MXPA98001515A - Prepared combination for application in the demen - Google Patents

Abstract

A combination preparation containing a compound that exhibits an inhibitory effect on acetylcholinesterase or a muscarinogenic effect, and a compound that increases the level of endogenous extracellular adenosine, is suitable for the treatment of dementia.

Description

Combination preparation for the application in dementia The invention relates to pharmaceutical combination preparations for the treatment of dementia by virtue of neurodegenerative diseases that are accompanied by a decline of cholinergic neurons and a cholinergic deficit (Tohgi et al., Neurosci Lett., 177 (1994) , pages 1939--1942). These combination preparations compensate for the cholinergic deficit by reinforcing the Ca2 + -dependent signal transduction triggered by the stimulation of muscarinic receptors. A reinforcement of this type can obviously be achieved by cooperative effects of adenosine, namely by a combination of substances that increase the extracellular concentration of adenosine with muscarinic receptor agonists or acetylcholinesterase inhibitors (ACHE inhibitors). The pharmacological strategy mainly followed until now for the therapy of senile dementias is the conservation of a muscarinic activation of receptors by the administration of muscarinogenic agonists or ACHE inhibitors that increase the concentration of the endogenous acetylcholine (ACH) in the receptor. It is doubtful whether, in the case of a progressive destruction of cholinergic neuron systems, it is certainly possible to achieve an increase in the level of ACH sufficient for a cellular function, according to the rule, by inhibition of ACHE. In addition, ACHE inhibitors show considerable side effects in the case of poor specificity. Therefore, an enhancement of the effect induced by muscarinic receptors of insufficient concentrations of ACH by other mechanisms would be desirable, and could be the basis for the development of a corresponding combination therapy. It is known that ACH affects not only nerve cell functions, but also glia cells (astrocytes) (Messamore et al., Neuroreport, 5 (1994), pages 1473-1476).

Since pathological reactions of glia cells obviously play an important role in the pathophysiology of dementia (Akiyama et al., Brain Res. 632 (1993), pages 249--259), cultured astrocytes were chosen as an in vitro model system. . The influence of the intracellular release of Ca2 + triggered by muscarinic receptors was investigated using the dynamic fluorescence imaging method. It was found that Cl-adenosine potentiates, depending on the concentration, the intracellular release of muscarinogenic Ca2 + in rat cultured astrocytes (see Figures 1 to 3, Tables 1 and 2). Thus, already in the presence of 1 μM of Cl-adenosine, a potentiation of approximately 30 times of the intracellular Ca + increase triggered by ACH is measured. This effect was not inhibitable by a nicotinic ACH receptor antagonist, but could be blocked by a muscarinic receptor antagonist (Table 3). Likewise, Cl-adenosine potentiated the intracellular release of Ca2 + triggered by the muscarinogen agonist oxotremorine-M (Table 4). A large part of the experiments to detect the enhancing effect of Cl-adenosine (n> 200) is carried out at an ACH concentration of 100 nM. At this low concentration, ACH alone is ineffective. Also Cl-adenosine alone is ineffective over the range of concentrations tested (3 nM to 3 μM). Dose-effect experiments for the determination of the Cl-adenosine concentration necessary for the triggering of a Ca2 + signal in cooperation with 100 nM ACH result in an enhancing effect at micromolar concentrations of Cl-adenosine. The threshold concentration is 1 μM. Since Cl-adenosine corresponds in its affinity for the receptor to endogenous adenosine (Daly et al., Life Sci., 28 (1981), pages 2083-2097), this means that, correspondingly, it is also sufficient an increase in the level of extracellular adenosine from the range of physiological nanomolar concentrations (Bailarín et al., Acta Physiol. Scand, 142 (1991), pages 97-103) to 1 μM, in order to make effective concentrations of ACH below the threshold in the muscarinic receptor. From these experimental results it turns out that the detriment of the cholinergic function induced through muscarinic receptors in the case of dementia can be improved by an increase in the extracellular adenosine concentration. The latter should be possible by a coupled application of an adenosine absorption inhibitor, such as propentofylline (Parkinson et al., Gem Pharmacol 25 (1994), pages 1053-1058). A pharmacological increase in the extracellular concentration of adenosine should also allow a lower dosage of the ACHE inhibitor or the muscarinic receptor agonist eventually used in combination therapy, which would reduce the risk of unwanted side effects. Therefore, the invention relates to a combination preparation, which contains at least l. a compound that has an inhibitory effect on acetylcholinesterase (so-called "ACHE inhibitors") or shows a muscarinérgico effect 2. a compound that increases the level of endogenous extracellular adenosine, and 3. a pharmaceutical support, with a supraadditive increase in effect muscarinic in the case of neurodegenerative diseases for simultaneous, separate or staggered application in time. Known compounds with an ACHE inhibitory effect are, for example, 9-amino-1, 2, 3, 4-tetrahydroacridine (tacrine, COGNEX) and l-benzyl-4- [(5,6-dimethoxy-1- inandon) -2-yl] -methyl-piperidine (E2020, ARICEPT). Known muscarinic agonists are, for example, miperlin. Compounds that increase the level of endogenous extracellular adenosine are, for example, xanthine derivatives of the formula I and / or physiologically compatible salts of the compound of the formula I, wherein R 1 represents a) oxoalkyl with 3 to 8 carbon atoms, whose carbon chain can be linear or branched, b) hydroxyalkyl with 1 to 8 carbon atoms, whose The carbon chain may be linear or branched and the hydroxy group of which represents a primary, secondary or tertiary alcohol function, or c) alkyl having 1 to 6 carbon atoms, the carbon chain of which may be linear or branched, R2 represents a) an hydrogen or b) alkyl having 1 to 4 carbon atoms, whose carbon chain can be linear or branched, R3 represents a) a hydrogen atom, b) alkyl having 1 to 6 carbon atoms, whose carbon chain can be linear or branched, c) alkyl with the ß carbon atoms, whose carbon chain is interrupted by an oxygen atom, d) oxoalkyl with 3 to 8 carbon atoms, whose carbon chain can be linear or branched. Preferably, compounds of the formula I are used, wherein R 1 represents a) oxoalkyl with 4 to 6 carbon atoms, whose carbon chain is linear, or b) C 3 -C 6 alkyl, R 2 represents alkyl with 1 to 4 carbon atoms. carbon atoms, R3 represents a) alkyl with 1 to 4 carbon atoms, or b) oxoalkyl with 3 to 6 carbon atoms. Particularly preferably, l- (5-oxohexyl) -3-methyl-7-n-propyl-xanthine is used. By way of example, the following compounds of the formula I can be mentioned: 1- (5-hydroxy-5-methyl-hexyl) -3-methyl-xanthine, 7- (ethoxymethyl-1- (5-hydroxy-5- methyl-hexyl) -3-methyl-xanthine, 1- (5-oxohexyl) -3,7-dimethylxanthine, 7- (2-oxopropyl) -1,3-di-n-butyl-xanthine or l-hexyl-3 7-dimethyl-xanthine Suitable physiologically compatible salts of the xanthine derivatives of the formula I are, for example, alkali metal, alkaline earth or ammonium salts, including those of physiologically compatible organic ammonium bases. compound of the formula I is carried out under standard conditions in a known manner (US Pat. No. 4,289,776, US Pat. No. 4,833,146, US Pat. No. 3,737,433). The starting substances of the reactions are known or can be easily prepared according to methods known per se. Bibliography By the term "supraadditive" are meant effects that are greater than the sum of the individual effects. Preferred compositions contain propento-phylline and l-benzyl-4- [(5,6-dimethoxy-l-inandon) -2-yl] -methyl--piperidine. The combination preparation according to the invention is suitable, for example, for the treatment of dementia, in particular senile dementia. The combination preparation according to the invention can also encompass combination containers or compositions, in which the components are located next to one another and, therefore, can be applied in the same human or animal body simultaneously, separately or in stepped form in time. The invention also relates to a process for the production of the combination preparation, which is characterized by 1) a compound having an acetylcholinesterase inhibitory effect or showing a muscarinogenic effect, 2) a compound that increases the level of endogenous extracellular adenosine, and 3) a pharmaceutical carrier is manufactured, in a usual manner, to give a pharmaceutical administration form. The combination preparation according to the invention can be presented as a dosage unit in the form of medicinal forms, such as capsules (including microcapsules which, in general, do not contain any pharmaceutical support), tablets (dragees and pills) or suppositories, in where, in the case of using capsules, the material of the capsule assumes the function of the support and the content can be presented, for example, in the form of powder, gel, emulsion, dispersion or solution. However, it is particularly advantageous and simple to prepare oral (peroral) formulations with the two active ingredient components 1) and 2), which contain the calculated amounts of the active ingredients together with any desired pharmaceutical support. A corresponding formulation (suppository) can also be used for rectal therapy. Likewise, transdermal application in the form of ointments or creams, and parenteral injection (intraperitoneal, subcutaneous, intravenous, intraarterial, intramuscular), or the infusion of solutions or the oral application of solutions containing the combinations according to the invention. Ointments, pastes, creams and powders may contain, together with the active ingredients, the usual support substances, for example animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicone, bentonites, talc, rust zinc, lactose, silicic acid, aluminum hydroxide, calcium silicate or polyamide powder, or mixtures of these substances. Tablets, pills or granule bodies can be prepared according to customary methods, such as compression, dip or fluidized bed processes or borage dragee formation, and contain customary carriers and other adjuvants, such as gelatin, agarose, starch (for example potato starch, corn or wheat), cellulose, such as ethyl cellulose, silicon dioxide, various sugars, such as lactose, magnesium carbonate and / or calcium phosphates. The dragee solution usually consists of sugar and / or starch syrup and contains, in most cases, also gelatin, gum arabic, polyvinylpyrrolidone, synthetic cellulose esters, surface-active substances, plasticizers, pigments and similar additives corresponding to the state of the technique. For the preparation of the drug forms, any usual flow regulator, lubricant or glidant, such as magnesium stearate, and separating agents may be used. Preferably, the preparations are in the form of shell / core tablets or multilayer tablets, the active component 2 being in the shell or in the core or in a layer, while the active component 1 is in the core or in the shell. envelope or in another layer. The active ingredient components can also be in the form of a delayed release or they can be adsorbed to the delayed release material or included in the delayed release material (for example, one based on cellulose or polystyrene resin, for example hydroxyethylcellulose). A delayed release of the active ingredients can also be achieved by providing the corresponding layer or the compartment of usual coatings, insoluble in the gastric juices. The dosage to be applied depends, of course, on different factors, such as the being to be treated (ie man or animal), the age, the weight, the general state of health, the degree of severity of the symptoms, the disease to be treated , eventual concomitant diseases, (if present) of the type of concomitant treatment with other medications, or of the periodicity of the treatment. The dosages are usually administered several times a day and, preferably, one to three times a day. The amounts used of the individual active ingredient are oriented in this case to the recommended daily dose of the respective individual active principle and, in general, they must be in the combination preparation in 10% to 100% of the recommended daily dose, preferably in 20% to 80%, in particular 50%. The appropriate therapy with the combinations according to the invention therefore consists, for example, in the administration of an individual dosage of the combination preparations according to the invention, consisting of 1) 100 mg to 600 mg, preferably 200 mg. mg to 400 mg of propentofylline, in particular 300 mg of propentofylline, and 2) 2 mg to 20 mg, preferably 5 mg to 10 mg of 1-benzyl-4- [(5,6-dimethoxy-1-inandon) -2-yl] -methyl-piperidine, the amount depending, natural form, the number of individual dosages and also the disease to be treated, and individual dosage may also consist of several dosage units administered at the same time.

Pharmacological examples Astrocyte cultures Astrocyte cultures of the cortex come from the cerebral cortex of 19-20-day-old Wistar rat embryos that were removed from the mother's body after being sacrificed under anesthesia with ether. After dissection of the brain, it was suctioned with a tissue pipette from the cortex and collected in Dulbecco's modified Eagle's medium (DEM) with the addition of 15% fetal calf serum.

After filtration through lenticular paper, the cell suspension was transplanted onto glass slides (coated with polyethylenimine) and cultured under standard conditions in the incubator in culture flasks, with a medium change twice a week. After about 7 days, the cells were harvested and, after trypsinization (in order to eliminate nerve cells), transplanted again at a concentration of 5 x 10 4 cells / cm 2 and cultured for another 6-8 days until the beginning of the trial. These cultures consisted, in more than 95%, of astrocytes that presented a positive immune reaction for the astrocyte marker GFAP (fibrillar protein glia acid).

Fluorescence imaging experiments for the measurement of intracellular Ca2 + concentration and its experimental influence At the beginning of the assay (6-8 days after the renewed transplant), the cultured astrocytes were loaded with a Ca2 + fluorescence marker, viz. by incubation with 5 μM fura-2-acetoxymethyl ester (molecular probes) in BHKR (Krebs-Ringer solution buffered with Hepes, bicarbonate) at 37 ° C for 1 hour. After loading, the glass supports containing the cultured astrocytes were transferred to a measuring chamber and were installed on the inverted fluorescence microscope belonging to the fluorescence imaging measurement site (Zeiss Axiovert 100, Zeiss Fluar objective). 40 x). Here, the chamber was perfused for the duration of the test (usually 20-30 min) continuously with BHKR controlled at temperature (37 ° C) at a flow rate of 600 μl / min. The measurement was carried out with the fluorescence imaging system FUCAL (TILL Photonics GmbH, Planegg), after excitation at two excitation wavelengths at 340 and 380 nm, the fluorescence emitted over the length of the fluorescence was measured. 420 nm wave with the help of a CCD camera (CS 90, Theta System, Gróbenzell) and the corresponding Ca2 + concentration was calculated. The measurements were carried out at time intervals of 12 s in each case before, during and after the addition of the different test substances to the perfused medium. The modifications of the intracellular concentration of Ca2 + were determined in the plane of the individual cell, namely in different measurement windows established in each case in an appropriate manner. The different test substances (acetylcholine or oxo-tremorine in the presence or absence of Cl-adenosine) were added to the perfusion medium, as a rule, for a time interval of 1 minute. Given that the intracellular increases of Ca2 + triggered were transitory, the modifications of the intracellular concentrations of Ca2 + recorded in the tables and curves of results, referred to the peak values measured in each case.

Table 1 Increase in the intracellular concentration of Ca2 + (nM) Mean values ± EMT, number of cells measured n = 45 (for each measurement value) Table 1 shows that the dose-effect curve of the intracellular increase of Ca2 + triggered by ACH in astrocytes, with the simultaneous action of Cl-adenosine 1 μM, is displaced considerably to the left. In this case, the ACH level should be only 100 nM, in order to create a Ca + signal of equal magnitude, for which an ACH concentration thirty times higher (more than 3 μM) would be necessary in the absence of a cooperating adenosine effect. The critical adenosine increase necessary for this cooperating effect is in a range that should be achieved pharmacologically by the therapy with the adenosine uptake blocker propentofylline.

Table 2: Mean values ± EMT, number of cells measured n = 40 (for each measurement value) Table 2 shows Cl-adenosine concentrations necessary for a Ca2 + mobilization in the presence of 100 nM ACH.

Table 3: Effect of nicotinic acetylcholine receptor antagonists (hexamethonium) and muscarinic acetylcholine receptor (pFHHSiD) on the increase of intracellular Ca2 + concentration by 100 nM acetylcholine and 1 μM Cl-adenosine in cultured bark astrocytes. pFHHSiD (hexahydro-sila-diphenidol hydrochloride, analogous to p-fluoro); manufacturer: RBI (Research Biochemicals International).

Mean values ± EMT as a percentage of the intracellular increase of Ca2 +, which was achieved in the absence of the antagonists by 100 nM acetylcholine and 1 μM Cl-adenosine (control value = 100%). An intracellular increase in Ca2 + of 98.4 ± 6.4 nM (n = 125) was measured as a control value. Table 3 shows that the effect of ACH potentiated by Cl-adenosine represents a representative ACH effect induced through muscarinic receptors that is antagonized by a muscarinic receptor blocker, but not by a nicotinic receptor blocker.

Table 4: Effect of Cl-adenosine and muscarinic acetylcholine receptor agonist oxotremorine-M on intracellular Ca2 + content in cultured bark astrocytes. to ores me ios ± EMT n = 6 Table 4 shows that Cl-adenosine also potentiates the Ca + signal triggered by a muscarinic receptor agonist. Figure 1 shows a typical fluorescence imaging experiment. 100 nM ACH as well as 1 μM Cl-adenosine are ineffective. Their combination leads to a drastic intracellular increase of Ca2 + within cultured astrocytes. The experiments carried out in Ca2 + -free medium allow the recognition of a scaled but still massive intracellular increase of Ca2 + within the cultured astrocytes, when ACH and Cl-adenosine are added together, this shows a catalytic mobilization of Ca2. Figure 2 shows that the dose-effect curve of the intraceular increase of Ca * 1 + triggered by ACH in astrocytes, • on the simultaneous action of Cl-adenosine i μM, '- > s displaced considerably to the left. In this case, the ACH level should be only 100 nM, in order to produce a Ca2 + signal of equal magnitude, for which a concentration of ACH thirty times higher (more than 3 μM) would be necessary in the absence of a cooperating adenosine effect. Figure 3 shows Cl-adenosine concentrations necessary for a homogenization of Ca2 + in the presence of ACH 100 nM.

Claims (6)

1. - Combination preparation, containing at least 1) a compound that has an acetylcholinesterase inhibitory effect or shows a muscarinogenic effect 2) a compound that increases the level of endogenous extracellular adenosine, 3) a pharmaceutical support with a supraadditive increase of the muscarinic effect in the case of neurodegenerative diseases, for simultaneous, separate or stepwise application in time.

2. The combination preparation according to claim 1, characterized in that the compound contained therein, which has an acetylcholinesterase inhibitory effect, is selected from the group of tetrahydroaminoacridine, l-benzyl-4- [(5,6-di-methoxy) 1-inandon) -2-yl] -methyl-piperidine and milamelin. 3.- Combination preparation according to claim 1, characterized in that the compound contained therein, which increases the level of endogenous extracellular adenosine, is chosen from the group of xanthine derivatives of the formula I and / or physiologically compatible salts of the compound of the formula I, wherein R 1 represents a) oxoalkyl with 3 to 8 carbon atoms, whose carbon chain can be linear or branched, b) hydroxyalkyl with 1 to 8 carbon atoms, whose carbon chain can be linear or branched and whose hydroxy group represents a primary, secondary or tertiary alcohol function, or c) alkyl with the ß carbon atoms, whose carbon chain can be linear or branched, R2 represents a) a hydrogen atom or b) alkyl with 1 to 4 carbon atoms, whose carbon chain can be straight or branched, R3 represents a) a hydrogen atom, b) alkyl with the β carbon atoms, whose carbon chain can be linear or branched, c) alkyl with the ß carbon atoms, whose carbon chain is interrupted by an oxygen atom, d) oxoalkyl with 3 to 8 carbon atoms, whose carbon chain can be linear or branched. 4. - Combination preparation according to one or more of claims 1 to 3, characterized in that the compounds contained therein are propentofylline and l-benzyl-4- [(5,6-di-methoxy-1-inandon) -2- il] -methyl-piperidine. 5. Use of the combination preparation according to one or more of claims 1 to 4 for the preparation of a medicament for the treatment of neurodegenerative diseases, in particular senile dementia. 6. Process for the production of the combination preparation according to one or more of claims 1 to 4, characterized in that 1) a compound that has an acetylcholinesterase inhibitory effect or shows a muscarinogenic effect, 2) a compound that increases the level of endogenous extracellular adenosine; and 3) a pharmaceutical carrier is processed, in the usual manner, to give a pharmaceutical administration form.