Metabolism and DNA interaction of 2',3'-didehydro-2',3'-dideoxythymidine in human bone marrow cells.

2',3'-Didehydro-2',3'-dideoxythymidine (D4T) is a potent inhibitor of human immunodeficiency virus (HIV), with low hematological toxicity. In the present study, the cellular pharmacology of D4T was investigated in human bone marrow cells (BMC), in an attempt to understand the mechanism of the observed low bone marrow toxicity. After exposure of human BMC to 10 microM [3H]D4T for 24 hr, D4T-5'-triphosphate (D4T-TP) was the predominant metabolite, reaching a concentration of 0.3 pmol/10(6) cells. The D4T-5'-monophosphate levels were slightly lower, whereas the D4T-5'-diphosphate levels were about 6-fold lower than those of D4T-TP at 24 hr. Nucleic acids of human BMC exposed to 10 microM [3H]D4T for 24 hr were purified and analyzed by cesium sulfate density gradient centrifugation. No radioactivity was detected in the RNA region, whereas a limited amount was associated with the DNA region. The amount of label incorporated into DNA correlated with the extracellular D4T concentration and the length of incubation time. Enzymatic hydrolysis of radiolabeled DNA and subsequent analysis by high performance liquid chromatography demonstrated incorporation of both D4T and thymidine (dThd) into DNA. Degradation of D4T to thymine and subsequent formation of labeled dThd was also detected in human BMC. Pulse (24 hr)-chase (48 hr) experiments with 10 microM [3H]D4T demonstrated that the amount of radiolabel from D4T in DNA decreased over time during the chase. Under similar conditions, [3H]3'-azido-3'-deoxythymidine (AZT) incorporated into DNA of human BMC did not decrease during the chase. Although D4T-TP standard was demonstrated to be unstable at 37 degrees and neutral pH, D4T was much more stable in solution when incorporated into newly synthesized DNA isolated from human BMC, suggesting that enzymatic excision may be the mechanism for D4T removal from DNA. In summary, although higher concentrations of D4T-TP, compared with AZT-5'-triphosphate, are observed in human BMC, after exposure of cells to similar extracellular concentrations of parent drug, steady state levels of D4T incorporated into DNA are 10-50-fold lower, compared with AZT. Competition with dTTP formed by D4T metabolism and excision of D4T from DNA may be responsible, in part, for these effects. This study further demonstrates that incorporation of 2',3'-dideoxynucleosides into nuclear DNA of human BMC may be related to the ability of these anti-HIV agents to induce hematological side effects.     

Assessment of the effect of phosphorylated metabolites of anti-human immunodeficiency virus and anti-hepatitis B virus pyrimidine analogs on the behavior of human deoxycytidylate deaminase

Deoxycytidylate deaminase, catalyzing the conversion of dCMP to dUMP, is an important enzyme in the de novo synthesis of thymidine nucleotides. It also may be involved in the action, as well as the metabolism of anticancer agents. Recently, several L- and D-configuration pyrimidine deoxynucleoside analogs were found to be potent antiviral and antitumor agents. Their interaction with dCMP deaminase as a monophosphate or a triphosphate metabolite is not clear. These include D-nucleoside analogs such as beta-D-2',3'-dideoxycytidine (ddC), beta-2'-fluoro-5-methyl-arabinofuranosyluracil (FMAU), 3'-azido-2',3'-dideoxythymidine (AZT), and 2',3'-didehydro-2',3'-dideoxythymidine (D4T) as well as L-nucleoside analogs such as beta-L-dioxolane-cytidine (L-OddC), beta-L-2',3'-dideoxy-3'-thiacytidine, beta-L-2',3'-dideoxy-5'-fluoro-3'-thia-cytidine (L-FSddC), beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine, and L-FMAU. None of the L-deoxycytidine analog monophosphates act as substrates or inhibitors. Among these pyrimidine deoxynucleoside analog monophosphates, D-FMAU monophosphate (MP) is the most potent competitive inhibitor, whereas L-FMAUMP has no inhibitory activity. Interestingly, AZTMP and D4TMP also have potent inhibitory activities on dCMP deaminase. Among the dCTP and TTP analogs examined, D- and L-FMAUTP were the most potent inhibitors and had the same extent of inhibitory effect. These results suggest that a chiral specificity for the substrate-binding site may exist, but there is no chiral specificity for the regulator-binding site. This is also supported by the observation that L-OddC and L-FSddC have inhibitory activities as triphosphates but not as monophosphates. None of the D- and L-dCTP analogs activated dCMP deaminase as dCTP. The biological activities of AZT and D4T could be partially attributable to their inhibitory activity against dCMP deaminase by their phosphorylated metabolites, whereas that of ddC and the L-deoxycytidine analogs may not involve dCMP deaminase directly.     

NDP kinase reactivity towards 3TC nucleotides

Nucleoside diphosphate (NDP) kinase is usually considered as the enzyme responsible for the last step of the cellular phosphorylation pathway leading to the synthesis of biologically active triphospho-derivatives of nucleoside analogs used in antiviral therapies and in particular in the treatment of AIDS. NDP kinase lacks specificity for the nucleobase and can use as substrate both ribo- or 2'-deoxyribonucleotides. However, only nucleoside analogs with a sugar moiety in the D-configuration (e.g. 3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T)) have so far been analyzed as substrates of NDP kinase. In contrast, beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), also called lamivudine, is a nucleoside analog that is now widely used in AIDS therapy and has a sugar moiety in the L-configuration. Using protein fluorescence to monitor the phosphotransfer between the enzyme and the nucleotide derivative at the presteady state, we have studied the reactivity of 3TC triphosphate and of other L-dideoxynucleotides with NDP kinase. We found that L-dideoxynucleoside triphosphates have a poor affinity for NDP kinase and that the catalytic efficiency of the phosphorylation of L-dideoxyderivatives is very low as compared with their D-enantiomers. We discuss these results using a computer model of 3TC diphosphate bound to the NDP kinase active site. NDP kinase may not seem to be the major enzyme phosphorylating 3TC-DP, in contrast to current opinion.     

Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5

The human multidrug resistance proteins MRP4 and MRP5 are organic anion transporters that have the unusual ability to transport cyclic nucleotides and some nucleoside monophosphate analogs. Base and nucleoside analogs used in the chemotherapy of cancer and viral infections are potential substrates. To assess the possible contribution of MRP4 and MRP5 to resistance against these drugs, we have investigated the transport mediated by MRP4 and MRP5. In cytotoxicity assays, MRP4 conferred resistance to the antiviral agent 9-(2-phosphonomethoxyethyl)adenine (PMEA) and high-performance liquid chromatography analysis showed that, like MRP5, MRP4 transported PMEA in an unmodified form. MRP4 also mediated substantial resistance against other acyclic nucleoside phosphonates, whereas MRP5 did not. Apart from low-level MRP4-mediated cladribine resistance, the cytotoxicity of clinically used anticancer nucleosides was not influenced by overexpression of MRP4 or MRP5. In contrast, MRP5 mediated efflux of the pyrimidine-based antiviral 2',3'-dideoxynucleoside 2',3'-didehydro-2',3'-dideoxythymidine 5'-monophosphate (d4TMP) and its phosphoramidate derivative alaninyl-d4TMP from cells loaded with the 2',3'-didehydro-2',3'-dideoxythymidine prodrugs cyclosaligenyl-d4TMP and aryloxyphosphoramidate d4TMP (So324), respectively. Moreover, only inside-out membrane vesicles derived from MRP5-overexpressing cells accumulated alaninyl-d4TMP. Cellular efflux and vesicular uptake studies were carried out to further compare transport mediated by MRP4 and MRP5 and showed that dipyridamole, dilazep, nitrobenzyl mercaptopurine riboside, sildenafil, trequinsin and MK571 inhibited MRP4 more than MRP5, whereas cyclic nucleotides and monophosphorylated nucleoside analogs were equally poor inhibitors of both pumps. These results strongly suggest that the affinity of MRP4 and MRP5 for nucleotide-based substrates is low.     

Fluorinated sugar analogues of potential anti-HIV-1 nucleosides

In order to obtain agents with therapeutic indices superior to those of AZT, FLT, or D4T, several analogues of anti-HIV-1 nucleosides were synthesized. These include 2',3'-dideoxy-2',3' -difluoro-5-methyluridine (13), its arabino analogue 19, arabino-5-methylcytosine analogue 21, 3'-deoxy-2',3'-didehydro-2' -fluorothymidine (25), 3'-azido-2',3'-dideoxy-2'-fluoro-5-methyluridine (29), 2'-azido-3'-fluoro-2',3'-dideoxy-5-methyluridine (31), and 2'3'-dideoxy-2' -fluoro-5-methyluridine (37). These new nucleosides were screened for their activity against HIV and feline TLV in vitro. None of the compounds showed significant activity. It is interesting to note that such a small modification in the sugar moiety of active anti-HIV nucleosides (i.e., displacement of hydrogen by fluorine) almost completely inactivate the agents.     

Characterization of the activation pathway of phosphoramidate triester prodrugs of stavudine and zidovudine.

The phosphoramidate triester prodrugs of anti-human HIV 2', 3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-monophosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2', 3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metabolism of triester prodrugs of d4T and AZT using pure enzymes or different biological media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-containing intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme preparations. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alkaline phosphatase, and phosphodiesterase.     

Suppression of murine retroviral disease by 2',3'-didehydro-2',3'-dideoxythymidine (D4T).

The thymidine analog, 2',3'-didehydro-2',3'-dideoxythymidine (D4T), and 3'-azido-3'-deoxythymidine (AZT) were evaluated for activity against Friend virus complex (FV) in Mus dunni cells using a focal immunoenzyme assay. The 50% effective doses were, respectively, 1.2 and 0.1 microM for the two compounds; the 50% cytotoxic doses using trypan blue dye exclusion were 25.4 and > 100 microM. Four FV inhibition experiments with D4T were run in F1 hybrid mice containing the Rfv-3r/s genotype. This mouse strain allows the study of treatment effects on development of specific neutralizing antibodies and on splenomegaly, splenic and plasma virus titers, and splenic viral RNA. In the first experiment, D4T was given by oral gavage (p.o.) three times daily (t.i.d.) for 14 days beginning 4 h post-virus inoculation. All dosages used (187.5, 375, 750 mg/kg/day) significantly inhibited all viral parameters. Other experiments used D4T p.o. twice daily, with dosages of 46.9, 93.8, 187.5 and 375 mg/kg/day or four times daily with a dose of 375 mg/kg/day. No significant disease inhibition was seen using the twice daily treatment schedule, but efficacy was apparent using the four times daily treatment. The final experiment repeated the initial study, extending the t.i.d. treatments to 25 days and using dosages of 46.9, 93.8, 187.5 and 375 mg/kg/day. All but the lowest dose reduced each virus parameter. None of the D4T treatment regimens caused death in toxicity controls, although moderate host weight loss or less weight gain was seen, and variable hematocrit decreases occurred, particularly in mice receiving the highest drug dosage. Inhibition of natural killer (NK) cell activity also was seen in these same animals, but in infected mice, FV-induced decrease in NK cell activity was prevented by D4T treatment. Virus-specific neutralizing antibodies developed in all infected, treated animals. These data indicate D4T has potential as a possible candidate for anti-human immunodeficiency virus evaluations in the clinic.