EP1448210A2 - REGULATION OF cGMP-SPECIFIC PHOSPHODIESTERASE 9A - Google Patents

REGULATION OF cGMP-SPECIFIC PHOSPHODIESTERASE 9A

Info

Publication number
EP1448210A2
EP1448210A2 EP02772410A EP02772410A EP1448210A2 EP 1448210 A2 EP1448210 A2 EP 1448210A2 EP 02772410 A EP02772410 A EP 02772410A EP 02772410 A EP02772410 A EP 02772410A EP 1448210 A2 EP1448210 A2 EP 1448210A2
Authority
EP
European Patent Office
Prior art keywords
pde9a
prophylaxis
cgmp
inhibitors
treatment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02772410A
Other languages
German (de)
French (fr)
Inventor
Frank Wunder
Peter Ellinghaus
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer AG
Original Assignee
Bayer Healthcare AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer Healthcare AG filed Critical Bayer Healthcare AG
Publication of EP1448210A2 publication Critical patent/EP1448210A2/en
Withdrawn legal-status Critical Current

Links

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
  • 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.
  • 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)
  • 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.

Abstract

The invention relates to the use of PDE9A inhibitors for producing a pharmaceutical for the treatment and/or prophylaxis of coronary heart diseases, especially stable and unstable angina pectoris, acute myocardial infarction, the prophylaxis of myocardial infarction, sudden cardiac death, heart failure, and hypertension, peripheral circulatory disturbances and arteriosclerosis.

Description

Regulation der cGMP-spezifischen Phosphodiesterase 9A Regulation of the cGMP-specific phosphodiesterase 9A
Die Erfindung betrifft die Verwendung von PDE9A-Inhibitoren zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe von koronaren Herzkrankheiten, insbesondere stabiler und instabiler Angina pectoris, akutem Myokardinfarkt, Myo- kardinfarktprophylaxe, plötzlichem Herztod, Herzinsuffizienz, sowie Bluthochdruck und den Folgen der Atherosklerose.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.
Das Herz benötigt als unablässig arbeitender Hohlmuskel zur Deckung seines Ener- giebedarfs eine besonders intensive Versorgung mit Sauerstoff. Nersorgungsstörun- gen betreffen daher in erster Linie den Sauerstofftransport, der bei verminderter Anpassungsfähigkeit der Durchblutung unzureichend sein kann. Eine Steigerung des Sauerstoffverbrauchs kann nur durch eine Zunahme der Herzdurchblutung abgedeckt werden.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.
Bei koronaren Herzkrankheiten wie stabile und instabile Angina pectoris, Herzinsuffizienz, Myokardinfarkt, plötzlichem Herztod, sowie den Folgen der Atherosklerose ist eine ausreichende Durchblutung von Teilen des Herzgewebes nicht mehr gewährleistet und es kommt zu Gewebsischämien, die zu Νekrose und Apoptosein den betroffenen Arealen führen. Dadurch kommt es zu einer myokardialen Dysfunktion, die sich bis zur Herzinsuffizienz hin entwickeln kann.With coronary heart diseases such as stable and unstable angina pectoris, heart failure, myocardial infarction, sudden cardiac death, as well as the consequences of atherosclerosis, adequate blood flow to parts of the heart tissue is no longer guaranteed and tissue ischemia occurs, leading to eczema and apoptosis in the affected areas. This leads to myocardial dysfunction, which can develop into heart failure.
Therapieverfahren und Wirkstoffe, die die koronare Durchblutung und somit das Sauerstoffangebot verbessern, aber auch solche, die den Sauerstoffverbrauch senken, sind zur Behandlung von Symptomen der oben genannten Erkrankungen geeignet.Therapy methods and agents that improve coronary blood flow and thus the oxygen supply, but also those that reduce oxygen consumption, are suitable for the treatment of symptoms of the above-mentioned diseases.
Dazu gehören die Dilatation größerer Koronargefäße, die Senkung der extravasalen Komponente des Koronarwiederstandes, die Senkung der intramyokardialen Wandspannung und die Dilatation der arteriolären Widerstandsgefäße in der systemischen Zirkulation. Substanzen und Verfahren, die zu einer Erhöhung des Koronarfmßes im Herzen und/oder zu einer Blutdrucksenkung fuhren, können therapeutisch genutzt werden (Forth, Henschler, Rummel; Allgemeine und spezielle Pharmakologie und Toxikologie; Urban & Fischer Verlag (2001), München)This includes dilating larger coronary arteries, lowering the extravascular component of coronary resistance, lowering intramyocardial wall tension, and dilating arteriolar resistance vessels in the systemic circulation. Substances and processes that lead to an increase in the coronary measurement in the heart and / or to a reduction in blood pressure can be used therapeutically (Forth, Henschler, Rummel; general and special pharmacology and toxicology; Urban & Fischer Verlag (2001), Munich)
Die oben beschriebenen Wirkungen können über die intrazelluläre Konzentration der sogenannten „second messenger" zyklisches Adenosinmonophosphat (cAMP) und zyklisches Guanosinmonophosphat (cGMP) gesteuert werden. Die intrazelluläre Konzentration von cGMP wird durch die Stimulation der löslichen bzw. membran- gebundenen Guanylatzyklasen erhöht. Die intrazelluläre Konzentration von cAMP kann durch die Aktivierung von sogenannten G Protein gekoppelten Rezeptoren moduliert werden. Die Aktivierung dieser Rezeptoren führt zur Aktivierung von G Proteinen und damit zur Aktivierung bzw. Inhibition der Adenylatzyklase.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). 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.
Am Abbau von intrazellulärem cAMP bzw. cGMP sind sogenannte Phosphodiestera-At the breakdown of intracellular cAMP or cGMP, so-called phosphodiestera-
■ sen beteiligt. Die Phosphodiesterasen werden nach ihren biochemischen bzw. phar- makologischen Eigenschaften in elf verschiedene Klassen unterteilt (Soderling and ■ involved. The phosphodiesterases are divided into eleven different classes according to their biochemical or pharmacological properties (Soderling and
Beavo, Current Opinion in Cell Biology, (2000) 174-179; Francis et. al., Prog. Nuc- leic Acid Res. Mol. Biol. (2000) 1-52).Beavo, Current Opinion in Cell Biology, (2000) 174-179; Francis et. al., Prog. Nuclear Acid Res. Mol. Biol. (2000) 1-52).
Die Phosphodiesterase 9A (PDE9A) ist eine cGMP-spezifische Phosphodiesterase. Das Enzym besitzt einen Km- Wert (Michaelis-Menten Konstante) von 70 nM (Soderling et. al. , J. Biol. Chem. (1998) 15553-15558), dies ist der niedrigste bekannte Km- Wert für cGMP aller bekannten Phosphodiesterasen. Daher ist die PDE9A an der Aufrechterhaltung bzw. Regulation des basalen, intrazellulären cGMP-Spiegels beteiligt.Phosphodiesterase 9A (PDE9A) 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.
Die DNA- und Protein-Sequenzen für die Phosphodiesterase 9A sind aus der MausThe DNA and protein sequences for phosphodiesterase 9A are from the mouse
(Soderling et. al., J. Biol. Chem. (1998) 15553-15558) und dem Menschen (Fisher et. al., J. Biol. Chem. (1998) 15559-15564; Guipponi et. al., Hum. Gen. (1998) 386-392) bekannt. Bisher konnten vier Spleißvarianten der PDE9A identifiziert werden (Guip- poni et. al. Hum. Gen. (1998) 386-392).(Soderling et. Al., J. Biol. Chem. (1998) 15553-15558) and humans (Fisher et. Al., J. Biol. Chem. (1998) 15559-15564; Guipponi et. Al., Hum Gen. (1998) 386-392) known. So far, four splice variants of PDE9A have been identified (Guiponi et. Al. Hum. Gen. (1998) 386-392).
In der Maus konnte eine Expression der PDE9A vor allem in der Niere, schwächer auch in Lunge und Leber nachgewiesen werden (Soderling et. al., J. Biol. Chem.In the mouse, expression of PDE9A could be detected primarily in the kidney, but also weaker in the lungs and liver (Soderling et. Al., J. Biol. Chem.
(1998) 15553-15558). Beim Menschen konnte eine starke Expression vor allem in Milz, Niere, Darm, Prostata und Gehirn gezeigt werden, eine schwächere Expression wurde auch in anderen Organen wie Lunge, Leber, Herz und Pankreas nachgewiesen (Fisher et. al, J. Biol. Chem. (1998) 15559-15564; Guipponi et. al., Hum. Gen. (1998) 386-392).(1998) 15553-15558). In humans, a strong expression could be shown especially in the spleen, kidney, intestine, prostate and brain, a weaker expression was also found in other organs such as the lungs, liver, heart and pancreas (Fisher et. Al, J. Biol. Chem. (1998) 15559-15564; Guipponi et al., Hum. Gen. (1998) 386-392).
Überraschenderweise wurde nun in der quantitativen Analyse der PDE9A rnRNA- Expression im Menschen gefunden, dass eine deutliche Expression der PDE9A in humanen Koronararterien vorhanden ist (Abbildungen 1 und 2).Surprisingly, it has now been found in the quantitative analysis of PDE9A rnRNA expression in humans that there is a clear expression of PDE9A in human coronary arteries (Figures 1 and 2).
Die Expression der PDE9A in der humanen Koronararterie ist dabei überraschenderweise sogar noch ca. 2,7-fach höher als die Expression der Phosphodiesterase 5A in diesem Gewebe (Abbildung 2).The expression of PDE9A in the human coronary artery is surprisingly even about 2.7 times higher than the expression of phosphodiesterase 5A in this tissue (Figure 2).
Für die Phosphodiesterase 5A ist aus der Literatur eine Rolle bei Blutversorgung desFor the phosphodiesterase 5A, a role in the blood supply of the
Herzens bekannt. Es konnte gezeigt werden, dass die Gabe von PDE5A-Inhibitoren zur Relaxation von Koronargefässen führt (Traverse et. al, Circulation (2000) 2997- 3002).Known from the heart. It was shown that the administration of PDE5A inhibitors leads to the relaxation of coronary vessels (Traverse et. Al, Circulation (2000) 2997-3002).
Die, auch im Vergleich zur PDE5A, hohe Expression der PDE9A in der humanenThe, also in comparison to the PDE5A, high expression of the PDE9A in the human
Koronararterie, sowie die extrem hohe Affinität der PDE9A zu cGMP (Km- Wert 70 nM) weisen nun darauf hin, dass die Phosphodiesterase 9A eine sehr bedeutende Rolle bei der Kontraktion bzw. Relaxation von Koronararterien und damit bei der Steuerung der Durchblutung des Herzens besitzt. Die Expression der PDE9A in Blutgefässen weist damit auch auf eine Rolle der PDE9A bei der Kontrolle des Blutdrucks und der Regulation der peripheren Durchblutung bin.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.
Die Wirkung von PDE9A-Inhibitoren auf den Koronarfluss kann am isoliert perfundierten Langendorff-Herz überprüft werden. Ein Inhibitor der PDE9A senkt den Perfusionsdruck am Langendorff-Herz der Ratte.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.
Da die Expression des humanen Phosphodiesterase 9A in Koronararterien eine Be- dingung für den Einsatz von Wirkstoffen, die die PDE9A hemmen, in Patienten mit koronaren Herzkrankheiten ist, schafft dieses Ergebnis die Voraussetzung für einen neuen Therapieansatz.Since the expression of human phosphodiesterase 9A in coronary arteries is a prerequisite for the use of active substances that inhibit PDE9A in patients with coronary artery disease, this result creates the prerequisites for a new therapeutic approach.
Aufgrund dieses neuen Ergebnis kamen wir zu dem Schluß, dass Substanzen, die die Phosphodiesterase 9A inhibieren, wegen der daraus resultierenden Erhöhung der intrazellulären cGMP-Konzentration und der damit verbundenen Erweiterung von Blutgefässen, speziell Koronararterien (und der damit verbundenen Erhöhung des Koronarflusses), zur Behandlung und/oder Prophylaxe von stabiler und instabiler Angina pectoris, akutem Myokardinfarkt, Myokardinfarktprophylaxe, Herzinsuffi- zienz, plötzlichem Herztod, sowie Bluthochdruck, peripherer Durchblutungsstörungen und den Folgen der Atherosklerose beim Menschen eingesetzt werden können.Based on this new result, we came to the conclusion that substances that inhibit phosphodiesterase 9A due to the resulting increase in the intracellular cGMP concentration and the associated dilation of blood vessels, especially coronary arteries (and the associated increase in coronary flow) Treatment and / or prophylaxis of stable and unstable angina pectoris, acute myocardial infarction, myocardial infarction prophylaxis, heart failure, sudden cardiac death, as well as hypertension, peripheral circulatory disorders and the consequences of atherosclerosis in humans can be used.
Die vorliegende Erfindung betrifft daher die Verwendung von Phosphodiesterase 9A Inhibitoren zur Herstellung eines Arzneimittels zur Behandlung und/oder der Pro- phylaxe der oben genannten Krankheiten.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.
Inhibitoren im Sinne der Erfindung sind alle Substanzen, die eine Aktivierung bzw. die biologische Aktivität des Enzyms verhindern (inhibieren). Die Inhibition kann z.B. im unten beschriebenen cGMP-Assay gemessen werden. Besonders bevorzugte Inhibitoren sind niedermolekulare Substanzen. Inhibition bedeutet für die Phosphodiesterase 9A eine mindestens 10%ige Abnahme der Aktivität bzw. eine mindestens 10%ige Zunahme der intrazellulären cGMP-Kon- zentration in einer Zelle, die die Phosphodiesterase 9A enthält. Inhibitoren können an aus geeignetem Gewebe gereinigter oder rekombinant exprimierter und gereinigter PDE9A getestet werden. Zusätzlich ist es möglich, die intrazelluläre cGMP -Konzentration einer Zelle, die die Phosphodiesterase 9A enthält, zu bestimmen. Bei diesen Zellen handelt es sich bevorzugt um Zellen aus der glatten Muskulatur von Gefässen oder aus Zellinien, die die PDE9A rekombinant exprimieren.For the purposes of the invention, 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. For phosphodiesterase 9A, 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. In addition, it is possible to determine the intracellular cGMP concentration of a cell that contains the phosphodiesterase 9A. These cells are preferably cells from the smooth musculature of vessels or from cell lines which express the PDE9A recombinantly.
Dabei werden solche PDE9A-Inhibitoren bevorzugt, die im unten angegebenen Aktivitätstest mit einem IC50 von 1 μM, bevorzugt weniger als 0,1 μM inhibieren.Preferred PDE9A inhibitors are those which inhibit in the activity test given below with an IC 50 of 1 μM, preferably less than 0.1 μM.
Vorzugsweise können die erfindungsgemäßen PDE9A-Inhibitoren die Blut/Hirn- Schranke nicht passieren und wirken systemisch und nicht zentral. The PDE9A inhibitors according to the invention preferably cannot pass the blood / brain barrier and act systemically and not centrally.
Kurzbeschreibung der Abbildungen:Brief description of the pictures:
1.) Relative Expression der humanen Phosphodiesterase 9A in humanen Geweben (Daten Siehe Tabelle 1)1.) Relative expression of human phosphodiesterase 9A in human tissues (data see table 1)
2.) Vergleich der relativen Expression der humanen PDE9A mit PDE5A in der humanen Koronararterie2.) Comparison of the relative expression of the human PDE9A with PDE5A in the human coronary artery
Inhibition der cGMP-spezifϊschen PDE9AInhibition of the cGMP-specific PDE9A
Die Wirkung von PDE9A-Inhibitoren wird am isolierten Enzym getestet. Dazu kann zum Beispiel der Phosphodiesterase [3H]cGMP SPA Enzyme Assay Kit der Firma Amersham verwendet werden. Die Durchführung des Tests erfolgt nach Herstellerangäben.The effect of PDE9A inhibitors is tested on the isolated enzyme. For example, the phosphodiesterase [3H] cGMP SPA enzyme assay kit from Amersham can be used. The test is carried out according to the manufacturer's instructions.
Für die Charakterisierung von Testsubstanzen werden auf einer 96-Loch Mikrotiter- platte eine geeignete Verdünnung des Enzyms, verschiedene Konzentrationen des Inhibitors (Verdünnungsreihen typischerweise von 10"9 -10"5M), sowie [3H]cGMP (0,05 μCi pro Ansatz) für 15 min bei 30°C inkubiert. Nach Abstoppen der Reaktion werden die „SPA-Beads" hinzugefügt und die Mikrotiterplatte für 30 Sekunden geschüttelt. Nach 60 min erfolgt die Messung mit Hilfe eines für Mikrotiterplatten geeigneten Szintillations-Meßgerätes (z. B. 1450 MicroBeta, Wallac).For the characterization of test substances, 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 μCi 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).
Der ICsö-Wert der Wirkung eines PDE9A-Inhibitors ist der Wert, bei dem 50 % des cGMP -Abbaus durch die PDE9A inhibiert werden. Quantifizierung der mRNA-Expression von PDE9A und PDE5A in humanen GewebenThe 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
Die relative Expression der PDE9A in humanen Geweben wird durch die Quantifi- zierung der mRNA mittels der Echtzeit-Polymerasekettenreaktion (PCR) ermitteltThe relative expression of PDE9A in human tissues is determined by quantifying the mRNA using the real-time polymerase chain reaction (PCR)
(sog. TaqMan-PCR, Heid et al., Genome Res 6 (10), 986-994). Gegenüber der klassischen PCR bietet die Echtzeit-PCR den Vorteil einer genaueren Quantifizierung durch Einführung eines zusätzlichen, fluoreszenzmarkierten Oligonukleotides. Diese sogenannte Sonde enthält am 5 '-Ende den Fluoreszenzfarbstoff F AM (6-Carboxy- Fluorescein) und am 3 '-Ende den Fluoreszenzquencher TAMRA (6-Carboxy-Tetra- methylrhodamin). Während der Polymerasekettenreaktion wird in der TaqMan-PCR durch die 5'-Exonukleaseaktivtät der Taq-Polymerase der Fluoreszenzfarbstoff FAM von der Sonde abgespalten und dadurch das vorher gequenchte Fluoreszenzsignal erhalten.(So-called TaqMan-PCR, Heid et al., Genome Res 6 (10), 986-994). Compared to classic PCR, real-time PCR offers the advantage of more precise quantification by introducing an additional, fluorescence-labeled oligonucleotide. This so-called probe contains the fluorescent dye F AM (6-carboxy-fluorescein) at the 5 'end and the fluorescent quencher TAMRA (6-carboxy-tetra-methylrhodamine) at the 3' end. During the polymerase chain reaction in the TaqMan PCR, the 5'-exonuclease activity of the Taq polymerase cleaves the fluorescent dye FAM from the probe, thereby obtaining the previously quenched fluorescence signal.
Als Templat für die PCR wird käuflich erworbene Gesamt-RNA verwendet (Fa. Clontech). Im Falle der Koronararterien werden kleine Stücke (ca. 0,5 g) von explantierten Herzen vom Deutschen Herzzentrum Berlin erhalten und nach Homogenisierung die Gesamt-RNA hieraus mittels Phenol/Chloroform-Extraktion isoliert. Je 1 μg Gesamt-RNA wird zur Entfernung von Kontaminationen genomischer DNA mit 1 Unit DNase I (Fa. Gibco) für 15 min bei Raumtemperatur inkubiert. Die Inak- tivierung der DNase I erfolgt durch Zugabe von 1 μl EDTA (25 mM) und nachfolgendem Erhitzen auf 65 °C (10 min). Anschließend wird im selben Reaktionsansatz die cDNA-Synthese gemäß der Anleitung zum „SUPERSCRTPT-II RT cDNA syn- thesis kit" (Fa. Gibco) durchgeführt und das Reaktionsvolumen mit dest. Wasser aufPurchased total RNA (from Clontech) is used as the template for the PCR. In the case of the coronary arteries, small pieces (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. To remove contaminations of genomic DNA, 1 μg of total RNA is incubated with 1 unit DNase I (Gibco) for 15 min at room temperature. DNase I is deactivated by adding 1 μl 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
200 μl aufgefüllt.Top up 200 μl.
Für die PCR wird zu je 5 μl der verdünnten cDNA-Lösung 7,5 μl Primer/Sondenmix sowie 12,5 μl TaqMan Universal Master Mix (Fa. Applied Biosytems) gegeben. Die Endkonzentration der Primer ist jeweils 300 nM, die der Sonde 150 nM. Die Sequenz des „forward"- und „reverse"-Primers für PDE9A lautet: 5C- TCCCGGCTACAACAACACGT-3' bzw. 5'-AGATGTCATTGTAGCGGACCG-3', die • Sequenz der fluoreszenzmarkierten Sonde 5'-6FAM-For the PCR, 7.5 μl of primer / probe mix and 12.5 μl of TaqMan Universal Master Mix (from Applied Biosytems) are added to 5 μl 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-
CCAGATCAATGCCCGCACAGAGCT-TAMRA-3'. Die Lage des Amplikons ist so gewählt, dass alle vier beschriebenen Spleißvarianten der PDE9A-mRNA (PDE9A1- ) detektiert werden. Für die PDE5A lautet die Sequenz des „forward"-CCAGATCAATGCCCGCACAGAGCT-TAMRA-3 '. The position of the amplicon is chosen so that all four described splice variants of the PDE9A mRNA (PDE9A 1- ) are detected. For the PDE5A, the sequence of the "forward" is
Primers: 5'-TGGCAAGGTTAAGCCTTTCAA-3', die des „reverse"-Primers: 5'- ATCTGCGTGTTCTGGATCCC-3' und die Sequenz der Sonde 5'-FAM- ATGACGAACAGTTTCTGGAAGCTTTTGTCATCTT-TAMRA-3r. Auch hier ist die Lage des Amplikons auf der mRNA so gewählt, daß beide Spleißvarianten (PDE5 A1-2) detektiert werden.Primer: 5'-TGGCAAGGTTAAGCCTTTCAA-3 ', that of the "reverse" primer: 5'ATCTGCGTGTTCTGGATCCC-3'. And the sequence of the probe 5'-FAM-TAMRA-3 ATGACGAACAGTTTCTGGAAGCTTTTGTCATCTT r Again, the location of the amplicon on the mRNA is selected so that both splice variants (PDE5 A 1-2 ) are detected.
Die PCR erfolgt auf einem ABI Prism SDS 7700 Gerät (Fa. Applied Biosystems) gemäß der Anleitung des Herstellers. Dabei werden standardmäßig 40 Zyklen durchgeführt. Für jedes Gewebe wird für jede Sonde ein sog. „treshold cycle" (Ct-Wert) er- halten. Der Ct-Wert entspricht dem Zyklus, in dem die Fluoreszenzintensität der freigesetzten Sonde das lOfache des Hintergrundsignals erreicht. Je niedriger der Ct- Wert, umso früher beginnt also die Amplifikation, d. h. je mehr mRNA ist in der ursprünglichen Probe enthalten. Zum Ausgleich eventueller Schwankungen bei der cDNA-Synthese wird in allen untersuchten Geweben auch die Expression eines sog. „housekeeping genes" analysiert. Dieses sollte in allen Geweben ungefähr gleich stark exprimiert werden. Für die Normierung der PDE9A- bzw. PDE5A-Expression wird hierfür ß-Actin verwendet. Die Sequenz des „forward"- bzw. „reverse" Primers für humanes cytosolisches ß-Actin ist 5'-TCCACCTTCCAGCAGATGTG-3' , und 5'-CTAGAAGCATTTGCGGTGGAC-3' die Sequenz der Sonde 5'-6FAM- ATCAGCAAGCAGGCAGTATGACGAGTCCG-TAMRA-3'. Die Auswertung derThe 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. For the normalization of PDE9A or PDE5A expression, β-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 '. The evaluation of the
Daten erfolgt durch die sog. ddCt-Methode entspechend der Anleitung zum ABI Prism SDS 7700 (Fa. Applied Biosystems). Für die graphische Darstellung der Gewebeverteilung der PDE9A-mRNA wird das Expressionsniveau des Gewebes mit dem höchsten Ct-Wert (= niedrigster Expression) willkürlich gleich 1 gesetzt und alle anderen Gewebe hierauf normiert. Langendorff-Herz der RatteData is generated by the so-called ddCt method in accordance with the instructions for the ABI Prism SDS 7700 (from Applied Biosystems). For the graphical representation of the tissue distribution of the PDE9A mRNA, the expression level of the tissue with the highest Ct value (= lowest expression) is arbitrarily set to 1 and all other tissues are standardized accordingly. Langendorff heart of the rat
Narkotisierten Ratten wird nach Eröffnung des Brustkorbes das Herz schnell entnommen und in eine konventionelle Langendorff-Apparatur eingeführt. Die Koro- nararterien werden volumenkonstant (lOml/min) perfundiert und der dabei auftretende Perfusionsdruck wird über einen entsprechenden Druckaufhehmer registriert. Eine Abnahme des Perfusionsdrucks in dieser Anordnung entspricht einer Relaxation der Koronararterien. Gleichzeitig wird über einen in die linke Herzkammer eingeführten Ballon und einen weiteren Druckaufhehmer der Druck (LVP) gemessen, der vom Herzen während jeder Kontraktion entwickelt wird. Die Frequenz des isoliert schlagenden Herzens wird rechnerisch aus der Anzahl der Kontraktionen pro Zeiteinheit ermittelt. Die Zugabe von Prüfsubstanzen erfolgt in einer aufsteigenden Konzentrationsreihe (üblicherweise 10-9 M bis 10-6 M) mit Hilfe eines Perfusors.After the chest is opened, 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. At the same time, 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.
PDE9A-Inhibitor FormulierungenPDE9A inhibitor formulations
Die PDE9A-Inhibitoren können in bekannter Weise in die üblichen Formulierungen überführt werden, wie Tabletten, Dragees, Pillen, Granulate, Aerosole, Sirupe, Emulsionen, Suspensionen und Lösungen, unter Verwendung inerter, nicht toxischer, pharmazeutisch geeigneter Trägerstoffe oder Lösungsmittel. Hierbei soll die therapeutischwirksame Verbindung jeweils in einer Konzentration von 0,5 bis 90 Gew.-% der Gesamtmischung vorhanden sein, d.h. in Mengen, die ausreichend sind, um den angegebenen Dosierungsspielraum zu erreichen.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. Here, 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.
Die Formulierungen werden beispielsweise hergestellt durch Strecken der Wirkstoffe mit Lösungsmitteln und/oder Trägerstoffen, gegebenenfalls unter Verwendung von Emulgiermitteln und/oder Dispergiermitteln, wobei z.B. im Fall der. Benutzung von Wasser als Verdünnungsmittel gegebenenfalls organische Lösungsmittel als Hilfs- . lösungsήiittel verwendet werden können. Die Applikation erfolgt in üblicher Weise, vorzugsweise oral, transdermal, intravenös oder parenteral, insbesondere oral oder intravenös. Sie kann aber auch durch Inhalation über Mund oder Nase, beispielsweise mit Hilfe eines Sprays erfolgen, oder topisch über die Haut.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. 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.
Im Allgemeinen hat es sich als vorteilhaft erwiesen, Mengen von etwas 0,001 bis 10 mg kg, bei oraler Anwendung vorzugsweise etwa 0,005 bis 3 mg/kg Körpergewicht zur Erzielen wirksamer Ergebnisse zu verabreichen.In general, it has been found to be advantageous to administer amounts of approximately 0.001 to 10 mg kg, preferably approximately 0.005 to 3 mg / kg of body weight when used orally in order to achieve effective results.
Trotzdem kann es gegebenenfalls erforderlich sein, von den genannten Mengen abzuweichen, und zwar in Abhängigkeit vom Körpergewicht bzw. der Art des Applikationsweges,, vom individuellen Verhalten gegenüber dem Medikament, der Art von dessen Formulierung und dem Zeitpunkt bzw. Intervall, zu welchen die Verabreichung erfolgt. So kann es in einigen Fällen ausreichend sein, mit weniger als der vorgenannten Mindestmenge auszukommen, während in anderen Fällen die genannte obere Grenze überschritten werden muss. Im Falle der Applikation größerer Mengen kann es empfehlenswert sein, diese in mehreren Einzelgaben über den Tag zu verteilen. Nevertheless, it may be necessary to deviate from the amounts mentioned, depending on the body weight or the type of application route, on the individual behavior towards the medicament, the type of its formulation and the time or interval at which the administration he follows. In some cases it may be sufficient to make do with less than the aforementioned minimum quantity, while in other cases the above upper limit must be exceeded. In the case of application of larger quantities, it may be advisable to distribute them in several individual doses over the day.

Claims

Patentansprüche claims
1. Verwendung von PDE9A-Inhibitoren zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe von koronaren Herzkrankheiten.1. Use of PDE9A inhibitors for the manufacture of a medicament for the treatment and / or prophylaxis of coronary heart diseases.
2. Verwendung von PDE9A-Inhibitoren zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe von Bluthochdruck.2. Use of PDE9A inhibitors for the manufacture of a medicament for the treatment and / or prophylaxis of high blood pressure.
3. Verwendung von PDE9A-Inhibitoren zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe peripherer Verschlußkrankheiten.3. Use of PDE9A inhibitors for the manufacture of a medicament for the treatment and / or prophylaxis of peripheral occlusive diseases.
4. Verwendung von PDE9A-Inhibitoren zur Herstellung eines Arzneimittels zur Behandlung und/oder Prophylaxe der Atherosklerose.4. Use of PDE9A inhibitors for the manufacture of a medicament for the treatment and / or prophylaxis of atherosclerosis.
5. Verwendung von Anspruch 1, wobei die koronaren Herzkrankheiten, stabile und instabile Angina pectoris, akuter Myokardinfarkt, Myokardinfarkt-pro- phylaxe, plötzlicher Herztod und Herzinsuffizienz sind.5. Use of claim 1, wherein the coronary artery disease, stable and unstable angina pectoris, acute myocardial infarction, myocardial infarction prophylaxis, sudden cardiac death and heart failure are.
6. Verwendung nach Anspruch 1-4, wobei der PDE9A-Inhibitor einen ICso-Wert von weniger als 1 μM hat.6. Use according to claims 1-4, wherein the PDE9A inhibitor has an IC 50 value of less than 1 μM.
7. Verwendung nach Anspruch 1-4, wobei der PDE9A-Inhibitor einen IC5o-Wert von weniger als 100 nM hat. 7. Use according to claim 1-4, wherein the PDE9A inhibitor has an IC 5 o value of less than 100 nM.
EP02772410A 2001-11-15 2002-11-11 REGULATION OF cGMP-SPECIFIC PHOSPHODIESTERASE 9A Withdrawn EP1448210A2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10156249A DE10156249A1 (en) 2001-11-15 2001-11-15 Regulation of the cGMP-specific phosphodiesterase 9A
DE10156249 2001-11-15
PCT/EP2002/012550 WO2003041725A2 (en) 2001-11-15 2002-11-11 Regulation of cgmp-specific phosphodiesterase 9a

Publications (1)

Publication Number Publication Date
EP1448210A2 true EP1448210A2 (en) 2004-08-25

Family

ID=7705937

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02772410A Withdrawn EP1448210A2 (en) 2001-11-15 2002-11-11 REGULATION OF cGMP-SPECIFIC PHOSPHODIESTERASE 9A

Country Status (6)

Country Link
US (1) US20040266736A1 (en)
EP (1) EP1448210A2 (en)
JP (1) JP2005511619A (en)
AU (1) AU2002337186A1 (en)
DE (1) DE10156249A1 (en)
WO (1) WO2003041725A2 (en)

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10238722A1 (en) 2002-08-23 2004-03-11 Bayer Ag Improving attention, concentration, cognition, learning and/or memory performance, using selective phosphodiesterase 9A inhibitors, preferably 4H-pyrazolo-(3,4-d)-pyrimidin-4-one derivatives
DE10238724A1 (en) 2002-08-23 2004-03-04 Bayer Ag New 6-alkyl-1,5-dihydro-4H-pyrazolo-(3,4-d)-pyrimidin-4-ones useful as selective phosphodiesterase 9A inhibitors for improving attention, concentration, learning and/or memory performance
DE10238723A1 (en) 2002-08-23 2004-03-11 Bayer Ag Phenyl substituted pyrazolyprimidines
DE10320785A1 (en) 2003-05-09 2004-11-25 Bayer Healthcare Ag 6-arylmethyl substituted pyrazolopyrimidines
US8044060B2 (en) 2003-05-09 2011-10-25 Boehringer Ingelheim International Gmbh 6-cyclylmethyl- and 6-alkylmethyl pyrazolo[3,4-D]pyrimidines, methods for their preparation and methods for their use to treat impairments of perception, concentration learning and/or memory
DE10328479A1 (en) 2003-06-25 2005-01-13 Bayer Ag 6-arylamino-5-cyano-4-pyrimidinones
DE102004001873A1 (en) 2004-01-14 2005-09-29 Bayer Healthcare Ag Cyanopyrimidinone
EP1829964A4 (en) * 2004-12-08 2009-03-04 Takeshi Yamamoto Method of examining gene sequence
DE102005024494A1 (en) * 2005-05-27 2006-11-30 Bayer Healthcare Ag Use of cyanopyrimidines
US8648085B2 (en) 2007-11-30 2014-02-11 Boehringer Ingelheim International Gmbh 1, 5-dihydro-pyrazolo (3, 4-D) pyrimidin-4-one derivatives and their use as PDE9A mudulators for the treatment of CNS disorders
UA105362C2 (en) 2008-04-02 2014-05-12 Бьорингер Ингельхайм Интернациональ Гмбх 1-heterocyclyl-1, 5-dihydro-pyrazolo [3, 4-d] pyrimidin-4-one derivatives and their use as pde9a modulators
PE20110383A1 (en) 2008-09-08 2011-07-15 Boehringer Ingelheim Int PYRAZOLOPYRIMIDINONES AS INHIBITORS OF PHOSPHODIESTERASE 9A (PDE9A)
JP5542196B2 (en) 2009-03-31 2014-07-09 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング 1-Heterocyclic-1,5-dihydro-pyrazolo [3,4-D] pyrimidin-4-one derivatives and their use as PDE9A modulators
TW201118099A (en) * 2009-08-12 2011-06-01 Boehringer Ingelheim Int New compounds for the treatment of CNS disorders
PL2603511T3 (en) 2010-08-12 2017-08-31 Boehringer Ingelheim Int 6-cycloalkyl-1, 5-dihydro-pyrazolo [3, 4-d] pyrimidin-4-one derivatives and their use as pde9a inhibitors
US8809345B2 (en) 2011-02-15 2014-08-19 Boehringer Ingelheim International Gmbh 6-cycloalkyl-pyrazolopyrimidinones for the treatment of CNS disorders
EP3939588A4 (en) * 2019-03-08 2023-01-11 Transthera Sciences (Nanjing), Inc. Uses of phosphodiesterase inhibitors

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4211239C2 (en) * 1992-04-03 1995-11-16 Max Planck Gesellschaft Medicines for cardiovascular diseases
US5922595A (en) * 1997-12-09 1999-07-13 Incyte Pharmaceuticals, Inc. Cyclic GMP phosphodiesterase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03041725A2 *

Also Published As

Publication number Publication date
WO2003041725A3 (en) 2004-03-18
JP2005511619A (en) 2005-04-28
AU2002337186A1 (en) 2003-05-26
US20040266736A1 (en) 2004-12-30
WO2003041725A2 (en) 2003-05-22
DE10156249A1 (en) 2003-05-28

Similar Documents

Publication Publication Date Title
EP1448210A2 (en) REGULATION OF cGMP-SPECIFIC PHOSPHODIESTERASE 9A
Galvin et al. Muscarinic M1 receptors modulate working memory performance and activity via KCNQ potassium channels in the primate prefrontal cortex
EP1534285B1 (en) Selective phosphodiesterase 9a inhibitors as medicaments for improving cognitive processes
EP1534711B1 (en) Phenyl-substituted pyrazolopyrimidines
Coull Pharmacological manipulations of the α 2-noradrenergic system: effects on cognition
EP1534713B1 (en) Alkyl-substituted pyrazolopyrimidines
DE69333874T2 (en) Use of four K-252a derivatives
DE69937198T2 (en) TREATMENT OF ALZHEIMER DISEASE BY INCREASING THE CYTIDINE MIRROR IN VIVO
EP0590551B1 (en) New therapeutic uses of phthalazinone-derivatives
DE60116352T2 (en) USE OF KCNQ CALIUM CHANNEL AGONISTS FOR THE MANUFACTURE OF A MEDICAMENT FOR MODULATING BLADDER FUNCTION
Nattie et al. Ventral medulla sites of muscarinic receptor subtypes involved in cardiorespiratory control
EP1416952A1 (en) Regulation of the apj receptor for use in the treatment or prophylaxis of cardiac diseases
Classen et al. Differential effects of orally versus parenterally administered qinghaosu derivative artemether in dogs
EP1644339B1 (en) 6-arylamino-5-cyano-4-pyrimidinones as pde9a inhibitors
WO2008043461A2 (en) Inhibition of pde2a
DE102005042877A1 (en) Use of PDE1A polypeptides, or nucleic acid, for identifying their specific inhibitors, which are useful for treatment and prevention of cardiac insufficiency
DE60125171T2 (en) METHOD FOR DETECTING AND QUANTIFYING HUMAN P450 MOLECULES AND PROBE AND KIT FOR THIS PROCEDURE
Ludlam et al. mRNAs encoding muscarinic and substance P receptors in cultured sympathetic neurons are differentially regulated by LIF or CNTF
DE4211239A1 (en) Medicines for cardiovascular diseases
DE60029110T2 (en) METHOD FOR IDENTIFYING NOVEL GENES THAT PLAY A ROLE IN THE REGULATION OF THE ANGIOGENESIS, ANALYSIS OF THESE GENES, AND THEIR USE FOR THERAPEUTIC USES
DE19740384A1 (en) Antisense oligonucleotides specific for protein kinase C isoforms
DE10332685A1 (en) Use of a modulator of specific potassium channels, expressed preferentially in the atrium, for treatment and prevention of arrhythmia, coronary disease and hypertension
Pepeu et al. The central cholinergic system during aging
Rinkel Pharmacodynamics of LSD and mescaline
DE10226934A1 (en) Regulation of sPLA2G2A

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LI LU MC NL PT SE SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

17P Request for examination filed

Effective date: 20040920

17Q First examination report despatched

Effective date: 20060807

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20061218