HUP0300032A2 - Use of compounds useful for the treatment or prevention of a disease mediated by the alpha-2b-adrenoreceptor for preparation of pharmaceutical composition - Google Patents

Abstract

The invention relates to compounds of formula (A), (B), (C), (D) or (E) and their pharmaceutically acceptable salts, which can be used as medicine. The invention covers the use of alpha-2B-adrenoreceptor antagonists for the production of pharmaceutical preparations suitable for the treatment or prevention of diseases mediated by alpha-2B-adrenoreceptors in mammals. HE

Description

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COMPOUNDS SUITABLE FOR THE TREATMENT AND PREVENTION OF DISEASES MEDIATED BY ALPHA-2B-ADRENORECEPTOR (- γά M2L ti te li tt ÉM/te

The invention relates to the use of selective alpha-2B-adrenoreceptor antagonists for the preparation of a pharmaceutical composition for the treatment or prevention of diseases mediated by alpha-2B-adrenoreceptors.

We refer to publications and other literature that shed light on the literary background of our invention and, in particular, further details regarding the implementation of our invention.

The selective alpha-2B-adrenoreceptor antagonists shown in the drawings are all known compounds, of which Compound A (No. AE-848/34956037), Compound C (No. AF399/36012031) and Compound D (No. AH-034/34347043) are available from SPECS and BioSPECS B.V. (Fleminglaan 16, 2289 CP Rijswijk, The Netherlands), and Compound B (No. 653716) and Compound E (No. 569063) are available from ChemBridge Corporation (16981 Via Tazon, Suite G, San Diego CA 92127).

The vasoconstrictor effect of alpha-2B-adrenoreceptors is known. Therefore, alpha-2B-adrenoreceptor antagonists are suitable for the treatment or prevention of diseases associated with vasoconstriction. A genetic polymorphism in the alpha-2B-adrenoreceptor gene has been detected in certain individuals. Meg97310-7676-FO-fa

- 2We observed that in some others, the alpha-2B-abrenoreceptor protein contains three glutamate deletions from a glutamic acid repeat unit containing 12 glutamate residues (amino acids 297-309) in a 17-amino acid trech located in the third intracellular loop of the receptor polypeptide [Heinonen et al., The Journal of Clinical Endocrinology & Metabolism 84, 2429 (1999)].

It has been found that the compounds of formulae (A), (B), (C), (D) and (E) are selective alpha-2B-adrenoreceptor antagonists.

Thus, the present invention relates to the use of selective alpha-2B-adrenoreceptor antagonists in the manufacture of a pharmaceutical composition for use in mammals for the treatment or prevention of alpha-2B-adrenoreceptor mediated diseases. The antagonists used in the present invention are compounds of formula (A), (B), (C), (D) or (E) or pharmaceutically acceptable salts thereof.

Alpha-2B-adrenoreceptor antagonists are useful for the treatment and/or prevention of a wide variety of diseases.

An important target group for treatment with selective alpha-2B-adrenoreceptor antagonists are individuals whose alpha-2B-adrenoreceptor protein contains a deletion, particularly the deletion/deletion genotype (D/D genotype) [Heinonen et al. (1999), supra].

In a five-year study, we found that the alpha-2B adrenoreceptor gene D/D genotype was associated with a significantly increased risk of acute myocardial infarction (AMI) in a population of middle-aged Finnish men. The risk of AMI was increased in those who had previously, at the time of the test,

- No coronary heart disease (CHD) was diagnosed before starting 3lat. Therefore, it was hypothesized that the D/D genotype is associated with an insufficient capacity to downregulate alpha-2B-adrenoceptor function during sustained receptor activation. Therefore, it can be hypothesized that alpha-2B-adrenoceptors are involved in the pathogenesis of a significant proportion of all AMI cases, especially in individuals with the D/D genotype, but also in individuals with l/D and l/l (where I stands for insertion, which refers to the normal allelomorphic gene).

The alpha-2B-adrenoreceptor antagonists of the present invention may be particularly useful in the treatment and prevention of coronary heart disease. Examples include:

a) Acute AMI

In certain cases of AMI, where the triggering factor is alpha-2B-adrenoreceptor-dependent vasoconstriction, antagonizing these receptors would preserve coronary circulation and reduce ischemic myocardial damage.

b) Unstable angina pectoris

Alpha-2B-adrenoreceptor antagonists reduce the vasoconstrictor component in a prolonged ischemic episode, thereby relieving symptoms and preventing AMI.

c) Prinzmetal's variant of angina pectoris

Vasoconstriction is a key factor in the pathogenesis of Prinzmetal's angina, and alpha-2B-adrenoreceptor antagonists can resolve this and prevent the attack.

d) Chronic angina pectoris and other forms of CHD

- 4Alpha-2B-adrenoreceptor antagonists help reduce the vasoconstrictor component in all types of CHD, thereby alleviating symptoms and providing protection against AMI. This is due to a general decrease in vascular tone by reducing venous return, cardiac workload and oxygen consumption (nitrate-type effect, see below).

e) Prevention of restenosis after coronary angioplasty in cases where vasoconstriction plays a role in restenosis

The alpha2B-adrenoreceptor antagonists used in the solution according to our invention are also suitable for the treatment or prevention of independent hypertension, especially in individuals with increased sympathetic activity and a hyperdynamic circulatory system.

In the aforementioned study, the association of the D/D variant of the alpha-2B-adrenoceptor gene with blood pressure is unclear. We believe that this is due to two main factors, 1) treatment aimed at lowering blood pressure and 2) the complex regulation of systemic blood pressure. In another study (Heinonen et al., supra), it was observed that the D/D genotype was associated with a lower basal metabolic rate and a lower heart rate. These associations probably reflect increased vascular resistance in the individuals.

Intravenous administration of alpha2B-adrenoceptor agonists to transgenic mice with a targeted inactivation of the alpha2B-adrenoceptor gene did not induce the characteristic increase in blood pressure that is seen in normal animals.

- 5furthermore, after administration of such drugs in humans in high doses [Link R. E. et al., Science, 273, 803 (1996)]. These drugs showed an increased antihypertensive effect. This shows that alpha-2B-adrenoceptors mediate vasoconstriction. Therefore, an antagonist should reduce blood pressure. This effect is not seen with alpha-2B-nonselective alpha-2B-adrenoceptor antagonists, since alpha-2A-adrenoceptor antagonism reduces sympathetic outflow, cardiac output and blood pressure. In mice with dysfunctional alpha-2B-adrenoceptors, alpha-2B-adrenoceptor agonists induce an increased hypertensive response and not hypotension [MacMillan L. B. et al., Science, 273, 801 (1996)].

It is hypothesized that alpha-2B-adrenoreceptor antagonists may have beneficial effects in hypertensive individuals through their effects on renal function, muscle blood flow, and vascular resistance in other blood vessels. The anti-AMI effect of this drug is an additional benefit, since hypertension is a significant risk factor for AMI. This protection is attributable to three factors: 1) reduction in systemic blood pressure, 2) reduced risk of coronary artery stenosis, and 3) nitrate-like effects on venous return, myocardial load, and oxygen consumption.

In addition to the above, the alpha-2B adrenoreceptor antagonists of our invention are also useful for the treatment or prevention of other vascular diseases. They may be particularly advantageous for the treatment or prevention of:

- vasoconstriction or hypoxic brain damage following subarachnoid hemorrhage,

- migraine,

- Raynaud's disease or cold intolerance,

- pre-eclampsia,

- male erectile dysfunction or

- obesity and metabolic syndrome.

The latter effect is due to the role of reduced muscle blood flow and reduced basal metabolic rate in the development of obesity and hypertension. Alpha-2B-adrenoreceptor antagonists increase energy expenditure and shift the calorie balance in a favorable direction by increasing muscle blood flow.

The alpha-2B-adrenoreceptor antagonists of our invention can also be used for anesthesia and sedation by enhancing the clinical efficacy of alpha-2B-adrenoreceptor agonists that are not selective for the alpha-2B-adrenoreceptor subtype. The alpha-2B-adrenoreceptor antagonists administered concomitantly block the vasoconstriction induced by the agonists, allowing the use of higher doses of these agonists, which can reach anesthetic dose levels that were previously only possible in veterinary anesthesia, not in humans.

Experimental part

Binding affinity for human alpha-2B-adrenoreceptor

- 7 The affinity of the test compounds for the three human _{α 2} -adrenoreceptor subtypes (α _2Α , α 2β and α _2C ) was determined by a competition binding assay with ³ H-rauwolscine. The biological materials used in the assays were membranes from Shionogi S115 cells stably transfected with one of the three human α 2 subtypes [Marjamáki, A., Ala-Uotila, S., Luomala, K., Perálá, M., Jansson, C., Jalkanen, M., Regan, JW and Scheinin, M., Biochem. Biophys. Acta, 1134, 169 (1992)]. The membrane (5-10 pg total protein per sample) is incubated with 1 nmol/l to 2 nmol/l ³ H-rauwolscine (specific activity 78 Ci/mmol) in 50 mmol/l potassium dihydrogen phosphate (pH 7.5) at six different compound concentrations. Each concentration is run in duplicate. Non-specific binding is determined with 100 pmol/l oxymetazoline, corresponding to 5-15% of total binding. After 30 min at room temperature, the incubation is terminated by rapid vacuum filtration through a GF/B glass fiber filter and washed three times with 5 ml of ice-cold incubation buffer. The filters are then dried, impregnated with scintillation and their radioactivity is determined in a scintillation counter. The analysis of the assays was performed by nonlinear least squares curve fitting. The experimentally determined IC50 values were converted to Ki values using the Cheng-Prusoff equation [Cheng, Y. and Prusoff, WH, Biochem. Pharmacol., 22, 3099 (1973)]. The experiments were repeated at least three times.

Table 1

Binding affinity at human _α2 -adrenoreceptor subtypes

Data are expressed as Ki values in nmol/L (mean + SEM), n is 3 unless otherwise stated.

Compound alpha-2A alpha-2B alpha-2C The 4100 ±200 30 ±4 4700 C >30,000 (n = 2) 1860 (n = 2) >30,000 (n = 2) D >30,000 530 ±90 >30,000 B >30,000 215 ±60 >30,000 This >100,000 2900 ±300 >100,000

Binding affinity for rat cortical a^adrenoreceptor

The affinity for rat neocortical α-adrenoreceptors is determined by a competition binding assay with ³ H-prazosin. In these assays, the biological material is membrane from rat neocortex. Membrane suspensions (100-200 pg total protein per sample) and ³ H-prazosin (specific activity 74 Ci/mmol) at concentrations ranging from 0.2 nmol/l to 0.25 nmol/l are incubated with six different concentrations of compound in a total volume of 0.25 ml (50 mM Tris, pH 7.7, 25 °C). Each concentration is run in duplicate. Nonspecific binding is determined with 10 pmol/l phentolamine methanesulfonate, which corresponds to 25-30% of total binding. At room temperature

- After 930 minutes, the incubation is terminated by rapid filtration through a GF/B glass fiber filter and washed three times with 10 mM ice-cold Tris (pH 7.7 at 4°C). After drying, the solid scintillate is fused onto the filter material and its radioactivity is determined using a scintillation counter.

Results

Compound A does not induce the adequate displacement of ³ H-prazosin up to a concentration of 30 pmol/l, which is necessary for determining the IC ₅₀ value. Therefore, the IC ₅₀ and Ki values of compound A are likely to be greater than 30,000 nmol/l.

Antagonist activity against human alpha-2B-adrenoceptor subtypes

Antagonist potency is defined as the ability of test compounds to competitively inhibit the binding of epinephrine-stimulated ³⁵ S-GTPγS G proteins [Tian, W.-N., Duzic, E., Lanier, S.M. and Deth, R.C., Mol. Pharmacol., 45, 524 (1993); Wieland, T. and Jakobs, K. H., Meth. Enzymol., 237, 3 (1994); Jasper, JR, Lesnick, JD, Chang, LK, Yamanashi, SS, Chang, TC, Hsu, SA 0., Daunt, DA, Bonhaus, DW and Egen, RM, Biochem, Pharmacol., 55., 1035 (1998)] in membranes of CHO cells stably transfected with one of the three α ₂ subtypes [Pohjanoska, J., Jansson, CC, Luomala, J., Marjamaki, A., Savoia, J.-Més Scheinin, M., Eur. J. Pharmacol., 35, 53 (1997); Marjamaki, A., Pihlavisto, M., Cockcroft, V., Heinonen, P., Savoia, J.-M. and Scheinin, M., Mol. Pharmacol., 53, 370 (1998)]. Membranes (2-6 pg protein per sample) and test compound 12

- 10 different concentrations are preincubated for 30 minutes with a defined concentration of epinephrine (5 pmol/l for _2A , 15 pmol/l for 2B, 5 mmol/l for _2C ) in a solution containing 50 mmol/l Tris, 5 mmol/l magnesium chloride, 150 mmol/l sodium chloride, 1 mmol/l DTT, 1 mmol/l EDTA, 10 pmol/l GDP, 30 pmol/l ascorbic acid at room temperature and pH 7.4. The binding of the radiolabel is initiated by adding a trace amount of ³⁵ S-GTPγS to the incubation mixture (between 0.08 nmol/l and 0.15 nmol/l, specific activity 1250 Ci/mmol). Incubate at room temperature for a further 60 minutes, which is terminated by rapid vacuum filtration through a glass fiber filter. The filter is washed three times with 5 ml of ice-cold wash buffer (20 mM Tris, 5 mM MgCl, 1 mM EDTA, pH 7.4 at room temperature), dried and its radioactivity is determined by scintillation counting. The analysis of the experiments is performed by nonlinear least squares fitting. The experiments are repeated at least three times.

Table 2

Antagonist effects of Compound A and Compound B on human alpha ₂ -adrenoreceptor subtypes

Data are presented as KB values in nmol/L (mean + SEM), n represents at least three experiments.

Compound alpha-2A* alpha-2B alpha-2C* The 2400 ±700 73 ±23 2400 ±900 B >10,000 250 ±80 >10,000

- 11 * only incomplete dose-response curves can be determined, KB values are minimum estimates

In the present invention, the alpha-2B-adrenoceptor antagonists of formula (A)-(E) or a pharmaceutically acceptable salt thereof may be administered in various ways. Suitable administration forms include oral formulations, parenteral injections including intravenous, intramuscular, intradermal and subcutaneous injections, and transdermal and rectal administration forms. The dosage of the alpha-2B-adrenoceptor antagonist compounds will depend on the condition being treated, the severity of the condition, the duration of treatment, the route of administration and the compound being used. A suitable dosage for an adult patient is between 5 pg/kg and 100 mg/kg of body weight per day.

Of course, the solution according to our invention can be implemented in various embodiments, only some of which have been shown. However, it will be obvious to a person skilled in the art that other embodiments are possible and do not depart from the principle of our invention. Therefore, the above embodiments are presented for illustrative purposes and our invention is not limited to them.

Claims (9)

- 12Patent claims Use of a compound of formula 1, (A), (B), (C), (D) or (E) or a pharmaceutically acceptable salt thereof as a medicament. 2. Use of a selective alpha-2B-adrenoreceptor antagonist for the manufacture of a pharmaceutical composition for the prevention or treatment of an alpha-2B-adrenoreceptor mediated disease in mammals, which antagonist is a compound of formula (A), (B), (C), (D) or (E) or a pharmaceutically acceptable salt thereof. The use according to claim 2, wherein the disease is coronary heart disease (CHD). 4. The use according to claim 3, wherein the disease - acute myocardial infarction (AMI), - unstable angina pectoris, - Prinzmetal variant of angina pectoris, - chronic angina pectoris and other forms of CHD or - restenosis after coronary angioplasty. 5. The use of claim 2, wherein the disease is independent hypertension. 6. The use according to claim 2, wherein the disease is a vascular disease, namely - vasoconstriction or hypoxic brain damage following subarachnoid hemorrhage, - migraine, - Raynaud's disease or cold intolerance, - pre-eclampsia, - male erectile dysfunction or - obesity. 7. Use of a selective alpha-2B-adrenoreceptor antagonist for the preparation of a pharmaceutical composition suitable for enhancing the clinical efficacy of an alpha-2B-adrenoreceptor agonist with hypnotic and/or analgesic effects, wherein the agonist is non-selective for the selective alpha-2B-adrenoreceptor subtype. 8. The use according to claim 2 for the preparation of a pharmaceutical composition for treating an individual having a three glutamate deletion from a glutamic acid repeat unit containing 12 glutamates in a 17-amino acid trech located in the third intracellular loop of the receptor polypeptide (amino acids 297-309). 9. The use of claim 8 for the preparation of a pharmaceutical composition for administration to an individual having a deletion/deletion genotype. 1!^· k instead of the notifier, the authorized person DANUBE Patent and Trademark Office Ltd. Dr. Flóra Fehérvári, patent attorney