Antiviral activity of low-dose alovudine in antiretroviral-experienced patients: results from a 4-week randomized, double-blind, placebo-controlled dose-ranging trial.

BACKGROUND: Alovudine inhibits replication of highly nucleoside reverse transcriptase inhibitor (NRTI)-resistant HIV strains in vitro. However, dose-dependent safety concerns resulted in its initial development being halted. Recently, a 4-week course of alovudine 7.5 mg/day added to a stavudine-free failing regimen yielded a significant decrease in viral load by -1.88 log(10) HIV-1 RNA copies/mL. The magnitude of the reduction in viral load suggested that lower doses might still be effective while offering adequate safety during long-term use. OBJECTIVE: To determine whether lower dosages of alovudine still provide significant antiviral activity in patients with broad NRTI resistance. METHODS: A randomized, double-blind, placebo-controlled trial investigating three doses of alovudine (0.5, 1 and 2 mg) or placebo added for 4 weeks to a failing regimen in patients with evidence of NRTI-resistant HIV strains [>or=2 thymidine-associated mutations (TAMs)]. The primary endpoint was the mean viral load reduction between baseline and week 4. RESULTS: Seventy-two patients were enrolled in the study: 21, 13, 18 and 20 in the placebo and 0.5, 1 and 2 mg arms, respectively. Baseline median CD4 count and viral load were 298 cells/microL (range 44-692 cells/microL) and 3.9 log(10) copies/mL (range 2.5-5.2 log(10) copies/mL), respectively. Baseline viral isolates harboured a median of four TAMs. Alovudine was added to a median four-drug failing regimen. At week 4, compared with placebo, mean viral load changes were -0.42 log(10) [95% confidence interval (CI) -0.67 to -0.18] and -0.30 log(10) (-0.55 to -0.06) in the 2 and 1 mg arms, respectively. There was no significant change in CD4 cell count. Alovudine was well tolerated. CONCLUSION: A 4-week course of alovudine 2 mg/day provided a modest but significant viral load reduction in patients harbouring viruses with a median of four TAMs.

Thyroid hormones selectively modulate human alcohol dehydrogenase isozyme catalyzed ethanol oxidation.

Thyroid hormones are potent, instantaneous, and reversible inhibitors of ethanol oxidation catalyzed by isozymes of class I and II human alcohol dehydrogenase (ADH). None of the thyroid hormones inhibits class III ADH. At pH 7.40 the apparent Ki values vary between 55 and 110 microM for triiodothyronine, 35 and greater than 200 microM for thyroxine, and 10 and 23 microM for triiodothyroacetic acid. The inhibition is of a mixed type toward both NAD+ and ethanol. The binding of the thyroid hormone triiodothyronine to beta 1 gamma 1 ADH is mutually exclusive with 1,10-phenanthroline, 4-methylpyrazole, and testosterone, identifying a binding site(s) for the thyroid hormones, which overlap(s) both the 1,10-phenanthroline site near the active site zinc atom and the testosterone binding site, the latter being a regulatory site on the gamma-subunit-containing isozymes and distinct from their catalytic site. The inhibition by thyroid hormones may have implications for regulation of ADH catalysis of ethanol and alcohols in the intermediary metabolism of dopamine, norepinephrine, and serotonin and in steroid metabolism. In concert with other hormonal regulators, e.g., testosterone, the rate of ADH catalysis is capable of being fine tuned in accord with both substrate and modulator concentrations.

Human class II (pi) alcohol dehydrogenase has a redox-specific function in norepinephrine metabolism.

Studies of the function of human alcohol dehydrogenase (ADH) have revealed substrates that are virtually unique for class II ADH (pi ADH). It catalyzes the formation of the intermediary glycols of norepinephrine metabolism, 3,4-dihydroxyphenylglycol and 4-hydroxy-3-methoxyphenylglycol, from the corresponding aldehydes 3,4-dihydroxymandelaldehyde and 4-hydroxy-3-methoxymandelaldehyde with Km values of 55 and 120 microM and kcat/Km ratios of 14,000 and 17,000 mM-1 X min-1; these are from 60- to 210-fold higher than those obtained with class I ADH isozymes. The catalytic preference of class II ADH also extends to benzaldehydes. The kcat/Km values for the reduction of benzaldehyde, 3,4-dihydroxybenzaldehyde and 4-hydroxy-3-methoxybenzaldehyde by pi ADH are from 9- to 29-fold higher than those for a class I isozyme, beta 1 gamma 2 ADH. Furthermore, the norepinephrine aldehydes are potent inhibitors of alcohol (ethanol) oxidation by pi ADH. The high catalytic activity of pi ADH-catalyzed reduction of the aldehydes in combination with a possible regulatory function of the aldehydes in the oxidative direction leads to essentially "unidirectional" catalysis by pi ADH. These features and the presence of pi ADH in human liver imply a physiological role for pi ADH in the degradation of circulating epinephrine and norepinephrine.

Human class I alcohol dehydrogenases catalyze the interconversion of alcohols and aldehydes in the metabolism of dopamine.

The class I human liver alcohol dehydrogenases (ADHs) catalyze the interconversion of the intermediary alcohols and aldehydes of dopamine metabolism in vitro, whereas those of the class II and class III do not. The individual, homogeneous class I isozymes oxidize (3,4-dihydroxyphenyl)ethanol and (4-hydroxy-3-methoxyphenyl)ethanol (HMPE) and ethanol with kcat/Km values in the range from 16 to 240 mM-1 min-1 and from 16 to 66 mM-1 min-1, respectively. They reduce the corresponding dopamine aldehydes (3,4-dihydroxyphenyl)acetaldehyde and (4-hydroxy-3-methoxyphenyl)acetaldehyde (HMPAL) with kcat/Km values varying from 7800 to 190,000 mM-1 min-1, considerably more efficient than the reduction of acetaldehyde with kcat/Km values from 780 to 4900 mM-1 min-1. For beta 1 gamma 2 ADH, ethanol competes with HMPE oxidation with a Ki of 23 microM. In addition, 1,10-phenanthroline inhibits HMPE oxidation and HMPAL reduction with Ki values of 20 microM and 12 microM, respectively, both quite similar to that for ethanol, Ki = 22 microM. Thus, both ethanol/acetaldehyde and the dopamine intermediates compete for the same site of ADH, a basis for the ethanol-induced in vivo alterations of dopamine metabolism.

Human alcohol dehydrogenases and serotonin metabolism.

Human liver alcohol dehydrogenases (ADH) may participate in serotonin (5-hydroxytryptamine) metabolism. Class I and II isozymes catalyze the oxidation of 5-hydroxytryptophol (5-HTOL) with kcat/Km values ranging from 10 to 100 mM-1 min-1 compared to 4-66 mM-1 min-1 for that of ethanol at pH 7.40, 25 degrees C. The product, 5-hydroxyindoleacetaldehyde, was purified as its semicarbazone and identified by mass spectrometry. Ethanol competitively inhibits 5-HTOL oxidation by beta 1 gamma 2 ADH with a Ki of 440 microM, a value similar to the Km of ethanol, 210 microM. The inhibition constants for 1,10-phenanthroline and 4-methylpyrazole are 20 microM and 80 nM respectively, essentially identical to those obtained with ethanol as substrate, 22 microM and 70 nM, respectively. The competition between ethanol and 5-HTOL for ADH can explain observations of ethanol induced changes in serotonin metabolism in vivo.

Testosterone allosterically regulates ethanol oxidation by homo- and heterodimeric gamma-subunit-containing isozymes of human alcohol dehydrogenase.

Testosterone and its physiologically active metabolite 5 alpha-dihydrotestosterone are selective, allosteric inhibitors of the gamma subunit-containing isozymes of class I human alcohol dehydrogenase (ADH) with apparent Ki values for testosterone at pH 7.4 between 3.5 and 16 X 10(-6) M. Testosterone inhibition is noncompetitive with respect to ethanol, NAD+, 1,10-phenanthroline, and 4-methylpyrazole, identifying a regulatory site distinct from the catalytic site. Testosterone does not inhibit the class I isozymes composed only of alpha and/or beta subunits and only weakly inhibits the class II and III isozymes. Importantly, none of these human ADH isozymes oxidize or reduce the steroids with the delta 4 double bond or 5 alpha configuration. The allosteric effect of testosterone, restricted to the gamma subunits of human ADH, suggests unique metabolic specificities and pathways for these isozymes, apart from all others. This inhibition may ultimately be critical to an identification of their function(s). Analogous considerations of other metabolic effectors might further lead to similar insights regarding the alpha and beta subunit-containing isozymes as well as the class II and III ADH.

Human class I alcohol dehydrogenases catalyze the oxidation of glycols in the metabolism of norepinephrine.

Investigations of the function of human liver alcohol dehydrogenase (ADH) in norepinephrine metabolism have revealed that class I ADH catalyzes the oxidation of the intermediary alcohols 4-hydroxy-3-methoxyphenyl glycol (HMPG) and 3,4-dihydroxyphenyl glycol (DHPG) in vitro. The kcat/Km values for the individual homogeneous class I isozymes are generally in the range from 2.0 to 10 mM-1 X min-1, slightly lower than those obtained for ethanol oxidation, 16-66 mM-1 X min-1, but considerably higher than those obtained for ethylene glycol oxidation, 0.23-1.5 mM-1 X min-1. Importantly, HMPG and DHPG are not substrates for the class II or class III ADHs. 4-Methylpyrazole and 1,10-phenanthroline inhibit the class I ADH-catalyzed oxidation of HMPG, DHPG, and ethanol with inhibition constants of 75-90 nM and 19-22 microM, respectively, indicating that these substrates interact at the same catalytic site of ADH. Moreover, ethanol inhibits the oxidation of HMPG. The competition of ethanol with HMPG for ADH provides a basis for the in vivo changes observed in norepinephrine metabolism after acute ethanol intake. Any assessment of norepinephrine function through the study of metabolites in peripheral body fluid must include monitoring the oxidation of HMPG by ADH.

Vanillic acid, a metabolite of 4-hydroxy-3-methoxyphenylglycol and 4-hydroxy-3-methoxymandelic acid in man.

4-Hydroxy-3-methoxyphenylglycol (HMPG) labelled with 14C was used to study the metabolic fate of HMPG in six healthy volunteers. Besides conjugation and oxidation to 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) a minor portion, 8.4 +/- 1.1% (mean +/- SEM) was excreted as 14C-labelled vanillic acid (VA). To study if VA was formed from HMPG or HMMA (VMA), deuterium-labelled HMPG [( 2H3]HMPG) and HMMA [( 2H6]HMMA) were simultaneously injected intravenously to seven healthy volunteers. The recovery of [2H3]VA from [2H3]HMPG was 8.3 +/- 2.1% and the recovery of [2H6]VA from [2H6]HMMA was 9.0 +/- 2.1%. The 2H-labelled VAs were probably formed by a decarboxylation reaction, in the case of HMPG after previous oxidation to HMMA.

Norepinephrine metabolism in man using deuterium labelling: origin of 4-hydroxy-3-methoxymandelic acid.

A double isotope labelling technique was used to simultaneously determine the in vivo turnover rates of 4-hydroxy-3-methoxyphenylglycol (HMPG) and 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) and the rate of HMPG oxidation to HMMA. Six healthy men were given intravenous injections of [2H3]HMPG and [2H6]HMMA and their plasma and urine samples analysed by gas chromatography--mass spectrometry (GC/MS) for the protium and deuterium species. HMPG and HMMA production rates were calculated by isotope dilution. The rate of HMPG oxidation to HMMA was obtained from the fraction of [2H3]HMPG recovered as [2H3]HMMA. The results showed that the entire production of HMMA, 1.11 +/- 0.21 mumol/h (mean +/- SE), could be accounted for by oxidation of HMPG, 1.49 +/- 0.31 mumol/h. In another experiment designed to avoid expansion of the HMPG body pool, a tracer dose of [14C]HMPG was given to the same subjects. The levels of [14C]HMPG and [14C]HMMA were measured in urine after extraction and separation by thin layer chromatography. Urinary excretion of endogenous HMPG and HMMA was determined by GC/MS. The results showed that the endogenous HMMA fraction of the total HMPG and HMMA urinary excretion rate, 0.57 +/- 0.04, was the same as the fraction of [14C]HMPG oxidized to [14C]HMMA, 0.62 +/- 0.01. Thus, HMPG is the main intermediate in the metabolic conversion of norepinephrine and epinephrine to HMMA in man.

Further studies on 4-hydroxy-3-methoxyphenylglycol oxidation in humans: effect of pool expansion and stereochemistry.

The in vivo oxidation of the norepinephrine metabolite 4-hydroxy-3-methoxyphenylglycol (HMPG) to 4-hydroxy-3-methoxymandelic acid was studied in man with two different doses of deuterium-labeled HMPG and a tracer dose of [14C]HMPG. HMPG oxidation appeared to be dose-dependent with an oxidation of 62-70% for doses below or equal to 2.2 mumol. With the use of a capillary column coated with an optically active phase (Chirasil-Val) and gas chromatography mass-spectrometry the human urinary excretions of the two stereoisomers of deuterium-labelled HMPG (free + conjugates) were found to be equal.

Norepinephrine metabolism in humans studied by deuterium labelling: turnover of 4-hydroxy-3-methoxyphenylglycol.

D,L(+/-)-4-Hydroxy-3-methoxyphenylglycol (HMPG) labelled with three deuterium atoms was used to study turnover of plasma free HMPG following an intravenous injection. Ten healthy men were given a pulse dose of either 4.3 mumol or 2.2 mumol of labelled HMPG ([2H3]HMPG piperazine salt). Plasma and urine levels of both endogenous and labelled HMPG were subsequently followed by gas chromatography-mass spectrometry with selected ion detection. Kinetic calculations based upon a single-compartment model were consistent with a monoexponential elimination of plasma free HMPG. The half-life of HMPG was 0.46 and 0.78 h (mean values in the two dose groups). The HMPG production rate was 2.01 and 2.35 mumol/hour, and the urinary excretion rate of HMPG (free and conjugated) was 0.48 and 0.47 mumol/h. The endogenous plasma level of free HMPG was 25 and 33 nmol/L. The results show that HMPG turns over rapidly and that HMPG is further metabolized extensively. About one-fourth of the HMPG produced is excreted in urine as free and conjugated HMPG.

Determination of endogenous ethanol in blood and breath by gas chromatography-mass spectrometry.

We describe methods for the determination of endogenous ethanol in biological specimens from healthy abstaining subjects. The analytical methods were headspace gas chromatography (GC) for plasma samples and gas chromatography-mass spectometry (GC/MS) with deuterium labelled species 2H3-ethanol and 2H5-ethanol as internal standards for breath analysis. Ethanol in rebreathed air was about 10% higher than in directly analysed end-expired alveolar air. Known volumes of rebreathed air were passed through a liquid-N2 freeze trap and the volatile constituents of breath were concentrated prior to analysis by GC or GC/MS. Besides endogenous ethanol, peaks were seen on the chromatograms for methanol, acetone and acetaldehyde as well as several as yet unidentified substances. The endogenous alcohols ethanol and methanol were confirmed from their mass chromatograms and the GC/MS profile also indicated the presence of endogenous propan-1-ol. The concentration of endogenous ethanol in plasma showed wide inter-subject variations ranging from below detection limits to 1.6 micrograms/ml (34.8 mumol/l) and with mean +/- SD of 0.39 +/- 0.45 micrograms/ml (8.5 +/- 9.8 mumol/l). We aim to characterise further the role of endogenous ethanol with the main focus on dynamic aspects such as the rate of formation and turnover.

Norepinephrine metabolism in man using deuterium labeling: turnover 4-hydroxy-3-methoxymandelic acid.

4-Hydroxy-3-methoxymandelic acid (HMMA; VMA) labeled with three deuterium atoms was used to study the turnover and fate of HMMA following intravenous injection. Five healthy men were given a pulse dose of 5.0 mumol of labeled HMMA. Plasma and urinary levels of both endogenous and labeled HMMA were subsequently followed by gas chromatography-mass spectrometry using selected ion detection. The kinetic parameters were determined both with and without compensation for the pool expansion caused by the injection of labeled HMMA. The urinary recovery of labeled HMMA was 85 +/- 10% (mean +/- SD). No conversion of HMMA to 4-hydroxy-3-methoxyphenyl glycol (HMPG) occurred. The biological half-life of HMMA was 0.54 +/- 0.22 h. The apparent volume of distribution was 0.36 +/- 0.11 L/kg. The production rate or body turnover was 1.27 +/- 0.51 mumol HMMA/h and urinary excretion rate was 0.82 +/- 0.22 mumol/h. These results show that HMMA is turnover over rapidly in a relatively small volume of distribution and that, unlike HMPG, it is an end metabolite of norepinephrine in man.

Norepinephrine metabolism in man using deuterium labelling: the conversion of 4-hydroxy-3-methoxyphenylglycol to 4-hydroxy-3-methoxymandelic acid.

4-Hydroxy-3-methoxyphenylglycol (HMPG) labelled with three deuterium atoms was used to study the disposition of peripherally administered HMPG. Five healthy men were given an intravenous pulse dose of 4.3 mumol of labelled HMPG and subsequent plasma and urine levels of endogenous and labelled HMPG as well as those of 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) were determined by gas chromatography-mass spectrometry, using selected ion detection. Approximately 40% of the injected amount of deuterium-labelled HMPG was recovered in the urine as HMMA and another 40% was eliminated as HMPG conjugates. Thus, the HMPG formed from norepinephrine either in the central or peripheral nervous system undergoes both conjugation and extensive oxidation.