism, a neurological problem caused by AIDS, cerebral hemorrhage, encephalomalitis, scephalomyelitis, complications, cerebral infections, shocks or increased intracranial pressure, and treatment of the effects of such a condition.