Effective antiplatelet drug concentrations in experimental arterial thromboembolism.

Although drugs that modify platelet function have been widely studied as antithrombotic agents in experimental and clinical studies, there is limited information regarding the relationship between in vivo drug blood concentrations and antithrombotic efficacy. This study compared the pharmacokinetics of three antiplatelet agents with their antithrombotic effects in an experimental model of arterial thromboembolism in baboons. Thrombus formation was measured as steady-state platelet utilization induced by thrombogenic arteriovenous cannulae. The drugs studied were aspirin, dipyridamole and sulfinpyrazone. Aspirin was administered in daily doses of 20 mg/kg, dipyridamole in daily doses of 2.5 and 10 mg/kg, and sulfinpyrazone in daily doses of 20 and 100 mg/kg; each drug was given in two equal doses per day. Multiple blood samples were collected for drug analysis after steady-state had been reached. The average concentrations of dipyridamole at steady-state were 26 +/- 15 and 79 +/- 69 ng/ml after 2.5 and 10 mg/kg/day. These concentrations were associated with 28 and 87% inhibition of cannula platelet consumption, respectively. The average steady-state concentrations of acetylsalicylic and salicylic acids were 0.67 +/- 0.80 and 3.76 +/- 2.60 micrograms/ml, respectively, after 20 mg/kg/day. Aspirin had no effect on platelet consumption. Average concentrations of sulfinpyrazone were 1.05 +/- 0.45 and 12.25 +/- 5.73 micrograms/ml after 20 and 100 mg/kg/day, with significant concentrations of the sulfide metabolite. These concentrations were associated with 23 and 85% inhibition of platelet consumption, respectively. No significant pharmacokinetic interactions were observed after concurrent administration of aspirin and dipyridamole or sulfinpyrazone. As the experimental model used involves thrombus formation on an artificial surface, it is likely that these results are most relevant to patients with arterial prosthetic devices.

The reduction of sulphinpyrazone and sulindac by intestinal bacteria.

1. Incubation of human or rabbit faeces with sulphinpyrazone gave greater reduction under anaerobic than under aerobic conditions. Reduction of sulindac by human faeces was more extensive than that of sulphinpyrazone. 2. Growth of mixed cultures of intestinal bacteria in nutrient media containing antibiotics produced a marked inhibition in their ability to reduce sulphinpyrazone. Sulphide formation was inhibited by metronidazole and lincomycin for human faeces and by tetracycline for rabbit faeces/caecal contents. 3. The formation of the sulphides of sulindac and sulphinpyrazone ex vivo was decreased in faeces from patients treated with metronidazole. Metronidazole, but not tetracycline, decreased the extent of reduction of sulphinpyrazone by rabbits in vivo. No reduction of either substrate occurred on incubation with ileostomy effluent. These data indicate that anaerobic intestinal bacteria are important in the reduction of these sulphoxide-containing drugs. 4. However, when incubated anaerobically with over 200 strains of bacteria isolated from human faeces, sulphinpyrazone was reduced by most of the aerobic but not the anaerobic organisms. Sulindac was reduced more extensively by the same aerobes and by some anaerobes. 5. The discrepancy between the apparent importance of anaerobes in vivo and in vitro may be due to their very large number present in the hind gut and to the production of an anaerobic environment suitable for the enzymic activity of other organisms, such as aerobes or facultative anaerobes.

Correlation between inhibitory effect on platelet aggregation and disposition of sulfinpyrazone and its metabolites in rabbits. Part II: Multiple dose study.

In a crossover study rabbits were given perorally sulfinpyrazone (SO) and the sulfide metabolite (S) every 24 h for 5 days on separate occasions and inhibition of aggregation was measured. The results showed: the dosage regimen is effective if the minimum effective concentration of S is defined to be between 0.5-1.0 microgram ml-1, and the repeated dosing did not cause changes in disposition kinetics except that the terminal half-life of S was reduced after dosing with S. No significant accumulations in trough concentration and inhibition of aggregation were observed. The results obtained in this study could provide some useful information for design of dosage regimen and blood level monitoring for humans.

Correlation between inhibitory effect on platelet aggregation and disposition of sulfinpyrazone and its metabolites in rabbits. Part I: Single dose study.

Simultaneous evaluation of inhibition of the sodium arachidonate-induced platelet aggregation and drug disposition was studied in rabbits receiving single doses of sulfinpyrazone (SO) and its sulfide metabolite (S). The metabolism of SO was found to be interconversible with that of S. Due to the parallelism of disposition profiles, the observed concentration-related inhibition not only strongly correlated with the much more potent sulfide, but also correlated with the p-OH-sulfide (OH-S) or with a summation of two substances. Exaggeration of inhibition at 24-30 h and rebound effect at 48 h were found after the substances were administered. There may exist a circadian rhythm of platelet aggregation.

Determination of sulfinpyrazone and four metabolites in plasma and urine by high pressure liquid chromatography.

An HPLC method for the simultaneous determination of sulfinpyrazone and its p-hydroxy, sulfone, sulfide and p-hydroxysulfide metabolites in human plasma and urine samples was developed. Optimization with regard to sample preparation and chromatographic conditions was investigated. In the final procedure samples were acidified with HCl, extracted with a 1 + 1 (vol/vol) mixture of 1-chlorobutane and chloroform; urine extracts were re-extracted with citrate buffer (pH 4.5). HPLC was performed on reversed phase (RP 8) columns with a 48 + 52 (vol/vol) mixture of ethanol and citrate buffer (pH 2.5) as the mobile phase. The lower limit of detection for sulfinpyrazone and the sulfide metabolite is 10 ng/ml, for the other metabolites it is 50 ng/ml. An example of application of this procedure to pharmacokinetic studies in a volunteer receiving a single oral dose of sulfinpyrazone is given.

Effects of platelet-modifying drugs on arterial thromboembolism in baboons. Aspirin potentiates the antithrombotic actions of dipyridamole and sulfinpyrazone by mechanism(s) independent of platelet cyclooxygenase inhibition.

To resolve questions of drug actions, efficacy, and interactions for platelet-modifying agents used clinically, we have compared the relative capacities and mechanisms of aspirin, dipyridamole, sulfinpyrazone, and dazoxiben to prevent arterial thromboembolism in a baboon model. In 136 studies the agents were given twice daily by oral administration both singly and in combination. The antithrombotic efficacy of a given therapy was determined by its capacity to interrupt steady-state platelet utilization induced by thrombogenic arteriovenous cannulae. When given alone, dipyridamole and sulfinpyrazone reduced the rate at which platelets were utilized by thrombus formation in a dose-dependent manner with essentially complete interruption by dipyridamole at 10 mg/kg per d. In contrast, neither aspirin (2-100 mg/kg per d) nor dazoxiben (20-100 mg/kg per d) decreased cannula platelet consumption detectably despite the striking reduction in the capacity of platelets to produce thromboxane B2. However, aspirin, but not dazoxiben, potentiated the antithrombotic effects of dipyridamole and sulfinpyrazone in a dose-dependent fashion without changing the pharmacokinetics for any of the agents. Complete potentiation required aspirin at 20 mg/kg per d to be given with each dose of dipyridamole. Because dazoxiben's blockade of platelet thromboxane A2 production was not associated with antithrombotic potentiation, and because complete potentiation by aspirin required a dose that fully inhibited vascular production of prostaglandin I2 (PGI2), we conclude that aspirin's potentiating effect on dipyridamole is independent of PGI2 production or inhibition of thromboxane A2 formation. In addition, because frequent repeated and synchronous dosing of aspirin was necessary, aspirin's potentiating effects appear to be produced by mechanism(s) unrelated to its potent, irreversible inhibition of platelet cyclooxygenase.

Cyclooxygenase inhibition, platelet function, and metabolite formation during chronic sulfinpyrazone dosing.

The inhibitory effects of sulfinpyrazone are more marked ex vivo than in vitro, suggesting biotransformation to potentially active metabolites such as the sulfide and sulfone metabolites. As a platelet inhibitor, the sulfide metabolite is 10 times as potent as the parent and because of its long t1/2, the former may lead to cumulative inhibition of platelet function in vivo during chronic sulfinpyrazone dosing. In our study, healthy subjects received sulfinpyrazone, 200 mg four times a day, for 6 days. Plasma levels of the sulfide metabolite rose slightly from 2.1 +/- 0.8 micrograms/ml 12 hr after the fourth dose to 2.8 +/- 0.8 microgram/ml 12 hr after the twenty-fourth dose. This was associated with increasing inhibition of ex vivo platelet aggregation induced by platelet-activating factor during the dosing period, but inhibition of arachidonic acid-induced aggregation did not increase cumulatively during dosing and collagen-induced aggregation was not inhibited. Inhibition of platelet aggregation was no longer evident 24 hr after the final dose of sulfinpyrazone. The effects of sulfinpyrazone on cyclooxygenase activity were assessed by measurement of thromboxane B2 production by thrombin-stimulated platelets ex vivo and urinary excretion of the major prostacyclin metabolite 2,3-dinor-6-keto-PGF1 alpha. During sulfinpyrazone dosing, thromboxane formation and prostacyclin biosynthesis were correspondingly lowered 50% to 60%. The extent of this depression was of the same order on days 2 and 5 of dosing.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of sulphinpyrazone and its major metabolites after a single dose and during chronic treatment.

The pharmacokinetics of sulphinpyrazone and its major metabolites (sulfide, sulfone, p-hydroxysulfone and p-hydroxy-sulphinpyrazone) were investigated in 9 volunteers after a single oral dose as well as after chronic treatment for 23 days. Chronic administration of sulphinpyrazone, in comparison with a single oral dose, led to significant changes in plasma AUC (115.86 to 42.90 mg/l . h), in renal clearance (1.06 to 1.80 l/h), in hepatic intrinsic clearance (319.0 to 598.0 l/h), and in the unbound fraction in plasma 1.15 to 1.69%) and in tissue (2.73 to 1.31%). The volume of distribution changed from 20.24 to 52.04 l. The steady state concentrations predicted from the single dose were significantly higher than the values found after chronic treatment. The results suggest that sulphinpyrazone induces its own metabolism. The metabolism of the sulfone, p-hydroxysulfone and the p-hydroxy-sulphinpyrazone to further degradation products was also induced. Chronic treatment with sulphinpyrazone reduced the plasma AUC of the sulfide and caused a decrease in its elimination half-life (20.9 to 14.3 h). Since considerable amounts of the sulfide are formed in the G.I. tract, it is suggested that besides the induction of metabolism, bacteria which reduce sulphinpyrazone to the sulfide may also be responsible for the observed pharmacokinetic changes.

Warfarin-sulfinpyrazone interaction on binding to human serum albumin.

Sulfinpyrazone displacement of warfarin from human serum albumin was studied in-vitro. At low sulfinpyrazone concentrations one molecule of warfarin is displaced on binding by one molecule of sulfinpyrazone. Clinical plasma concentrations of sulfinpyrazone are, however, too low to cause significant displacement.

The site of reduction of sulphinpyrazone in the rabbit.

Comparison of oral and i.v. administration of sulphinpyrazone (10 mg/kg) to rabbits showed that the oral route was associated with an incomplete bioavailability and a six-fold greater formation of the active sulphide metabolite. The bile was an important route of elimination of unchanged sulphinpyrazone in rabbits (18% of an i.v. dose in four hours). Only small amounts of the sulphide appeared in the bile after i.v. administration. Pretreatment with oral antibiotics decreased the area under the plasma concentration-time curve (AUC) for the sulphide but increased that of the parent drug. Excretion of the p-hydroxysulphide metabolite in urine was decreased 30-fold by antibiotic treatment. The contents of the caecum showed the greatest capacity for sulphinpyrazone reduction in vitro. The liver possessed a slight ability to reduce sulphinpyrazone in vitro under anaerobic, but not aerobic, conditions. The gut bacteria are the main site of reduction of sulphinpyrazone to the active sulphide metabolite in the rabbit. These findings contrast with those obtained for sulindac which was reduced extensively under both aerobic and anaerobic conditions by rabbit-liver soluble fraction in vitro. The sulphide metabolites of both sulphinpyrazone and sulindac were oxidized to the parent drug by rabbit-liver microsomes.

Sudden death related to advanced coronary atherosclerosis in mini-pigs: influence of some drugs.

Advanced coronary atherosclerosis was produced in 30 mini-pigs by a combination of a hypercholesterolaemic diet and X-irradiation to the precordial region. Within 11-25 weeks after the irradiation, 13 of the 30 animals died a sudden death probably caused by coronary atherosclerosis. The contents of free and ester-bound cholesterol in the right coronary artery were significantly higher in the animals which died spontaneously than in surviving animals. In an untreated group of 12 animals 7 died whereas in a group treated with beta-pyridylcarbinol only 1 out of 5 died. In the coronary arteries, the contents of both free and ester-bound cholesterol were significantly lower in the beta-pyridylcarbinol-treated animals. In a sulfinpyrazone-treated group 3 out of 8, and in a metoprolol-treated group 2 out of 5 animals died. None of these drugs reduced the accumulation of cholesterol in the coronary arteries. The rate of sudden death was 26 +/- 6% (P less than 0.05) lower in the combined group of treated animals than in the untreated ones. By regular ECG recordings, signs which could predict the fatal outcome of the experiment were looked for. Although depressed ST segments were present before death in a few animals, this was not a regular phenomenon. It is concluded that advanced coronary atherosclerosis in mini-pigs often leads to sudden death and that this animal model seems suitable for testing the potential therapeutic effects of drugs.

High-performance liquid chromatographic determination of sulfinpyrazone and its metabolites in plasma.

A specific sensitive reversed-phase high-performance liquid chromatographic method for simultaneous determination of sulfinpyrazone (Anturane) and five of its metabolites (p-hydroxy-sulfinpyrazone, p-hydroxy-sulfone, sulfone, p-hydroxy-sulfide, and sulfide) in plasma is described. The compounds were extracted from plasma, back-extracted, re-extracted and separated on a 10-micron muBondapak C18 column using 10 mmol/l orthophosphoric acid-acetonitrile-ethanol as the mobile phase. Phenylbutazone was used as internal standard, which was totally separated from all other compounds. The lower limit of sensitivity for sulfinpyrazone, sulfone, p-hydroxy-sulfide and sulfide is 30 ng/ml, and 100 ng/ml for p-hydroxy-sulfinpyrazone and p-hydroxy-sulfone. Concentrations of sulfinpyrazone between 0.5 and 25 micrograms/ml were measured with an average coefficient of variation of 5.1%. Examples of application of this assay are given.

Kinetics and metabolism of sulfinpyrazone.

Six normal subjects (three men and three women) took 200 mg sulfinpyrazone in two oral preparations, a capsule and a suspension. Plasma and urine levels of sulfinpyrazone and the sulfide, p-hydroxy, and sulfone metabolites were measured over three days. The plasma sulfinpyrazone/time concentration profiles indicated a postabsorptive biexponential decline with a mean terminal half life (t1/2) of 299 +/- 107 min. There was intersubject variation in the formation of the metabolites, the greatest being with the sulfide metabolite. Mean t1/2 of the sulfide metabolite was 659 +/- 192 min. The apparent fraction of sulfinpyrazone absorbed was 0.93 +/- 0.24 and the free fraction in plasma was 1.26 +/- 0.04%. Since the sulfide metabolite has a more potent antiplatelet effect and its formation in normal subjects is variable, direct administration of the sulfide may provide a more predictable antithrombotic effect in patients.

Plasma levels of sulfinpyrazone and of two of its metabolites after a single dose and during the steady state.

The pharmacokinetics of sulfinpyrazone, and the plasma levels of its sulfide and sulfone metabolites, have been determined after a single oral dose (400 mg) and during steady-state conditions (4 x 200 mg daily for 6 days) in healthy female volunteers. The plasma half-lives of sulfinpyrazone, the sulfone and the sulfide were 3.7, 3.2 and 14.7 h, respectively, during steady-state. After a single dose and during steady state conditions the half-lives of sulfinpyrazone and the sulfone did not differ significantly. The trough plasma levels of the sulfide metabolite exceeded those of the parent compound in four of the six volunteers on the last day of the study. The data suggest that in man the most likely candidate for the prolonged inhibition of platelet aggregation observed after treatment with sulfinpyrazone is its sulfide metabolite, because of its prolonged elimination.

Paired-ion high-performance liquid chromatographic assay for sulfinpyrazone in plasma.

A specific and sensitive liquid chromatographic method is reported for the assay of sulfinpyrazone in plasma utilizing ion pairing between the tetrabutylammonium cation and the sulfinpyrazone anion. The method is rapid in that conventional extraction procedures are avoided in favor of using disposable cartridges packed with an octade-cylsilane bonded phase as a means of separating the drug from plasma. The samples were chromatographed on a C18 reversed-phase column using a mobile phase consisting of 0.005 M tetrabutylammonium phosphate in methanol-water (56:44). The coefficient of variation obtained was 4.5% and the response was linear over a range of 0.2-80 micrograms/ml.