Metabolism of Zidovudine

1. The anti-HIV drug zidovudine (3'-azido-2',3'-dideoxythymidine; ZDV) has three important pathways of metabolism. ZDV is a prodrug and must be phosphorylated in lymphocytes in order to exert its antiviral action. However, in quantitative terms this is a minor pathway probably accounting for less than 1% of the overall metabolic profile. The predominant pathway of metabolism is glucuronidation to GZDV and the metabolite is renally excreted. A further metabolite, derived by reduction of the azido moiety is 3'-amino-3'-deoxythymidine (AMT). 2. Zidovudine glucuronidation has been characterised in human liver microsomes. A number of drugs (e.g., naproxen, indomethacin and probenecid) have been shown to inhibit the in vitro conjugation of ZDV. Some of these drugs have also been co-administered with ZDV in HIV-positive patients. Significant pharmacokinetic interactions have been demonstrated with probenecid, naproxen and fluconazole. 3. 3'-amino-3'-deoxythymidine formation is probably mediated by both cytochrome P450 isozymes and NADPH-cytochrome P450 reductase. Peak plasma concentrations of AMT are approximately 10-15% of ZDV in patients. This is a potentially important metabolite because of its alleged cytotoxicity. 4. Measurement of intracellular ZDV phosphates in patients provides the key to our understanding of both the efficacy and toxicity of ZDV. Important recent work has demonstrated that as patients deteriorate (i.e., CD4 counts decrease below 100 x 10(6)/L), there is a corresponding increase in intracellular ZDV-monophosphate. This could have toxicological implications.     

CTLA4 mediates antigen-specific apoptosis of human T cells

The regulation of T cell-mediated immune responses requires a balance between amplification and generation of effector function and subsequent selective termination by clonal deletion. Although apoptosis of previously activated T cells can be induced by signaling of the tumor necrosis factor receptor family, these molecules do not appear to regulate T-cell clonal deletion in an antigen-specific fashion. We demonstrate that cross-linking of the inducible T-cell surface molecule CTLA4 can mediate apoptosis of previously activated human T lymphocytes. This function appears to be antigen-restricted, since a concomitant signal T-cell receptor signal is required. Regulation of this pathway may provide a novel therapeutic strategy to delete antigen-specific activated T cells.     

DNA-Directed alkylating agents. 7. Synthesis, DNA interaction, and antitumor activity of bis(hydroxymethyl)- and bis(carbamate)-substituted pyrrolizines and imidazoles

A series of bis(hydroxymethyl)-substituted imidazoles, thioimidazoles, and pyrrolizines and related bis(carbamates), linked to either 9-anilinoacridine (intercalating) or 4-(4-quinolinylamino)benzamide (minor groove binding) carriers, were synthesized and evaluated for sequence-specific DNA alkylation and cytotoxicity. The imidazole and thioimidazole analogues were prepared by initial synthesis of [(4-aminophenyl)alkyl]imidazole-, thioimidazole-, or pyrrolizine dicarboxylates, coupling of these with the desired carrier, and reduction to give the required bis(hydroxymethyl) alkylating moiety. The pyrrolizines were the most reactive alkylators, followed by the thioimidazoles, while the imidazoles were unreactive. The pyrrolizines and some of the thioimidazoles cross-linked DNA, as measured by agarose gel electrophoresis. Strand cleavage assays showed that none of the compounds reacted at purine N7 or N3 sites in the gpt region of the plasmid gpt2Eco, but the polymerase stop assay showed patterns of G-alkylation in C-rich regions. The corresponding thioimidazole bis(carbamates) were more selective than the bis(hydroxymethyl) pyrrolizines, with high-intensity bands at 5'-NCCN, 5'-NGCN and 5'-NCGN sequences in the PCR stopping assay ( indicates block sites). The data suggest that these targeted compounds, like the known thioimidazole bis(carbamate) carmethizole, alkylate exclusively at guanine residues via the 2-amino group, with little or no alkylation at N3 and N7 guanine or adenine sites. The cytotoxicities of the compounds correlated broadly with their reactivities, with the bis(hydroxymethyl)imidazoles being the least cytotoxic (IC50s >1 microM; P388 leukemia) and with the intercalator-linked analogues being more cytotoxic than the corresponding minor-groove-targeted ones. This was true also for the more reactive thioimidazole bis(carbamates) (IC50s 0.8 and 11 microM, respectively), but both were more active than the analogous "untargeted" carmethizole (IC50 20 microM). The bis(hydroxymethyl)pyrrolizine analogues were the most cytotoxic, with IC50s as low as 0.03 microM.     

Sex and age differences in pulmonary mechanics in normal nonsmoking subjects

This study was performed to determine whether stimulation of the carotid chemoreceptors increases total or regional cerebral blood flow and whether activation of arterial chemoreceptors contributes to cerebral vasodilation during systemic hypoxemia. In anesthetized and ventilated dogs, carotid chemoreceptors were stimulated with nicotine or hypoxic and hypercapnic blood. To measure total and regional cerebral blood flow, we used labeled 15-mu microspheres. Stimulation of chemoreceptors did not increase cerebral blood flow or produce significant redistribution of cerebral blood flow, even though the chemoreflex was intact in these animals (as manifested by vasoconstriction in muscle, kidney, and small bowel) and the cerebral vessels dilated in response to systemic hypercapnia. In other studies in anesthetized, ventilated dogs and rhesus monkeys, cerebral vasodilator responses to systemic hypoxemia were observed before and after denervation of carotid and aortic chemoreceptors. Systemic hypoxemia produced large and equivalent increases in cerebral blood flow before and after chemodenervation. We conclude that stimulation of carotid chemoreceptors does not produce cerebral vasodilation and that chemoreceptors do not contribute significantly to cerebral vasodilation during systemic hypoxemia.     

The influence of CSF calcium and magnesium on the ventilatory response to carbon dioxide during hyperoxia

Respiratory responses to inhaled carbon dioxide were measured in anaesthetized cats during perfusion of the ventriculocisternal system with artificial cerebrospinal fluid. A study was performed to evaluate the effect of changes in the magnesium and/or calcium concentration of the CSF on the CO2 response curve which was described as VE = S (PCSF, CO2 -- B). A decrease of S was observed when the magnesium concentration of the perfusion fluid was increased; the B-value remaining the same. The reverse was true down to magnesium concentrations of 0.6 mmol-1-1. Below this concentration S remained the same or decreased; the B-value was lowered. When both the calcium and magnesium concentrations of the CSF were changed, the relation between S and these concentrations could be described as to be proportional to CCAa-CMgb. The effect of changes in the calcium concentration was much more pronounced than comparable changes of the magnesium concentration as reflected by the magnitude of the exponents a and b which were found to be -2.80 (S.D. 0.11) and -0.60 (S.D. 0.03) respectively.     

Binding of radioactively labeled carboxyatractyloside, atractyloside and bongkrekic acid to the ADP translocator of potato mitochondria

1. The inhibition of the ADP-stimulated respiration of potato mitochondria by carboxyatractyloside is relieved by high concentration of ADP or by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Atractyloside is a much less potent inhibitor than carboxyatractyloside. The inhibition of the ADP-stimulated respiration required about 60-times more atractyloside than carboxyatractyloside. 2. [35S]carboxyatractyloside and [3H]bongkrekic acid bind to potato mitochondria with high affinity (Kd = 10 to 20 nM, n=0.6-0.7 nmol per mg protein). Added ADP competes with carboxyatractyloside for binding; on the contrary ADP increases the amount of bound bongkrekic acid. [3H]atractyloside binds to potato mitochondria with a much lower affinity (Kd=0.45 muM) than carboxyatractyloside or bongkrekic acid. 3. Bound [3H]atractyloside is displaced by ADP, carboxyatractyloside and bongkrekic acid. The displacement of bound [35S]carboxyatractyloside by bongkrekic acid and of bound [3H]bongkrekic acid by carboxyatractyloside is markedly increased by ADP. 4. Bongkrekic acid competes with [35S]carboxyatractyloside for binding. Addition of a small concentration of ADP considerably enhances the inhibitory effect of bongkrekic acid on [35S]carboxyatractyloside binding. 5. The adenine nucleotide content of potato mitochondria is of the order of 1 nmol per mg protein. ADP transport in potato mitochondria is inhibited by atractyloside 30- to 40-times less efficiently than by carboxyatractyloside.