Thymidine phosphorylase as a target for imaging and therapy with thymine analogs.

PURPOSE: Thymidine phosphorylase (TPase; platelet-derived endothelial cell growth factor) is an attractive target for imaging and therapy because of the strong relationship between its expression in tumor biopsies and clinical outcome in many tumor types. Although the mechanism has yet to be explained, expression of TPase is highly associated with angiogenesis. METHODS: Tumor cells were phenotyped for TPase activity, and incubated with thymine or its analogs (5-X-Ura). After intracellular conversion to thymidine analogs via the reverse reaction for TPase, these molecules were phosphorylated and incorporated into DNA. RESULTS: Preferential localization was found in cells with high TPase, e.g. U937. Incorporation was enhanced in cells with high TPase by coincubation with modulators such as deoxyuridine. CONCLUSIONS: 5-X-Ura molecules can be readily labeled with positron emitters, and this finding provides support for further evaluation in vivo of their potential as probes for noninvasive external imaging of TPase, both at the time of diagnosis and during maneuvers intended to manipulate TPase. If the 5-X-Ura molecules were labeled with a therapeutic isotope, e.g. 125I or 211At, selective cytotoxicity would be expected in cells with high TPase expression. However, direct evaluation of the safety in vivo of the therapeutic approach is required. The 5-X-Ura compounds constitute a novel approach to both imaging and therapy directed towards TPase. Further, there are distinct advantages to using the imaging mode to identify tumors likely to benefit from therapy with the same set of molecules.

Suicide prodrugs activated by thymidylate synthase: rationale for treatment and noninvasive imaging of tumors with deoxyuridine analogues.

Most tumors are resistant to therapy by thymidylate synthase (TS) inhibitors due to their high levels of TS. Instead of inhibiting TS, we hypothesized that it was possible to use this enzyme to activate suicide prodrugs (deoxyuridine analogues) to more toxic species (thymidine analogues). Tumors with high levels of TS could be particularly sensitive to deoxyuridine analogues because they would be more efficient in producing the toxic methylated species. Furthermore, the accumulation of methylated species within tumors could be visualized externally if a tracer dose of the deoxyuridine analogue was tagged with an isotope, preferably a positron emitter, such as 18F. Higher accumulation of isotope indicates higher activity of TS and lower sensitivity of the tumor to TS inhibitors, but perhaps more sensitivity to therapy with deoxyuridine analogues as suicide prodrugs. 2'-F-ara-deoxyuridine (FAU) was used as a prototype to demonstrate these concepts experimentally. FAU readily entered cells and was phosphorylated, methylated, and subsequently incorporated into cellular DNA. Among different cell lines, FAU produced varying degrees of growth inhibition. Greater DNA incorporation (e.g., for CEM and U-937 cells) was reflected as increased toxicity. FAU produced less DNA incorporation in Raji or L1210 cells, and growth rate was minimally decreased. As the first demonstration that cells with high levels of TS activity can be more vulnerable to therapy than cells with low TS activity, this preliminary work suggests a new therapeutic approach for common human tumors that were previously resistant. Furthermore, it appears that the TS activity of tumors could be noninvasively imaged in situ by tracer doses of [18F]FAU and that this phenotypic information could guide patient therapy.

Glucuronidation of 3'-azido-3'-deoxythymidine (zidovudine) by human liver microsomes: relevance to clinical pharmacokinetic interactions with atovaquone, fluconazole, methadone, and valproic acid.

Zidovudine (3'-azido-3'-deoxythymidine [AZT]), an antiviral nucleoside analog effective in the treatment of human immunodeficiency virus infection, is primarily metabolized to an inactive glucuronide form, GAZT, via uridine-5'-diphospho-glucuronosyltransferase (UGT) enzymes. UGT enzymes exist as different isoforms, each exhibiting substrate specificity. Published clinical studies have shown that atovaquone, fluconazole, methadone, and valproic acid decreased GAZT formation, presumably due to UGT inhibition. The effect of these drugs on AZT glucuronidation was assessed in vitro by using human hepatic microsomes to begin understanding in vitro-in vivo correlations for UGT metabolism. The concentrations of each drug studied were equal to those reported with the usual clinical doses and at concentrations at least 10 times higher than would be expected with these doses. High-performance liquid chromatography was used to assess the respective metabolism and formation of AZT and GAZT. All four drugs exhibited concentration-dependent inhibition of AZT glucuronidation. The respective concentrations of atovaquone and methadone which caused 50% inhibition of GAZT were > 100 and 8 micrograms/ml, well above their usual clinical concentrations. Fluconazole and valproic acid exhibited 50% inhibition of GAZT at 50 and 100 micrograms/ml, which are within the clinical ranges of 10 to 100 and 50 to 100 micrograms/ml, respectively. These data suggest that inhibition of AZT glucuronidation may be more clinically significant with concomitant fluconazole and valproic acid. Factors such as inter- and intraindividual pharmacokinetic variability and changes in AZT intracellular concentrations should be considered as other mechanisms responsible for changes in AZT pharmacokinetics with concomitant therapies.

Predicting drug interactions in vivo from experiments in vitro. Human studies with paclitaxel and ketoconazole.

This study was performed to evaluate whether concomitant treatment with ketoconazole could reduce the clearance of paclitaxel given to ovarian cancer patients. Paclitaxel, 175 mg/m2, was given as a 3-hour continuous intravenous infusion and repeated every 21 days. Initially, ketoconazole, 100 to 1600 mg, was given as a single oral dose 3 hours after paclitaxel. Later, ketoconazole, 200 mg, was given perorally 3 hours before paclitaxel. Plasma drug concentrations were measured by high-pressure liquid chromatography (HPLC), and cytochrome P450 3A (CYP3A) activity was measured with the erythromycin breath test (ERMBT). Ketoconazole did not alter plasma concentrations of paclitaxel or its principal metabolite, 6 alpha-hydroxypaclitaxel. Although there was marked inter- and intrapatient variability in ketoconazole pharmacokinetics, peak plasma concentrations in all but one course were below the 50% inhibitory concentration (IC50) point determined for inhibition of paclitaxel metabolism in vitro. Therefore, paclitaxel and ketoconazole can be coadministered safely without dose adjustments. There was no correlation between ERMBT measurements and serial plasma concentrations of paclitaxel. The erythromycin breath-test measurements did correlate with the corresponding ketoconazole plasma concentrations. The erythromycin breath test is a valuable tool for measuring instantaneous CYP3A activity in vivo. This clinical study confirms the results of prior studies with human-derived materials in vitro, reinforcing the notion that such studies are useful predictors of drug pharmacokinetics and interactions in vivo.

Rifampin and rifabutin and their metabolism by human liver esterases.

1. The main metabolites of rifampin and rifabutin in man are their respective 25 deacetylated derivatives, but the enzyme(s) responsible for these biotransformations are not known. 2. In experiments with human liver slices and human liver microsomes, the 25 deacetylated derivatives of these drugs were the main metabolites observed. Slices and microsomes metabolized rifabutin 3-6-fold faster than rifampin, in agreement with their relative clearance in patients. Rifabutin partitioned into slices more avidly than rifampin. 3. In microsomal incubations, deacetylation did not require NADPH, but the amount of metabolite at the end of incubation was affected by NADPH. With NADPH the amount of 25 deacetyl rifabutin decreased, whereas the amount of 25 deacetyl rifampin increased slightly. A panel of liver microsomes from seven donors showed a 3-4-fold difference in the formation of 25 deacetyl rifabutin or 25 deacetyl rifampin, with strong correlation between the production of the two metabolites (r2 = 0.94). 4. The production of 25 deacetyl rifabutin and 25 deacetyl rifampin by human liver microsomes was not significantly affected by 1 microM 4 chloromercuricbenzoic acid or bis-(4-nitrophenyl) phosphate, but was completely inhibited by 1 microM paraoxon or 1 microM diisopropylfluorophosphate. These results indicate that in man rifampin and rifabutin are deacetylated to their main metabolites by B-esterases.

Stereoselective metabolism of fenoldopam and its metabolites in human liver microsomes, cytosol, and slices.

Fenoldopam is a racemic mixture (R-FEN, S-FEN) that is a selective dopamine (DA-1) receptor agonist with pronounced cardiovascular and renal effects in humans. Metabolism of fenoldopam in human liver microsomes, cytosol, and slices was stereoselective for glucuronidation, sulfation, and methylation. Microsomal and cytosolic fractions were supplemented with appropriate cofactors to obtain enzyme activity. There was no evidence of metabolism of fenoldopam by cytochrome P-450. R-FEN was metabolized to fenoldopam-8-sulfate (8-SO4), 7-methoxy fenoldopam (7-MeO), 8-methoxy fenoldopam (8-MeO), and two glucuronidated products. The 7-MeO formed with incubation of R-FEN in human liver slices was further metabolized to an unknown sulfated product. S-FEN was metabolized to fenoldopam-7-sulfate (7-SO4), a second unknown sulfated product, 7-MeO, 8-MeO, and two glucuronidated products. Metabolism of S-FEN and R-FEN in human liver slices to 7-MeO occurred at the same rate, whereas further metabolism of 7-MeO was stereospecific and slower for the S-isomer of 7-MeO. Fenoldopam has served as an excellent model compound for comparison of metabolism in human liver slices with metabolism in subcellular fractions. The parallel pathways of fenoldopam metabolism lessen the possible impact of drug-drug interactions.

Metabolism of rifabutin and its 25-desacetyl metabolite, LM565, by human liver microsomes and recombinant human cytochrome P-450 3A4: relevance to clinical interaction with fluconazole.

Rifabutin and fluconazole are often given concomitantly as therapy to prevent opportunistic infections in individuals infected with the human immunodeficiency virus. Recent reports have shown increased levels of rifabutin and its 25-desacetyl metabolite, LM565, in plasma when rifabutin is administered with fluconazole. Since fluconazole is known to inhibit microsomal enzymes, this study was undertaken to determine if this rifabutin-fluconazole interaction was due to an inhibition of human hepatic enzymes. The metabolism of both rifabutin and LM565 was evaluated in human liver microsomes and recombinant human cytochrome P-450 (CYP) 3A4 in the presence of fluconazole and other probe drugs known to inhibit CYP groups 1A2, 2C9, 2D6, 2E1, and 3A. The concentrations of rifabutin (1 microg/ml), LM565 (1 microg/ml), and fluconazole (10 and 100 microg/ml) used were equal to those observed in plasma after the administration of rifabutin and fluconazole at clinically relevant doses. High-performance liquid chromatography was used to assess the metabolism of rifabutin and LM565. Rifabutin was readily metabolized to LM565 by human microsomes, but the reaction was independent of NADPH and was not affected by the P-450 inhibitors. No rifabutin metabolism by recombinant CYP 3A4 was found to occur. LM565 was also metabolized by human microsomes to two products, but metabolism was dependent on NADPH and was affected by certain P-450 inhibitors. In addition, LM565 was readily metabolized by the recombinant CYP 3A4 to the same two products found with its metabolism by human microsomes. Therefore, rifabutin is metabolized by human microsomes but not via cytochrome P-450 enzymes, whereas LM565 is metabolized by CYP 3A4.

Phase I and pharmacokinetic study of the multidrug resistance modulator dexverapamil with EPOCH chemotherapy.

PURPOSE: Dexverapamil is a competitive inhibitor of the P-glycoprotein (Pgp) efflux pump, a potent mechanism of multidrug resistance (mdr-1) in vitro. We performed a phase I study to determine the maximum-tolerated dose (MTD) and pharmacokinetics of dexverapamil with etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (EPOCH) chemotherapy. PATIENTS AND METHODS: Eligible patients had relapsed or refractory lymphoma or sarcoma. Patients initially received EPOCH alone, and those with stable or progressive disease were crossed-over to received dexverapamil on subsequent cycles of EPOCH. Dexverapamil was administered orally for 6 days and escalated over eight dose levels ranging from 240 to 1,200 mg/m2/d. Pharmacokinetics of dexverapamil and its active metabolite, nor-dexverapamil, were obtained in most patients. In seven patients, pharmacokinetics of doxorubicin, doxorubicinol, and etoposide were determined on paired cycles of EPOCH with or without dexverapamil. RESULTS: Sixty-five patients received 130 cycles of dexverapamil/EPOCH chemotherapy. The MTD of dexverapamil was 150 mg/m2 every 4 hours (900 mg/m2/d), and hypotension was the principal dose-limiting toxicity. The dexverapamil area under the curve (AUC) increased proportionally with dexverapamil dose, but significant interpatient variation occurred. At the MTD, the median plasma average concentrations of dexverapamil and nor-dexverapamil were 1.2 and 1.4 mumol/L, respectively. Dexverapamil did not affect the steady-state concentration (Css) of etoposide, but increased the Css of doxorubicin and doxorubicinol nearly twofold. The absolute neutrophil and platelet nadirs were significantly lower on the dexverapamil cycles compared with cycles of EPOCH alone, but other chemotherapy-related toxicities did not change. CONCLUSION: The phase II recommended dose of dexverapamil with EPOCH is 150 mg/m2 every 4 hours. This dose was well tolerated on an outpatient basis and achieved plasma concentrations of dexverapamil and nor-dexverapamil within the effective range for Pgp inhibition in vitro. Although dexverapamil increased the hematopoietic toxicity of EPOCH, it was mild, readily reversible, and offset by EPOCH dose reductions. Dexverapamil should be considered for further study.

Metabolism of taxol by human and rat liver in vitro: a screen for drug interactions and interspecies differences.

Human liver slices, human liver microsomes, and rat liver microsomes were used to investigate the metabolism of 3H-taxol. The effects of drugs frequently coadministered with taxol and the effects of several cytochrome P450 system probes were studied. In all, 16 compounds were screened. After incubation with liver slices or with microsomal protein, 3H-taxol was converted into several radioactive species resolved by HPLC. There were qualitative and quantitative species differences in the metabolism of taxol. The pattern of metabolism was similar for both human-derived preparations, with 6 alpha-hydroxytaxol being the major metabolite peak. In drug interaction studies performed with human liver microsomes, cimetidine 80 microM, and diphenhydramine 200 microM, had little or no effect on 6 alpha-hydroxytaxol formation. Quinidine, ketoconazole, dexamethasone and Cremophor EL inhibited 6 alpha-hydroxytaxol formation with IC50 values of 36 microM, 37 microM, 16 microM and 1 microliter/ml, respectively, but these concentrations exceed the usual clinical range. Cremophor EL also inhibited microsomal metabolism of taxol, but at 2 microliters/ml it had little or no effect on 6 alpha-hydroxytaxol production by human liver slices. These results suggest that: (1) taxol is metabolized by the cytochrome P450 system; (2) taxol metabolism is different in humans than in rats; (3) taxol metabolism in humans is unlikely to be altered by cimetidine, dexamethasone, or diphenhydramine, drugs regularly coadministered with taxol; (4) taxol metabolism can be indirectly affected by Cremophor EL, the formulation vehicle; (5) taxol metabolism may be altered by concentrations of ketoconazole achievable in humans only at very high doses; and (6) taxol metabolism and drug interaction studies of clinical relevance can be performed in vitro with human liver microsomes and human liver slices, but not with rat liver preparations.

Toxicity, metabolism, DNA incorporation with lack of repair, and lactate production for 1-(2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl)-5-iodouracil in U-937 and MOLT-4 cells.

Two cell lines, U-937 and MOLT-4, were used to investigate the toxicity, DNA incorporation, and effect on mitochondria of 1-(2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) and its putative metabolite 1-(2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl)-uracil (FAU). After 72-hr incubation, the IC50 values for FIAU were 6.4 microM for U-937 cells and 26 microM for MOLT-4 cells. IC50 values for FAU were 10-fold higher in both cell lines. Incubation for 24 hr with 10 microM [2-14C]FIAU led to 2.1% and 0.93% replacement of thymidine in DNA of U-937 and MOLT-4 cells, respectively. The predominant radioactive species measurable in DNA was FIAU. A similar incubation with [2-14C]FAU resulted in 4-fold lower DNA incorporation of a single radioactive species that coeluted with 1-(2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl)-5-methyluracil (FMAU). There was no evidence of a selective repair process after DNA incorporation of FIAU or FAU (FMAU). Increased intracellular concentrations of FIAU triphosphate and incorporation into DNA were associated with an increase in cellular toxicity. Continuous exposure to a clinically achievable concentration of FIAU, 0.44 microM, produced a constant DNA incorporation of 0.80% and 0.11% for U-937 and MOLT-4 cells, respectively. FIAU was not readily metabolized to FAU or iodouracil by human liver in vitro. Compared with 2',3'-dideoxycytidine as a positive control, after 12 days of continuous exposure of U-937 and MOLT-4 cells to FIAU there was no evidence of increased lactate production. These data negate several possible mechanisms (DNA chain termination, DNA polymerase inhibition, one form of selective mitochondrial poisoning, and FAU-mediated toxicity) and provide clues for possible mechanisms (FIAU triphosphate concentration and DNA incorporation). Further work is needed to develop a complete explanation for the delayed hepatic toxicity observed in the investigational clinical trials of FIAU.

Analysis of the active lactone form of 9-aminocamptothecin in plasma using solid-phase extraction and high-performance liquid chromatography.

We have developed a sensitive HPLC assay to quantitate the active lactone form of 9-aminocamptothecin (9AC) in human plasma over the concentration range 10-0.25 nM (0.091 ng/ml). Solid-phase extraction separated 9AC lactone from its less active metabolite, 9AC carboxylate, allowing samples to be stored for up to two months prior to reversed-phase HPLC analysis. An acidic (pH 2.55) isocratic HPLC mobile phase was used to enhance 9AC fluorescence resulting in an over 50-fold increase in assay sensitivity compared to previous methods. This assay was able to measure steady-state 9AC lactone concentrations even at the lowest dose level of 9AC used in our Phase I clinical trial.

Effect of cimetidine, probenecid, and ketoconazole on the distribution, biliary secretion, and metabolism of [3H]taxol in the Sprague-Dawley rat.

Male Sprague-Dawley rats had their bile ducts cannulated and were dosed with [3H]taxol (2 mg/kg, 68-77 microCi/mg) as a continuous intravenous infusion for 6 hr so that the plasma concentrations, tissue distribution, metabolism, and biliary secretion of taxol could be studied. Defining potential drug-drug interactions of taxol with cimetidine (90 mg/kg), probenecid (360 mg/kg), and ketoconazole (50 mg/kg) was motivated by frequent concomitant clinical use or the potential to reduce clearance of taxol so that lower doses could be used. At 6 hr, rats were killed. Samples of blood (plasma), lung, spleen, liver, kidney, heart, skeletal muscle, brain, testes, and fat were obtained. Taxol and metabolites were measured by total radioactivity counting and HPLC separation using on-line radioactivity detection. Concentrations of taxol in plasma increased to 0.19 microM in the control rats and did not reach steady-state by 6 hr. Lung, spleen, liver, and kidneys had the greatest tissue taxol concentrations [4.7-5.7 nmol/g (microM)] and were > 25-fold higher than the simultaneous 6-hr plasma taxol concentration. Taxol concentrations in brain and testes were negligible, 0.06 and 0.07 nmol/g, respectively. Radioactive metabolites were not found in plasma or most tissues. Only liver had appreciable concentrations of taxol and metabolites; however, > 80% of hepatic radioactivity was parent taxol. Through 6 hr of collection, 24% of the dose was secreted in the bile approximately 38% of which was as parent taxol. Cimetidine had no effect on the distribution, metabolism, or elimination of [3H]taxol. Probenecid did not effect tissue distribution or plasma concentrations of taxol.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the mutation of tyrosine 713 in p93c-fes on its catalytic activity and ability to promote myeloid differentiation in K562 cells.

The protooncogene protein-tyrosine kinase c-fes plays an active role in the induction of terminal myeloid differentiation in myeloid leukemia cells. Although p93c-fes contains two autophosphorylation sites, it is not known what role they play in its catalytic or biological activities. To address this question, the major autophosphorylation site at tyrosine 713 was mutated to phenylalanine (YF713), and the mutated cDNA was expressed in a baculovirus system to assess catalytic activity, as well as in an inducible retrovirus to determine its biological activity. The major phosphopeptide in p93c-fes in vitro contained Y713 and was absent in the YF713 mutant, which exhibited an 85% loss of autophosphorylation activity. The catalytic activity of p93c-fesYF713 with either RCM-lysozyme or poly(Glu,Tyr)4:1 as substrate was reduced by 85 and 78%, respectively, in comparison to p93c-fes. Retroviral infection of K562 cells with the c-fes cDNA under the control of the mouse metallothionein promoter increased superoxide formation, phagocytosis, CD13 and CD33 antigen expression, and doubling time 4-6 days after induction. Cells infected with c-fesYF713 exhibited 40% less superoxide formation but similar levels of phagocytosis, CD13/CD33 antigen, and doubling time in comparison to cells infected with c-fes. The level of phosphotyrosine-containing proteins did not markedly differ between K562 cells expressing either neo, c-fes, or c-fesYF713, with the exception of a reduction in the level of a 210-kDa protein specifically in both c-fes-expressing cell lines. The p210 was tentatively identified as bcr-abl, whose level was also reduced in cells expressing c-fes or c-fesYF713.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal of resistance to adriamycin by 8-chloro-cyclic AMP in adriamycin-resistant HL-60 leukemia cells is associated with reduction of type I cyclic AMP-dependent protein kinase and cyclic AMP response element-binding protein DNA-binding activities.

8-Chloro-cyclic AMP (8-Cl-cAMP) produces growth-inhibitory and differentiating activity in the promyelocytic leukemia cell line HL-60. Adriamycin (ADR)-resistant HL-60 (HL-60/AR) cells exhibit the multidrug-resistant phenotype but do not express the mdr1 gene product P-glycoprotein. To explore potential signaling processes that may be involved in this atypical form of drug resistance, 8-Cl-cAMP was used as a modulator of the cAMP second messenger signal transduction pathway. Treatment for 48 hr with a 10% inhibitory concentration of 8-Cl-cAMP potentiated ADR cytotoxicity 14-fold in HL-60/AR cells but not in the parental cell line. 8-Cl-cAMP was stable to hydrolysis in the medium after 48 hr and was present intracellularly predominantly as phosphorylated metabolites (70%) and the parent compound (30%). No difference occurred in ADR accumulation in HL-60/AR cells after treatment with 8-Cl-cAMP. Accompanying the 8-Cl-cAMP-mediated increase in ADR cytotoxicity in HL-60/AR cells was a reduction in the cytosolic type I cAMP-dependent protein kinase (PKA) and disappearance of the nuclear PKA holoenzyme. Coincident with these changes in drug-resistant cells was a marked reduction in the DNA-binding activity of the cAMP response element-binding protein to levels equivalent to those in sensitive cells. This effect appears to result from reduced phosphorylation of the cAMP response element-binding protein. These results suggest that the potentiation by 8-Cl-cAMP of ADR cytotoxicity in HL-60/AR cells occurs through down-regulation of nuclear type I PKA and cAMP response element-binding factors whose activities are regulated by PKA.

Steady-state plasma concentrations and effects of taxol for a 250 mg/m2 dose in combination with granulocyte-colony stimulating factor in patients with ovarian cancer.

Taxol, a natural product initially isolated from the stem bark of the western yew Taxus brevifolia, is undergoing phase II and III evaluation due to its reported activity against a variety of tumors. Previous studies have described correlations between plasma concentrations and toxicity when taxol is given (a) at lower doses, (b) for shorter infusion times, and (c) without granulocyte-colony-stimulating factor. Because the 24-h infusion schedule is most commonly used in current clinical trials, we attempted to correlate steady-state plasma concentrations of taxol achieved with a 24-h continuous i.v. infusion with toxicities and responses. Plasma samples from 48 refractory ovarian cancer patients were obtained 1-2 h prior to the end of the first taxol infusion. Taxol concentrations were measured by high-performance liquid chromatography (HPLC). Interpatient variation of taxol plasma concentrations was small (mean +/- SD, 0.85 +/- 0.21 microM. Total taxol body clearance was 256 +/- 72 ml min-1 m-2 (mean +/- SD). Taxol plasma protein binding was 88.4% +/- 1.3% (mean +/- SD, n = 9). Grade 3-4 hematologic toxicity, mainly leukopenia, occurred in 92% of the patients. The leukopenia was transient and did not warrant a reduction in the dose of taxol. Grade 3-4 nonhematologic toxicity occurred in 8% of the patients. No severe hypersensitivity reaction or grade 3-4 neurotoxicity was observed. Correlations of plasma concentrations and toxicities were not feasible due to the high frequency of hematologic effects and the low frequency of nonhematologic toxicity. The low degree of interpatient variation in plasma concentrations hindered the development of correlations with response.

Dextran sulfate is poorly absorbed after oral administration.

STUDY OBJECTIVE: To determine whether dextran sulfate (molecular weight, 7000 to 8000 daltons; 17% to 20% sulfur), a synthetic heparin analogue with anti-human immunodeficiency virus (HIV) activity in vitro, is absorbed after oral administration. DESIGN: Open-label, single-center study in two parts. The first part was a bioavailability study in which six subjects received a single 1800-mg oral dose and a single 225- or 300-mg intravenous dose. The second part was a study of the dose-response relation between dextran sulfate and total plasma lipolytic activity in which twelve subjects were given a single infusion of either 0.05, 0.5, 5, or 50 mg of dextran sulfate. SUBJECTS: Eighteen healthy volunteers. MEASUREMENTS AND MAIN RESULTS: In the bioavailability study, plasma and urine dextran-sulfate concentrations were measured by a competitive binding assay after each dose. In addition, two bioassays were used to assess plasma concentrations: plasma lipolytic activity and activated partial thromboplastin time (APTT). After the oral dose, plasma concentrations were not measurable with the competitive binding assay (lower limit of sensitivity, 1 microgram/mL); less than 0.5% of the dose was recovered in the urine, the APTT did not increase, and the median increase in the plasma lipolytic activity was only twofold (maximum increase, 11-fold). In contrast, after the intravenous dose of 225 mg, peak plasma concentrations by competitive binding assay were 26 to 35 micrograms/mL (median, 28 micrograms/mL); 25% to 29% (median, 25%) of the dose was recovered in the urine; the APTT increased to 3.5 to 9.2 times the baseline value (median increase, 6.9 times), and the plasma lipolytic activity increased by 185 to 548 times the baseline value (median increase, 438 times). In the dose-response study, intravenous doses as low as 0.5 mg produced significant increases in the plasma lipolytic activity. There was a steep dose-response curve between 0.5 and 50 mg. CONCLUSION: Dextran sulfate is very poorly absorbed after oral administration.

Iododeoxyuridine (IdUrd) incorporation into DNA of human hematopoietic cells, normal liver and hepatic metastases in man: as a radiosensitizer and as a marker for cell kinetic studies.

Iododeoxyuridine (IdUrd) was administered as a continuous infusion for 14 days to patients with glioblastoma and sarcoma, and for 3 days to patients with metastatic colorectal carcinoma. In the first group, the maximum incorporation of IdUrd into DNA was determined, taking granulocytes as parameter. In the second group, selective incorporation into DNA of normal liver and hepatic metastases of colorectal cancer was investigated. The highest dose of 675 mg/sq.m./day for 14 days produced IdUrd plasma concentrations of 1.8 +/- 0.3 microM, and a substitution of dThd by IdUrd in the range of 7.1-11.7%. Coadministration of fluorodeoxyuridine did not show significant enhancement of IdUrd-incorporation in granulocytes. Three-day intravenous infusions of IdUrd 1000 mg/sq.m./day produced 1.7-4.5% IdUrd-incorporation in hepatic metastases. Three-day intraarterial infusions (hepatic artery) produced 3.8-10.5% dThd-replacement, whereas, in 9/10 patients this was less than 1% in normal liver. In tumor tissue there was a trend towards FdUrd-modulated enhancement of IdUrd-incorporation, although there was considerable scatter. Cell kinetic studies revealed that IdUrd-incorporation in monocytes and granulocytes was very similar. In lymphocytes, a much lower fraction incorporated IdUrd. Liver tumor contained a considerably higher fraction of IdUrd-labeled cells, compared with normal liver. Potential doubling times for the tumors were estimated to be 10 days.

Selective incorporation of iododeoxyuridine into DNA of hepatic metastases versus normal human liver.

Fourteen patients received 5-iodo-2(1)-deoxyuridine (IdUrd) before surgery for placement of a hepatic arterial catheter. Biopsy specimens were obtained at the time of surgery and incorporation of IdUrd into deoxyribonucleic acid (DNA) in tumor and normal hepatic tissue was measured by HPLC and used as an index of drug selectivity. Over a 3-day intravenous infusion of IdUrd at 1000 mg/m2/day, substitution for thymidine in tumor DNA averaged 3.1%. Normal hepatic DNA contained less than 1% substitution by IdUrd. Arterial delivery of IdUrd increased levels in DNA, whereas modulation with fluorodeoxyuridine produced mixed results. In six patients, flow cytometric analysis showed that the tumor contained a median of 32% of tumor cells that had incorporated IdUrd in 3 days, corresponding to a potential doubling time of only 10 days. Thymidylate synthetase activity in tumors was 20-fold greater than in normal liver tissue, whereas thymidine kinase activity was twofold greater in tumors. These pharmacologic studies encourage further clinical trials of IdUrd as a cytotoxic agent or radiosensitizer.

Pharmacokinetics of 2',3'-dideoxycytidine in patients with AIDS and related disorders.

The clinical pharmacokinetics of 2',3'-dideoxycytidine (DDC) were determined after oral and intravenous administration in ten patients with AIDS or AIDS-related complex. A high performance liquid chromatography (HPLC) analysis procedure using cation exchange extraction columns was used to measure DDC levels as low as 0.1 microM (21 ng/mL) in plasma and urine. The kinetics of DDC were linear over the dose range of 0.03 to 0.5 mg/kg. Total body clearance was 227 mL/min/m2 and did not change after 6 to 14 days of dosing. The volume of distribution at steady state was 0.54 L/kg. Plasma half-life was 1.2 hours, and bioavailability was 88%. Most (75%) of the parent drug was found unchanged in the urine. As a result, renal function could play a role in dose adjustment of DDC. Comparison is made between the kinetics of DDC and 3'-azido-2',3'-dideoxythymidine (AZT). Similarities are noted in half-life and bioavailability. However, differences are observed for total body clearance, cerebrospinal fluid penetration, volume of distribution, metabolism, and recovery in urine.

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.