Stereoelectronic factors in the binding of substrate analogues and inhibitors to purine nucleoside phosphorylase isolated from human erythrocytes.

Several aspects of the stereoelectronic requirements of substrates of human erythrocytic purine nucleoside phosphorylase (E.C. 2.4.2.1) were elucidated providing the following information: (a) the N1 position cannot have a nonhydrogen substituent; (b) the 5'-OH group must be present for catalytic activity to be exhibited but is not an essential functional group for inhibitory action to be observed; (c) on the C8 position groups larger than -NH2 or -Br cannot be accommodated; (d) the syn-glycosyl conformation (i.e., 8-bromoguanosine) is acceptable but may not be an absolute requirement for phosphorolysis; (e) among nucleic base inhibitors methylation at N3, N7, or N9 vastly decreases the inhibitory properties as does a nitrogen in lieu of C-H in the 8 position. The results clearly indicate that this enzyme differs in its stereoelectronic requirements from the Escherichia coli enzyme.

The efflux of purine nucleobases and nucleosides from the rat brain.

The efflux of purine nucleobases and their nucleosides from the rat brain was investigated using the brain efflux index (BEI) method. Calculated BEI values showed that purine nucleobases had very rapid initial efflux after the intracerebral injection, which was followed by the slower efflux due to the intracellular trapping of labelled molecules and confirmed by the capillary depletion technique. The efflux of ribonucleosides was much slower than the efflux of nucleobases and the structure of the sugar moiety seemed to be important, since a significant difference in the efflux velocity between ribo- and deoxyribonucleosides was observed. The results of self- and cross-inhibition studies suggested that the efflux of test molecules was saturable and that purines shared the same transport system on the abluminal side of the blood-brain barrier.

Spectrophotometric determination of the cardioprotective drug GP531 in human plasma.

A simple spectrophotometric assay has been developed for determining concentrations of the new cardioprotective agent, GP531, in human plasma. The method is adapted from the Bratton-Marshall assay (BMA) for the detection of primary aromatic amines. The assay can be used to measure plasma concentrations of the drug during i.v. infusion administration to patients. The limit of quantitation of the assay is 1 microgram/ml using a 0.8 ml sample ultrafiltrate of plasma. The colorimetric assay correlates well with a previously described high performance liquid chromatographic (HPLC) procedure.

A sensitive assay for the aminoimidazole-containing drug GP531 in plasma using liquid chromatography with amperometric electrochemical detection: a new class of electroactive compounds.

Aminoimidazole-containing compounds have been found to be electroactive and can be detected by amperometric electrochemical detection (ECD) with a high degree of sensitivity. A liquid chromatography (LC) method using ECD was developed for measuring plasma concentrations of the aminoimidazole-containing drug GP531, a potent adenosine-regulating agent. Plasma samples were extracted with 2-propanol and analyzed by LC under isocratic conditions using a mobile phase of methanol-sodium phosphate (pH 6.3; 3.3 mM) (32:68, v/v). The potential of the glassy carbon working electrode was set at +800 mV. The limit of quantitation was 12.5 ng ml-1 of GP531 using 100 microliters of plasma. The method was used to define the pharmacokinetics of GP531 in monkey following i.v. administration.

Development and validation of an assay for the simultaneous determination of zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma using liquid chromatography-tandem mass spectrometry.

This paper describes the development and validation of an assay for the simultaneous quantification of the antiviral and antiretroviral drugs zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma using liquid chromatography coupled to tandem mass spectrometry. Sample pretreatment consisted of protein precipitation with 0.1% (v/v) formic acid in methanol, evaporation and reconstitution. Chromatographic separation was performed on a Synergy Polar reversed phase C18 column (150 mm × 2.0 mm ID, particle size 4 µm) using a stepwise gradient with 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in methanol at a flow rate of 300 µL/min. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for drug detection and quantification. Isotopically labeled zidovudine, lamivudine, tenofovir and ribavirin were used as internal standards. The method was validated over a clinical range of 20-2500 ng/mL for zidovudine, lamivudine and tenofovir, 4-500 ng/mL for abacavir and emtricitabine and 160-20,000 ng/mL for ribavirin. The inter and intra-assay accuracies and precisions were between -8.47% and 14.2% for zidovudine, emtricitabine and ribavirin. For abacavir, lamivudine and tenofovir, the inter and intra-assay accuracies and precisions at the lower limit of quantification were between -11.0% and 18.3%, whereas at all other levels these accuracies and precisions were between -11.7% and 12.0%. The described method is suitable for the determination of zidovudine, abacavir, emtricitabine, lamivudine, tenofovir and ribavirin in human plasma in clinical practice to monitor plasma concentrations in selected cases to optimize therapy.

Pyrimidine dideoxyribonucleosides: selectivity of penetration into cerebrospinal fluid.

Cerebrospinal fluid (CSF)/plasma ratios of 1 to 30% were obtained in rhesus monkeys for the 3'-azido- and 2',3'-dideoxy-analogs of thymidine, deoxycytidine and deoxyuridine. Penetration of thymidine and deoxyuridine analogs was much greater than for deoxycytidine analogs. Octanol/buffer partition coefficients varied more than 30-fold, but did not correlate with CSF entry. Plasma protein binding was insignificant for all compounds. The presence or absence of the azido group at position 3' did not appear to influence the extent of CSF penetration. Although we do not fully understand the mechanistic basis for the penetration of these nucleosides into the CSF, it is apparent that the structural specificity is related more closely to the nucleobase than the sugar. Based upon elimination rates from the CSF after direct intrathecal injection, the differences in net penetration are determined by influx rather than efflux processes.

[Possibility of biological indication of radiation injury from the blood levels of deoxynucleosides and deoxynucleotides in combined radiation-mechanical injuries]. / Vozmozhnost' biologicheskoi indikatsii luchevogo porazheniia po soderzhaniiu dezoksinukleozidov i dezoksinukleotidov v krovi pri radiatsionno-mekhanicheskom porazhenii.

A study was made of the dependence of a total content of deoxynucleosides and deoxynucleotides in blood serum and blood clot leukocytes upon radiation dose and time lapsed after radiation- and radiation-mechanical affection. It was established that after radiation-mechanical affection this dependence was different from that observed after the effect of radiation alone. From the analysis of the data obtained it was inferred that deoxynucleosides and deoxynucleotides could be used for biological indication of radiation damage in conditions of radiation-mechanical affection.

Determination of ribonucleosides, deoxyribonucleosides, and purine and pyrimidine bases in adult rabbit cerebrospinal fluid and plasma.

Purine and pyrimidine base and nucleoside levels were measured in adult rabbit cisternal CSF and plasma by reversed-phase high-performance liquid chromatography. The concentrations of bases, nucleosides, and nucleoside phosphates were similar in plasma and CSF except for the adenosine phosphates and uracil which were higher in the plasma. In plasma and CSF, adenosine levels were low (0.12 microM) and guanosine, deoxyadenosine, deoxyguanosine, and deoxyinosine were not detectable (less than 0.1 microM); inosine and xanthine concentrations were 1-2 microM and hypoxanthine concentrations were approximately 5 microM; uridine (approximately 8 microM), cytidine (2-3 microM), and thymidine, deoxyuridine, and deoxycytidine (0.5-1.4 microM) were easily detectable. In both plasma and CSF, guanine, and thymine were undetectable (less than 0.1 microM), adenine and cytosine were less than 0.2 microM, but uracil was present (greater than 1 microM). Adenosine, inosine, and guanosine phosphates were also detectable at low concentrations in CSF and plasma. These results are consistent with the hypothesis that purine deoxyribonucleosides are synthesized in situ in the adult rabbit brain. In contrast, pyrimidine deoxyribonucleosides and ribonucleosides, and purine and pyrimidine bases are available in the CSF for use by the brain.

High-performance liquid chromatographic determination of purine and pyrimidine bases, ribonucleosides, deoxyribonucleosides and cyclic ribonucleotides in biological fluids.

A method is presented for the separation and quantitative determination of compounds normally related to purine and pyrimidine metabolism in biological material. The retention behaviour of nucleobases, ribonucleosides, deoxyribonucleosides and cyclic ribonucleotides has been systematically investigated by reversed-phase high-performance liquid chromatography using a non-linear gradient. Ultimately a separation of the purine and pyrimidine compounds was achieved in a 35-min run with an average detection limit of 5-10 pmol per injection. Recoveries of standards added to urine, plasma or serum were 96 +/- 5%.

Effect of iron deficiency and desferrioxamine on DNA synthesis in human cells.

Desferrioxamine (10(-3) M) caused a fall in the deoxyadenosine triphosphate level after 4 h incubation in normal phytohaemagglutinin-stimulated lymphocytes. There was a rise in the concentrations of the other three deoxyribonucleoside triphosphates (deoxythymidine-,deoxycytidine-and deoxyguanosine-triphosphate). The changes are similar to those caused by hydroxyurea, a known inhibitor of ribonucleotide reductase. Desferrioxamine (10(-3 M) was found to inhibit human lymphocyte ribonucleotide reductase to a mean of 11% of control activity after 45 min incubation. Both drugs, desferrioxamine and hydroxyurea, inhibited incorporation of [3H]thymidine DNA into lymphocytes in the presence or absence of deoxyuridine, and inhibited production of lymphocytic thymidine kinase, having opposite effects to methotrexate on both [3H]thymidine incorporation and thymidine kinase activity. Phytohaemagglutinin-stimulated lymphocytes from patients with chronic iron deficiency showed lower levels of all our deoxyribonucleoside triphosphates than normal lymphocytes. It is suggested that this may be due to reduced ribnucleotide reductase activity of the iron-deficient cells.