Raymond W. Klecker

Plasma and cerebrospinal fluid pharmacokinetics of 3′‐azido‐3′‐deoxythymidine: A Novel pyrimidine analog with potential application for the treatment of patients with AIDS and related diseases

We investigated the clinical pharmacokinetics of azidothymidine (N3TdR) as part of a phase I/II trial in the treatment of acquired immunodeficiency syndrome and related diseases. During the 6‐week course of therapy, drug levels in plasma, cerebrospinal fluid, and urine were determined by HLPC. The plasma half‐life of N3TdR was 1.1 hour. The total body clearance was 1.3 L/kg/hr. At intravenous doses of 5 mg/kg or oral doses of 10 mg/kg, plasma levels were continuously maintained above the target level of 1 μmol/L. Oral bioavailability was 63% ± 13%. Substantial penetration of N3TdR into cerebrospinal fluid was demonstrated. At doses of 5 mg/kg intravenously or 10 mg/kg orally, cerebrospinal fluid drug levels exceeded and were maintained close to 1 μmol/L. Nineteen percent of the administered dose was excreted unchanged into the urine. Renal clearance was 0.23 L/kg/hr. N3TdR possesses pharmacokinetic properties that would facilitate the long‐term treatment of patients with acquired immunodeficiency syndrome: it can be given orally and it penetrates the central nervous system.

Clinical Pharmacokinetics of Suramin in Patients With HTLV-III/LAV Infection

Suramin has been reported to inhibit the reverse transcriptase activity of a number of retroviruses and to reduce the in vitro infectivity and cytopathic effect of HTLV‐III/LAV, the etiologic agent of acquired immune deficiency syndrome (AIDS). The clinical pharmacokinetics of suramin were investigated as part of a pilot study to evaluate the safety and efficacy of this drug for the treatment of patients with diseases caused by HTLV‐III/LAV. A dose of suramin 6.2 g was given intravenously over a five‐week period to four patients. After the last dose, the plasma half‐life of suramin was 44 to 54 days. This is among the longest half‐lives reported for any therapeutic substance given to humans. Total plasma levels of suramin were greater than 100 μg/mL for several weeks. In vitro activity of suramin was found at concentrations as low as 50 μg/mL. Metabolites were not found in plasma, and urinary excretion accounts for elimination of most of the drug. Suramin is approximately 99.7% bound to plasma proteins. The results from these initial clinical pharmacokinetic studies might assist the design of further therapeutic trials of suramin, especially the selection of frequency of dosing and adjustments for renal impairment.

Pharmacology and phase I/II study of continuous intravenous infusions of iododeoxyuridine and hyperfractionated radiotherapy in patients with glioblastoma multiforme.

Forty-seven adult patients with glioblastoma multiforme (GBM) were treated in a phase I/II study combining continuous intravenous (IV) infusions of iododeoxyuridine (IdUrd) and hyperfractionated radiation therapy. IdUrd was administered as a continuous infusion (24 h/d) for two separate 14-day infusion periods. The dose of IdUrd was escalated from 500 to 1,200 mg/m2/d. The initial wide-field tumor volume was treated to 45 Gy at 1.5 Gy fractions twice daily over 3 weeks. Following a planned 2-week break, a reduced-field boost of 25 Gy was delivered using 1.25 Gy fractions twice daily over 2 weeks (total dose, 70 Gy over 9 weeks). The IdUrd infusion preceded both the wide-field and reduced-field irradiation by 1 week. All treatment was performed on an outpatient basis. Dose-limiting systemic toxicity to the bone marrow (primarily thrombocytopenia) and gastrointestinal (GI) tract (both stomatitis and diarrhea) established the maximum tolerable dose (MTD) at 1,000 mg/m2/d for a 14-day infusion. Significant local toxicity (within the radiation field) was not seen. The kinetics of IdUrd were linear with dose escalation and reached steady-state plasma concentrations of 1.3 to 3.4 mumol/L. The total body clearance of IdUrd was .82 L/min/m2. The primary metabolite, 5-iodouracil (IUra) approached steady state by day 6 of the infusion when plasma levels were 60 times higher than IdUrd. Plasma levels of uracil and thymine, but not thymidine, were elevated throughout the infusion. With a minimum follow-up of 1 year, ten patients remain alive, while 33 patients died of progressive disease, and four patients died of other causes (including one treatment-related death). The median survival for all 47 patient and for the 40 patients receiving the MTD was 45 and 47 weeks, respectively, with 12% and 14% survivals at 24 months. Using a Cox regression analysis, age (less than or equal to 50 years v greater than 50 years) and pretreatment performance status (PS) (Eastern Cooperative Oncology Group [ECOG]-PS 0 to 1 v PS 2 to 3) were independent, statistically significant (P2 less than .05) predictors of survival, with the ECOG status being a better predictor. Patients with a PS 0 to 1 (28 patients) had a median survival of 64 weeks with 21% survival at 24 months, compared with a median survival of 29 weeks and 0% survival at 12 months in the 19 patients with PS 2 to 3. The overall and subgroup survival data are at least comparable to other combined modality treatment approaches in patients with GBM.

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 μM (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.

Clinical pharmacology of 5-iodo-2'-deoxyuridine and 5-iodouracil and endogenous pyrimidine modulation.

We describe the clinical pharmacology and metabolism of 5‐iodo‐2′‐deoxyuridine (IdUrd) during and after a 12‐hour infusion. The kinetics of IdUrd were linear between 250 and 1200 mg/m2. The plasma IdUrd concentration reached steady state in <1 hour. Total body clearance of IdUrd was 750 ml/min/ m2 and the disappearance t½ at the end of the infusion was <5 minutes. The primary metabolite, 5‐iodouracil (IUra), did not reach steady state during the infusion. At the end of the 1200 mg/m2 infusion, the maximum plasma IUra concentration was 100 µmol/L, or about 10 times the simultaneous IdUrd plasma concentration. During the infusion there was at least a fifty‐ to 100‐fold increase in uracil and thymine plasma concentrations. After the infusion, IUra disappearance from plasma was nonlinear, with an apparent Michaelis constant of 30 µmol/L. Plasma uracil and thymine levels slowly decreased after the IdUrd infusion until IUra fell to <30 µmol/L. There was subsequently a parallel and more rapid decrease in the plasma concentrations of uracil and thymine. Uridine, 2′‐deoxyuridine, and thymidine plasma levels did not change significantly as a result of IdUrd therapy. These changes in endogenous pyrimidine pools are consistent with competitive inhibition of dihydrouracil dehydrogenase by IUra. An in vitro human bone marrow assay was used to determine the relative toxicity of IdUrd and IUra. Although exposure to IUra was tenfold higher than that to IdUrd, IdUrd was at least 100 times more cytotoxic to marrow cells.

Quantification of Suramin by Reverse-Phase Ion-Pairing High-Performance Liquid Chromatography

Abstract A specific and sensitive method has been developed for the separation and quantification of suramin and trypan blue (internal standard) in human plasma. Plasma samples were extracted by centrifugation after the addition of ion-pairing reagent (tetra-butylammonium phosphate, TRAP) and methanol. Extracts were injected directly onto a reverse-phase ion-pairing HPLC system with 5 mM TBAP in the mobile phase. There was nearly 100% extraction efficiency after 3 cumulative extracts of each sample. The limit of quantitation was 0.5 μg/ml at a detection wavelength of 313 nm. Analysis of 3 post-therapy samples from a patient with AIDS was used to determine a plasma half-life for suramin of at least 3 weeks.

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

Fourteen patients received 5‐iodo‐21‐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 < 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 pharmacologie studies encourage further clinical trials of IdUrd as a cytotoxic agent or radiosensitizer.

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.

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.

Distribution of 1-(2-Deoxy-2-fluoro-β-d-arabinofuranosyl) Uracil in Mice Bearing Colorectal Cancer Xenografts Rationale for Therapeutic Use and as a Positron Emission Tomography Probe for Thymidylate Synthase

Purpose: In colorectal, breast, and head and neck cancers, response to 5-fluorouracil is associated with low expression of thymidylate synthase. In contrast, tumors with high expression of thymidylate synthase may be more sensitive to prodrugs such as 1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl) uracil (FAU) that are activated by thymidylate synthase. These studies were designed to evaluate FAU as a potential therapeutic and diagnostic probe. Experimental Design: [18F]-FAU and [3H]-FAU were synthesized with >97% radiochemical purity. [3H]-FAU or [18F]-FAU was administered intravenously to severe combined immunodeficient mice bearing either HT29 (low thymidylate synthase) or LS174T (high thymidylate synthase) human colon cancer xenografts. Four hours after [3H]-FAU dosing, tissue distribution of total radioactivity and incorporation of 1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl) 5-methyluracil (FMAU), derived from thymidylate synthase activation of FAU, into tumor DNA was measured. Positron emission tomography (PET) images were obtained for 90 minutes after injection of [18F]-FAU. Thymidylate synthase activity was determined in vitro in tumors from untreated mice by [3H] release from [3H]dUMP. Each cell line was incubated in vitro with [3H]-FAU or [3H]-FMAU in the absence or presence of 5-fluoro-2′-deoxyuridine (FdUrd) and then was analyzed for incorporation of radiolabel into DNA. Results: Thymidylate synthase enzymatic activity in LS174T xenografts was ∼3.5-fold higher than in HT29 xenografts, and incorporation of radioactivity derived from [3H]-FAU into LS174T DNA was ∼2-fold higher than into HT29 DNA. At 240 minutes, radioactivity derived from [3H]-FAU was ∼2-fold higher in tumors than in skeletal muscle. At times up to 90 minutes, PET imaging detected only small differences in uptake of [18F]-FAU between the tumor types. Fluorine-18 in skeletal muscle was higher than in tumor for the first 90 minutes and plateaued earlier, whereas [18F] in tumor continued to increase during the 90-minute imaging period. For both cell lines in vitro, FdUrd decreased the rate of incorporation of [3H]-FAU into DNA, whereas the incorporation of [3H]-FMAU was increased. Conclusions: These results for FAU incorporation into DNA in vitro and in vivo further support clinical evaluation of FAU as a therapeutic agent in tumors with high concentrations of thymidylate synthase that are less likely to respond to 5-fluorouracil treatment. The high circulating concentrations of thymidine reported in mice may limit their utility in evaluating FAU as a PET probe.