Cellular and molecular events leading to mitochondrial toxicity of 1-(2-deoxy-2-fluoro-1-beta-D-arabinofuranosyl)-5-iodouracil in human liver cells.

We have explored the mechanism(s) related to FIAU-induced liver toxicity, particularly focusing on its effect on mitochondrial function in a human hepatoma cell line-HepG2. The potential role of FMAU and FAU, metabolites detected in FIAU-treated patients were also ascertained. FIAU and FMAU inhibited cell growth and were effectively phosphorylated. A substantial increase in lactic acid production in medium of cells incubated with 1-10 microM FIAU or FMAU was consistent with mitochondrial dysfunction. Slot blot analysis demonstrated that a two week exposure to 10 microM FIAU or FMAU was not associated with a decrease in total mitochondrial (mt) DNA content. However, FIAU and FMAU were incorporated into nuclear and mtDNA and relative values suggest that both compounds incorporate at a much higher rate into mtDNA. Electron micrographs of cells incubated with 10 microM FIAU or FMAU revealed the presence of enlarged mitochondria with higher cristae density and lipid vesicles. In conclusion, these data suggest that despite the lack of inhibition of mtDNA content, incorporation of FIAU and FMAU into mtDNA of HepG2 cells leads to marked mitochondrial dysfunction as evidenced by disturbance in cellular energy metabolism and detection of micro- and macrovesicular steatosis.

Dihydrofluorouracil, a fluorouracil catabolite with antitumor activity in murine and human cells.

Dihydrofluorouracil (FUH2), the initial catabolite of 5-fluorouracil (FUra), was examined to determine whether this derivative had antitumor activity or host cell (bone marrow) toxicity. Studies were undertaken with Ehrlich ascites tumor and bone marrow cells isolated from CF-1 mice. Cells were exposed for 1 h either to no drug (control) or to varying concentrations, ranging from 1 to 250 microM, of either FUra, FUH2, or alpha-fluoro-beta-alanine. Cells were then cultured and colony formation was assessed after 10 to 14 days. Ehrlich ascites tumor cells were more sensitive to FUra [50% lethal dose (LD50) = 18 microM] than to FUH2 [LD50 = 50 microM], with no sensitivity to alpha-fluoro-beta-alanine even at 250 microM. Bone marrow cells had a toxicity profile similar to that of FUra (LD50 = 10 microM) but were relatively insensitive to FUH2 (LD50 greater than 250 microM), with no sensitivity to alpha-fluoro-beta-alanine. Subsequent studies examined colony formation of the human breast carcinoma cell line MCF-7 following 1 h exposure to varying concentrations of FUra and FUH2. These cells were less sensitive to both FUra (LD50 approximately 80 microM) and FUH2 (LD50 approximately 350 microM). Initial studies on the mechanism of toxicity of FUH2 demonstrated that this FUra catabolite could produce inhibition of thymidylate synthase activity in Ehrlich ascites tumor cells with a pattern similar to that resulting from exposure to FUra. This is the first study to demonstrate that FUH2 (a quantitatively important catabolite of FUra) is cytotoxic, and it suggests that FUH2 may contribute to the toxicity of FUra in vivo, possibly by being anabolized to FUra.

Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma, urine, and bile.

Kinetics of 5-fluorouracil (FUra) and FUra metabolites in plasma and urine were investigated in 10 cancer patients following i.v. bolus administration of 500 mg/m2 FUra with 600 microCi of [6-3H]FUra. Biliary excretion was examined in two patients with external biliary catheters. Quantitation of unchanged drug and metabolites was assessed by a highly specific high-performance liquid chromatographic method. FUra plasma levels declined rapidly with an apparent elimination half-life of 12.9 +/- 7.3 min. Dihydrofluorouracil was detected within 5 min in most patients, demonstrating rapid catabolism and reached maximum peak levels of 23.7 +/- 9.9 microM at approximately 60 min. The apparent elimination half-life of dihydrofluorouracil (61.9 +/- 39.0 min) was consistently greater than that of the unchanged drug. The apparent elimination half-lives of the subsequent metabolites alpha-fluoro-beta-ureidopropionic acid and alpha-fluoro-beta-alanine were prolonged with values of 238.9 +/- 175.4 min and 1976 +/- 358 min, respectively. Approximately 60-90% of the administered dose was excreted in urine within 24 h, primarily as alpha-fluoro-beta-alanine. Biliary excretion accounted for 2-3% of total administered radioactivity. The major fraction of this radioactivity eluted on high-performance liquid chromatography as a previously unrecognized FUra metabolite. Analysis of its structure is currently ongoing in our laboratory. In conclusion, this study provides the first comprehensive analysis of the formation and excretion of FUra metabolites in plasma, urine, and bile following i.v. bolus administration of FUra in humans.

Kinetics and metabolism of a new fluoropyrimidine, 5'-deoxy-5-fluorouridine, in humans.

5'-Deoxy-5-fluorouridine (5'-dFUrd) is a new antineoplastic agent which possesses a higher therapeutic index in several experimental tumors compared to other fluoropyrimidines. During a Phase I trial, 5'-dFUrd, 1 to 15 g/sq m/week, was administered to patients as a 25- to 35-min i.v. infusion. Plasma kinetics and metabolism of 5'-dFUrd were investigated. The unmetabolized drug was measured by a high-performance liquid chromatography assay. 5-Fluorouracil and 5,6-dihydrofluorouracil, the two detected plasma metabolites, were quantitated by a gas chromatography-mass spectrometry methodology with a detection limit of 0.07 microM for both metabolites. The disposition of 5'-dFUrd in humans at therapeutic doses followed a nonlinear kinetic process. Plasma concentrations of 5-fluorouracil generated in vivo represented approximately 6% of 5'-dFUrd concentrations and the 5-fluorouracil half-life ranged from 8.8 to 27.1 min. High plasma values of 5,6-dihydrofluorouracil (14.5 to 30 microM) were observed in patients, indicating the importance of this pathway in humans.

Disposition and metabolism of the angiogenic moderator O-(chloroacetyl-carbamoyl) fumagillol (TNP-470; AGM-1470) in human hepatocytes and tissue microsomes.

The biotransformation of O-(chloroacetyl-carbamoyl) fumagillol (TNP-470; AGM 1470), a potent in vitro inhibitor of angiogenesis, was investigated in primary cultured human hepatocytes and microsomal fractions of various human tissues. Exposure of human hepatocytes to 5 microM [3H]TNP-470 led to a rapid metabolism of unchanged drug to six metabolic derivatives within 30 min. The predominant extracellular metabolites were M-II and M-IV, attaining a maximum level of 3.23 +/- 0.34 and 0.88 +/- 0.10 microM, respectively. M-II leveled off, while M-IV rapidly declined to 0.06 +/- 0.05 microM by 3 h. TNP-470 was undetectable after 60 min. M-V and M-VI slowly reached maximal concentrations of 0.26 +/- 0.12 and 0.32 +/- 0.16 microM, respectively. M-I only reached a concentration of 0.18 +/- 0.07 microM at 60 min and leveled at 0.13 +/- 0.06 microM for the remaining time of the experiment. The intracellular profile was different, with M-III and M-V representing the major metabolites detected. Studies using human liver microsomes demonstrated that M-IV formation was associated with an esterase-like enzymatic cleavage of TNP-470 and that this metabolite was then further metabolized by microsomal epoxide hydrolase to M-II, as evidenced by inhibition of this metabolic step by cyclohexene oxide, a microsomal epoxide hydrolase inhibitor. Extrahepatic metabolism of TNP-470 was also demonstrated using different sites of human intestinal, stomach, and kidney microsomes, with metabolite M-IV as the principal derivative detected in these tissues. Hepatic microsomal samples from seven different donors demonstrated large interindividual variations in the formation of both M-II and M-IV. In summary, this study demonstrates a rapid and extensive metabolism of TNP-470 in human tissues. The data emphasize the need to evaluate the in vivo formation and extent of TNP-470 metabolites to adequately assess the pharmacodynamic effects of this novel anticancer drug with a novel mechanism of action.

Modulation of 5-fluorouracil catabolism in isolated rat hepatocytes with enhancement of 5-fluorouracil glucuronide formation.

The catabolism of 5-fluorouracil (FUra), which accounts for 90% of the elimination of this antimetabolite in vivo, has recently been characterized in freshly isolated rat hepatocytes in suspension using a highly specific high-performance liquid chromatographic methodology. The present study evaluates the effect of thymine and uracil, which are thought to be catabolized by the same enzymes as FUra, on the metabolism and transmembrane distribution of FUra in isolated rat hepatocytes. Following simulataneous exposure of cells for 5 min to 30 microM [6-3H]FUra and increasing concentrations of either thymine or uracil, dihydrofluorouracil (FUH2) levels decreased in a concentration-dependent manner, and the concentration determined for 50% inhibition of FUra catabolism was 8.0 +/- 0.3 (S.D.) and 67.8 +/- 15.6 microM for thymine and uracil, respectively. Analysis of intracellular and extracellular 3H from 1 min to 2 hr after simultaneous incubation of the hepatocytes with 30 microM FUra and thymine (or uracil) in a 1:7 molar ratio resulted in a decrease of intracellular and extracellular FUH2 and alpha-fluoro-beta-alanine (FBAL), while alpha-fluoro-beta-ureidopropionic acid (FUPA) was enhanced. Unmetabolized FUra (not detected in the absence of thymine or uracil) was detected intracellularly in the presence of thymine or uracil and was accompanied by the appearance of a novel metabolite, preliminarily identified as a glucuronide of the FUra base which reached intracellular levels of 44 +/- 9.76 and 27.45 +/- 1.35 microM in the presence of thymine or uracil, respectively, within 1 hr. This metabolite, which penetrates the cell membrane only slowly, accounted for approximately 60% of the intracellular 3H in the presence of 300 microM FUra and 2 mM thymine, whereas FUra catabolism was inhibited by more than 99% under these conditions. The formation of FUra anabolites was insignificant in the presence of thymine and uracil, and incorporation of FUra into RNA was not enhanced. The lack of anabolism of FUra in isolated hepatocytes exposed to either high initial concentrations of FUra or high intracellular FUra concentrations resulting from modulation (inhibition) of FUra catabolism is consistent with the clinical observation of minimal hepatotoxicity with FUra, despite exposure of the liver to high blood levels. These studies indicate that thymine is a more potent modulator of FUra catabolism in hepatocytes than is uracil. Further studies are needed to clarify the biological importance of the glucuronide of the base FUra which accumulates intracellularly as the concentration of FUra increases within the hepatocytes.

Evidence from rat hepatocytes of an unrecognized pathway of 5-fluorouracil metabolism with the formation of a glucuronide derivative.

Isolated rat hepatocytes in suspension were exposed to [3H]-5-fluorouracil for intervals over 2 h, following which the cells were removed from the media and sonicated, and the cytoplasm was sampled. High-performance liquid chromatography was used to separate 5-fluorouracil (FUra) from its known anabolites and catabolites, with subsequent quantitation of these metabolites by measurement of radioactivity. As the extracellular concentration of FUra was increased above 30 microM, the intracellular levels of FUra increased, with detection of a new peak of radioactivity distinct from any of the known anabolites or catabolites. This new metabolite, "G," increased in concentration as the extracellular concentration of FUra was raised above 1 mM. Inhibition of FUra catabolism by 2 mM thymine resulted in a further increase in intracellular FUra (approaching the extracellular FUra concentration) and was accompanied by a further increase in the intracellular concentration of "G," demonstrating that "G" was not formed via the catabolic pathway. The increase in intracellular FUra and "G" was not accompanied by an increase in intracellular anabolites, suggesting that "G" was formed via a novel metabolic pathway. "G" was retained within the hepatocytes, although it was not bound to intracellular macromolecules. "G" was converted to FUra in the presence of beta-D-glucuronidase; this reaction was inhibited with the addition of saccharo-1,4-beta-lactone, a specific inhibitor of the beta-D-glucuronidase. This data, together with evidence from hepatocyte homogenates in which formation of "G" was shown to be dependent on the concentration of uridine-5'-diphosphoglucuronic acid, demonstrates that "G" is a glucuronide of FUra. The formation of "G" suggests that FUra is metabolized via a previously unrecognized metabolic pathway.

Fumagillin analog in the treatment of Kaposi's sarcoma: a phase I AIDS Clinical Trial Group study. AIDS Clinical Trial Group No. 215 Team.

PURPOSE: Angiogenesis is a major component of Kaposi's sarcoma (KS) and a critical process in tumor growth. The present study was designed primarily to test the toxicity and pharmacokinetics (PK) of the angiogenesis inhibitor TNP-470 and secondarily to evaluate tumor response in patients with early AIDS-related KS. PATIENTS AND METHODS: Patients with AIDS-related KS were required to have cutaneous disease with > or = 5 measurable lesions and no evidence of pulmonary, symptomatic gastrointestinal, or acutely life-threatening KS. Thirty-eight patients received TNP-470 by weekly intravenous infusion over 1 hour at one of six dose levels for up to 24 weeks. RESULTS: The dose levels tested included 10, 20, 30, 40, 50 and 70 mg/m2. Median CD4 count was 24 cells/microl (range, 0 to 460). Fourteen patients (36%) had > or = 50 cutaneous lesions and 19 (49%) had oral lesions. Adverse events included neutropenia (n = 2), hemorrhage (n = 3), and urticaria (n = 1). PK studies showed wide interpatient and intrapatient variability. Elimination half-life values were short (range, 0.01 to 0.61 hours). Seven patients (18%) achieved a partial response. The median time to partial response was 4 weeks (range, 2 to 25), and the median duration of response was 11 weeks (range, 3 to 26+). CONCLUSION: TNP-470, administered as a weekly, 1-hour infusion to patients with early AIDS-KS is well-tolerated at doses up to and including the highest dose tested. Tumor responses were observed in a substantial number of cases and occurred at various dose levels. TNP-470 should be evaluated further in patients with AIDS-KS as a single agent and in combination with other biologic response modifiers in early disease or after initial response to cytotoxic chemotherapy.

HIV protease inhibitors: pharmacologic and metabolic distinctions.

In the earlier era of nucleoside analogue antiretroviral therapy, considerations of pharmacologic drug-drug interactions were only a small factor in choosing optimal therapies for patients with HIV infection. However, pharmacologic considerations are critical for selecting and managing the current multiple-drug antiretroviral regimens that include one or more HIV protease inhibitors (HPIs). Each of the available HPIs, both those that are licensed and those still under investigation, has distinct pharmacokinetic and metabolic properties that result in different dosing regimens and potential for interactions with food and other drugs. Although drug interactions are usually considered undesirable, some of the drug interactions between HPIs have beneficial effects on drug levels and total drug exposure. The non-nucleoside reverse transcriptase inhibitors also interact with HPIs. This article briefly reviews pharmacokinetic and metabolic distinctions among the newer antiretroviral agents, especially HPIs, up to the end of 1997, including selected agents that are still under investigation. As current treatment guidelines emphasize, HIV/AIDS patients typically receive several drugs; therefore, the potential for drug interactions, particularly those mediated by the P450 system in patients receiving HPIs or non-nucleoside reverse transcriptase inhibitors or both, must be appreciated by all physicians who care for patients with HIV disease.

Cellular nucleoside pharmacokinetics and pharmacology: a potentially important determinant of antiretroviral efficacy.

Among the potential reasons for treatment failure in patients receiving therapy for HIV-1 infection, one that has received only limited attention is intracellular interactions between nucleosides that may reduce their metabolism (phosphorylation) to active forms necessary for antiretroviral activity. Results reviewed in this paper indicate that there is considerable potential for interaction among nucleosides used to suppress viral replication in patients with HIV-1 infection. Zidovudine (ZDV) and zalcitabine (ddC) have both been shown to inhibit their own metabolism. ZDV also inhibits the phosphorylation of stavudine (d4T) and lamivudine (3TC) and increases that of didanosine (ddI). d4T has been shown to slightly decrease the phosphorylation of ZDV. ddC decreases the formation of triphosphates of both d4T and 3TC, and 3TC decreases the phosphorylation of ddC. ddI has no significant effects on the intracellular metabolism of any of the nucleoside analogues currently used to treat patients with HIV-1 disease. Thus, the pharmacokinetics of antiretroviral agents and intracellular phosphorylation/activation of nucleosides may be important determinants of the virologic and clinical effectiveness of therapy. They must be considered along with characteristics of the virus, such as phenotypic and genotypic resistance, and those of the patient, including motivation to adhere to therapy, in individualizing antiretroviral treatment regimens for individuals with HIV-1 disease.

Plasma population pharmacokinetics and penetration into cerebrospinal fluid of indinavir in combination with zidovudine and lamivudine in HIV-1-infected patients.

OBJECTIVES: To evaluate plasma population pharmacokinetics and penetration into cerebrospinal fluid (CSF) by indinavir (IDV) in HIV-infected individuals receiving IDV, zidovudine and lamivudine. METHODS: Plasma population pharmacokinetic analysis was performed on 805 IDV plasma values from 171 patients, using a non-linear mixed-effects modeling approach. CSF data from 19 patients were analyzed using an individual approach. RESULTS: Mean individual Bayesian estimates for oral clearance (CL) and volume of distribution (V) by the final model that incorporated interoccasion variability were 0.75 l/h per kg [coefficient of variation (CV) 54.8%] and 1.74 l/kg (CV 82.7%), respectively. Mean model-predicted plasma IDV level at 8 h, maximal level, area under the plasma level-time curve up to 8 h and plasma half-life were 0.42 micromol/l (CV 57.5%), 9.51 micromol/l (CV 47.3%), 29.56 micromol/l x h (CV 46.9%) and 1.50 h (CV 20.9%), respectively. The mean IDV CSF level was 0.11 micromol/l (CV 49.7%) and the mean CSF:plasma concentration ratio was 0.017. CONCLUSIONS: Population estimates of pharmacokinetic parameters of IDV and its CSF penetration were in excellent agreement with previously reported data from individual analyses. Intraindividual interoccasion variability of IDV pharmacokinetics was estimated to be of similar order of magnitude to its interindividual variability, which may affect response to long-term antiretroviral therapy involving IDV. CSF levels of IDV exceeded its in vitro 95% inhibitory concentration of HIV replication. Given that CSF is virtually free of protein, viral suppression in the central nervous system should be achievable with an IDV-containing regimen.

Enhanced penetration of indinavir in cerebrospinal fluid and semen after the addition of low-dose ritonavir.

OBJECTIVE: Penetration of antiretroviral drugs into anatomical HIV-1 reservoirs such as the male genital tract and the central nervous system is important. Data on indinavir (IDV) concentrations in seminal plasma are lacking and IDV concentrations in cerebrospinal fluid are at best borderline. DESIGN: Thirteen patients were treated with zidovudine (or stavudine), lamivudine, abacavir, nevirapine and IDV (1000 mg three times daily). When nevirapine led to low IDV concentrations, IDV was changed into the combination IDV/ritonavir (RTV) 800/100 mg twice daily to improve the pharmacokinetic profile of IDV. METHODS: A serum pharmacokinetic profile, a semen sample and a cerebrospinal fluid sample were collected at weeks 8, 24, 48 and 72. RESULTS: Addition of RTV increased the median IDV trough concentration in serum from 65 to 336 ng/ml (P = 0.005). Median IDV concentration in seminal plasma increased from 141 to 1634 ng/ml (P = 0.002) (n = 9) and in cerebrospinal fluid from 39 (n = 12) to 104 (n = 7) ng/ml (P < 0.001). In six patients with samples collected both before and after the addition of RTV, the IDV concentration in seminal plasma increased 8.2 times [95% confidence interval (CI) 5.2-11.6], and in cerebrospinal fluid 2.4 times (95% CI 1.8-3.9). CONCLUSIONS: IDV penetrates well into the male genital tract. The addition of low-dose RTV not only increases IDV concentrations in serum but also in seminal plasma and cerebrospinal fluid, thereby probably improving the potency of the regimen in these anatomical HIV reservoirs. Higher serum trough levels alone can not sufficiently explain the observed increases in seminal plasma and cerebrospinal fluid concentrations. Inhibition of P-glycoprotein-mediated transport by RTV might be an additional mechanism.

Lamivudine in combination with zidovudine, stavudine, or didanosine in patients with HIV-1 infection. A randomized, double-blind, placebo-controlled trial. National Institute of Allergy and Infectious Disease AIDS Clinical Trials Group Protocol 306 Investigators.

OBJECTIVE: To study the antiviral activity of lamivudine (3TC) plus zidovudine (ZDV), didanosine (ddl), or stavudine (d4T). DESIGN: Randomized, placebo-controlled, partially double-blinded multicenter study. SETTING: Adult AIDS Clinical Trials Units. PATIENTS: Treatment-naive HIV-infected adults with 200-600x10(6) CD4 T lymphocytes/l. INTERVENTIONS: Patients were openly randomized to a d4T or a ddl limb, then randomized in a blinded manner to receive: d4T (80 mg/day), d4T plus 3TC (300 mg/day), or ZDV (600 mg/day) plus 3TC, with matching placebos; or ddl (400 mg/day), ddl plus 3TC (300 mg/day), or ZDV (600 mg/day) plus 3TC, with matching placebos. After 24 weeks 3TC was added for patients assigned to the monotherapy arms. MAIN OUTCOME MEASURE: The reduction in plasma HIV-1 RNA level at weeks 24 and 48. RESULTS: Two hundred ninety-nine patients were enrolled. After 24 weeks the mean reduction in plasma HIV-1 RNA copies/ml from baseline was 0.49 log10 (d4T monotherapy) versus 1.03 log10 (d4T plus 3TC; P = 0.001), and 0.68 log10 (ddl monotherapy) versus 0.82 log10 (ddl plus 3TC; P>0.22). After 48 weeks the mean reduction was 1.08 log10 (d4T plus 3TC) versus 1.01 log10 (ZDV plus 3TC) in the d4T limb (P = 0.66), and 0.94 log10 (ddl plus 3TC) versus 0.88 log10 (ZDV plus 3TC; P = 0.70) in the ddl limb. CONCLUSIONS: 3TC added significantly to the virologic effects of d4T, but not ddl, in treatment-naive patients. 3TC plus d4T produced virologic changes comparable to those of 3TC plus ZDV. These results support the use of 3TC with either ZDV or d4 as a component of initial combination antiretroviral therapy.

Continued lamivudine versus delavirdine in combination with indinavir and zidovudine or stavudine in lamivudine-experienced patients: results of Adult AIDS Clinical Trials Group protocol 370.

OBJECTIVE: To compare the virologic activity of continued lamivudine (3TC) versus a switch to delavirdine (DLV) when initiating protease inhibitor therapy in nucleoside-experienced patients. DESIGN: Randomized, open-label, multi-center study. SETTING: Adult AIDS clinical trials units. PATIENTS: Protease and non-nucleoside reverse transcriptase inhibitor-naive patients who had received 3TC plus zidovudine (ZDV), stavudine (d4T), or didanosine (ddl) for at least 24 weeks. INTERVENTIONS: Patients with plasma HIV-1 RNA levels > 500 copies/ml who previously received d4T + 3TC or ddI + 3TC were randomized to ZDV + 3TC + indinavir (IDV) or ZDV + DLV + IDV. MAIN OUTCOME MEASURES: Primary endpoints were the proportion of patients with plasma HIV-1 RNA levels < or = 200 copies/ml at 24 weeks, and occurrence of serious adverse events. The proportion of patients with plasma HIV-1 RNA levels < or = 200 copies/ml at week 48 was a secondary endpoint. RESULTS: At week 24, 58% of subjects in the ZDV + 3TC + IDV arm and 73% in the ZDV + DLV + IDV arm had plasma HIV-1 RNA levels < or = 200 copies/ml (P = 0.29). At week 48, plasma HIV-1 RNA levels were < or = 200 copies/ml in 48% and 83%, respectively (P = 0.007). Rash and hyperbilirubinemia occurred more frequently in the DLV arm than in the 3TC arm. Steady-state plasma IDV levels were higher among patients in the DLV arm as compared with the 3TC arm. CONCLUSIONS: Substituting DLV for 3TC when adding IDV improved virologic outcome in nucleoside-experienced patients. This result might be explained, in part, by the positive effect of DLV on IDV pharmacokinetics.

Symptomatic lactic acidosis in hospitalized antiretroviral-treated patients with human immunodeficiency virus infection: a report of 12 cases.

We retrospectively investigated the clinical and histopathologic features of hospitalized patients infected with human immunodeficiency virus who had symptomatic lactic acidosis syndrome at a university teaching hospital during 1995-2000. Twelve patients were identified, 11 during 1998-2000; of these, 5 died with rapid progression to otherwise unexplained multiple-organ failure. All had extensive prior exposure to nucleoside analog reverse-transcriptase inhibitors (NRTIs). At presentation, the most commonly identified NRTI component of antiretroviral regimens was stavudine plus didanosine. Eleven patients presented with abdominal pain, nausea, and/or emesis. Eight patients had prior acute weight loss (mean [+/-SD], 12+/-5.3 kg). Median venous plasma lactate levels were > or =2-fold greater than the upper limit of normal (2.1 mmol/L). Serum transaminase levels were near normal limits at presentation. Histopathologic studies confirmed hepatic macrovesicular and microvesicular steatosis in 6 patients. Concurrent chemical pancreatitis was identified in 6 patients. The increasing number of cases identified during the study period suggests that physicians better recognize symptomatic lactic acidosis and/or that cumulative NRTI exposure may increase the risk for this syndrome.

Reduction of 3'-azido-3'-deoxythymidine to 3'-amino-3'-deoxythymidine in human liver microsomes and its relationship to cytochrome P450.

The formation of 3'-amino-3'-deoxythymidine (AMT) in patients receiving 3'-azido-3'-deoxythymidine (zidovudine) and the potential role of this metabolite in zidovudine-induced toxicity was recently demonstrated by our laboratory. This study evaluated the formation of AMT versus cytochrome P450 (P450) content, cytochrome B5 (B5) content and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase activity in human liver microsomes obtained from 24 different donors. Significant interindividual differences in total P450 content and P450 reductase activity were observed, whereas no variation was observed in B5 content. Of particular importance, metabolism of zidovudine to AMT varied widely and correlated with P450 content but not with B5 content or P450 reductase activity. The apparent values for the Michaelis-Menten constant and the maximum rate of metabolism of the reaction were 46.1 mmol/L and 3.5 nmol/min/mg microsomal protein. These large variations of AMT levels as a function of P450 suggest that major interindividual differences may be observed in the pharmacokinetics and formation of this metabolite that may affect the pharmacodynamic properties of zidovudine. Potential drug-drug interactions may occur with therapeutic agents that interact with or induce P450 (zidovudine).

Clinical pharmacokinetics of 3'-azido-3'-deoxythymidine (zidovudine) and catabolites with formation of a toxic catabolite, 3'-amino-3'-deoxythymidine.

This study investigated pharmacokinetics and metabolism of 3'-azido-3'-deoxythymidine (zidovudine) in patients after a 1-hour intravenous infusion of 2.5 mg/kg zidovudine with a radiolabeled tracer amount of [5-3H]-zidovudine. In addition to unchanged drug and its 5'-O-glucuronide (zidovudine glucuronide), two novel catabolites of zidovudine were detected as 3'-amino-3'-deoxythymidine (AMT), and its 5'-O-glucuronide (GAMT). The AMT apparent plasma elimination half-life (2.70 +/- 0.7 hours) was longer than that of zidovudine (1.20 +/- 0.30 hours) and zidovudine glucuronide (1.60 +/- 0.5 hours). The zidovudine/AMT plasma peak concentration and area under the concentration-time curve ratios were approximately 8 and 5, respectively. Urinary recovery of radioactivity was essentially complete within 24 hours. AMT glucuronide was not detected in urine or plasma, and only low levels of this catabolite were detected in bile. In contrast, AMT was not detected in bile. The substantial levels of AMT in the plasma of patients after zidovudine administration suggests that this catabolite may affect the pharmacodynamic properties of zidovudine in relation to its activity against human immunodeficiency virus replication and cytotoxicity to host cells.

Pharmacological considerations in antiretroviral therapy.

Suboptimum drug exposure arising from pharmacological and pharmacokinetic factors is an important reason that combination antiretroviral therapy fails in patients with human immunodeficiency virus type 1 infection. With all three available drug classes, the major causes are drug-drug interactions and interpatient pharmacokinetic variability. Inadequate concentrations of protease inhibitors and non-nucleoside reverse transcriptase inhibitors occur when concurrently administered drugs interfere with their metabolism by the hepatic cytochrome P450 enzyme system, the major metabolic pathway for these drugs. Combined therapy with certain nucleoside reverse transcriptase inhibitors can reduce systemic drug exposure by interfering with the intracellular conversion of administered drugs to their active metabolites. Important causes of interpatient variability in pharmacokinetic parameters include low oral drug bioavailability and individual differences in drug disposition and metabolism. Careful selection and monitoring of combination drug therapy along with individualized rather than standard dosage regimens may minimize these pharmacological problems and help ensure optimum antiviral activity.

Synthesis, biotransformation, and pharmacokinetic studies of 9-(beta-D-arabinofuranosyl)-6-azidopurine: a prodrug for ara-A designed to utilize the azide reduction pathway.

As a part of our efforts to design prodrugs for antiviral nucleosides, 9-(beta-D-arabinofuranosyl)-6-azidopurine (6-AAP) was synthesized as a prodrug for ara-A that utilizes the azide reduction biotransformation pathway. 6-AAP was synthesized from ara-A via its 6-chloro analogue 4. The bioconversion of the prodrug was investigated in vitro and in vivo, and the pharmacokinetic parameters were determined. For in vitro studies, 6-AAP was incubated in mouse serum and liver and brain homogenates. The half-lives of 6-AAP in serum and liver and brain homogenates were 3.73, 4.90, and 7.29 h, respectively. 6-AAP was metabolized primarily in the liver homogenate microsomal fraction by the reduction of the azido moiety to the amine, yielding ara-A. However, 6-AAP was found to be stable to adenosine deaminase in a separate in vitro study. The in vivo metabolism and disposition of ara-A and 6-AAP were conducted in mice. When 6-AAP was administered by either oral or intravenous route,the half-life of ara-A was 7-14 times higher than for ara-A administered intravenously. Ara-A could not be found in the brain after the intravenous administration of ara-A. However, after 6-AAP administration (by either oral or intravenous route), significant levels of ara-A were found in the brain. The results of this study demonstrate that 6-AAP is converted to ara-A, potentially increasing the half-life and the brain delivery of ara-A. Further studies to utilize the azide reduction approach on other clinically useful agents containing an amino group are in progress in our laboratories.

In vitro and in vivo evaluation of 6-azido-2',3'-dideoxy-2'-fluoro-beta-D-arabinofuranosylpurine and N6-methyl-2',3'-dideoxy-2'-fluoro-beta-D-arabinofuranosyladenine as prodrugs of the anti-HIV nucleosides 2'-F-ara-ddA and 2'-F-ara-ddI.

In an effort to improve the pharmacokinetic properties and tissue distribution of 2'-F-ara-ddI, two lipophilic prodrugs, 6-azido-2'-3'-dideoxy-2'-fluoro-beta-D- arabinofuranosylpurine (FAAddP, 4) and N6-methyl-2'-3'-dideoxy-2'-fluoro-beta-D-arabinofuranosyladenine (FMAddA, 5), were synthesized and their biotransformation was investigated in vitro and in vivo, in mice. Compounds 4 and 5 were synthesized via the intermediate 2. For the in vitro studies, FAAddP and FMAddA were incubated in mouse serum, liver homogenate, and brain homogenate. FAAddP was metabolized in liver homogenate by the reduction of the azido to the amino moiety followed by deamination, yielding 2'-F-ara-ddI. The conversion of FAAddP to 2'-F-ara-ddA was mediated by microsomal P-450 NADPH reductase system, as shown by the liver microsomal assay. FAAddP was also converted to 2'-F-ara-ddI at a slower rate in the brain than in the liver. FMAddA, however, was stable in brain homogenate and was slowly metabolized in the liver homogenate. Metabolic conversion of FMAddA in vitro was stimulated by the addition of adenosine deaminase. In the in vivo metabolism study, FAAddP underwent reduction to 2'-F-ara-ddA followed by deamination to 2'-F-ara-ddI. FMAddA did not result in increased brain delivery of 2'-F-ara-ddI in vivo, probably due to the slow conversion as observed in the in vitro studies. However, there was an increase in the half-life of 2'-F-ara-ddI produced from FMAddA. This report is the first example in the design of prodrugs using the azido group for adenine- and hypoxanthine-containing nucleosides. This interesting and novel approach can be extended to other antiviral and anticancer nucleosides.