Characterization of WR-1065 absorption in the rat small intestine.

A circulating in situ rat small intestine absorption model was used to study the lumenal metabolism and absorption of [14C]WR-1065. WR-1065 was found to be more tissue reactive and toxic than its phosphorylated form, ethiofos, at equimolar perfusate concentrations. The disappearance profiles of the radiolabeled drug and free WR-1065 indicate that WR-1065 is extensively metabolized in the intestinal lumen prior to absorption. Coadministration of disodium ethylenediaminetetraacetic acid enhances the absorption of the free thiol although not to the same extent as seen with ethiofos. Perfusion of WR-1065 in citrate buffer decreased lumenal degradation of the drug but resulted in decreased absorption. The total material converted to WR-1065 portal blood profiles following ethiofos and WR-1065 perfusion were altered possibly due to distribution and metabolism differences. This study coupled with earlier work completed on ethiofos have increased our understanding of the significant barriers to absorption observed following oral administration of these compounds.

Characterization of ethiofos absorption in the rat small intestine.

The absorption characteristics of ethiofos were studied using the rat in situ intestine circulating perfusion technique. Slow absorption kinetics were observed for ethiofos with varying rates of absorption and metabolism/degradation in situ as a function of buffer and absorption enhancers. In most cases less than 10 per cent of the radiolabeled compound is lost from the circulating perfusate in 90 min. In addition, over the same time period greater than 40 per cent of the intact parent compound was lost by degradation. Much of the difference can be accounted for in the formation of the free thiol metabolite. WR-1065, suggesting ester hydrolysis or metabolic activity. Good stability was observed in all perfusate systems ex vivo indicating that the degradation occurs in situ. The disodium salt of ethylenediaminetetraacetic acid (EDTA) was shown to be an effective absorption enhancer of ethiofos. The enhancement of intestinal absorption by EDTA was dose-dependent resulting in a 20-fold increase in blood levels of ethiofos in the portal blood. Follow-up studies in the rhesus monkey confirm this observation. Salicylate and dimethylsulfoxide (DMSO) also resulted in absorption enhancement although to a lesser degree than that seen after EDTA treatment. Addition of several alkaline phosphatase inhibitors did not significantly improve absorption of ethiofos in the rat small intestine. Proposed mechanism(s) for intestinal absorption and absorption enhancement of ethiofos are discussed.

Pharmacokinetics and disposition of WR-1065 in the rhesus monkey.

The pharmacokinetics of WR-1065 [S-2-(3-aminopropylamino)ethanethiol] were investigated following iv, intraduodenal, and intraportal administrations in the rhesus monkey. Pharmacokinetic parameters were estimated by compartmental modeling of plasma concentration data from 10-min and 120-min iv infusions. Higher apparent volumes of distribution (Vc and Vss) and higher mean residence time (MRT) were observed at the slower infusion rate but a constant total dose. The values reflect a change in the distribution of WR-1065, possibly due to to saturation of binding in plasma and tissue. However, clearance remained unchanged. For a monkey administered approximately twice the 60 mg/kg dose infused over 120 min, data analysis indicates a disproportional increase in AUC and a substantial decrease in clearance. Low and erratic plasma concentrations of free drug (analytically determined without reductive cleavage) were observed following intraduodenal administration of WR-1065, demonstrating the drug's poor oral bioavailability. Results of intraduodenal administrations of radiolabeled drug indicated than an appreciable amount of the radiolabel in the dose reached the systemic circulation. However, after either intraduodenal or iv administration, only 31% of the AUC (radiolabel) could be accounted for as total (free and disulfide-bound) WR-1065 by specific analysis in separate experiments. Low levels of total cysteamine strongly suggest it to be a minor contributor to the disposition of the drug. Free WR-1065 AUC values following intraportal administration were similar to values obtained after iv administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Intraduodenal administration of ethiofos (WR-2721): dose proportionality study in the rhesus monkey.

A dose progression crossover study of ethiofos (WR-2721) was conducted in three healthy male rhesus monkeys. Each subject was tested with three single intraduodenal doses containing 150, 300, and 600 mg/kg. Blood samples were drawn as a function of time and the concentrations of ethiofos, WR-1065 (free thiol metabolite), and total drug convertible to the free thiol (total WR-1065) were determined by HPLC using electrochemical detection. Ethiofos levels in plasma were usually below quantifiable limits of detection (0.23 mumol/L) at all three dose levels, but free WR-1065 plasma levels increased with increasing dose. Analysis of the free WR-1065 bioavailability values indicated large variability and an unpredictable dose response among subjects. Bound WR-1065 appears to reach saturable levels over the dose range, suggesting a saturable pool of binding sites in plasma. The time-to-peak plasma levels for WR-1065 were variable regardless of the administered dose and ranged from 1.0-2.5 hours. The high variability in the data may be a result of poor permeability or absorption of the parent compound (ethiofos), saturable binding to a variable pool of binding sites in plasma and/or high first-pass metabolism of ethiofos involving the gut lumen, gut wall (epithelium), and liver.

Disposition of the radioprotector ethiofos in the rhesus monkey. Influence of route of administration.

Plasma concentrations of ethiofos [S-2-(3-aminopropylamino)ethyl phosphorothioic acid, WR-2721] were compared following iv, ip, intraduodenal, and portal administration to the rhesus monkey. Plasma samples were analyzed for ethiofos, free WR-1065, [2-(3-aminopropylamino)ethanethiol], and total material convertible to WR-1065 (total WR-1065). In separate experiments, total radioactivity in plasma was compared following iv, ip, and intraduodenal administration of [14C]ethiofos; excretion of the radiolabel was measured in urine and in feces. Intraduodenal administration of unlabeled ethiofos rarely gave measurable levels of unchanged drug in plasma. In contrast, intraduodenal administration of [14C]ethiofos produced an average AUC for total radioactivity that was 62% of that for a 10-min iv infusion of [14C]ethiofos. Urinary excretion of radioactivity following iv and intraduodenal administration of [14C]ethiofos was 78.9 +/- 14.0% and 43.8 +/- 12.4%, respectively, whereas 1.9 +/- 0.5% and 9.7 +/- 6.3% was excreted in feces. After an ip dose of either labeled or unlabeled ethiofos, absorption of the dose was prolonged, but AUC values for total radioactivity or ethiofos and total WR-1065 were similar to those observed after the corresponding 10-min iv experiments. For either iv or portal routes, increases in ethiofos AUC values were observed for the same total dose when the infusion rate was increased from 1.25 to 15 mg/kg/min.(ABSTRACT TRUNCATED AT 250 WORDS)

The disposition of ethiofos (WR-2721) in the isolated perfused rat liver.

We have investigated the disposition of ethiofos (20 mg, 4 microCi [14C]ethiofos) in the isolated perfused rat liver preparation to determine the hepatic contribution to the poor oral bioavailability of the drug. Ethiofos clearance (10.6 +/- 3.3 ml h-1) was only a small fraction (1.2 +/- 0.03%) of the perfusate flow rate. The elimination half-life was calculated at 7.1 +/- 1.9 h. The area under curve, AUC0-4 h, for ethiofos (2858 +/- 314 nM h ml-1) was not significantly different from that of 14C (3038 +/- 692 nM h ml-1) or total material convertible to WR-1065 (total WR-1065, 3324 +/- 612 nM h ml-1), indicating a low level of metabolism. The AUC0-4 h for free WR-1065 (37.5 +/- 23.3 nM h ml-1) was less than 2% of ethiofos. Biliary elimination of ethiofos, WR-1065, and 14C was below 1%. At 4 h postdose, 7.9 +/- 1.9% of the dose of radioactivity remained in the liver. Less than 1.5% could be identified as ethiofos (0.12 +/- 0.09%) or total WR-1065 (1.09 +/- 0.05%). Ethiofos, 14C, and total WR-1065 were approximately evenly distributed between the 10,000-g pellet and supernatant. However, significantly more ethiofos, WR-1065, and 14C were recovered from the 105,000-g supernatant compared with the pellet. In summary, both the metabolism and biliary elimination of ethiofos and its derivatives were sparing. Hence it is likely that in the rat, the contribution of the liver to the presystemic biotransformation and poor bioavailability of ethiofos is relatively minor.

Measurement of WR-1065 in plasma: preliminary pharmacokinetics in the beagle.

Described is a high-performance liquid chromatographic (HPLC) method for the quantification of WR-1065 [2-(3-aminopropylamino)-ethanethiol] in plasma. After a brief sample preparation, the drug and internal standard were separated in less than 15 minutes using a reversed-phase column and ion-pairing reagent. An electrochemical detector provided a minimum quantifiable limit of 0.05 microgram/mL. The analytical method was applied in three experiments to measure drug concentrations in the plasma of beagle dogs following IV administration of WR-1065 (60 mg/kg). The concentration-time data, best described by a three-compartment open kinetic model, were used to estimate pharmacokinetic parameters. Mean values were: steady state volume of distribution--2.27 L X kg-1; clearance--0.064 L X min-1 X kg-1; and terminal elimination half-life--81.4 min. The high clearance is consistent with the formation of mixed disulfides in the circulation.

A method for the combined measurement of ethiofos and WR-1065 in plasma: application to pharmacokinetic experiments with ethiofos and its metabolites.

An analytical method for the combined measurement of ethiofos (WR-2721) and a major metabolite (WR-1065) in plasma is described. Plasma samples were subjected to conditions which quantitatively converted both ethiofos and bound WR-1065 to free WR-1065 which was subsequently separated by HPLC and detected electrochemically using established procedures. Although bound WR-1065 in plasma is thought to exist mainly in the form of mixed disulfides, the symmetrical disulfide, WR-33278, also was quantitatively converted to the free thiol form. Standard curves were linear over the range 0.10 to 25 micrograms/mL (0.75 to 186 mumol/L). Mean precision over the range was 5.4% (coefficient of variation, CV) and recoveries of various mixtures of ethiofos, WR-1065 and WR-33278 averaged 102% (CV = 6.6%). This analytical procedure and others specific for ethiofos, free WR-1065 and WR-33278 were applied to dosing experiments in which the parent drug and its major metabolites were variously administered to beagle dogs and rhesus monkeys. Following i.v. administration of ethiofos (120-150 mg per kg body weight) to monkeys, plasma concentrations of unchanged drug ranged from 477 micrograms/mL (2.23 mM) down to the minimum detectable limit of the analytical procedure (0.05 micrograms/mL, 0.23 microM) 2-3 hours postinfusion. Clearances averaged 43.5 +/- 13.4 (SD) mL min-1 kg-1 and half-lives observed in the 20-60 minute postinfusion period were 8-15 min.

An improved HPLC assay for S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721) in plasma.

An HPLC assay is presented for the detection and quantitation of the radioprotective drug S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721, ethiofos) present in plasma. Improved selectivity and a 40-fold increase in sensitivity have been demonstrated over the method previously reported by this laboratory. Using precolumn derivatization with fluorescamine and S-3-(4-aminobutylamino)propyl phosphorothioate (WR-80855, a homolog of WR-2721) as the internal standard, drug levels of 0.05 to 1000 micrograms/mL were determined with excellent precision (CV less than or equal to 5% over the concentration range). An isocratic mobile phase of acetonitrile/ethanol/water (16:7:77) modified with 0.01 M tetrabutylammonium phosphate eluted the drug and the internal standard from the C-18 reverse-phase column in 23 minutes and 26 minutes, respectively. Detector response was linear over the entire range. The assay uses 150 microL of plasma and requires a total chromatography time of about 50 minutes. The method was found suitable for pharmacokinetic studies in a preliminary experiment with a beagle dog in which no interferences due to plasma constituents or drug metabolites were observed.

HPLC assay for 2-(3-aminopropylamino)ethanethiol (WR-1065) in plasma.

A high pressure liquid chromatography (HPLC) plasma assay for WR-1065 is described which is both precise (coefficient of variation less than 5%) and accurate (% average deviation less than or equal to 6.1) throughout the concentration range from 1 to 500 micrograms/mL of plasma. The analyte is separated by HPLC and detected with a thiol specific electrochemical transducer cell. The detector response is linear over the ranges 1 to 10 micrograms/mL (R2 = 0.995), 10 to 100 micrograms/mL (R2 = 0.995), and 100 to 500 micrograms/mL (R2 = 0.974). The absolute retention times for WR-1065 and WR-1729 are 9 and 12 minutes, respectively. The assay uses 100 microL of plasma and requires a total chromatography cycle time of 40 minutes. The method has been found suitable for the determination of WR-1065 in plasma from a beagle dog after i.v. administration of S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721).