3'-Azido-3'-deoxythymidine (AZT) is a competitive inhibitor of thymidine phosphorylation in isolated rat heart and liver mitochondria

Long-term use of 3'-azido-3'-deoxythymidine (AZT) is associated with various tissue toxicities, including hepatotoxicity and cardiomyopathy, and with mitochondrial DNA depletion. AZT-5'-triphosphate (AZTTP) is a known inhibitor of the mitochondrial DNA polymerase gamma and has been targeted as the source of the mitochondrial DNA depletion. However, in previous work from this laboratory with isolated rat heart and liver mitochondria, AZT itself was shown to be a more potent inhibitor of thymidine phosphorylation (IC50 of 7.0+/-1.0 microM AZT in heart mitochondria and of 14.4+/-2.6 microM AZT in liver mitochondria) than AZTTP is of polymerase gamma (IC50 of >100 microM AZTTP), suggesting that depletion of mitochondrial stores of TTP may limit replication and could be the cause of the mitochondrial DNA depletion observed in tissues affected by AZT toxicity. The purpose of this work is to characterize the nature of AZT inhibition of thymidine phosphorylation in isolated rat heart and rat liver mitochondria. In both of these tissues, AZT was found to be a competitive inhibitor of the phosphorylation of thymidine to TMP, catalyzed by thymidine kinase 2. The inhibition constant (Ki) for heart mitochondria is 10.6+/-4.5 microM AZT, and for liver mitochondria Ki is 14.0+/-2.5 microM AZT. Since AZT is functioning as a competitive inhibitor, increasing thymidine concentrations may be one mechanism to overcome the inhibition and decrease AZT-related toxicity in these tissues.     

3'-Azido-3'-deoxythymidine (AZT) inhibits thymidine phosphorylation in isolated rat liver mitochondria: a possible mechanism of AZT hepatotoxicity

3'-azido-3'-deoxythymidine (AZT) is a staple of highly active antiretroviral therapy (HAART). Prior to HAART, long-term use of high-dosage AZT caused myopathy, cardiomyopathy, and hepatotoxicity, associated with mitochondrial DNA depletion. As a component of HARRT, AZT causes cytopenias and lipodystrophy. AZT-5'-triphosphate (AZTTP) is a known inhibitor of the mitochondrial polymerase gamma and has been targeted as the source of the mitochondrial DNA depletion. However, in previous work from this laboratory with isolated rat heart mitochondria, AZT phosphorylation beyond AZT-5'-monophosphate (AZTMP) was not detected. Rather, AZT was shown to be a more potent inhibitor of thymidine phosphorylation (50% inhibitory concentration (IC50) of 7.0+/-1.0 microM) than AZTTP is of polymerase gamma (IC50 of >100 microM), suggesting that depletion of mitochondrial stores of TTP may limit replication. This work has investigated whether an identical mechanism might account for the hepatotoxicity seen with long-term use of AZT. Isolated rat liver mitochondria were incubated with labeled thymidine or AZT, and the rate and extent of phosphorylation were determined by HPLC analysis of acid-soluble extracts of the incubated mitochondria. The results showed that in the phosphorylation of thymidine to TMP, liver mitochondria exhibit a higher Vmax and Km than heart mitochondria, but otherwise heart and liver mitochondria display similar kinetics. AZT is phosphorylated to AZTMP, but no further phosphorylated forms were detected. In addition, AZT inhibited the production of TTP, with an IC50 of 14.4+/-2.6 microM AZT. This is higher, but comparable to, the results seen in isolated rat heart mitochondria.     

In vitro glucuronidation of 3'-azido-3'-deoxythymidine by human liver. Role of UDP-glucuronosyltransferase 2 form.

The glucuronidation of 3'-azido-3'-deoxythymidine (AZT) by human liver microsomes and human hepatocytes in culture has been studied in vitro to determine the UDP-glucuronosyltransferase (UDPGT) form conceivably involved in the AZT biotransformation process. The glucuronide of AZT was preliminarily identified through hydrolysis by beta-D-glucuronidase. Brij 58 was shown to be the best activator of AZT glucuronidation by human liver microsomes, as it increased the rate of glucuronide formation 3-fold. The UDPGT activities toward AZT measured in 29 different microsomal fractions was slightly variable among samples (79 to 268 nmol/hr/mg protein). The apparent KM value for AZT glucuronidation was about 5 mM. We sought to determine if various known UDPGT activities (i.e. p-nitrophenol UDPGT, 4-hydroxybiphenyl UDPGT, and DT1-UDPGT) in 18 microsomal samples were correlated with AZT-UDPGT activity. Experiments revealed that only 4-hydroxybiphenyl UDPGT activity was strongly correlated (r = 0.815, p less than 0.001) with AZT-UDPGT activity, whereas no correlation was found for the other UDPGT activities. To determine the isozyme conceivably involved in AZT glucuronidation, we studied the effect of various compounds on AZT glucuronidation. AZT glucuronidation was inhibited by numerous substrates of the UDPGT2, form: morphine (Ki = 1.8 mM), 4-hydroxybiphenyl (Ki = 0.92 mM), and ketoprofen (Ki = 0.75 mM), but also oxazepam, codeine, and chloramphenicol. p-Nitrophenol appeared to be an inhibitor, whereas acetaminophen had no effect. Bilirubin, aspirin, cimetidine, and acyclovir did not inhibit AZT glucuronidation. Since all the inhibitors tested except p-nitrophenol are known to be glucuronidated by the UDPGT2 form, our results strongly suggest the involvement of this isozyme in AZT glucuronidation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Doses and time-dependent effects of 3'-azido-3'-deoxythymidine on T47D human breast cancer cells in vitro

The objective of the present work was to study the effects of 3'-azido-3'-deoxythymidine (azidothymidine, Zidovudine) on human breast cancer cells by using, as a model, the T47D cell line (typified as oestrogen-dependent and p53-mutated). Low azidothymidine doses (3.125 microM) increase the percentage of cells in S-phase, with the effect reversing after 24 hr of incubation; as azidothymidine doses increase, the magnitude and duration of its effect increase proportionally, although, even with the highest concentrations (50-100 microM) the effects decline after 48 hr of incubation. If media (containing azidothymidine or vehicle) are daily renewed, the azidothymidine effects (accumulation of cells in S-phase) are higher and decline later than when media and drug are not changed during the whole culture period, thereby suggesting that the reversion of azidothymidine effects could be related with a degradation of the drug or accumulation in media of substances which counteract its effects. Azidothymidine inhibits T47D cell proliferation at concentrations higher than 50 microM. The exposure to 50 or 100 microM azidothymidine induced cell apoptosis after 48 hr or more of incubation. We conclude that: a) azidothymidine, with appropriate doses and duration of treatment, synchronizes cells in S-phase, inhibits proliferation, and induces apoptosis, b) the discontinuous application of the drug rather than continuous exposure to it increases its efficiency to synchronize the T47D cell cycle. This in vitro anti-breast cancer activity suggests that a possible clinical usefulness of azidothymidine, either alone or associated with other drugs with cycle-specific antitumoural activity circumscribed to the S-phase of cell cycle, is worthy of investigation.     

In vitro inhibition of human polymorphonuclear cell function by cloricromene

Summary Cloricromene, a coumarin derivative with antiaggregating and vasodilating properties, was tested in vitro on polymorphonuclear cell (PMN) adhesion to the endothelium, superoxide anion generation and chemotaxis. PMN adhesion was measured using cultured human umbilical vein endothelial cells (EC) either untreated or previously activated with interleukin-1 (IL-1). Cloricromene (5-50 M) induced dose-related inhibition of PMN adhesion to untreated and IL-1 treated EC. Cloricromene also inhibited PMN superoxide generation induced by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or by N-formyl-methionyl-leucyl-phenylalanine (FMLP).PMN and monocyte chemotaxis was evaluated by a modification of the Boyden chamber technique. Cloricromene inhibited both types of cell motility induced by FMLP in a concentration-dependent fashion. The major cloricromene metabolite (cloricromene acid) had no effect on any of the biological parameters studied up to a concentration of 500M. HPLC measurement showed that cloricromene accumulated in PMN within a few minutes and levels of the drug were still high after 60 min. In contrast its acid metabolite was not taken up in a significant amount during incubation periods up to 60 min. We conclude that cloricromene inhibits a series of PMN activities in vitro. This effect might be of pharmacological interest in view of the role of PMN activation in different pathophysiological conditions.Abbreviations PMN Polymorphonuclear cells - IL-1 interleukin-1 - EC human umbilical vein endothelial cells - BSA bovine serum albumin - HBSS Hanks' balanced salt solution - FMLP N-formylmethionyl-leucyl-phenylalanine - TPA tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate - TNF tumor necrosis factor Send offprint requests to E. Dejana at the above address     

Glucuronidation of 3'-azido-3'-deoxythymidine catalyzed by human liver UDP-glucuronosyltransferase. Significance of nucleoside hydrophobicity and inhibition by xenobiotics

The enzymatic glucuronidation of 3'-azido-3'-deoxythymidine (AZT) catalyzed by human liver microsomal UDP-glucuronosyltransferase (EC 2.4.1.17, UDPGT) was inhibited by a number of nucleoside analogs. The inhibitory potency of these nucleoside analogs correlated with their hydrophobicity (r2 = 0.90, N = 13). Since similar results were obtained with solubilized UDPGT (r2 = 0.87, N = 7), the affinity of the nucleosides for UDPGT was probably being assessed rather than the ability of the compounds to access the membrane-bound enzyme. Three homologous inhibitors, 3'-azido-2',3'-dideoxyuridine (AzddU), 5-ethyl-AzddU, and 5-propyl-AzddU, were also studied as substrates of UDPGT. The substrate efficiency (Vmax/Km) of these three compounds and AZT also correlated with their hydrophobicity (r2 = 0.94). Sixteen drugs that are structurally unrelated to nucleosides also inhibited the glucuronidation of AZT. The mechanism of inhibition was competitive for seven compounds tested. Ki values were estimated from Dixon plots for nine other less soluble inhibitors; their mechanism of inhibition was assumed to be competitive. Since the peak physiological drug concentrations of the tested inhibitors are considerably less than their Ki values, none of these compounds are expected to strongly inhibit AZT glucuronidation in humans. However, the rank order of these drugs with respect to their inhibitory potential is probenecid greater than chrloramphenicol greater than naproxen greater than phenylbutazone much greater than other drugs tested.     

In vitro effects of an immunostimulating bacterial lysate on human lymphocyte function

MLBL is an oral immunostimulating vaccine consisting of bacterial standardized lysates obtained by mechanical lysis of different strains of Gram-positive and Gram-negative bacteria that can cause acute and chronic infections of the respiratory tract. Previous studies suggested a stimulating effect of MLBL both on humoral and cellular immune responses. In the present study, the in vitro effects of MLBL on human lymphocyte effector functions and its mechanisms of action were evaluated. The results show that the most remarkable effects of MLBL on the immune system are: i) activation of the IL-2 receptor (IL-2Ralpha) on different lymphocyte subsets (B, CD4+ T and CD8+ T cells) involved both in humoral and cellular immune responses; ii) induction of cytokine synthesis (IL-2, IL-10, IL-12, IFNgamma) in the immune competent cells that induce and regulate immune responses; iii) generation of CD4+ and CD8+ effector T cells. Overall, these results suggest that the therapeutic effect of MLBL on acute and recurrent infections of the respiratory tract is related to its ability to activate the responses of different subsets of immune competent cells both for humoral and cellular immunity. Moreover, these effects can be induced either by direct immune cell activation or through the generation and activation of immune effector cells.     

Glucuronidation of 3'-azido-3'-deoxythymidine: human and rat enzyme specificity

Since preclinical studies indicated that 3'-azido-3'-deoxythymidine (AZT, zidovudine, Retrovir, BW A509U), a potent anti-HIV agent, is not metabolized extensively in rats, rabbits, mice, guinea pigs, cats, or dogs, the extensive biotransformation of AZT observed in humans was not expected. On average, approximately 75% of an oral AZT dose is recovered in human urine as a single metabolite while only 14-18% of the dose is recovered unchanged. Ultraviolet, infrared, nuclear magnetic resonance, and mass spectra and enzymatic degradation characterized the isolated major metabolite as a 5'-O-glucuronide (3'-azido-3'-deoxy-5'-beta-D-glucopyranuronosylthymidine, GAZT), a very unique nucleoside metabolite. These observations suggest that UDP-glucuronosyltransferase (UDPGT), EC2.4.1.17, mediates the in vivo biotransformation of AZT to GAZT. Since glucuronidation is one of the major conjugation reactions involved in the metabolic conversion of xenobiotics to more polar, water-soluble metabolites, it is an important detoxification pathway in humans. Therefore, it is important to understand the enzymatic basis for the discrepancy between metabolism of AZT in laboratory mammals and humans. This is especially relevant in light of the use of laboratory mammals to predict the metabolism of novel pharmaceutical agents in humans. The study presented herein confirms that liver UDPGT does catalyze the glucuronidation of AZT and that the higher substrate efficiency of AZT with human enzyme compared to rodent enzyme may account for metabolic differences observed in vivo.     

Glucuronidation of 3'-azido-3'-deoxythymidine by rat and human liver microsomes

The glucuronidation of 3'-azido-3'-deoxythymidine (AZT) by rat and human liver microsomes has been studied in vitro. The AZT-glucuronide was preliminarily identified through specific hydrolysis by beta-glucuronidase and rigorous product identification was performed by high-field proton nuclear magnetic resonance and fast-atom-bombardment mass spectrometry. A beta-linked 5'-O-glucuronide was the exclusive product formed in liver microsomes. Rat and human liver microsomal uridine 5'-diphosphoglucuronyltransferase activities toward AZT were investigated. These studies revealed that AZT had a lower Km and a 5-6-fold higher relative catalytic efficiency for uridine 5'-diphosphoglucuronyltransferase in human as compared to rat liver microsomes which may play a role in the quantitative differences observed in the degree of AZT glucuronidation between rat and human.     

3',3'-Difluoro-3'-deoxythymidine: comparison of anti-HIV activity to 3'-fluoro-3'-deoxythymidine.

3',3'-Difluoro-3'-deoxythymidine (3) has been synthesized in four steps from thymidine, and characterized by 1H NMR and NOE experiments. The JHF coupling constants support a conformation in solution that is predominantly 2'-endo (S). Although conformationally and sterically nucleoside 3 may resemble other thymidine analogs which are active against HIV-1, 3 is virtually inactive.     

Aphidicolin inhibition of gamma-radiation-induced DNA repair in human lymphocyte subpopulations

1. DNA repair was measured in 3 Gy gamma-irradiated human peripheral lymphocyte subpopulations by means of nucleoid sedimentation. 2. The influence of aphidicolin (an inhibitor of DNA polymerase) on the repair process was investigated. 3. Repair of 40-44% of the DNA lesions induced by gamma-irradiation was blocked by aphidicolin. 4. Enriched B- and T-lymphocyte fractions were affected by aphidicolin to the same extent.     

Influence of beta-adrenoceptor blocking agents on lymphocyte function in vitro

1 In order to assess whether beta-adrenoceptor blocking agents may have an immunomodulatory influence, the in vitro effect of propranolol metoprolol and atenolol on mitogenic activation of human peripheral blood lymphocytes was studied. To investigate further the mechanism of this influence, the effects of quinidine and two other membrane stabilizing agents were examined. 2 At 100 and 1000 mumol/l, both (+)-propranolol, (-)-propranolol and (+/-)-propranolol evoked a strong reduction of mitogenic lymphocyte activation. A similar effect was obtained with quinidine. Even at 1000 mumol/l metoprolol, had only a slight suppressive effect and atenolol had no suppressive influence at all. 3 It appears that propranolol may suppress mitogenic activation of lymphocytes, and that the effect may be ascribed to membrane stabilization rather than to beta-adrenoceptor blockade.     

3'-azido-3'-deoxythymidine drug interactions. Screening for inhibitors in human liver microsomes.

Zidovudine is a widely used antiretroviral drug active against human immunodeficiency virus. The drug interactions of this compound, which are primarily eliminated as a glucuronide, have not yet been extensively studied. Because zidovudine is frequently combined with other drugs, complete knowledge of interactions is essential to optimize AIDS therapy. We therefore screened the effect of 55 molecules, representative of 20 different therapeutic classes, on 3'-azido-3'-deoxythymidine (AZT) glucuronidation by human liver microsomes. We demonstrate that many drugs caused more than 15% inhibition of AZT glucuronidation in vitro, whereas major antibiotics (ceftazidine, isoniazid, aminoglycosides, macrolides, and sulfamides), antivirals (2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and acyclovir), flucytosine, metronidazole, acetaminophen, and ranitidine had no effect. For compounds that appeared to inhibit AZT glucuronidation, extrapolation to the clinical situation must take into account both the in vitro apparent Ki values and the usual expected plasma level for the coadministered drug. By considering these parameters, this work indicates that clinically relevant inhibition of AZT glucuronidation may be observed with the following drugs: cefoperazone, penicillin G, amoxicilin, piperacillin, chloramphenicol, vancomycin, miconazole, rifampicin, phenobarbital, carbamazepine, phenytoin, valproic acid, quinidine, phenylbutazone, ketoprofen, probenecid, and propofol. Complementary clinical and pharmacokinetic studies should be performed to validate these assumptions.     

Effect of praziquantel on human lymphocyte proliferation in vitro

Summary The antischistosomal drugs tartar emetic and niridazole exert immunosuppression both in vitro and in vivo. In the present study the influence of praziquantel (Biltricide), a potent schistosomicidal drug, on human lymphocyte proliferation in vitro was investigated. Praziquantel 80 µg/ml significantly reduced thymidine incorporation by human blood mononuclear cells stimulated with mitogens and antigens, whereas no effect could be detected in concentrations below 40 µg/ml. Thus, praziquantel, in concentrations as high as 30 times the peak serum concentration during therapy, has no suppressive effect on human lymphocyte proliferation in vitro.