Felix Helmut Schmidt

The pharmacological profile of the thromboxane A2 antagonist BM 13.177. A new anti-platelet and anti-thrombotic drug

BM 13.177 (4-[2-(benzenesulfonamido)-ethyl]-phenoxyacetic acid) is a representative of a new class of sulfonamidophenylcarboxylic acids which possess platelet-inhibitory and anti-thrombotic activity and inhibits the contraction of rabbit aorta stimulated by PG endoperoxides and TXA2. BM 13.177 5 mg/kg body weight p.o. protected rabbits from arachidonate-induced sudden death and greater than or equal to 10 mg/kg dose-dependently reduced the experimental thrombus formation induced in the rabbit aorta by perivascular administration of silver nitrate. In guinea-pigs, the collagen-induced bronchoconstriction was inhibited in a dose- and time-dependent fashion. The formation of TXA2 and the TXA2-induced platelet aggregation and smooth muscle contraction are probably crucial events in these experimental models. The protective effect of BM 13.177 may, therefore, be due to the TXA2-antagonizing effect of BM 13.177, which has been conclusively demonstrated in human platelets (PATSCHEKE and STEGMEIER, Thrombosis Res., 33, 277-288 (1984). The antagonism of TXA2 is supported by the observation that BM 13.177 also specifically inhibits the contraction of isolated arterial strips from rabbits which were stimulated with the thromboxane A2 mimetic U 46619. Schild-plot with a slope close to unity suggests a competitive type of antagonism. BM 13.177 exhibited neither anti-inflammatory nor ulcer-inducing activity of cyclooxygenase inhibitors. Furthermore it did not block the TXB2 formation in spontaneously clotting blood from rabbits and did not inhibit the release of prostacyclin-like activity from rabbit aortas. The lack of toxicological effects in long-term toxicity studies in rat and dog, together with the absence of objective and subjective side effects in the first human studies have encouraged us to initiate clinical trials in order to evaluate the therapeutic benefit of this new approach in humans.

The effects of BM 15.766, an inhibitor of 7-dehydrocholesterol δ7-reductase∗, on cholesterol biosynthesis in primary rat hepatocytes

The effect of the piperazine derivative BM 15.766 (4-(2-[1-(4-chlorocinnamyl)piperazin-4-yl]ethyl]-benzoic acid) on the biosynthesis of sterols was investigated in adult rat hepatocytes in primary monolayer culture. The substance led to a dose-dependent reduction of cholesterol in the serum of various species of animals such as rat, dog and marmoset. BM 15.766 showed a dose-dependent action on the incorporation of 14C-acetate in neutral, nonsaponifiable lipids. The inhibition of the overall incorporation was 10-12% (10(-5) M). No inhibition was observed in the hepatocytes over the entire dose range of 10(-8) M to 2 X 10(-5) M, while the release of the neutral lipids from the hepatocytes into the culture medium was reduced by up to 40%. The biosynthesis of cholesterol could be reduced by more than 90%. Simultaneously, 7-dehydrocholesterol levels rose in the cells and, to a less marked extent, in the medium. This can be interpreted as an indication that 7-dehydrocholesterol is incorporated into the cell membrane, which results in a lower release of 7-dehydrocholesterol into the medium in comparison with controls. The site of attack is the inhibition of the delta 5.7-sterol delta 7-reductase. The formation of desmosterol and cholestatrienol as well as other 7-dehydrocholesterol precursors could also be demonstrated. After longer incubation, there was an additional accumulation of squalene and lanosterol with simultaneous reduction of 7-dehydrocholesterol by BM 15.766, whereas the total 14C-acetate incorporation in neutral lipids was increased.

Enzymatic ammonia determinations in the blood and cerebrospinal fluid of healthy persons

Abstract In 200 healthy adults (80 females, 120 males) the ammonia concentration of the V. cubitalis blood was determined enzymatically. The distribution of the values is log-normal, the 95% range of the female values lying between 36 and 139 μg% and that of the males between 29 and 116 μg%. There is no correlation between body weight, height, surface and age. In 20 probands the ammonia concentration in the liquor was 11–37 μg%.

Thin-layer chromatography of radioactively labelled cholesterol and precursors from biological material

SummaryThe investigation methods of the action of xenobiotics on sterol biosynthesis from 14C-acetate in rat hepatocyte cultures can be developed, with regard to extraction using Extrelut and the separation of the sterol pattern by thin-layer chromatography, in such a way that they are suitable for wider application, e.g., screening. Good visualisation and recognition of changes in the sterol pattern are possible using autoradiography of the thin-layer chromatogram.

Studies on the mechanism of action of the hypoglycemic agent, 2-(3-methylcinnamylhydraznoo)-propionate (BM 42.304)

A new hypoglycemic agent, 2-(3-methylcinnamylhydrazono)-propionate MCHP (BM 42.304) was shown to be an inhibitor of the transfer of long-chain fatty acids across the mitochondrial inner membrane. The following data support this conclusion: the drug, at already 5 microM, inhibited ketogenesis from oleate but not from octanoate in the perfused guinea-pig liver; likewise, ketogenesis from L-(-)-palmitoylcarnitine and palmitoyl-CoA + L-(-)-carnitine, but not from octanoate, was depressed in isolated guinea-pig liver mitochondria. Oxigraphic measurements of the oxygen uptake by isolated mitochondria showed that the drug impaired oxygen uptake with the long-chain fatty acid derivatives but not with octanoate. Finally, in vivo effects of the drug such as hypoketonemia and an increased concentration of free fatty acids in blood are in agreement with the above formulated mechanism of action. A comment is given on the relationships between fatty acid oxidation and gluconeogenesis in the guinea-pig liver.

In vivo and in vitro effects of a new hypoglycemic agent, 2-(3-methylcinnamylhydrazono)-propionate (BM 42.304) on glucose metabolism in guinea pigs

A new compound, 2-(3- methylcinnamylhydrazono )-propionate (BM 42.304), showed a dose dependent hypoglycemic effect in starved guinea pigs after both oral and intraperitoneal administration. In contrast to biguanides (phenformin and metformin) the new compound produced only a moderate increase in blood lactate concentration and did not alter the content of adenine nucleotides in the freeze-clamped liver in vivo. Gluconeogenesis from a variety of precursors in the perfused guinea-pig liver was also inhibited by BM 42.304. These properties suggest that the compound deserves further investigation in connection with its potential usefulness for the treatment of diabetes.

A new inhibitor of the long-chain fatty acid transfer across the mitochondrial membrane: 2-(3-methylcinnamylhydrazono)-propionate (BM 42.304)

Using two different assay systems to distinguish between overt and inner forms of carnitine palmitoyl transferase (CPT, EC 2.3.1.21) of intact guinea-pig liver mitochondria, we have shown that the hypoglycemic agent 2-(3-methylcinnamylhydrazono)-propionate (BM 42.304) inhibits the activity of carnitine-acylcarnitine translocase of liver mitochondria. The results offer an explanation for the inhibitory effect of the compound on ketogenesis with oleate but not with octanoate in the perfused guinea-pig liver, previously reported by us (Biochem. Pharmacol. 32, 3405-3412, 1983).

Blood-glucose-lowering activity of 2-(3-phenylpropoxyimido)-butyrate (BM 13.677).

A single oral or intraperitoneal application of 2-(3-phenylpropoxyimido)-butyrate (BM 13.677) resulted in a dose-dependent blood-glucose-lowering effect in fasted guinea-pigs. The threshold dose and the EC50 were estimated as 25 mg/kg and 63 mg/kg, respectively, which is between that of the biguanides phenformin and metformin. A rise in blood lactate concentrations was observed only at high doses of BM 13.677, but was not related to an irreversible metabolic inhibition. Among several rodent species studied the potency of the drug decreased in the order guinea-pig much greater than mouse greater than rat = rabbit. Inhibition of hepatic gluconeogenesis by the drug was demonstrated in the perfused liver or hepatocytes of guinea-pigs. Inhibition of glucose production by the perfused liver in the presence of 0.1 mM BM 13.677 was dependent on the substrate and decreased in the order: lactate greater than pyruvate greater than alanine much greater than propionate greater than glycerol = fructose. This suggests a specific interaction of the drug with a mitochondrial key reaction of gluconeogenesis. Stimulation of glucose oxidation in rat diaphragm by the compound (EC50 = 0.85 mM) suggests that besides inhibition of gluconeogenesis also extrahepatic effects contribute to the blood-glucose-lowering effects of the drug.

Reduction of BM 15.766-induced 7-dehydrocholesterol accumulation by bezafibrate and mevinolin in rats

SummaryThe effects of mevinolin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor and bezafibrate, a modulator of lipoprotein metabolism, were measured on BM 15.766-induced 7-dehydrocholesterol (7-DHC) accumulation in liver and serum of rats. BM 15.766, an inhibitor of Δ7 sterol reductase, leads to an accumulation of 7-DHC, which can be used as a measure of cholesterol (CH) synthesis de novo. The investigations were carried out to evaluate the usefulness of this new non-isotopic in vivo method for testing compounds that affect directly and indirectly the CH-biosynthetic pathway.Mevinolin showed a dose-dependent reduction of BM 15.766-induced 7-DHC accumulation after a single oral dose. The dose range for reduction of 7-DHC in the liver of rats was comparable with that for serum CH-lowering in humans. Bezafibrate reduced the BM 15.766-induced 7-DHC accumulation in liver in a dose- and time-dependent manner. These findings agree with the reported reduced activity of HMG-CoA reductase and support the view, that bezafibrate reduces CH biosynthesis by modulation of lipoprotein metabolism. The 7-DHC levels in serum do rot reflect those in the liver and cannot be used as a measure of CH biosynthesis. The investigations show that BM 15.766-induced 7-DHC accumulation in liver of rats is an appropriate measure for CH de novo synthesis and can be used for testing compounds that interfere directly and indirectly with the CH-biosynthetic pathway. In contrast to previously described methods, no radiolabelled precursors are necessary.The extraction of 7-DHC and its photometric determination is, in comparison with separation and measurement of radioactive cholesterol, a relatively simple analytical procedure.