Comparison of Echinacea alkylamide pharmacokinetics between liquid and tablet preparations

The relative oral bioavailability of alkylamides from two different Echinacea dosage forms (liquid and tablet) were compared in a small two-way crossover study in humans (n=3). The liquid preparation investigated contained a mixture of Echinacea purpurea root (300 mg/ml) and Echinacea angustifolia root (200 mg/ml) extracted in 60% ethanol. The tablet preparation investigated was also a mixture of E. purpurea root (675 mg/tablet) and E. angustifolia root (600 mg/tablet), but was prepared from the dried 60% ethanolic extracts of these two Echinacea species. Alkylamides were found to be rapidly absorbed and measurable in plasma from both preparations. No significant differences in the tetraene alkylamide pharmacokinetic parameters for T(1/2), AUC(t-lin) and C(max) in the two different preparations were found. T(max) increased from 20 min for the liquid to 30 min for the tablet, which is not unexpected as the tablet required time for disintegration before absorption could occur. These results suggested that there was no significant difference in the bioavailability of alkylamides from the liquid and tablet Echinacea formulations. Furthermore, the results also indicated that the absorption site and any alkylamide loss due to digestive processes were similar in both preparations.     

Alkamides from Echinacea inhibit cyclooxygenase-2 activity in human neuroglioma cells

During past years inhibition of the cyclooxygenase-2 (COX-2) enzyme has been proven as an effective strategy to suppress pain and inflammation. Based on this and other mechanistic findings, interest has also renewed in the molecular pathways underlying the anti-inflammatory effects of herbal drugs. The present study addressed this issue and investigated the impact of several polyunsaturated alkamides isolated from a CO2 extract of the roots of Echinacea angustifolia DC. on both activity and expression of COX-2. A 48-h treatment of H4 human neuroglioma cells with the CO2 extract led to a significant suppression of prostaglandin (PG) E2 formation. Analysis of eight different alkamides revealed a contribution of undeca-2Z-ene-8,10-diynoic acid isobutylamide (A5), dodeca-2E-ene-8,10-diynoic acid isobutylamide (A7), and dodeca-2E,4Z-diene-8,10-diynoic acid 2-methylbutylamide (A8) to this response. Using an established short-term COX-2 activity assay, all three alkamides were shown to interfere with COX-2 activity. In contrast, none of the COX-2-suppressing nor any other tested alkamide was found to inhibit COX-2 mRNA and protein expression. Instead, increased COX-2 mRNA and protein levels were registered in the presence of the CO2 extract and most of the analyzed alkamides which caused, however, no stimulation of PG formation. Overall, our results suggest that certain alkamides derived from E. angustifolia roots may contribute to the pharmacological action of the herbal extract by inhibiting COX-2-dependent PGE2 formation at sites of inflammation.     

Alkamide production from hairy root cultures of <Emphasis Type="Italic">Echinacea</Emphasis>

Hairy root cultures of Echinacea, one of the most important medicinal plants in the US, represent a valuable alternative to field cultivation for the production of bioactive secondary metabolites. In this study, the three most economically important species of Echinacea (Echinacea purpurea, Echinacea pallida, and Echinacea angustifolia) were readily transformed with two strains of Agrobacterium that produce the hairy root phenotype. Transformed roots of all three species exhibited consistent accelerated growth and increased levels of alkamide production. Optimization of the culture of Echinacea hairy roots was implemented to enhance both growth and alkamide production concomitantly. The use of half-strength Gamborg’s B5 medium supplemented with 3.0% sucrose was twice as effective in maintaining hairy root production than any other media tested. The addition of indolebutyric acid increased the growth rate of roots by as much as 14-fold. Alkamide production increased severalfold in response to the addition of the elicitor, jasmonic acid, but did not respond to the addition of indolebutyric acid. Induced accumulation of the important bioactive compounds, alkamides 2 and 8, was observed both in transformed roots and in response to jasmonic acid treatments. The results of this study demonstrate the efficacy of hairy root cultures of Echinacea for the in vitro production of alkamides and establish guidelines for optimum yield.     

Multidisciplinary perspectives on mechanisms of activity of popular immune-enhancing herbal supplements used by athletes

This paper examines cellular and molecular mechanisms that may underpin the purported effects of five herbal supplements in the context of athlete immune function. Ginseng and echinacea are used frequently by athletes, whereas astragalus and elderberry are used infrequently and pequi is just emerging as a possible supplement. In vivo studies of these products on athlete immune function have yielded heterogeneous results, likely due to experimental design differences. Ginseng, echinacea, elderberry, and pequi are considered asterids sensu lato. Ginseng appears to exert strongest effects on components of adaptive immunity, in particular maintaining Th1/Th2 balance of CD4⁺T cells and their downstream effects, via its ginsenosides, flavonoids, and polysaccharides. Echinacea alkamides, caffeic acid derivatives, and polysacchardies may target both innate and adaptive immunity, though perhaps the former more consistently. Elderberry harbors anthocyanins and lectins which may modulate innate immunity. Data on pequi is limited but suggests that carotenoids, phenols, and fatty acids may alter circulating leukocyte populations. More phylogenetically distant, astragalus is a rosid sensu lato and may influence the innate immune system through flavonoids, polysaccharides, and saponins. Supplements generally demonstrate no effects on physiologic parameters such as lactate, oxygen dynamics, or athletic performance. Bioavailability studies indicate that purported bioactive molecules of these supplements may reach circulation in low but therapeutically-relevant quantities. Difficulties in crosscomparisons due to study design differences, coupled with an overall dearth of research on the topic, currently hamper any formal conclusions regarding the efficacy of these supplements as immunoregulators for athletes.     

Optimization and use of a spectrophotometric method for determining polysaccharides in <Emphasis Type="Italic">Echinacea purpurea</Emphasis>

Echinacea purpurea is one of the most widely used immunostimulant plants. Its main active compounds are polysaccharides, glycoproteins, caffeic acid derivatives, alkamides, and melanins. The article describes an optimized extraction procedure that enables spectrophotometric quantification of polysaccharides from Echinacea purpurea. The extraction procedure can be widely applied as it demonstrated to be useful for determining polysaccharide content in flowers and leaves, in summer and autumn plants, in plants with green and red stem, and in plants from two different plantations. A significantly higher content of polysaccharides in flowers in comparison to leaves, as well as in plants with green stems in comparison to plants with red stems was determined. Statistical differences were absent in plants collected in different seasons and growing at different plantations.     

Bioavailability of omega-3 essential fatty acids from perilla seed oil

Increased dietary intake of fish oil omega-3 fatty acids, eicosapentanoic acid and docosohexanoic acid, and their precursor, alpha-linolenic acid (ALA), is associated with various health benefits. Enteric-coating (Entrox), which improves stability of omega-3 capsules, has been shown to facilitate fish oil absorption after chronic treatment. To assess the effect of Entrox coating on the short-term bioavailability of ALA administered in the form of ALA-rich Perilla seed oil, 12 healthy subjects (6 males and 6 females) received in a random order Entrox-coated and non-coated ALA formulations, each as a single 6g dose separated by a 3-week washout period. Measurements of plasma ALA concentrations from 0 to 24h showed no difference in ALA pharmacokinetics between the two formulations. However, significantly greater increases in plasma ALA levels from baseline to 24h were observed after ingestion of Entrox vs. non-coated product, suggesting a possible benefit of Entrox with long-term treatment.     

Chitosan oligomers as drug carriers for renal delivery of zidovudine

Chitosan oligomers (COS) are water soluble and can be a potential drug carrier for renal targeting delivery. Zidovudine (AZT), a FDA approved antiretroviral drug, has a very short half life and is eliminated very quickly in human plasma and kidney after administration. In this study, AZT-COS conjugates were prepared and evaluated in terms of renal targeting. The in vitro release of AZT from AZT-COS conjugates was confirmed in mice plasma and renal homogenate. The pharmacokinetics study indicated longer mean retention time of AZT-COS conjugates with the values of about 1.5 h than 0.59 h of AZT. The AZT-COS conjugates were found accumulated in kidney other than heart, liver, spleen, lung and brain after i.v. administration, in line with the evidence of the fluorescence imaging of FITC labeled COS in 12 h. In conclusion, AZT-COS conjugates have the potential to be developed into a renal-targeting drug delivery system.     

Bioactivity of alkamides isolated from Echinacea purpurea (L.) Moench.

Alkamides from the roots of Echinacea purpurea (L.) Moench were examined for anti-inflammatory activity in an in vitro model system. Cyclooxygenase-I (COX-I) and cyclooxygenase-II (COX-II) inhibitory activities were assessed at pH 7 for alkamides isolated from E. purpurea roots to compare inhibitory activities between the two cyclooxygenase isozymes. At 100 microg/ml, several E. purpurea alkamides inhibited COX-I and COX-II enzymes in the range of 36-60% and 15-46%, respectively, as compared to controls. Mosquitocidal activity was assessed at 100 and 10 microg/ml, with 100% mortality against Aedes aegyptii L. larvae noted for several E. purpurea alkamides at 100 microg/ml.     

Pharmacokinetics and metabolism of RU 486

The effects of dose on the initial pharmacokinetics and metabolism of an antiprogesterone steroid RU 486 (mifepristone) were studied in healthy female volunteers after administration of RU 486 as a single dose of 50-800 mg. The concentrations of RU 486 and its monodemethylated, dimethylated and hydroxylated non-demethylated metabolites were measured specifically after Chromosorb-column chromatography by HPLC. Their relative binding affinities to the human uterine progesterone receptor were also determined. Micromolar concentrations of the parent compound in blood were reached within the first hour after oral administration. The pharmacokinetics of RU 486 followed two distinct patterns in a dose-dependent fashion. With a low dose of 50 mg the pharmacokinetics followed an open two-compartment model with a half-life of over 27 h. With the doses of 100-800 mg the initial redistribution phase of 6-10 h was followed by zero-order kinetics up to 24 h or more. Importantly, after ingestion of doses higher than 100 mg of RU 486 there were no significant differences in plasma concentrations of RU 486 within the first 48 h, with the exception of plasma RU 486 concentrations at 2 h. After single oral administration of 200 mg unchanged RU 486 was found 10 days later in two subjects. The elimination phase half-life with this dose, calculated between day 5 and 6, was 24 h. Micromolar concentrations of monodemethylated, didemethylated and non-demethylated hydroxylated metabolites were measured within 1 h after oral administration of RU 486. In contrast to plasma RU 486 concentrations, circulating plasma concentrations of metabolites increased in a dose-dependent fashion. With higher doses the metabolite concentrations were close to, or even in excess to the parent compound. The relative binding affinities of RU 486, monodemethylated, didemethylated and hydroxylated metabolites (progesterone = 100%) to the human progesterone receptor were 232, 50, 21, and 36, respectively. The existence of a high affinity-limited capacity serum binding protein would explain the long half-life and the observed diverging dose-dependent pharmacokinetics. The extravasation of RU 486 after the saturation of serum binding sites would explain the blunted serum peak concentrations of RU 486 with higher doses. The return of the drug back to circulation thereafter explains the zero-order kinetics. High concentrations of circulating metabolites capable of binding to the progesterone receptor suggest a significant contribution of these steroids in the overall antiprogestational action.     

Alkamides from Echinacea disrupt the fungal cell wall-membrane complex

We tested the hypothesis that alkamides from Echinacea exert antifungal activity by disrupting the fungal cell wall/membrane complex. Saccharomyces cerevisiae cells were treated separately with each of seven synthetic alkamides found in Echinacea extracts. The resulting cell wall damage and cell viability were assessed by fluorescence microscopy after mild sonication. Membrane disrupting properties of test compounds were studied using liposomes encapsulating carboxyfluorescein. Negative controls included hygromycin and nourseothricin (aminoglycosides that inhibit protein synthesis), and the positive control used was caspofungin (an echinocandin that disrupts fungal cell walls). The results show that yeast cells exposed to sub-inhibitory concentrations of each of the seven alkamides and Echinacea extract exhibit increased frequencies of cell wall damage and death that were comparable to caspofungin and significantly greater than negative controls. Consistent with effects of cell wall damaging agents, the growth inhibition by three representative alkamides tested and caspofungin, but not hygromycin B, were partially reversed in sorbitol protection assays. Membrane disruption assays showed that the Echinacea extract and alkamides have pronounced membrane disruption activity, in contrast to caspofungin and other controls that all had little effect on membrane stability. A Quantitative Structure-Activity Relationship (QSAR) analysis was performed to study the effect of structural substituents on the antifungal activity of the alkamides. Among the set studied, diynoic alkamides showed the greatest antifungal and cell wall disruption activities while an opposite trend was observed in the membrane disruption assay where the dienoic group was more effective. We propose that alkamides found in Echinacea act synergistically to disrupt the fungal cell wall/membrane complex, an excellent target for specific inhibition of fungal pathogens. Structure-function relationships provide opportunities for synthesis of alkamide analogs with improved antifungal activities.     

Methyl jasmonate increases reported alkamides and ketoalkene/ynes in Echinacea pallida (Asteraceae)

Methyl jasmonate (MJ), a naturally-occurring plant cellular signal molecule, was found to induce production of lipophilic secondary metabolites in Echinacea pallida seedlings. Seedling aerial parts were sprayed with 100 ppm MJ, and roots were harvested and extracted 24 h later. Lipophilic root extracts, separated by HPLC, revealed significant increases (P< 0.05) in six alkamides or related ketoalkene/ynes produced by 34 day-old plants and in seven compounds produced by 58 day-old plants.     

Influence of Echinacea purpurea intake during pregnancy on fetal growth and tissue angiogenic activity

The process of angiogenesis and control of blood vessels sprouting are fundamental to human health, as they play key roles in many physiological and pathological conditions. Intake of different pharmaceuticals with antiangiogenic activity by pregnant women may lead to severe developmental disturbances as it was described in case of thalidomide. It may also cause immunomodulatory effects as it was shown for antibiotics, theobromine, caffeic acid or catechins on the pregnant mice model. At present, Echinacea purpurea-based phytoceuticals are among the most popular herbals in the marketplace. Many compounds of Echinacea extracts (polysaccharides, alkamides, polyphenols, glycoproteins) exert immunomodulatory, anti-oxidative and anti-inflammatory activity. Echinacea is one of the most powerful and effective remedies against many kinds of bacterial and viral infections. In previous studies we shown significant inhibitory effect of the Echinacea purpurea based remedy on tumour angiogenic activity using cutaneous angiogenesis test, and an inhibitory effect on L-1 sarcoma growth was observed . The aim of the present study was to establish whether pharmaceuticals containing alcoholic extracts of Echinacea purpurea given to pregnant mice influence angiogenic activity and tissue VEGF and bFGF production of their fetuses. We showed that angiogenic activity of tissue homogenates was increased in Esberitox group and diminished in case of Immunal forte as compared to standard diet group. In case of Echinapur group we did not find significant differences in angiogenic activity. VEGF and bFGF concentration were lower in all groups compared to the control. In the case of Echinapur and Esberitox number of fetuses in one litter were slightly lower as compared to control group, but the difference is on the border of statistical significance. In conclusion, there is some possibility that pharmaceuticals containing Echinacea purpurea might influence fetal development in human also, because they may interfere with embrional angiogenesis , and should not be recommended for pregnant women.     

Comparative metabolomics approach coupled with cell- and gene-based assays for species classification and anti-inflammatory bioactivity validation of Echinacea plants

Echinacea preparations were the top-selling herbal supplements or medicines in the past decade; however, there is still frequent misidentification or substitution of the Echinacea plant species in the commercial Echinacea products with not well chemically defined compositions in a specific preparation. In this report, a comparative metabolomics study, integrating supercritical fluid extraction, gas chromatography/mass spectrometry and data mining, demonstrates that the three most used medicinal Echinacea species, Echinacea purpurea, E. pallida, and E. angustifolia, can be easily classified by the distribution and relative content of metabolites. A mitogen-induced murine skin inflammation study suggested that alkamides were the active anti-inflammatory components present in Echinacea plants. Mixed alkamides and the major component, dodeca-2E,4E,8Z,10Z(E)-tetraenoic acid isobutylamides (8/9), were then isolated from E. purpurea root extracts for further bioactivity elucidation. In macrophages, the alkamides significantly inhibited cyclooxygenase 2 (COX-2) activity and the lipopolysaccharide-induced expression of COX-2, inducible nitric oxide synthase and specific cytokines or chemokines [i.e., TNF-α, interleukin (IL)-1α, IL-6, MCP-1, MIP-1β] but elevated heme oxygenase-1 protein expression. Cichoric acid, however, exhibited little or no effect. The results of high-performance liquid chromatography/electron spray ionization/mass spectrometry metabolite profiling of alkamides and phenolic compounds in E. purpurea roots showed that specific phytocompound (i.e., alkamides, cichoric acid and rutin) contents were subject to change under certain post-harvest or abiotic treatment. This study provides new insight in using the emerging metabolomics approach coupled with bioactivity assays for medicinal/nutritional plant species classification, quality control and the identification of novel botanical agents for inflammatory disorders.     

Stereoselective pharmacokinetics of indobufen from tablets and intramuscular injections in man

The influence of administration route (oral or intramuscular (i.m.)) on the pharmacokinetics of total indobufen (INDB) enantiomers was studied in healthy volunteers after a 200 mg dose as a single oral tablet or i.m. injection. Enantiospecific reversed phase (RP) HPLC with UV detection was used for the determination of INDB enantiomers in serum. INDB enantiomers were isolated from serum by solid phase extraction (SPE) procedure using C(18) columns. INDB enantiomers were converted to their L-leucinamide diastereoisomers and separated on a C(18) HPLC column. INDB from i.m. injections is absorbed faster (t(max) = 0.6-0.9 h) than from tablets (t(max) = 1.3-1.8 h). The area under curve (AUC) after administration of the tablet was slightly higher than after i.m. injection. The pharmacokinetic behaviour of (+)-S- and (-)-R-INDB after administration of the tablet was different from i.m. injection of racemic INDB. The (+)-S-enantiomer is more rapidly eliminated than its (-)-R-antipode. Statistically significant differences also occurred between enantiomers in AUC, first order elimination rate constant (k), clearance (Cl). The ratio AUC(R):AUC(S) was similar for the tablet (1.57-1.62) and i.m. injection (1.59-1.62). It was concluded that the formulation and extent of ionisation of rac-INDB (acid or sodium salt of INDB) do not significantly influence its stereoselective pharmacokinetics.     

Agrobacterium rhizogenes-mediated transformation of Echinacea purpurea

Summary Echinacea purpurea seedlings were inoculated with several Agrobacterium rhizogenes strains in order to obtain hairy roots. Infection with A. rhizogenes strains LMG63 and LMG150 resulted in callus formation. Upon infection with strains ATCC 15834 and R1601 hairy roots were obtained. Opine detection confirmed transformation of E. purpurea. Comparative HPLC fingerprint analysis of the alkamides from natural plant source, control tissues, and transformed callus and roots indicated that transformed callus and hairy roots might be a promising source for continuous and standardized production of the dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide and related amides.Abbreviations HPLC high-pressure liquid chromatography - MS Murashige and Skoog culture medium     

Absorption and excretion of conjugated flavonols, including quercetin-4'-O-β-glucoside and isorhamnetin-4'-O-β-glucoside by human volunteers after the consumption of onions

Flavonols are polyphenols found ubiquitously in plants and plant-products. Flavonols, particularly quercetin, are potent antioxidants in vitro and their intake has been associated inversely with the incidence of coronary heart disease. The aim of this study was to investigate the accumulation in plasma and excretion in urine of flavonol glucosides following ingestion of lightly fried onions. Five healthy volunteers followed a low-flavonoid diet for 3 days. On day 4, after an overnight fast, subjects were given 300 g of lightly fried yellow onions which contain conjugates of quercetin and isorhamnetin, including quercetin-3,4'-diO-β-glucoside, isorhamnetin-4'-O-β-glucoside and quercetin-4'-O-β-glucoside. Blood collection was carried out at 0 min, 0.5, 1.0, 1.5, 2, 3, 4, 5 and 24h after the supplement. In addition, subjects collected all their urine for 24h following the onion supplement. Isorhamnetin-4'-O-β-glucoside and quercetin-4'-O-β-glucoside accumulated in plasma with maximum levels, defined as proportion of intake, of 10.7 ± 2.6% and 0.13 ± 0.03% respectively. The time of the quercetin-4'-glucoside peak plasma concentration was 1.3 ± 0.2 h after the ingestion of onions while a value of 1.8 ± 0.7 h was obtained for isorhamnetin-4'-glucoside. Excretion in urine, as a proportion of intake, was 17.4 ± 8.3% for isorhamnetin-4'-O-β-glucoside and 0.2±0.1% for quercetin-4'-O-β-glucoside. Possible reasons for the accumulation and excretion of isorhamnetin-4'-glucoside in proportionally much higher amounts than quercetin-4'-glucoside are discussed. It is concluded that flavonols are absorbed into the bloodstream as glucosides and minor structural differences affect markedly both the level of accumulation and the extent to which the conjugates are excreted.     

Effects of acute tryptophan depletion on plasma and cerebrospinal fluid tryptophan and 5-hydroxyindoleacetic acid in normal volunteers

Brain serotonin synthesis and metabolism (turnover), as indicated by CSF concentrations of 5-hydroxyindoleacetic acid (5-HIAA), may depend on plasma concentrations of the essential amino acid L-tryptophan (TRP). We investigated the biochemical effects of acute plasma TRP depletion (ATD) in normal volunteers undergoing a 36-h CSF collection via lumbar drain. Six subjects who were in good health were put on a low-TRP diet (160 mg/day) 24 h before lumbar puncture; this diet was continued for the first 22 h of the CSF collection. At hour 22, subjects ingested a TRP-deficient 15-amino acid drink shown previously to deplete plasma TRP. Total plasma TRP, free plasma TRP, and CSF TRP subsequently decreased 86.3, 86.5, and 92.3%, respectively. CSF 5-HIAA decreased by 32.8%. Plasma total and free TRP concentrations were both decreased at approximately 2 h following ingestion of the TRP-free amino acid drink and were lowest approximately 6 h after ATD; CSF TRP and 5-HIAA were decreased at 2.5 h and approximately 4 h after ATD, respectively. CSF TRP was lowest 8.0 h later. CSF 5-HIAA continued to decrease 14 h after the TRP-deficient amino acid drink was given.     

Simultaneous quantitative determination of the major phase I and II metabolites of ibuprofen in biological fluids by high-performance liquid chromatography on dynamically modified silica

Ibuprofen has previously, after ingestion by man, been demonstrated to yield four major phase I metabolites, which are excreted in the urine partly as glucuronic acid conjugates. However, in previous investigations the quantitative determinations of the conjugates were performed by indirect methods. The purpose of the present investigation was to develop a high-performance liquid chromatographic (HPLC) system for the simultaneous determination of the major phase I and II metabolites of ibuprofen in biological fluids. The separation was performed using bare silica dynamically modified with N-cetyl-N,N,N-trimethylammonium hydroxide ions contained in the mobile phase. The separation of the metabolites of ibuprofen is greatly improved with this system compared to other published reversed-phase HPLC systems intended for the same purpose. The method developed makes it possible to simultaneously determine the intact glucuronic acid conjugates of ibuprofen as well as its phase I metabolites in human urine. In a study involving four healthy volunteers, a total recovery in urine of the dose given was found to be 58–86% within 8 h. This may be compared to an average of 67% earlier reported in the literature.     

Determination of apovincaminic acid in human plasma by high-performance liquid chromatography using solid-phase extraction and ultraviolet detection

A new, simple and rapid high-performance liquid chromatography (HPLC) method with UV detection has been developed for the determination of apovincaminic acid in human plasma. Apovincaminic acid and internal standard were isolated from plasma samples by solid-phase extraction with OASIS HLB cartridges. The chromatographic separation was accomplished on a reversed-phase C(18) column and UV detection was set at 311 nm. The calibration curves were linear in the concentration range of 2.4-240.0 ng/ml, and the limits of quantification was 2.4 ng/ml. The precision and accuracy ranged from 0.84 to 8.54% and 91.5 to 108.3%, respectively. The developed method was subsequently applied to study the pharmacokinetics of apovincaminic acid in a group of 20 human subjects at a single oral dose of 10mg of vinpocetine tablet.     

Sodium 5-(3′-pyridinylmethyl)benzofuran-2-carboxylate (U-63557A), a new,selective thromboxane synthase inhibitor: Intravenous and oral pharmacokintics in dogs and correlations with exsitu thromboxane B2 production

The pharmacokinetics of a new, selective thromboxane synthase inhibitor, sodium 5-(3′-pyridinylmethyl)benzofuran-2-carboxylate were determined for single dose, bolus intravenous injections (1, 3, and 10 mg/kg); for continuous 24 hr infusions (10 and 30 μg/kg/min); and for oral doses of gelatin encapsulated powdered drug (3, 10, and 30 mg/kg). Drug disappeared biexponentially after intravenous administration, and plasma concentrations were proportional to the dose. Absorption of drug occurred rapidly after its oral administration; peak plasma levels occurred 1–2 hours after ingestion, and circulating drug was detectable within 30 minutes. For all experiments, inhibition of cellular thromboxane B₂ production, $\text{ex situ}$, corresponded with plasma drug levels and its reactivation corresponded with disappearance of the drug indicating that it was not accumulated by platelets.