DE3805744C2 - Phenylcarbamate for the inhibition of acetylcholinesterase - Google Patents

Description

The present invention relates to the use of (S) -N-ethyl-3 - [(1-dimethylamino) ethyl] -N-methyl-phenyl-carbamate of the formula I.

in the form of the free base or as acid addition salt according to the Claim.

As can be seen from this formula is in the form of the free Base is the characteristic of the rotation of the compound of formula I. (-). However, it may be in the form of the acid addition salt (+) or (-) his. For example, the hallmark of the rotation of the hydrogen tartrates (+). The present invention independently covers their rotational characteristics the free base form as well as the Acid addition salt forms.

The racemic mixture (±) -N-ethyl-3 - [(1-dimethylamino) ethyl] -N-methyl-phenyl-carbamate in the form of its hydrochloride is known from European Patent Application 193,926 A2, where it is identified as RA ₇ HCl ,

In accordance with this disclosure, the racemate in Free base form by amidation of α-m-hydroxyphenylethyl dimethylamine with a corresponding carbamoyl halide hold. The resulting compound and its pharmacological acceptable acid addition salts, which in known manner from the free base can be prepared as acetylcholine esterase inhibitor in the CNS.

It has now surprisingly been found that the (-) - enantio Formula I and its pharmacologically acceptable acid addition salts, hereinafter referred to as compounds used in the invention denotes a particularly pronounced and have selective inhibition of acetylcholinesterase.

These findings are unexpected, especially since one did not believe that the Dialkylaminoalkylseitenkette, which is the optically active Center is responsible for the main cause of the Acetylcholinesterase inhibiting activity of phenylcarbamates.

The free base can be isolated from the racemate by separation of the enantiomers in Agreement with known methods, eg. B. by using Di-O, O'- p-toluene-tartaric acid. The acid addition salts may be prepared from the free base in a manner known per se become. These include, for example, the hydrogen tartrate.

The compounds used according to the invention show pharma Ecological effect as displayed in standard tests and are therefore useful as pharmaceuticals. They reach the CNS quickly according to s.c., i.p. or p.o administration in rats. They practice one  Brain region selective inhibition of acetylcholinesterase activity which hippocampus and cortical enzymes are more inhibited as derived from the striatum and pons / medulla acetylcholine esterase. Moreover, they have a long duration of action.

For example, the following results illustrate the pharma Ecological profile of the compounds used in the invention Comparison with the corresponding isomers and racemates. Ver Compound A is the compound of formula I in the form of its hydrogen tartrate. Compound B is the optical isomer of said Salt. C denotes the racemic mixture of the compound of Formula I and its optical isomer in the form of the hydrochloride.

In vitro experiments Electrically induced ³ H-acetylcholine release from hippocampal slices of the rat

Electrically evoked ³ H-acetylcholine ( ³ H-ACh) release from rat hippocampal slices is a convenient in vitro model for the study of presynaptic muscarinic autoreceptor agonists and antagonists. This model can also be used as an indirect method of evaluating drugs that inhibit acetylcholinesterase (AChE). The inhibition of AChE activity leads to the accumulation of endogenous ACh, which then interacts with presynaptic muscarinic autoreceptors and inhibits the further release of ³ H-ACh.

Rat hippocampal slices (Wistar strain, 180-200 g) are prepared by crisscrossing whole hippocampus with a Mc Ilwain tissue chopper at a distance of 0.3 mm. Hippocampus sections obtained from 3 rats are incubated for 30 minutes at 23 ° C in 6 ml Krebs Ringer containing 0.1 μCi of ³ H-choline and transferred to the superfusion chamber. The superfusion is carried out with Kreb's medium containing 10 μM hemicholinium-3 at a rate of 1.2 ml / min. at 30 ° C instead. The collection of 5-minute fractions of the superfusate begins 60 minutes after the superfusion. Two periods of electrical stimulation (2 Hz rectangular pulses 2 msec, 10 mA, 2 min) are applied after 70 minutes (S ₁ ) and after 125 minutes (S ₂ ) superfusion. Test substances are added 30 minutes prior to S _2, and are present in the superfusion medium to 145 minutes superfusion. At the end of the experiments, the sections are dissolved in concentrated formic acid and the tritium content in the superfusate and in the dissolved sections is determined. The tritium effluent is expressed as a fraction of the tritium outflow per minute. Electrically induced tritium outflow is calculated by subtracting the extrapolated basal tritium outflow from the total tritium outflow during the two minutes of electrical foreheading and the following 13 minutes, expressed as percent of the tritium content at the beginning of the sample collection. Drug effects in stimulation-induced tritium outflow are expressed as ratios S ₂ / S ₁ . All experiments are performed in duplicate using a programmable 12-channel superfusion system. A computer program is used for the calculation.

In this assay, compound A inhibits electrically induced ³ H-ACh release from hippocampal slices in approximately 40% (100 μM) while racemate C (100 μM) inhibits approximately 25%. The inhibitory effects of compound A and racemate C can be antagonized by atropine. These results are consistent with AChE inhibitory activity. Compound B is inactive in this model.

Acetylcholinesterase inhibition in different regions of the rat brain

In this test AChE preparations from different regions of the rat brain (cortex, hippocampus and striatum) are used and the IC ₅₀ (inhibitory concentrations in μM) is determined. The enzyme preparations are pre-incubated with the inhibitor 15 minutes before the determination.

The AChE activity is determined according to the procedure described by Ellman (Arch. Biochem. Biophys. 82, 70, 1959). Rat brain tissue is suspended in cold phosphate buffer pH 7.3 (0.25 mM) containing 0.1% Triton X-100, homogenized. After Centrifugation will be aliquots of clear supernatant as Enzyme source used. The enzyme comes with different concentrations preincubated concentrations of the inhibitor. After different times the substrate (acetylthiocholine iodide 0.5 mM) is added and the remaining activity measured.

The results are shown in Table 1 below:

Table 1

As can be seen from this table, the AChE inhibition is with Compound A slightly higher than with racemate C, while Ver B is significantly less active.  

Acetylchilinesterase inhibition ex vivo in different regions of the rat brain

Thirty minutes after administration of various doses of Compound A, AChE activity is measured ex vivo in various regions of the rat brain. The method is described above. The IC ₅₀ values found are 7 μmol / kg po in the striatum, 4 μmol / kg po in the hippocampus and 2 μmol / kg po in the cortex. The IC ₅₀ obtained after the administration of racemate C are 2 to 3 times higher for all tested regions. Six hours after administration of Compound A (10 μmol / kg po), the AChE in the striatum is still inhibited by 16%, while at the same time activities in the cortex and hippocampus are inhibited by 39% and 44%, respectively.

In vivo experiments Influence of dopamine metabolism

Male OFA rats (150-200 g) are used both for acute and also used for subchronic experiments. The animals will be held for 12 hours periods of light and dark. The animals are always killed between 11.00 and 13.00 o'clock. The Brains are cut out immediately, then after the method by GLOWINSKI and IVERSEN, J Neurochem. 13, 655 (1966) on ice dissected, frozen on dry ice and the tissue samples to the Analysis stored at -80 ° C.

Dopamine and its metabolite DOPAC (3,4-Dihydroxyphenyl-acetic acid) and HVA (homovanillic acid) are in brain tissue extracts certainly. These are obtained by homogenization of stored brain tissue samples in 0.1 N HCl containing 0.05 mM Acorbic acid, and subsequent centrifugation. It will used striatal and cortical tissues.  

The determination of the metabolites is carried out either under Application of Gas Chromatography / Mass Fragmentation (GCMS) - Technique described by KAROUM et al., J. Neurochem. 25, 653 (1975) and CATABENI et al., Science 178, 166 (1972) or the fluorometric method as described by WALDMEIER and MAITRE, Analyt. Biochem. 51, 474 (1973). For the GCMS method Tissue extracts are prepared by adding known amounts deuterated monoamines and their respective metabolites as internal standards.

The dopamine metabolism in the striatum is after administration of the Compounds A and B and the racemate C increases (This Eigen is a consequence of the compounds mentioned induced acetylcholine accumulation). Nevertheless, Ver Compound A is more active than compound B and racemate C in increasing the striatal dopamine metabolite concentrations.

Muscarinic and nicotinic effects on brain glucose utilization

Changes in the functional activity of the CNS are ver associated with altered deoxyglucose (DOG) utilization in the Brain. These can be used simultaneously in different regions of the world Brains are visualized when using the car radio graphic method of Sokoloff et al., J. Neurochem. 28, 897 (1977). The administration of cholinergic drugs either directly (muscarinic agonists) or indirectly (accumulation of Acetylcholine) causes a characteristic in this model "Fingerprint" pattern by changing the regional glucose metabolism.

Male Wistar rats (150-200 g) are used. The Wirk Substances are given to the animals in various doses and on ver various routes (i.v., p.o., i.p.) administered. [14C] -2-deoxy glucose (125 μC / kg) is taken 45 minutes before the animals are killed  be injected. The brains are cut out immediately, frozen at -80 ° C and then sliced with a thickness cut from 20 microns. The optical densities of radiographic Images are taken according to a modification of Sokoloff et al. measured.

After p.o. Administration of Compounds A and B (7.5 μmol / kg) Significant changes in DOG recovery in ver observed in different regions of the rat brain. The effect of Compound A is stronger than the first 30 minutes of connection B. The most pronounced changes will be in the facial regions and the anteroventral thalamus as well found in the lateral habenula nucleus.

Acetylcholine levels in different regions of the rat brain

The effects of compounds A and B and of racemate C as AChE inhibitor in vivo is determined by measuring ACh levels in different regions of the rat brain at different times after administration of the active substance.

OFA rats (200-230 g) are used. The animals are killed by microwave irradiation adjusted upside down (6 kW operating power 2450 MHz exposure 1.7 sec., Pueschner Mikro Wellen-Energietechnik, Bremen). The brains are removed, dissected according to Glowinski and Iversen (1966) and stored at -70 ° C until analysis. The brain parts are homogenized in 0.1 M perchloric acid containing internally deuterated standards of ACh-d ₄ and Ch (choline) -d ₄ . After centrifugation, endogenous ACh and Ch along with their deuterated variants are extracted with dipicrylamine (2,2 ', 4,4', 6,6'-hexanitrodiphenylamine) in dichloromethane as ion pairs. The Ch portions are derivatized with propionyl chloride and the resulting mixture of ACh and propyl choline derivatives are demethylated with sodium benzenethiolate and analyzed by mass fragmentation according to Jenden et al., Anal. Biochem., 55, 438-448, (1973).

A single administration of 25 μmol / kg p.o. boosts the ACh concentration in striatum, cortex and hippocampus. The maximum effect will be approximately 30 minutes after oral administration reached and decreases in the next 3-4 hours. in the Cortex and hippocampus are still ACh levels at 4 hours significantly higher compared to controls. The effects are dose-dependent manner. The influence of Compound B is significant weaker than the one triggered by the racemate C, and the influence of racemate C is significantly weaker than the one that is triggered by connection A.

Furthermore, the compounds used in the invention well tolerated, and they have a long Duration of action, z. In the above and other standard tests.

The compounds used in the invention are therefore useful for the treatment of senile dementia, Alzheimer's Disease, Huntington's Chorea, Dyskinesia tarda, hyperkinesia, mania, acute confusion, Down's syndrome, and Friedreich's ataxia.

An indicated daily dosage is within the range of approx. 0.1 to about 25 mg, z. From about 0.1 to about 5 mg, according to the invention used compound, together with a fixed or liquid carrier or diluent.  

In the following example the temperatures are uncorrected and in Celsius degrees indicated.  

synthesis example (S) -N-ethyl-3 - [(1-dimethylamino) ethyl] -N-methyl phenyl-carbamate

130 g of (±) -N-ethyl-3 - [(1-dimethylamino) ethyl] -N-methylphenyl carbamate and 210 g of (+) - di-O, O'-p-toluenoic acid monohydrate are dissolved while heating in 1.3 liters of methanol / water (2: 1). After cooling, the precipitated salt is filtered and recrystallized three times from methanol / water (2: 1). The (S) -enantiomers are released by distribution between 1 N NaOH and ether. [α] 20 | D = -32.1 ° (c = 5 in ethanol).

The hydrogen tartrate of the title compound (from ethanol) melts at 123-125 °. [α] 20 | D = + 4,7 ° (c = 5 in ethanol).  

The present invention relates to the systemic trans dermal application of the phenylcarbamates of the formula I.  

EP 0 155 229 A2 relates to transdermal systemic administration bopindolol (beta-blocker), methysergide (serotoninantago nist), tizanidine (myotonolytic agent) as well as clemastine and Ketotifen (Antihistaminica) (see abstract in connection with Page 2, paragraph 3, and page 5, paragraph 1). The document mentions but not carbamate or phenyl carbamate, and certainly not that S-phenylcarbamate of claim 1 of the present application.  

It has now surprisingly been found that the compounds of the formula I as free bases or in the form of pharmaceutical acceptable acid addition salts have unexpectedly good skin penetration when percutaneously be administered.

The penetration of the skin of the compounds used in the invention can be in standard in vitro or in vivo Tests are observed.

An in vitro test is the well-known diffusion test that can be carried out in accordance with the principles described in GB 2098865 A and by TJ Franz in J. Invest. Dermatol. (1975) 64, 194-195. Solutions containing the active agent in unlabelled or radiolabeled form are applied to one side of isolated pieces of intact human skin or hairless rat skin over an area of about 2 cm ² . The other side of the skin is in contact with physiological saline. The amount of active substance in the saline is measured by methods known per se, for. By HPLC or spectrophotometric techniques, or by determination of radioactivity. For example, in this test using rat skin the following penetration rates are found:

Compound A defined above: 23.6 ± 14.9%
Compound of the formula I in the form of the free base: 28.0 ± 8.2%.

Furthermore, it has been found that the transdermal administration of the Compounds used in the invention long-lasting and constant inhibition of acetylcholinesterase effect as displayed in standard tests with  slow onset of action, which in terms of the contracts The sensitivity of these compounds is particularly advantageous.

For example, acetylcholinesterase inhibition in ver various regions of rat brain ex vivo measured transdermal administration of the compounds used according to the invention and compared with the inhibition, how It was obtained after administration in various ways.

The compounds are dissolved in or diluted with n-heptane up to a concentration of 1 or 3 mg / 20 μl. male Rats (OFA strain, approx. 250 g) are shaved in the neck region and the solution is applied to the skin with a micropipette. The Application point is immediately with a thin plastic film and covered by a plaster. The animal has no access to the pavement. At various times after administration, the animals become killed by beheading and the remaining AChE effect measured.

For example, causes the transdermal administration of the above-defined Compound A is a long-lasting, dose-dependent inhibition of AChE activity. In contrast to the rapid onset of action either after oral or subcutaneous use (max 30 minutes) AChE inhibition follows this route of application (max <2 hours) only slowly without the brain region selective To influence AChE inhibition.

The results are shown in Table 2 below. 24 hours after transdermal application is the AChE effect still inhibited in central and peripheral regions. After this same period, the orally administered compound A has no Effect on the enzyme while after the s.c. Administration only the enzyme in the heart is significantly inhibited.  

The present invention sees therefore a pharmaceutical composition for the systemic transdermal administration comprising a compound of Formula I as a free base or in the form of a pharmaceutical Acceptable acid addition salt, with a pharmaceutical Carriers or diluents which are suitable for systemic transdermal administration.

In addition, the present invention provides the use of a compound of formula I 'as the free base or in the form of a pharmaceutically acceptable acid addition salt Active ingredient in the production of a systemic trans dermal administration of appropriate pharmaceutical composition in front.

The active ingredients can be found in any conventional liquid or solid transdermal pharmaceutical composition be administered, for. As described in Remington's Pharma ceutical Sciences 16th Edition Mack; Sucker, Fox and Spieser, Pharmaceutical Technology 1st Edition, Springer and in GB 2098865 A or DE 32 12 053 A1.

Suitably, the composition is in the form of a viscous Liquid, an ointment, a solid reservoir or a solid matrix. For example, the active ingredient is a by fixed reservoir or a solid matrix distributed. The latter will made of a gel or a solid polymer, e.g. B. one hydrophilic polymer as in the European patent application 155,229 A2.

The active ingredient may also be incorporated in a patch.

The compositions for transdermal administration may from about 1 to about 20% by weight of active ingredient of  Formula I as a free base or in the form of a pharmaceutical contain acceptable acid addition salt.

The pharmaceutical compositions for transdermal administration may be for the same indications as for the oral or intravenous administration. The amount of too administering pharmaceutical agent is personalized depend on the characteristics of the drug release of the pharmaceutical compositions, in vitro and in vivo Tests observed penetration rate of the drug, the starch of the active substance, the extent of the skin contact surface, the part of the Body, which is occupied with the formulation and the duration the required effect. The amount of active ingredient and the Area of the pharmaceutical composition, etc. can be determined are routinely performed bioavailability tests, wherein the blood levels of the active substance after administration of the Active substance on intact skin in a pharmaceutical Composition according to the present invention are compared with the blood levels of the drug as they are after oral or intra venous administration of a therapeutically effective dose of the pharmacologically active agent can be observed.

Is the daily dose of a drug for oral administration given, the choice of a suitable amount of the drug by means of which in a transdermal composition according to lying invention, depend on the pharmako kinetic properties of the active substance, including the Liver passage effect; from the amount of drug passing through the skin can be absorbed by the matrix in question for a given application area and in a given time room; and from the period in which the composition is applied shall be. So can a medicine with a high liver passage effect a relatively small amount in the transdermal Need composition compared with the daily oral  Dose as the liver passage effect is avoided. on the other hand is generally a maximum of only approximately 50% of the Drug in the matrix through the skin in a 3-day Period released.

The pharmaceutical compositions according to the present invention find in general z. B. an effective contact surface of the drug reservoir on the skin of about 1 to about 50 square centimeters, preferably about 2 to 20 square centimeters, and should be applied during 1-7 days, preferably 1-3 days.

For example, Compound A can be administered in a 10 mg dose to a patch of approximately 10 cm ² , once every three days.

The following example illustrates the invention.  

formulation example Preparation of a Transdermal Composition Containing a Hydrophobic Polymer

Compound of formula I ', z. B. Compound A 20% Hydrophilic polymer, e.g. Eudragit E 100 * 30% Non-swelling acrylate polymer, e.g. B. Durotack 280-2416 ** 44% Plasticizer, z. B. Brij 97 *** 6% *: Registered Trademark, available through Röhm, Darmstadt, W. Germany @ **: Registered Trademark, available from Delft National Chemie Zutphen, Holland @ ***: Registered trademark available from Atlas Chemie, W. Germany.

The components are acetone or ethanol or another added to suitable volatile organic solvents and mixed, whereby a viscous mass is obtained. The crowd will on an aluminized polyester film (23 micron thick) under Using a standard apparatus scattered, using a film of 0.2 mm thick (wet) is formed. One lets in the film Room temperature for 4 to 6 hours to dry. The aluminum foil is then cut into pieces of about 10 square centimeters cut.

Claims (1)

Use of (S) -N-ethyl-3 - [(1-dimethylamino) -ethyl] -N- methyl phenyl carbamate as the free base or in the form of a pharma ceutically acceptable acid addition salt for the systemic transdermal application for acetylcholinesterase inhibition, esp for the treatment of senile dementia, Alzheimer's disease, Huntington's chorea, dyskinesia tarda, hyperkinesia, mania, acute Confusion states, Down syndrome or Friedreich's ataxia.