Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/08—Vasodilators for multiple indications
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives

Definitions

• the invention relates to the use of PDE9A inhibitors for the manufacture of a medicament for the treatment and / or prophylaxis of coronary heart diseases, in particular stable and unstable angina pectoris, acute myocardial infarction, myocardial infarction prophylaxis, sudden cardiac death, heart failure, and high blood pressure and the consequences of atherosclerosis.
• the heart as a continuously working hollow muscle, needs a particularly intensive supply of oxygen to cover its energy needs.
• Supply disorders therefore primarily concern oxygen transport, which can be insufficient if the blood circulation is less adaptable.
• An increase in oxygen consumption can only be covered by an increase in cardiac blood flow.
• the effects described above can be controlled via the intracellular concentration of the so-called “second messenger” cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP).
• cAMP cyclic adenosine monophosphate
• cGMP cyclic guanosine monophosphate
• the intracellular concentration of cGMP is increased by stimulating the soluble or membrane-bound guanylate cyclases.
• the intracellular The concentration of cAMP can be modulated by activating so-called G protein-coupled receptors, which activate G proteins and thus activate or inhibit the adenylate cyclase.
• the phosphodiesterases are divided into eleven different classes according to their biochemical or pharmacological properties (Soderling and
• Phosphodiesterase 9A is a cGMP-specific phosphodiesterase.
• the enzyme has a Km (Michaelis-Menten constant) of 70 nM (Soderling et. Al., J. Biol. Chem. (1998) 15553-15558), this is the lowest known Km for cGMP of all known phosphodiesterases , Therefore, the PDE9A is involved in the maintenance or regulation of the basal, intracellular cGMP level.
• the DNA and protein sequences for phosphodiesterase 9A are from the mouse
• Coronary artery as well as the extremely high affinity of PDE9A for cGMP (Km value 70 nM) now indicate that phosphodiesterase 9A has a very important role in the contraction or relaxation of coronary arteries and thus in controlling the blood flow to the heart.
• the expression of PDE9A in blood vessels thus also indicates a role for PDE9A in the control of blood pressure and the regulation of peripheral blood flow.
• PDE9A inhibitors The effect of PDE9A inhibitors on coronary flow can be checked on the isolated perfused Langendorff heart.
• An inhibitor of PDE9A lowers the perfusion pressure on the rat's Langendorff heart.
• the present invention therefore relates to the use of phosphodiesterase 9A inhibitors for the production of a medicament for the treatment and / or prophylaxis of the abovementioned diseases.
• inhibitors are all substances which prevent (inhibit) activation or the biological activity of the enzyme.
• the inhibition can be measured, for example, in the cGMP assay described below.
• Particularly preferred inhibitors are low molecular weight substances.
• inhibition means an at least 10% decrease in activity or an at least 10% increase in intracellular cGMP concentration in a cell which contains phosphodiesterase 9A.
• Inhibitors can be tested on PDE9A purified or recombinantly expressed and purified from suitable tissue.
• These cells are preferably cells from the smooth musculature of vessels or from cell lines which express the PDE9A recombinantly.
• Preferred PDE9A inhibitors are those which inhibit in the activity test given below with an IC 50 of 1 ⁇ M, preferably less than 0.1 ⁇ M.
• the PDE9A inhibitors according to the invention preferably cannot pass the blood / brain barrier and act systemically and not centrally.
• PDE9A inhibitors The effect of PDE9A inhibitors is tested on the isolated enzyme.
• the phosphodiesterase [3H] cGMP SPA enzyme assay kit from Amersham can be used. The test is carried out according to the manufacturer's instructions.
• a suitable dilution of the enzyme various concentrations of the inhibitor (dilution series typically of 10 “9 -10 “ 5 M) and [3H] cGMP (0.05 ⁇ Ci per batch) are used on a 96-well microtiter plate ) incubated for 15 min at 30 ° C. After the reaction has stopped, the “SPA beads” are added and the microtiter plate is shaken for 30 seconds. After 60 minutes, the measurement is carried out using a scintillation measuring device suitable for microtiter plates (eg 1450 MicroBeta, Wallac).
• a scintillation measuring device suitable for microtiter plates (eg 1450 MicroBeta, Wallac).
• the ICs ö value of the action of a PDE9A inhibitor is the value at which 50% of the cGMP degradation is inhibited by the PDE9A. Quantification of mRNA expression of PDE9A and PDE5A in human tissues
• the relative expression of PDE9A in human tissues is determined by quantifying the mRNA using the real-time polymerase chain reaction (PCR)
• RNA Purchased total RNA (from Clontech) is used as the template for the PCR.
• small pieces approximately (approx. 0.5 g) of explanted hearts are obtained from the German Heart Center Berlin and after homogenization the total RNA is isolated therefrom by means of phenol / chloroform extraction.
• 1 ⁇ g of total RNA is incubated with 1 unit DNase I (Gibco) for 15 min at room temperature.
• DNase I is deactivated by adding 1 ⁇ l EDTA (25 mM) and then heating to 65 ° C. (10 min).
• the cDNA synthesis is then carried out in the same reaction mixture in accordance with the instructions for the “SUPERSCRTPT-II RT cDNA synthesis kit” (from Gibco) and the reaction volume is diluted with distilled water
• PCR For the PCR, 7.5 ⁇ l of primer / probe mix and 12.5 ⁇ l of TaqMan Universal Master Mix (from Applied Biosytems) are added to 5 ⁇ l of the diluted cDNA solution. The final concentration of each primer is 300 nM, that of the probe 150 nM.
• the sequence of the "forward" and “reverse” primer for PDE9A is: 5 C - TCCCGGCTACAACAACACGT-3 'or 5'-AGATGTCATTGTAGCGGACCG-3', the • sequence of the fluorescence-labeled probe 5'-6FAM-
• the position of the amplicon is chosen so that all four described splice variants of the PDE9A mRNA (PDE9A 1- ) are detected.
• PDE9A 1- the sequence of the "forward" is
• the PCR is carried out on an ABI Prism SDS 7700 device (from Applied Biosystems) according to the manufacturer's instructions. 40 cycles are carried out as standard. For each tissue, a so-called “threshold cycle” (Ct value) is obtained. The Ct value corresponds to the cycle in which the fluorescence intensity of the released probe reaches 10 times the background signal. The lower the Ct value The amplification therefore begins earlier, ie the more mRNA is contained in the original sample. To compensate for any fluctuations in cDNA synthesis, the expression of a so-called “housekeeping gene” is also analyzed in all examined tissues. This should be expressed approximately equally strongly in all tissues.
• ⁇ -actin is used for this.
• the sequence of the "forward” or “reverse” primer for human cytosolic ß-actin is 5'-TCCACCTTCCAGCAGATGTG-3 ', and 5'-CTAGAAGCATTTGCGGTGGAC-3' the sequence of the probe 5'-6FAM-ATCAGCAAGCAGGCAGCCATGACGAGAG '.
• anesthetized rats are quickly removed from the heart and inserted into a conventional Langendorff apparatus.
• the coronary arteries are perfused at a constant volume (10 ml / min) and the resulting perfusion pressure is registered using a suitable pressure transducer.
• a decrease in the perfusion pressure in this arrangement corresponds to a relaxation of the coronary arteries.
• the pressure (LVP) which is developed by the heart during each contraction, is measured via a balloon inserted into the left ventricle and another pressure transducer.
• the frequency of the isolated beating heart is calculated from the number of contractions per unit of time.
• the test substances are added in an increasing concentration series (usually 10-9 M to 10-6 M) with the help of a perfuser.
• the PDE9A inhibitors can be converted in a known manner into the customary formulations, such as tablets, dragées, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert, non-toxic, pharmaceutically suitable excipients or solvents.
• the therapeutically active compound should in each case be present in a concentration of 0.5 to 90% by weight of the total mixture, i.e. in amounts sufficient to achieve the dosage range indicated.
• the formulations are prepared, for example, by stretching the active ingredients with solvents and / or carriers, optionally using emulsifiers and / or dispersants, for example in the case of.
• solvents and / or carriers optionally using emulsifiers and / or dispersants, for example in the case of.
• Use of water as a diluent, optionally organic solvents as auxiliary. solvents can be used.
• the application is carried out in the usual way, preferably orally, transdermally, intravenously or parenterally, in particular orally or intravenously. However, it can also be done by inhalation via the mouth or nose, for example with the aid of a spray, or topically via the skin.

Landscapes

• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Chemical & Material Sciences (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Cardiology (AREA)
• Organic Chemistry (AREA)
• General Chemical & Material Sciences (AREA)
• Heart & Thoracic Surgery (AREA)
• Gastroenterology & Hepatology (AREA)
• Immunology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Epidemiology (AREA)
• Hospice & Palliative Care (AREA)
• Urology & Nephrology (AREA)
• Vascular Medicine (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (2)

Family

ID=7705937

Family Applications (1)

Country Status (6)

Cited By (13)

Families Citing this family (5)

Family Cites Families (2)

• 2001
  • 2001-11-15 DE DE10156249A patent/DE10156249A1/de not_active Withdrawn
• 2002
  • 2002-11-11 WO PCT/EP2002/012550 patent/WO2003041725A2/de not_active Ceased
  • 2002-11-11 US US10/495,638 patent/US20040266736A1/en not_active Abandoned
  • 2002-11-11 EP EP02772410A patent/EP1448210A2/de not_active Withdrawn
  • 2002-11-11 JP JP2003543612A patent/JP2005511619A/ja not_active Withdrawn
  • 2002-11-11 AU AU2002337186A patent/AU2002337186A1/en not_active Abandoned

Also Published As

Similar Documents

Legal Events