Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2001-05-15
2002-05-07
Coleman, Brenda (Department: 1624)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Heterocyclic carbon compounds containing a hetero ring...
C544S066000
Reexamination Certificate
active
06384029
ABSTRACT:
TECHNICAL FIELD
The invention relates to an optically active form of a pyridyl-4H-1,2,4-oxadiazine derivative, to the therapeutical use thereof and to pharmaceutical compositions containing the compound as active ingredient. More particularly, the invention relates to the (−) enantiomer of 5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)4H-1,2,4-oxadiazine and its acid addition salts as well as the use of these chemical compounds in the treatment of vascular diseases and pharmaceutical compositions containing the said compounds as active ingredients.
BACKGROUND ART
The racemic 5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)4H-1,2,4-oxadiazine is known. Its preparation and hsp expression enhancing effect on cells exposed to heat shock is described in WO 97/16349 and its protective and regenerating effect on vascular endothelial cells is described in WO 98/06400. This compound is suitable mainly for fending off the damages caused by ischemia and in the treatment of cardiovascular and cerebrovascular diseases.
The optically active forms of 5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine were not described in the literature.
During our recent experiments, the optically active forms of 5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)4H-1,2,4-oxadiazine were prepared and their biological activities studied. It has been found that the (−)-5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)4H-1,2,4-oxadiazine has a substantially stronger vasoprotective and cardioprotective effect than the (+) enantiomer and the racemic compound. It is expressly more efficient in preventing the damages of the endothelium caused by ischemia.
Due to these properties the (−)-5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)4H-1,2,4-oxadiazine is prominently suitable to the treatment of vascular diseases and diseases connected to vascular abnormalities.
DISCLOSURE OF INVENTION
In course of experiments we recognized, unexpectedly, that (−)-5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)4H-1,2,4-oxadiazine, in contrast to the (+)-enantiomer and the racemic compound, has a positive inotropic effect i. e. it is capable of increasing the contractile force of the heart. This activity makes (−)-5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)4H-1,2,4-oxadiazine suitable for the treatment of patients with cardiac failure, contrary to the (+)-enantiomer and the racemic compound, the application of which being risky in case of cardiac failure.
BEST MADE OF CARRYING OUT THE INVENTION
The above mentioned advantageous biological properties of (−)-5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazine are verified in the following tests.
Langendorff Perfused Rat Heart
Protocol for Assesment of Endothelial Function Before and After Ischemia
Rats were heparinised with i.p. heparin sodium (2500 IU) and anesthetized with i.p. pentobarbital (60 mg/kg). Hearts from spontaneously hypertensive (SH) rats were quickly removed and immediately perfused via the aorta using a gravity-flow, non-recirculating Langendorff apparatus (Experimetria Ltd) at a constant perfusion pressure (100 cm H
2
O) with Krebs-Henseleit Solution (KHS) containing in mM: NaCl 120; KCl 5.4; CaCl
2
2.7; MgCl
2
1.1; NaHCO
3
24; D-glucose 11. The KHS was gassed with carbogen (95% O
2
; 5% CO
2
) resulting pH 7.4.
We used the so-called non-working heart model and measured the mean coronary flow by a transonic flow meter (Type T206, Transonic Systems Inc.). The flow probe was placed above the aortic cannula. Coronary flow was monitored throughout the experiment and registered by a potentiometric recorder. Hearts were allowed to beat spontaneously throughout the experiment. After 20-30 min of equilibration period the coronary flow reached a baseline value.
Endothelial function was assessed through observations of preischemic and postischemic coronary flow responses to serotonin (5-HT). In response to serotonin, the progress of dilatation of coronary artery depends on the soundness of the endothelium.
The Langendorff infusion was switched to the another column containing additional 10
−7
M serotonin (5-HT, Sigma Chemical Co.). The ensuing dilatation of coronary artery was monitored and when the steady state had been reached the 5-HT was washed out by switching back to the ordinary KHS. The heart was then subjected to a global ischemia for 10 min by the clamping the aortic cannula. At the end of the ischemic period, the heart was reperfused. When the baseline coronary flow had been reestablished, the heart was again subjected to the same protocol of sequential infusion of 5-HT and KHS as in the preischemic period. Control hearts were perfused with pure KHS and the hearts of the another three groups were perfused with KHS containing additional 10
−6
M racemic 5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)4H-1,2,4-oxadiazine, (−)-5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)4H-1,2,4-oxadiazine and (+)-5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)4H-1,2,4-oxadiazine, respectively. 20-30 min after the onset of hemodynamic stability, perfusion of the drugs were initiated and continued until reperfusion with the exception of the occlusion period.
The results are shown in the following table. The potischemic coronary responses are expressed in percentage of the preischemic vasodilator responses.
SH control
racemic compound
(−) enantiomer
(+) enantiomer
37.13 ± 9.5
63.08 ± 11.7
88.41 ± 10.9
49.78 ± 9.9
p = 0.07
p = 0.016
p = 0.45
Perfusion of the rat heart with the (−)-5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-pyridyl)4H-1,2,4-oxadiazine (10
−6
M) preserved endothelial function after 10 min of global ischemia in contrast with the racemic compound, the (+) enantiomer and the racemic compound or the SH control.
Myocardial Infarction in Spontaneously Hypertensive Rats
Induction of Infarction
Myocardial ischemia was induced by a temporary occlusion of the main left coronary artery, according to Griswold et al (J. Pharmacol. Methods 1988, 20: 225-235). SH rats were anaesthetized with sodium pentobarbital (60 mg/kg i.p.). After tracheotomy, the animals were ventilated with room air by a respirator for small rodents (model: Harvard 552), with a stroke volume of 1,5-2 ml/100 g and a rate of 55 strokes/min to maintain normal pO
2
, pCO
2
and pH parameters. The right carotid artery was catheterized and connected to a pressure transducer (P236B Stetham) for the measurement of systemic arterial blood pressure (BP) by means of a preamplifier (Hg-O2D Experimetria®). Heart rate (HR) was measured by a cardiotachometer (HR-01, Experimetria®). The electrocardiogram (ECG standard lead III) was recorded on a devices recorder (ER- 14, Micromed®) by means of subcutaneous steel needle electrodes. The chest was opened by a left thoracotomy and the heart was exteriorized by a gentle pressure on the right side of the rib cage. A 4/0 silk ligature was quickly placed under the main left coronary artery. The heart was replaced in the chest and the animal left to recover. Rectal temperature was monitored and was maintained constant at 37° C. The experiment was initiated with a 15 min stabilization period during which the observation of a sustained blood pressure less than 70 mmHg and/or the occurrence of arrhythmias lead to exclusion. Myocardial ischemia was induced with coronary artery occlusion for 1 h and reperfusion allowed for 1 hour. The drugs were administered orally 6 hours before occlusion.
Quantification of Myocardial Infarction
At the end of experiment, the heart was quickly removed. The left ventricle was then sliced into 2 mm thick sections parallel to the atrioventricular groove. The slices were incubated in a 0.1% solution of p-Nitroblue Tetrazolium (NBT) grade III, pH 7,4 for 15 min. The non-infarcted area was colored blue due to formation of a precipitate that results from reaction of NBT with dehydrogenase enzymes. Loss of these enzymes in the infarcted myocardium p
Acsai Károly
Bácsy Ernö
Barabás Mihály
Csákai Zita
Dénes Lászió
Biorex Kutató Fejlesztö RT.
Coleman Brenda
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