Zidovudine azido-reductase in human liver microsomes: activation by ethacrynic acid, dipyridamole, and indomethacin and inhibition by human immunodeficiency virus protease inhibitors.

AZT (zidovudine, 3'-azido-3'-deoxythymidine), although metabolized primarily to AZT-glucuronide, is also metabolized to 3'-amino-3'-deoxythmidine (AMT) by reduction of the azide to an amine. The formation of the myelotoxic metabolite AMT has not been well characterized, but inhibition of AMT formation would be of therapeutic benefit. The aim of this study was to identify compounds that inhibit AMT formation. Using human liver microsomes under anaerobic conditions and [2-14C]AZT, K(m) values of AZT azido-reductase, estimated by radio-thin-layer chromatography, were 2.2 to 3.5 mM (n = 3). Oxygen completely inhibited this NADPH-dependent reduction. Thirteen of the 28 compounds tested inhibited the formation of AMT. In addition to the CYP3A4 inhibitors ketoconazole, fluconazole, indinavir, ritonavir, and saquinavir, metyrapone strongly inhibited AMT formation. An unexpected finding was the more-than-twofold increase in AMT formation in the presence of ethacrynic acid, dipyridamole, or indomethacin. Such activation of toxic metabolite formation would impair drug therapy.

Inhibition of haloperidol reduction by non-steroidal anti-inflammatory drugs in human liver cytosol.

A thorough knowledge of drug-drug interactions is crucial as the practice of multiple drug therapy escalates. In vitro studies using human liver enzymes are a valuable and non-invasive tool for predicting potential drug interactions in vivo. 1. A simple radio-TLC method was developed to monitor the formation of reduced haloperidol from haloperidol in human liver cytosol. 2. Indomethacin, known to be a potent inhibitor of 3a-hydroxysteroid dehydrogenase, was chosen as a reference for the evaluation of several arylpropionic acid derived non-steroidal anti-inflammatory drugs, ketoprofen, tiaprofenic acid, fenbufen, Ibuprofen, d-naproxen and 1-naproxen. The IC50 ranged from 0.4-6.0 mM with indomethacin the most potent inhibitor of haloperidol carbonyl reductase. 3. The carbonyl reduction of haloperidol was inhibited significantly by these most commonly used non-steroidal anti-inflammatory drugs and the degree of inhibition reflected their pharmacological potency. 4. Sephadex G-100 fractionation of human liver cytosol yielded a fraction with haloperidol reductase activity at a molecular weight of about 32,000.

Effects of perfusate flow rate on measured blood volume, disse space, intracellular water space, and drug extraction in the perfused rat liver preparation: characterization by the multiple indicator dilution technique.

The effect of hepatic blood flow on the elimination of several highly cleared substrates was studied in the once-through perfused rat liver preparation. A constant and low input concentration of ethanol (2.0 mM), [14C]-phenacetin and [3H]-acetaminophen (0.36 and 0.14 microM, respectively), or meperidine (8.1 microM) was delivered once-through the rat liver preparation in five flow periods (greater than 35 min each); control flow periods at 12 ml/min were interrupted by flow changes to 8 or 16 ml/min. The steady-state hepatic availabilities (F or outflow survivals) at 12 ml/min were ethanol, 0.075 +/- 0.038; [14C]-phenacetin, 0.15 +/- 0.059; [3H]-acetaminophen, 0.34 +/- 0.051; meperidine, 0.047 +/- 0.017. Flow-induced changes were different among the compounds: with reduced flow (8 ml/min), F was decreased for ethanol (0.061 +/- 0.032) and [3H]-acetaminophen (0.28 +/- 0.051), as expected, but was increased for [14C]-phenacetin (0.20 +/- 0.068) and meperidine (0.05 +/- 0.03); with an elevation of flow (to 16 ml/min), F was increased for all compounds, as expected of shorter sojourn times: ethanol, 0.13 +/- 0.065; [14C]-phenacetin, 0.22 +/- 0.062; [3H]-acetaminophen, 0.43 +/- 0.063; meperidine, 0.055 +/- 0.022. A marked increase in F for ethanol had occurred when flow changed from 12 to 16 ml/min due to nonlinear metabolism; the latter was confirmed by a reduction in the extraction ratios at increasing concentrations (1.8 to 11.4 mM); this condition was not present for the other compounds. In order to explain the observations, we used the multiple indicator dilution technique to investigate the flow-induced behaviors of tissue distribution spaces of vascular and intracellular references in the perfused rat liver preparation.

Relevance of plasma, glandular and urinary kallikrein in renal hypertrophy in streptozotocin-diabetic rats.

The relevance of plasma, glandular and renal kallikrein as an intrarenal hemodynamic regulator, in renal hypertrophy, in 1-5 weeks streptozotocin diabetic rats has been investigated. The fasting plasma glandular kallikrein level significantly decreased with increasing duration of diabetes (p less than 0.05). Glandular kallikrein correlated negatively with kidney weight (r = 0.76, p = 0.05). The 24 hour urinary kallikrein excretion significantly increased with increasing duration of diabetes (p less than 0.05), but this level was not correlated with glucose level, nor with kidney weight. Aprotinin (a kallikrein inhibitor) injected (10 X 10(3) KIU/kg) twice daily for 2 weeks in diabetic rats, significantly decreased plasma glucose levels by 28%, 24 hour urinary kallikrein by 37% (p less than 0.05) and kidney weight by 6%. These results suggest that plasma, glandular and renal kallikrein did not play an important role in the renal hypertrophy observed in streptozotocin diabetic rats.

Absorption and metabolism of acetaminophen by the in situ perfused rat small intestine preparation.

The in situ perfused rat small intestine preparation was used to examine the extents of segmental absorption and metabolism of acetaminophen (A). Additionally, the preparation was employed to investigate any intestinal excretion of A and its conjugates from the circulation to the intestinal lumen. In this preparation, blood perfusate (300 ml) recirculated the intestinal preparation at 7.5 ml/min, entering via the superior mesenteric artery and returned to the reservoir via the portal vein. To demonstrate the extent of segmental absorption, metabolism, and excretion by different segments of the intestine, tracer doses of 3H-A (0.41 to 0.55 mumol in 0.3 ml of saline) were administered into the (a) entire intestine; (b) segments (first, second, and third) of one-third the length of the intestine, by instillation of the dose into the lumens of the segments; and (c) reservoir of perfusate. Exudates of luminal fluid from the injected segment and segments not exposed to drug were monitored for A and its conjugates during the experiment and at the end of 2 hr. Absorption of A was usually complete by 60 min; the extent of absorption of A at the end of 2 hr by the entire length of the intestine and by its three (first, second, and third) individual segments were 71.7 +/- 2.6, 50.5 +/- 4.0, 73.9 +/- 2.1, and 58.8 +/- 6.1% of dose (mean +/- SE), respectively. At the end of 2 hr, the total amount of acetaminophen glucuronide in perfusate and luminal fluid accounted for 3.1-5.5% and 0.14-0.1% of dose, respectively, among these preparations; acetaminophen sulfate was present only as a small percentage of dose in the lumen. Glucuronidation activity, when expressed as a percentage of the absorbed dose, was fairly constant for the entire intestine and first and second segments (8%) but decreased slightly for the third segment (7%). When A was present in blood perfusing the intestine, no metabolite was detected in perfusate or luminal fluid. Instead, unchanged A was excreted (5.6% dose) into the lumen. The effect of dose and vehicle on the extents of absorption and metabolism of A in the preparation was investigated by the instillation of different doses of A (0.16, 99.2, and 396.9 mumol in 0.3 ml of polyethene glycol 400) into the entire intestine at the duodenum.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetics of meperidine N-demethylation in the perfused rat liver preparation.

Meperidine and normeperidine elimination was studied in the once-through perfused rat liver preparation. Biliary excretion of these bases was minimal, and nonlinear metabolism of meperidine (extraction ratio of 1.0 to 0.89 at 1 to 19 micrograms/ml) and normeperidine (extraction ratio of 0.6 to 0.1 at 1 to 25 micrograms/ml) was observed when input concentration was increased. The rate of efflux of normeperidine in hepatic venous blood represented an increasing proportion of the rate of presentation and the rate of loss of meperidine (34 to 92%) at increasing meperidine input concentration. But when the data were corrected for the nonlinear sequential metabolism of normeperidine, the rate of N-demethylation accounted completely for the rate of metabolism of meperidine. These N-demethylation rates obeyed Michaelis-Menten behavior, and appeared to be saturated at input meperidine concentration greater than 5 micrograms/ml.

Inhibition of acetaminophen sulfation by 2,6-dichloro-4-nitrophenol in the perfused rat liver preparation. Lack of a compensatory increase of glucuronidation.

The effectiveness of 2,6-dichloro-4-nitrophenol ( DCNP ) as an inhibitor of sulfation of acetaminophen, and the effect of DCNP on other conjugation reactions such as glucuronidation and glutathione conjugation were investigated in the once-through perfused rat liver preparation. The formation of glucuronide and glutathione conjugates of acetaminophen was insignificant under conditions when sulfation was suppressed either by the absence of inorganic sulfate or the presence of DCNP , suggesting that acetaminophen was a poor substrate for glucuronidation (high Km) and for the formation of the reactive metabolite leading to the formation of acetaminophen glutathione conjugate. DCNP was most effective in suppressing sulfation at low concentrations of acetaminophen, and the degree of inhibition increased with DCNP concentration, possibly due to a competitive mechanism of inhibition. Subsequent studies with tracer concentration of 3H-acetaminophen and 40 microM DCNP indicated that the steady state hepatic extraction ratio of acetaminophen decreased by 16%, sulfation was reduced by 19%, whereas glucuronidation increased by 28% in the presence of DCNP ; glutathione conjugation was not affected. Because acetaminophen is a poor substrate for glucuronidation and because glucuronidation remains a minor metabolic pathway in the biotransformation of acetaminophen, the changes in glucuronidation of acetaminophen with DCNP (28% above control value) failed to effect gross changes in acetaminophen disposition. Rather, the suppression of acetaminophen sulfation by DCNP is responsible for the decreased hepatic extraction of acetaminophen. This inhibition of acetaminophen sulfation by DCNP is readily reversible.

Kinetics of procainamide N-acetylation in the rat in vivo and in the perfused rat liver preparation.

The kinetics of procainamide N-acetylation were studied in the rat in vivo and in vitro. For the in vivo studies, first order kinetics for procainamide and N-acetylprocainamide were found among their respective iv doses of 20, 50, and 70 mg/kg, and 10, 20, and 30 mg/kg. The fraction of total body clearance of procainamide that forms N-acetylprocainamide was found to be 0.22, and very insignificant sequential elimination of N-acetylprocainamide occurred during its formation. By contrast, results from once-through liver perfusion indicated that the steady state hepatic extraction ratio of procainamide was highly dependent on the steady state input concentration delivered under constant hepatic blood flow (10 ml/min). The rate of N-acetylation, however, was a constant percentage of the rate of presentation of procainamide at less than or equal to 80 micrograms/min among the preparations and became apparently saturated at higher rates of input of procainamide. Interestingly, the rate of N-acetylation accounted for an increasing proportion of the rate of loss of procainamide at greater than 80 micrograms/min, and suggested that alternate metabolic routes of procainamide are more easily saturable than N-acetylation. The comparative in vivo and in vitro data suggested that a region of nonlinearity existed during the early periods immediately following iv injection of procainamide into the rat in vivo. Because of rapid distribution, the region of nonlinearity was transient, and was not reflected by area under the curve measurements, which is a time-averaged parameter. Total body clearance, which bears a reciprocal relationship with the area under the curve, hence remained constant and was dose-invariant. The trend of nonlinearity may be more evident on chronic dosing of the drug when accumulation sets in.

The release of plasticizer from polyvinyl chloride haemodialysis tubing.

The extent of migration of plasticizer from haemodialysis lines into saline and plasma has been examined and a number of different types of tubing have been compared. A model system designed to simulate in vivo haemodialysis was evolved. The method uses a continuous cyclohexane extraction in order to mimic uptake of plasticizer by body tissues. Although not an ideal system, the results obtained are indicative of the extent to which the plasticizers are leached.