SK58198A3 - Treatment of traumatic brain injury - Google Patents

Abstract

The compound 5-(2-ethyl-2H-tetrazol-5-yl)-1,2,3,6-tetrahydro-1-methylpyridine of formula (I) improves cognitive performance and attenuates injury-reduced reductions of cholinergic neurones in traumatic brain injury models and is useful for the manufacture of a pharmaceutical preparation for the treatment of traumatic brain injury.

Description

EP-A1 0 296 721 discloses a class of piperidine or 1,2,3,6-tetrahydropyridine compounds substituted at the 5-position by a five-membered heterocyclic group including a subclass of optionally substituted 5-tetrazolyl-1,2,3,6-tetrahydropyridine compounds. The compounds have been found to have a high affinity for central cholinergic receptors, in particular a high affinity for central muscarinic M1 receptors, and are therefore suitable for the treatment of Alzheimer's disease, age dementia and reduced educational and memory capabilities.

The structure-activity relationship of this subclass is described by Moltzen et al., J. Med. Chem., 1994, 37, 4085-4099. One of the compounds, ie 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine, was shown to be selective for muscarinic receptors with several times higher affinity for M 1 receptors compared to M ₂ and M3 receptors (Muscarinic receptor subtypes, 6th International Symposium, Nov. 9-12, 1994, Fort Lauderdale). In view of the mechanism of action it was found to acts as a partial agonist at the mu receptor and as an antagonist at _M2 and M3 receptors. In addition, the only significant in vivo effect observed was the effect on spatial memory acquisition in young and old rats.

IPM caused by physical or neurological damage or other diseases is becoming increasingly important in the population and there is a great need for effective and safe drugs to treat such conditions and their consequences

It has now surprisingly been found that the compound 5- (2-ethyl-2H-tetrazol-5-yl) 1,2,3,6-tetrahydro-1-methylpyridine shows good effects in the treatment of PMI and its consequences.

2. Summary of the Invention

The present invention relates to the use of 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine of formula I

(I) or an acid addition salt thereof for the manufacture of a medicament for the treatment of traumatic brain injury (CPM) or its sequelae.

The term brain injury (CPM) in this specification includes all disease states associated with brain or spinal cord injury, e.g. through the effect of physical forces on the skeleton of the head or spinal cord, ischemia, stroke, respiratory arrest, cardiac arrest, cerebral thrombosis or embolism, neurological problems caused by AIDS, cerebral hemorrhage, encephalomyelitis, hydrocephalus, postoperative infections, cerebral infections, cerebral infarction.

Pharmaceutically acceptable acid addition salts of the compounds used in the invention are those formed with non-toxic organic or inorganic acids. Examples of such organic salts are those derived from maleic acid, fumaric acid, benzoic acid, ascorbic acid, pamoic acid, succinic acid, oxalic acid, bis-methylenesalicylic acid, methanesulfonic acid, ethanedisulfonic acid, acetic acid, propionic acid, tartaric, almond, cinnamic, , glycol, p-aminobenzoic, glutamic, benzenesulfonic and theophylline acetic, as well as 8-halothiophins, for example 8-bromothiophilin. Examples of such inorganic salts are those with hydrochloric, hydrobromic, sulfuric, sulfamide, phosphoric and nitric acids.

The -3U compound of the invention has been found to be useful in the treatment of IPM. Thus, for example, it improves cognitive performance after moderate traumatic brain injury and attenuates the injury-induced decrease in cholinergic neurons. Furthermore, it has been found that at doses considered clinically effective, it has no cardiac side effects or other side effects.

In another aspect, the present invention provides a method for the prevention or treatment of UPM in a human by administering a therapeutically effective amount of 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine or an acid addition thereof salts to the patient with indication.

The compound used according to the invention and its pharmaceutically acceptable acid addition salts can be administered by any suitable route, i. j. orally or parenterally, and the compounds may be in any suitable dosage form for such administration, e.g. as tablets, capsules, powders, syrups or solutions or as dispersions for injectables.

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

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

pharmacology

The compound of the invention was tested by the following recognized and reliable method:

Cognitive ability after ÚPM

Rats were subjected to brain damage by rapidly injecting a volume of fluid into the cranial cavity as reported by Dixon, C.E. et al., J. Neurosurgery, 67 (1987), 110-119. Damaged animals were 1 to 15 days post-injury s. c. treated with saline or with 5 (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methyl-4-pyridine starting treatment 24 hours after injury at a dose of 3.6 μΐηοΙ / kg or 15 pmol / kg.

Suppression of upright posture reflex was determined according to Dixon et al., 1987, as described above.

Body weight was recorded prior to the injury and at 1-5 days after the injury and the rotational test was determined 1-5 days after the injury according to Hamm, RJ et al., J. Neurotrauma, 11, (1994), 187 -196. The pivot test was used to measure motor ability after the PMI.

Finally, the mean time (+ S.E.M.) was measured until the rats reached the target plateau in the Morris maze of water, on days 11-15 after the injury. The results were analyzed by ANOVA. During the water maze testing, all animals were injected 10 minutes prior to the maze assessment.

Simulated injury animals (animals prepared for injury but without injecting a volume of fluid into the cranial cavity) were included for comparison.

Quantification of ChAT neurons after IPM

Rats were subjected to traumatic brain injury by injecting a volume of fluid into the cranial cavity and were treated as above. They were with. c. injected with saline or test compound (5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine, 15 μιτιοΙ / kg) (n = 5). Simulated lesion animals were injected (s.c.) with saline (n = 4) or test compound, 15 μιτιοΙ / kg) (n = 4).

The suppression of upright reflex was determined as above. Possible loss of cholinergic neurons as a result of PMI was determined by quantitation of choline acetyltransferase (ChAT) immunoreactive neurons in the basal forebrain. On the 15th day after the injury (2-4 hours after the last injection) all animals were anesthetized with pentobarbital (90 mg / kg, ip) and were transaortically perfused with 200-250 ml isotonic saline followed by 500 ml 4.0% paraformaldehyde / 0. 2% picric acid in 0.1 M phosphate buffer at a rate of 500 ml / 30 minutes at room temperature. After perfusion, the brains were divided into 2 portions and stored in the same solution at 10 ° C for 24 hours. Coronal 40μ sections were deposited on a vibrator through the forebrain and

-5 every 5th section was evaluated for ChAT immunoreactivity. The parallel sections were stained for Nissi substance with cresyl violet for quantitative determination of any possible losses of cholinergic and non-cholinergic neurons in the basal nuclei of the forebrain.

Free-flowing forebrain sections were incubated at a final ChAT antibody concentration (1:50) of 1.0 µg / ml in 0.01 M phosphate buffered saline (PBS) containing 0.1% triton X-100. The forebrain sections were split and incubated in primary antibody for 24 hours at room temperature on culture plates (4 sections / 300 μΙ / well).

After 4 washes in PBS, sections were incubated in secondary antibody (horse anti-mouse IgG; Vector Laboratories, Burlingame, CA) for 1 hour at 37 ° C. After 3 washes in PBS, the sections were incubated with the mouse avidin-biotin peroxidease (ABC) complex technique (Vector), the floating sections were treated with glucose oxidase-diaminobenzidine-nickel method as described by Shu et al., Neurosci. Lett., 85, 1988, 169-171. The reaction was terminated by transferring the sections to TBS. Slices were coated onto gelatin-chrome-aluminum coated glass slides and allowed to dry overnight at room temperature, dehydrated with increasing concentrations of ethanol and xylene and then overlaid with Permount.

ChAT immunoreactive neurons in the middle septal nucleus (MSN), the vertical limbal nucleus of the diagonal beam (VDB), and the basalis magnocellularis (NMB) nucleus were counted using a microcomputer imaging system (MCID) (Imaging Research Inc., Ontario, Canada). The boundaries between MSN and VDB were defined as the front link. NMB was defined as immunolabelled neurons located in the globulus pallidus and in the adjacent part of the inner capsule. Cell numbers were determined for each animal, obtained at a 0.2 mm distance in the forebrain nuclei. Cell numbers are expressed as group averages of 10,000 pm ² for each forebrain nucleus.

MSN, VDB, and NMB neurons from parallel sections were stained with cresyl violet and also counted for MCID. Due to the large number of total cholinergic and non-cholinergic neurons that stained with cresyl violet in these regions, a bilateral sample of neurons in each nucleus was counted at each null. A grid was used to count the cells for each nucleus

-6with pre-marked areas. For MSN and VDB nuclei, three 2,000 μπι ² regions were counted on each side (ie the total area for each nucleus was 12,000 pm ² for each of the four sections counted). For NMB, one 12,000 pm ² area was counted on each side (total area = 24,000 pm ² in each of the four counted sections). Only large cells (> 24,000 pm ² diameter) with visible somatic Nissi bodies and a clear neuronal type of nucleus were quantified.

The results

There are no significant differences between individual injured animals in suppression of upright reflex after PMI. This indicates that in each experiment the severity of the injury was the same in the groups of animals. Seemingly injured animals erected faster than any post-APM animals (p <0.0001 for each comparison). Upright suppression in a group of apparently injured animals is due to inhalation anesthesia used prior to injury.

There was no significant difference between any of the injured groups with respect to the reduction in body weight after IPM, again indicating the same severity of injury among the injured groups.

There were no significant differences in performance in the pivot test at 1-5 for the injured groups. the day after the injury. Since the test substance was administered on days 1 to 15 post-injury, these tests also indicate that the drug had no effect on body weight or performance in the rotational test after injury.

Anova's test indicated that wounded animals injected with test compound (s.c.) daily had better performance in the Morris water maze test than wounded animals treated with saline. Injured animals treated with test compound at 15 pmoles / kg had a significantly improved performance with p <0.01.

IPM caused a significant decrease in the number of Chat-IR neurons in VDB and NMB in rats treated with saline and test compound. However, the compound of the invention significantly attenuated the reduction in Chat-IR reduced after injury

-7 (32% reduction compared to the saline treated group in VDB and 51% in NMB). The parallel sections stained with cresyl violet showed no reduction in the number of cells in MSN, VDB or NMB, suggesting that the loss of Chat-IR neurons is not the result of cell death.

DETAILED DESCRIPTION OF THE INVENTION

Examples of preparations

The pharmaceutical compositions of the invention may be prepared by conventional methods known in the art.

For example, tablets may be made by mixing the active ingredient with conventional excipients and / or diluents and subsequently compressing the mixture in a tabletting machine. Examples of excipients or diluents are: corn starch, lactose, talc, magnesium stearate, gelatin, lactose, resins, and the like. Any other auxiliary or additive colorants, flavors, preservatives, etc. may be used, provided that they are compatible with the active ingredients.

Solutions for injections may be made by dissolving the active ingredient and possible additives in a portion of the vehicle, preferably sterile water, adjusting the solution to the desired volume, sterilizing the solution and filling it in suitable ampoules or vials. Any suitable commonly used additives such as ionizing agents, preservatives, antioxidants, and the like can be added.

Typical examples of formulations for the formulations of the invention are given below (amounts of active ingredient are recalculated to the free base):

1) Tablets:

(5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1- methylpyridine 20 mg lactose 60 mg maize starch 30 mg hydroxypropyl 2,4mg microcrystalline cellulose 19.2 mg

- croscarmellose sodium, type A, magnesium stearate

2) Tablets:

(5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine lactose maize starch povidone • microcrystalline cellulose croscarmellose sodium type A magnesium stearate

3) syrup:

(5- (2-ethyl-2 H -tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine sorbitol hydroxypropylcellulose glycerol methylparaben propylparaben ethanol flavor sodium saccharin water

4) solution:

(5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine sorbitol acetic acid aqua for injection)

2.4 mg 0.84 mg mg 46.9 mg 23.5 mg

1.8 mg 14.4 mg

1.8 mg 0.63 mg

5.0 mg 500 mg mg 50 mg mg 0.1 mg 0.005 ml 0.05 mg 0.5 mg ad 1 ml

1.0 mg 5.1 mg 0.08 mg ad 1 ml

Claims (4)

Use of 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine or a pharmaceutically acceptable acid addition salt thereof for the manufacture of a medicament for the treatment of traumatic brain injury. Use according to claim 1, characterized in that the medicament comprises 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine or a pharmaceutically acceptable acid addition salt thereof in unit dosage form. Use according to claim 1, characterized in that the medicament comprises 5- (2-ethyl-2H-tetrazol-5-yl) -1,2,3,6-tetrahydro-1-methylpyridine or a pharmaceutically acceptable acid addition salt thereof. in an amount of 500 µg to 600 mg / day, preferably 1.0 mg to 100 mg / day. Use according to any one of claims 1 to 3, characterized in that the medicament is intended for the treatment of traumatic brain injury caused by the effect of physical forces on the skeleton of the head or spinal cord, ischemia, stroke, respiratory arrest, cardiac arrest, cerebral thrombosis or embolism, neurological problems caused by AIDS, cerebral hemorrhage, encephalomyelitis, hydrocephalus, postoperative events, cerebral infections, concussions or intracranial pressure, and / or for treating the consequences of such a condition.