Edward E. Nishizawa

Prostaglandin d2 as a potential antithrombotic agent.

Prostaglandin D2 was found to be a potent inhibitor of platelet aggregation. Aggregation of human platelets by ADP, collagen and prostaglandin G2 was inhibited more strongly by PGD2 than by PGE1. Although ADP-induced aggregation of rabbit platelets was inhibited more strongly by PGE1 than by PGD2 the latter prostaglandin gave a more long-lasting inhibitory effect on platelet aggregation following intravenous or oral administration. These results coupled with the finding that PGD2 has less hypotensive effects on the cardiovascular system than PGE1 suggest the possible use of PGD2 as an antithrombotic agent.

Flurbiprofen, a new potent inhibitor of platelet aggregation

Abstract Flurbiprofen [dl-2-(2-fluoro-4-biphenylyl)propionic acid] is a non-steroidal anti-inflammatory compound whose inhibitory activity on collagen-induced platelet aggregation approaches the activity seen with prostaglandin E1 (PGE1). However, unlike PGE1, it does not cause an increase in platelet cAMP levels, is orally active when given to animals or man, and is without side effects at platelet anti-aggregating doses. An oral dose of 1 mg/kg in rats inhibited collagen-induced platelet aggregation for 48 hours. At this dose, the mesenteric bleeding time in rats was prolonged from 197.2±24.4 sec. to 470.4±48.4 sec. The compound prevented death in mice due to pulmonary thromboembolism following collagen injection and inhibited platelet aggregation in man following oral administration. The drug appears to function by blocking the interaction of platelets with surfaces such as collagen but does not bind to the platelet membrane. It has no effect on the uptake of glucose or serotonin. The platelet anti-aggregating effect of flurbiprofen resides in the d-isomer while the l-isomer is without platelet anti-aggregating effect and does not counteract or enhance the effect of the d-isomer. These studies suggest that flurbiprofen may be a potentially useful antithrombotic drug.

Collagen-induced pulmonary thromboembolism in mice

Abstract Injection of an aqueous collagen suspension into the caudal-caval vein of normal rats or mice resulted in about 75% mortality due to the formation of platelet aggregates which subsequently lodged in the pulmonary circulation. Animals pretreated with inhibitors of platelet aggregation [aspirin (ASA), phenylbutazone] or animals made thrombocytopenic were protected from death. Based on these observations a 2-stage, group sequential screen for antithrombotic activity was developed in which 6 mice were tested in each stage. Compounds possessing little or no activity could be eliminated after testing in the first stage. Aspirin was used as a positive standard to monitor the test system. A secondary test for platelet aggregation in rat platelet-rich plasma was used to confirm activity.

Inhibitory effect of ibuprofen (motrin®) on platelet function

Abstract Ibuprofen was found to be slightly more active than acetylsalicyclic acid (ASA) as an inhibitor of collagen-induced platelet aggregation in human and rat PRP. This inhibition was demonstrable only while the drug was present in the plasma. It did not inhibit thrombin-induced aggregation but inhibited the second wave of ADP and epinephrine-induced platelet aggregation. It inhibited arachidonic acid-induced aggregation at much lower concentrations than those required to inhibit collagen-induced aggregation. The release of radioactivity from platelets prelabeled with 3 H-serotonin as well as the oxygen-burst were inhibited by the compound when platelets were stimulated with collagen. The compound was found to be orally active in reducing the number of deaths in mice following injection of collagen suspension and it inhibited platelet aggregation after oral administration of 10 mg/kg to rats.

Synthesis of dl-4,5,6-trinor-3,7-inter-m-phenylene-3-oxaprostaglandins including one which inhibits platelet aggregation

The synthesis of dl-4,5,6-trinor-3,7-inter-m-phenylene-3-oxaprostaglandins of the E1 and F1alpha series from 6-endo-(1-heptenyl)-bicyclo[3:1:0]hexan-3-one (III), is described. Preliminary biological screening data for gerbil colon smooth muscle stimulation, rat blood pressure and substrate specificity toward 15-hydroxyprostaglandin dehydrogenase is presented. Platelet function studies, both in vitro and in vivo of dl-4,5,6-trinor-3,7-inter-m-phenylene-3-oxa-PGE1, methyl ester (VIII) are presented.

Duration of activity of the acid, methyl ester and amide of an orally active platelet aggregation inhibitory prostanoid in the rat

The prostanoid 3-oxa-4,5,6-trinor-3,7-inter-m-phenylene PGE1 (OI-PGE1) has been shown to be a more potent inhibitor of ADP-induced human platelet aggregation than PGE1. OI-PGE1 inhibits ex vivo ADP-induced platelet aggregation for 60 minutes after an oral dose of 20 mg/kg to rats. Present studies compare duration of ex vivo inhibition of ADP-induced platelet aggregation in the rat by OI-PGE1, its methyl ester and amide after administration by various routes. All oral (p.o.) and intraduodenal (i.d.) doses were 20 mg/kg and all intravenous (i.v.) doses were 1 mg/kg. OI-PGE1 and its methyl ester had the same duration of activity after i.v. (60 min.) and p.o. (60 min.) administration, however, the methyl ester, when administered i.d., had a longer duration of activity than the free acid i.d. (greater than 90 min. vs. 60 min.). OI-PGE1-amide had significantly longer duration than the acid or methyl ester after i.v. (greater than 120 min.), p.o. (greater than 240 min.) or i.d. (greater than 240 min.) administration. Present data suggest that in the rat (1) intestinal absorption of OI-PGE1-methyl ester is more efficient than it is for the free acid and (2) due to metabolic and/or distributional differences between OI-PGE1 and its amide, the amide has a much greater duration of activity.

Hydrolysis of an orally active platelet inhibitory prostanoid amide in the plasma of several species.

The prostanoid 3-oxa-4,5,6-trinor-3,7-inter-m-phenylene-PGE1-amide (OI-PGE1-amide) has a prolonged duration of oral platelet aggregation inhibitory activity when compared to the parent free acid (OI-PGE1) in the rat. When incubated in rat plasma at 1 microgram/ml for 30 seconds prior to addition of ADP, OI-PGE1-amide inhibits in vitro rat platelet aggregation approximately 50%. OI-PGE1 inhibits at 1 ng/ml. Inhibition of platelet aggregation by plasma incubated with OI-PGE1-amide (1 microgram/ml) increases with time and the rate of this increase differs with species. Incubation of OI-PGE1 in plasma does not result in an increase of platelet inhibitory activity with time. The increase of platelet inhibitory activity was assumed to indicate hydrolysis of OI-PGE1-amide to the more active OI-PGE1. A compound, different from OI-PGE1-amide, was isolated by an ion exchange/silica gel separation sequence from an incubation of OI-PGE1-amide in rat plasma. It had potent platelet aggregation inhibitory activity. This material was shown to be OI-PGE1 by thin-layer chromatography, gas chromatography and mass spectral analysis. Studies with [3H]-OI-PGE1-amide confirmed the formation of OI-PGE1 in plasma incubations. Amide hydrolytic activity was significantly different between species, the rank order being: rat greater than guine pig greater than monkey = human greater than dog. This relationship corresponded with that determined by measuring the increase in platelet inhibitory activity with time in plasma incubations of OI-PGE1-amide reported above. Present data indicate that (a) OI-PGE1-amide is hydrolyzed to the parent acid by plasma enzymes of several species and (b) hydrolytic activity of plasma varies widely between species.

The formation of a thrombin-like material (TLM) following stimulation of leukocytes.

When thrombin, tissue thromboplastin or Russells viper venom was added to a suspension of either lymphocytes or neutrophils containing normal plasma, aggregation of these cells ensued. The aggregate formed one gelatinous mass which was readily separable from the cell free supernatant, an aliquot of which caused platelet aggregation. This leukocyte derived platelet aggregatory substance had characteristics similar to thrombin but not AGEPC. When plasma deficient in Factor V was substituted for normal plasma, the platelet stimulatory substance was not produced. Substitution with plasma deficient in Factor VII, VIII, IX, X or XI was without effect. Thrombin clotting time measurements indicated a generation of activity, relative to thrombin, of about 3.0 U/5 X 10(6) cells.