The glimmer of HIV proteinase inhibitors.
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HIV Infections↗
Biomedical subjects
J J Lipsky
Publications and source records attributed to J J Lipsky.
S-methyl N,N-diethylthiocarbamate sulfone, a potential metabolite of disulfiram and potent inhibitor of low Km mitochondrial aldehyde dehydrogenase.
Disulfiram inhibits hepatic aldehyde dehydrogenase (ALDH) causing an accumulation of acetaldehyde after ethanol ingestion. It is thought that disulfiram is too short-lived in vivo to directly inhibit ALDH, but instead is biotransformed to reactive metabolites that inhibit the enzyme. S-Methyl N,N-diethylthiocarbamate (MeDTC) sulfoxide has been identified in the blood of animals given disulfiram and is a potent inhibitor of ALDH (Hart and Faiman, Biochem Pharmacol 46: 2285-2290, 1993). MeDTC sulfone is a logical metabolite of MeDTC sulfoxide. Therefore, we investigated the effects of MeDTC sulfone on the activity of rat hepatic low Km mitochondrial ALDH, the major enzyme in the metabolism of acetaldehyde. MeDTC sulfone inhibited the low Km mitochondrial ALDH in vitro with an IC50 of 0.42 +/- 0.04 microM (mean +/- SD, N = 5) compared with disulfiram, which had an IC50 of 7.5 +/- 1.2 microM under the same conditions. The inhibition of ALDH by MeDTC sulfone was time dependent. The decline in ALDH activity followed pseudo first-order kinetics with an apparent half-life of 2.1 min at 0.6 microM MeDTC sulfone. Inhibition of ALDH by MeDTC sulfone was apparently irreversible; dilution of the inhibited enzyme did not restore lost activity. The substrate (acetaldehyde, 80 microM) and cofactor (NAD, 0.5 mM) together completely protected ALDH from inhibition by MeDTC sulfone; substrate alone partially protected the enzyme. Addition of either thiol-containing compound glutathione (GSH) or dithiothreitol (DTT) to MeDTC sulfone before incubation with the enzyme increased the IC50 of MeDTC sulfone by 7- to 14-fold. Neither GSH nor DTT could restore lost ALDH activity after exposure of the enzyme to MeDTC sulfone. Results of these studies indicate that MeDTC sulfone, a potential metabolite of disulfiram, is a potent, irreversible inhibitor of low Km mitochondrial ALDH.
Aldehyde Dehydrogenase↗
Simultaneous structure-activity determination of disulfiram photolysis products by on-line continuous-flow liquid secondary ion mass spectrometry and enzyme inhibition assay.
Disulfiram (DSF) is used in the treatment of recovering alcoholics and exerts its effect by inhibiting the enzyme aldehyde dehydrogenase (ALDH). We analyzed a mixture of products derived photochemically from DSF with on-line microbore HPLC-continuous-flow liquid secondary ion mass spectrometry (HPLC-CF-LSI-MS). By utilizing the post-HPLC column split of solvent flow, a small proportion (ca. 5%) was sent directly into the mass spectrometer, and the remainder was collected. Simultaneous MS analysis and enzyme inhibition studies on ALDH were then possible. Furthermore, using HPLC-CF-LSI-MS-MS, we were able to structurally characterize an interesting sulfine compound that inhibited ALDH.
Aldehyde Dehydrogenase↗
An intravenous loading dose of azathioprine decreases the time to response in patients with Crohn's disease.
BACKGROUND & AIMS: Azathioprine, an effective therapy for Crohn's disease, is limited by a prolonged time to response. The aim of this study was to determine the safety and utility of a loading dose of azathioprine to decrease the time to response in patients with Crohn's disease. METHODS: Twelve patients were studied: 6 with 13 fistulae and 6 with inflammatory disease. All patients received an intravenous infusion of azathioprine (50 mg/h for 36 hours). Response was determined by physical and radiographic examination for fistulae and by the Crohn's Disease Activity Index for inflammatory disease. Erythrocyte concentrations of azathioprine metabolites were measured by chromatography. RESULTS: Seven of 13 fistulae closed by week 4, and three had a temporary decrease in drainage. One fistula improved at week 16. Two fistulae failed to improve. Four of 6 patients with inflammatory disease achieved remission, and 1 improved temporarily. Improvement was rapid (< or = 4 weeks). Peak concentrations of azathioprine metabolites occurred within 3 days. Clinical response did not correlate with azathioprine metabolite concentrations at the azathioprine dose studied. No adverse events occurred. CONCLUSIONS: An 1800-mg intravenous loading dose of azathioprine is safe and may decrease the time to response to < or = 4 weeks in patients with Crohn's disease. Correlation between clinical response and azathioprine metabolite concentrations at larger azathioprine doses should be determined.
Adult↗
Utility of a "swish and spit" technique for the collection of buccal cells for TAP haplotype determination.
OBJECTIVE: To demonstrate the utility of a "swish and spit" technique as a nucleated cell source for transporter associated with antigen processing (TAP) haplotype determination by molecular methods in large-scale clinical trials. DESIGN: Twenty normal volunteers were recruited for this prospective feasibility study. From each subject, buccal or blood cells (or both) were collected for use in TAP haplotype assignment by molecular methods and subjected to various storage conditions. MATERIAL AND METHODS: As an alternative to use of lymphocytes obtained by venipuncture, we developed a swish and spit technique for collecting buccal cells for assigning TAP haplotype. For this technique, the subject vigorously swishes isotonic saline in the mouth and expectorates it into a collection container. DNA is extracted from the buccal cells by proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. In addition, we compared DNA extracted from mouthwash specimens stored under various conditions to which a specimen might be exposed if mailed. RESULTS: DNA extracted from buccal cells obtained by the swish and spit technique provided excellent templates for the polymerase chain reaction (PCR), and subject acceptance of this method was universal. In all cases, assigning TAP haplotype by PCR amplification of specific alleles with use of buccal or blood-derived specimens was successful. The integrity of the specimens was unaffected by storage at -20 degrees C, 4 degrees C, 25 degrees C, or 37 degrees C, and we were able to use the DNA from cells stored under any of these conditions for TAP haplotying. CONCLUSION: We conclude that DNA from buccal cells collected by the swish and spit technique for TAP haplotype assignment is an excellent substitute for DNA obtained from nucleated blood cells, and the technique is useful for large-scale clinical studies that require DNA from subjects geographically distant from the research site.
DNA↗
Transcutaneous drug delivery: a practical review.
OBJECTIVE: To describe the advantages, disadvantages, practical considerations, and future developments of transcutaneous drug delivery. MATERIALS AND METHODS: The physiochemical properties of the drug preparation that are factors in the effectiveness of transcutaneous transport are drug stability or volatility, use of a solvent carrier or vehicle, use of a penetration enhancer, and type of delivery device. Because a drug should remain on the skin without evaporating or becoming otherwise inactive, it is suspended in a vehicle--any gel, lotion, or paste used to apply the drug to the skin. Penetration enhancers include several compounds that are mixed into vehicles to alter the molecular environment of the epidermis and facilitate absorption. The delivery system itself occasionally proves to be the ultimate determinant of transdermal drug flow. RESULTS: The advantages of transcutaneous drug delivery are avoidance of the gastrointestinal tract and hepatic first-pass biotransformation and metabolism, control of absorption, availability of multiple skin sites to avoid local irritation and toxicity, and improved patient compliance. The disadvantages include the potential for localized irritant and allergic cutaneous reactions, systemic toxicity, and difficulties associated with the time necessary for a drug to diffuse through the skin. CONCLUSION: Transdermal drug regimens are safe and effective. They provide clinicians the opportunity to offer more therapeutic options to their patients to optimize their care.
Administration, Cutaneous↗
Photolysis of sulfiram: a mechanism for its disulfiram-like reaction.
Sulfiram, a drug applied topically to treat scabies, produces effects similar to those of disulfiram after subsequent ingestion of ethanol. Disulfiram, used in aversion therapy in the treatment of alcoholism, inhibits hepatic aldehyde dehydrogenase (ALDH) causing an accumulation of acetaldehyde after ethanol ingestion. The increased tissue levels of acetaldehyde cause a spectrum of undesirable side-effects including flushing, nausea, vomiting, and tachycardia, which are referred to as the disulfiram reaction. Previous studies have shown that in vitro sulfiram is a very weak inhibitor of ALDH, but solutions of sulfiram markedly increase in potency with time. In the present study, fresh solutions of sulfiram were exposed to fluorescent room light under ambient conditions and analyzed at timed intervals by HPLC. At least eight products, including disulfiram, were formed in the light-exposed sulfiram solutions, but not in solutions kept in the dark. Structural characterization of two of the photolysis products was obtained by on-line microbore HPLC-mass spectrometry (mu LC-MS) and on-line microbore HPLC-tandem mass spectrometry (mu LC-MS/MS) using continuous flow-liquid secondary ion mass spectrometry (CF-LSIMS) as the primary ionization method. Sulfiram was converted to disulfiram at an initial rate of 0.7%/hr, and the formation of disulfiram correlated with the increase in ALDH inhibition in vitro. The results of this investigation show that while sulfiram is a weak inhibitor of ALDH in vitro, it is readily photoconverted to disulfiram, a very potent inhibitor of ALDH, which may explain the adverse reaction to ethanol after sulfiram therapy.
Aldehyde Dehydrogenase↗
Monosulfiram, disulfiram, and light.
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Administration, Cutaneous↗
Nutritional sources of vitamin K.
OBJECTIVE: To review the function of vitamin K in clotting and methods of its analysis, to present results of previous studies on the role of dietary vitamin K in humans and animals, and to reanalyze these data in light of current methods. DESIGN: A review of assumptions stated in the literature is presented, including the incorrect theory that a diet-induced deficiency of vitamin K is nonexistent and the unsubstantiated hypothesis that antibiotics can cause vitamin K deficiency by destroying intestinal bacteria. CONCLUSION: The insistent belief that intestinal bacteria are an important source of vitamin K has led to erroneous conclusions about the sources of vitamin K for human nutrition. In the future, the importance of various sources of vitamin K, their pathways of absorption, and their susceptibility to administration of antibiotics should be evaluated without recourse to current assumptions.
Animals↗
Diethyldithiocarbamate S-methylation: evidence for catalysis by human liver thiol methyltransferase and thiopurine methyltransferase.
Disulfiram is used in the treatment of alcoholism to inhibit the enzyme aldehyde dehydrogenase. Disulfiram is rapidly reduced in vivo to form diethyldithiocarbamate (DDC), and DDC can undergo methyl conjugation to form S-methyl-DDC. Human tissues contain two separate genetically regulated enzymes that can catalyze thiol S-methylation. Thiol methyltransferase (TMT) is a microsomal enzyme that preferentially catalyzes, the S-methylation of alipathic sulfhydryl compounds, whereas thiopurine methyltransferase (TPMT) is a cytoplasmic enzyme that preferentially catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds. Our experiments were performed to determine whether human liver microsomal and/or cytosolic preparations could catalyze the S-methylation of DDC, and, if so, to determine whether TMT or TPMT might be the enzymes involved. We found that both human liver microsomes and cytosol could catalyze DDC S-methylation. The microsomal activity displayed biphasic substrate kinetics, with apparent Km values for DDC of 7.9 and 1500 microM for the high- and low-affinity activities, respectively. The high-affinity activity had an apparent Km value for S-adenosyl-L-methionine, the methyl donor for the reaction, of 5.8 microM. The thermal inactivation profile and response to methyltransferase inhibitors of the high-affinity microsomal DDC S-methyltransferase activity were similar to those of human liver microsomal TMT. In addition, TMT activity and the activity catalyzing the S-methylation of DDC were highly correlated in 19 individual liver samples (rs = 0.956; P < .0001). Hepatic cytosolic DDC S-methyltransferase activity had an apparent Km value for DDC of 95 microM. The cytosolic enzyme which catalyzed DDC S-methylation and TPMT activity had similar thermal inactivation profiles, similar patterns of response to methyltransferase inhibitors and the two activities coeluted during ion exchange chromatography. Furthermore, the activities of TPMT and cytosolic DDC S-methyltransferase were highly correlated in 20 individual liver samples (rs = 0.963; P < .0001). These results were compatible with the conclusion that both TMT and TPMT could catalyze the S-methylation of DDC in the human liver. Because the activities of both TMT and TPMT are controlled by inheritance, our observations raise the possibility of pharmacogenetic variation in the biotransformation and therapeutic effect of DDC in humans.
Aldehyde Dehydrogenase↗
Cephalosporin-induced alteration in hepatic glutathione redox state. A potential mechanism for inhibition of hepatic reduction of vitamin K1,2,3-epoxide in the rat.
Hypoprothrombinemia is a serious adverse effect of antimicrobial therapy that occurs after administration of some second- and third-generation cephalosporins which contain the methyltetrazole-thiol (MTT) group. Previous studies have shown that in vitro MTT directly inhibits microsomal gamma-carboxylation of a synthetic pentapeptide. Since MTT is a thiocarbamide, a type of compound that can increase oxidation of glutathione, the present studies were carried out to determine whether alterations in hepatic glutathione redox state might interfere with vitamin K metabolism. Dose-related increases in biliary efflux and hepatic concentration of oxidized glutathione (GSSG) occurred after intravenous administration of MTT or MTT-containing antibiotics to rats. This finding suggested that these compounds could alter the hepatic glutathione redox state in vivo. Microsomal reduction of vitamin K epoxide occurred in the presence of 100 microM dithiothreitol (DTT), but was inhibited by preincubation with GSSG at concentrations as low as 10 microM. At higher concentrations of DTT (1.0 mM) inhibition by GSSG persisted, but higher concentrations were required, suggesting that the thiol/disulfide ratio, rather than the absolute concentration of GSSG was important. By contrast, GSSG did not effect microsomal gamma-carboxylation of a pentapeptide, using either vitamin K1 or its hydroquinone as a cofactor. These findings suggest a novel mechanism for the hypoprothrombinemia occurring after administration of MTT-containing antibiotics.
Animals↗
Ability of 1-methyltetrazole-5-thiol with microsomal activation to inhibit aldehyde dehydrogenase.
Antibiotics that contain the 1-methyltetrazole-5-thiol (MTT) leaving group are associated with an adverse effect when alcohol is ingested after their administration. Therefore, the ability of MTT to inhibit an enzyme in alcohol metabolism, aldehyde dehydrogenase (ALDH), was examined. In the absence of microsomes, MTT did not inhibit ALDH obtained from either yeast or rat liver. In the presence of rat hepatic microsomes, MTT was able to inhibit the enzyme from both sources. The characteristics of the inhibition were studied, using the yeast enzyme, and found to be dependent upon the length of incubation with the hepatic microsomes and upon the concentration of MTT. Inhibition required the presence of NADH and was not detected if the microsomes were heat treated. Dilution did not reverse the inhibition. Intact antibiotics which contain the MTT moiety did not cause an inhibition of yeast ALDH unless the antibiotics were first treated with potassium hydroxide and then incubated with microsomes. Inhibition of ALDH activity measured in the mitochondrial plus microsomal fractions of rat liver also required NADH and was prevented by glutathione and heat treatment of the microsomes. These results indicate that microsomal activation of MTT is necessary for inhibition of aldehyde dehydrogenase. The behavior of MTT described here may explain the adverse effect observed if alcohol is ingested following administration of MTT-containing antibiotics.
Aldehyde Dehydrogenase↗
Effect of biliary diversion on the ability of cefamandole to inhibit vitamin K metabolism.
The effect of biliary diversion on the ability of cefamandole, a methyltetrazole-thiol (MTT) containing antibiotic, to alter both hepatic vitamin K metabolism and the gamma-carboxylation of glutamic acid were examined in the rat, in order to understand the hypoprothrombinemia associated with MTT-containing antibiotics. At a dose of 3 gm/kg, cefamandole decreased the activity of hepatic vitamin K epoxide reductase at 24 but not at 4 hours after its administration. This inhibition occurred with or without diversion of the bile duct from the intestine. When vitamin K was used as the cofactor in the enzymatic reaction, carboxylation of glutamic acid was also found to be reduced in both biliary diverted as well as in biliary intact rats. Carboxylation of glutamic acid was not reduced when vitamin K hydroquinone was used. These results suggest that part of the mechanism underlying the hypoprothrombinemia associated with MTT containing antibiotics is linked to the ability of the MTT group to inhibit vitamin K metabolism. Furthermore, biliary secretion of the intact antibiotic may not be required for the effect upon vitamin K metabolism to be observed.
Animals↗
Antibiotic-associated hypoprothrombinaemia.
Hypoprothrombinaemia is regarded as a serious adverse effect of antimicrobial therapy. This effect has commonly been attributed to the potential of these drugs to kill intestinal bacteria, a possible source of vitamin K, which is a necessary co-factor in the synthesis of four of the clotting factors. This review examines the evidence for and against this hypothesis, first in experimental animals, then in humans, and assesses the reports of antibiotic associated hypoprothrombinaemia in man, with particular attention to information about the mechanism. The hypothesis that it is the destruction of intestinal bacteria that ultimately results in hypoprothrombinaemia may not be justified. Certain antibiotics, which contain thiol-leaving groups, may produce hypoprothrombinaemia because the thiol group inhibits the vitamin K-dependent step in clotting factor synthesis.
Animals↗
Mechanism of thioamide antithyroid drug associated hypoprothrombinemia.
The thioamide class of antithyroid drugs has been associated with the development of hypoprothrombinemia. Two drugs in this class, propylthiouracil and methimazole, resemble the methyltetrazole-thiol leaving group of certain cephalosporin antibiotics. Both were found in vitro to inhibit the vitamin K dependent step in clotting factor synthesis, the gamma-carboxylation of glutamic acid with 50 per cent inhibitory concentrations of 2 mM for propylthiouracil and 0.1 mM for methimazole. Methimazole was also found to inhibit vitamin K epoxide reductase, an enzyme related to the carboxylation reaction, with a 50 per cent inhibitory concentration of 25 uM. In vivo methimazole, administered twice at a dose of 500 mg/kg to rats on a vitamin K deficient diet produced hypoprothrombinemia. These results suggest that the mechanism of hypoprothrombinemia associated with thioamide antithyroid drugs may be similar to the mechanism of hypoprothrombinemia associated with cephalosporins which contain the methyltetrazole-thiol leaving group.
Amides↗
Cefotetan and hypoprothrombinemia.
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Adult↗