James M. Brennan

Phase I trial of buthionine sulfoximine in combination with melphalan in patients with cancer.

PURPOSE AND METHODS Resistance to alkylating agents and platinum compounds is associated with elevated levels of glutathione (GSH). Depletion of GSH by buthionine sulfoximine (BSO) restores the sensitivity of resistant tumors to melphalan in vitro and in vivo. In a phase I trial, each patient received two cycles as follows: BSO alone intravenously (i.v.) every 12 hours for six doses, and 1 week later the same BSO as cycle one with melphalan (L-PAM) 15 mg/m2 i.v. 1 hour after the fifth dose. BSO doses were escalated from 1.5 to 17 g/m2 in 41 patients. RESULTS The only toxicity attributable to BSO was grade I or II nausea/vomiting in 50% of patients. Dose-related neutropenia required an L-PAM dose reduction to 10 mg/m2 at BSO 7.5 g/m2. We measured GSH in peripheral mononuclear cells (PMN), and in tumor biopsies when available, at intervals following BSO dosing. In PMNs, GSH content decreased over 36 to 72 hours to reach a nadir on day 3; at the highest dose, recovery was delayed beyond day 7. The mean PMN GSH nadirs were approximately 10% of control at BSO doses > or = 7.5 g/m2; at 13 and 17 g/m2, all but two patients had nadir values in this range. GSH was depleted in sequential tumor biopsies to a variable extent, but with a similar time course. At BSO doses > or = 13 g/m2, tumor GSH was < or = 20% of starting values on day 3 in five of seven patients; recovery had not occurred by day 5. We measured plasma concentrations of R- and S-BSO by high-performance liquid chromatography (HPLC) in 22 patients throughout the dosing period. Total-body clearance (CLt) and volume of distribution at steady-state (Vss) for both isomers were dose-independent. The CLt of S-BSO was significantly less than that of R-BSO at all doses, but no significant differences in Vss were observed between the racemates. Harmonic mean half-lives were 1.39 hours and 1.89 hours for R-BSO and S-BSO, respectively. CONCLUSION A biochemically appropriate dose of BSO for use on this schedule is 13 g/m2, which will be used in phase II trials to be conducted in ovarian cancer and melanoma.

Determination of antisense phosphorothioate oligonucleotides and catabolites in biological fluids and tissue extracts using anion-exchange high-performance liquid chromatography and capillary gel electrophoresis

Chemically modified phosphorothioate oligodeoxynucleotides (ODNs) have become critical tools for research in the fields of gene expression and experimental therapeutics. Bioanalytical assays were developed that utilized fast anion-exchange high-performance liquid chromatography (HPLC) and capillary gel electrophoresis (CGE) for the determination of 20-mer ODNs in biological fluids (plasma and urine) and tissues. A 20 mer ODN in the antisense orientation directed against DNA methyltransferase (denoted as MT-AS) was studied as the model ODN. The anion-exchange HPLC method employed a short column packed with non-porous polymer support and a ternary gradient elution with 2 M lithium bromide containing 30% formamide. Analysis of the MT-AS is accomplished within 5 min with a detection limit of approximately 3 ng on-column at 267 nm. For plasma and urine, samples were diluted with Nonidet P-40 in 0.9% NaCl and directly injected onto the column, resulting in 100% recovery. For tissue homogenates, a protein kinase K digestion and phenol-chloroform extraction were used, with an average recovery of about 50%. Since the HPLC assay cannot provide one-base separation, biological samples were also processed by an anion-exchange solid-phase extraction and a CGE method to characterize MT-AS and its catabolites of 15-20-mer, species most relevant to biological activity. One base separation, under an electric field of 400 V/cm at room temperature, was achieved for a mixture of 15-20-mer with about 50 pg injected. Assay validation studies revealed that the combined HPLC-CGE methods are accurate, reproducible and specific for the determination of MT-AS and its catabolites in biological fluids and tissue homogenates, and can be used for the pharmacokinetic characterization of MT-AS.

Phase I trial of fluorouracil modulation by N-phosphonacetyl-L-aspartate and 6-methylmercaptopurine ribonucleoside

Inhibition of pyrimidine and purine synthesis has been demonstrated to potentiate 5-fluorouracil (5-FU) activity in preclinical models. Low-dose phosphonacetyl-l-aspartate (PALA) potentiates the incorporation of 5-FU into RNA, without detectably increasing its toxicity. 6-Methylmercaptopurine riboside (MMPR) results in inhibition of purine biosynthesis with elevation of phosphoribosyl pyrophosphate (PRPP), which in turn is believed to increase the phosphorylation and intracellular retention of 5-FU. We conducted a phase I clinical trial to determine the maximum tolerated dose of 5-FU in combination with low-dose PALA and a biochemically-optimized dose of MMPR. The regimen consisted of PALA 250 mg/m2 given on day 1, followed 24 h later by MMPR 150 mg/m2, and escalating doses of 5-FU from 1625 to 2600 mg/m2 by 24 h continuous infusion. This regimen was repeated weekly. A group of 29 patients with a diagnosis of malignant solid tumor were entered; their median performance status was 1. The dose-limiting toxicity was mucositis, while other gastrointestinal toxicity was minimal. Two patients also experienced ischemic chest pain during the 5-FU infusion. The maximum tolerated dose of 5-FU in this combination was 2600 mg/m2. Several responses were observed including a complete remission in a previously treated breast cancer patient and two partial responses in breast and colon cancer. MMPR pharmacokinetics were obtained from urine analyses in 21 patients on this trial; there was no correlation between the pharmacokinetics of MMPR and the toxicity observed. This regimen was well tolerated and phase II trials are warranted using PALA 250 mg/m2, MMPR 150 mg/m2, and 5-FU 2300 mg/m2 by continuous infusion over 24 h.

Phase I trial of fluorouracil modulation by n-phosphonacetyl-l-aspartate and 6-methylmercaptopurine ribonucleoside (MMPR), and leucovorin in patients with advanced cancer

The results of several clinical trials support the hypothesis that biochemical modulation may enhance the antitumor activity of 5-Fluorouracil (5-FU). We have performed a phase I trial using a combination of three different biochemical modulators at the optimal dose established in previous clinical trials. The modulators include: phosphonacetyl-l-aspartate (PALA), which may increase 5-FU incorporation into RNA; leucovorin, which potentiates thymidylate synthase inhibition; and 6-methylmercaptopurine riboside (MMPR), which promotes the intracellular retention of fluorinated nucleotides. The treatment regimen consisted of PALA 250 mg/m2 day 1, followed 24h later by MMPR 150 mg/m2 as an iv bolus, and the initiation of a 24 hour infusion of 5-FU along with leucovorin 50 mg/m2. This regimen was repeated weekly. Doses of 5-FU were escalated in cohorts of four or more patients from 2,000 to 2,600 mg/m2. Among 20 patients entered, the majority had colorectal cancer, and most had received prior 5-FU treatment. Toxicity was predominantly gastrointestinal, and diarrhea was dose-limiting at a 5-FU dose of 2600 mg/m2. There were three partial remissions observed, two of whom had colorectal cancer. Emerging data that casts doubt on the modulation value of PALA at this dose and schedule suggests that revision of this regimen be considered before Phase II trial.

Phase I study of phosphonacetyl-L-aspartate, 5-fluorouracil, and leucovorin in patients with advanced cancer.

Low-dose phosphonacetyl-l-aspartate (PALA) may potentiate both 5-fluorouracil (5-FU) incorporation into RNA and thymidylate synthase inhibition by 5-fluorodeoxyuridylate (5-FdUMP). The gastrointestinal toxicity of 5-FU is not increased by PALA administration. Exogenous leucovorin, on the other hand, which enhances thymidylate synthase inhibition, appears to increase the clinical toxicity of 5-FU in a dose-dependent manner. As a result, the clinical use of high-dose leucovorin requires a marked dose reduction of 5-FU. Extracellular leucovorin levels of 1 μM suffice to maximize the enhancement of thymidylate synthase inhibition in several models. We conducted a trial to add leucovorin to the PALA/5-FU regimen. We chose a leucovorin dose that was predicted to yield end-infusion total reduced folate concentrations of 1 μM. The major endpoint was to determine the maximum tolerated dose of 5-FU in this combination. The regimen consisted of 250 mg/m2 PALA given on day 1 and, 24 h later, escalating 5-FU doses ranging from 1,850 to 2,600 mg/m2 admixed with 50 mg/m2 leucovorin and given by 24-h infusion. Courses were repeated weekly. A total of 24 patients with a median performance status of 1 were entered at three dose levels. Diarrhea was dose-limiting; 6/13 patients had grade II or worse diarrhea at 2,600 mg/m2. Dose modification resulted in a mean dose intensity of 2,300 mg/m2 at both the 2,600- and 2,300-mg/m2 dose levels. The 2,300-mg/m2 dose is suitable for phase II testing of this regimen. Three patients (two with breast cancer and 1 with sarcoma) had a partial remission. We measured steady-state concentrations (Css) of 5-FU in 23 patients. The mean Css increased with dose from 0.738 to 1.03 μg/ml. Total body clearance did not vary with dose in this range. Patients with grade II or worse diarrhea had a higher mean Css (1.10±0.19) than those with grade O or I toxicity (0.835±0.25,P<0.02). Total bioactive folates (bound and free) were measured using a biological assay. Pretreatment values ranged from 2 to 52 nM and were not predictive of toxicity. End-infusion (23-h) values were somewhat lower than predicted and ranged from 400 to 950 nM. The risk of diarrhea was positively correlated with end-infusion total folate values. In a logistic regression analysis, total folate values obtained at 23 h were a more powerful predictor of diarrhea than were 5-FU Css values. These results confirm the contribution of leucovorin to the toxicity of the 5-FU/leucovorin combination and suggest that interpatient differences in folate pharmacology may contribute to the therapeutic index of the 5-FU/leucovorin combination.

High-performance liquid chromatographic determination of ethacrynic acid in human plasma

A high-performance liquid chromatographic method for the determination of ethacrynic acid (EA) in human plasma is described. Plasma was prepared for analysis by addition of 4-(2,4-dichlorophenoxy)-butyric acid as an internal standard followed by acidification with hydrochloric acid and extraction with ethyl acetate. Separation was by isocratic reversed-phase chromatography, the column effluent was monitored at 280 nm and quantitation was performed using peak-area ratios. The linear range for EA determination was from 0.5 to 25 micrograms/ml with a lower limit of detection of 0.1 microgram/ml. The reported method is convenient, sensitive and reproducible, illustrating its usefulness for pharmacokinetic studies.

High-performance liquid chromatographic determination of the S- and R-diastereoisomers of L-buthionine (SR)-sulfoximine in human plasma and urine

A sensitive and reproducible HPLC procedure was developed for the simultaneous determination of the diastereoisomers of the synthetic amino acid L-buthionine-(SR)-sulfoximine (BSO) in human plasma and urine. Plasma samples were prepared for analysis by addition of internal standard (L-norleucine) followed by ultrafiltration using disposable centrifugal filtration units. Urine samples received internal standard followed by solid phase extraction using disposable C18 cartridges. All samples were derivatized with phenylisothiocyanate (PITC). The derivatized amino acids were separated by HPLC on an octyldecyl column (250 mm x 4.6 mm I.D., 5 microns particle size) using a mobile phase of sodium acetate-acetonitrile-triethylamine-ethylaminediaminetetraacetic acid. The column effluent was monitored at 254 nm and quantitation was performed using peak areas. The linear range for each diastereoisomer of L-(SR)-BSO was from 2 to 100 micrograms/ml in plasma and from 10 to 1000 micrograms/ml in urine. The method is reproducible, convenient and sensitive, illustrating its utility for application in pharmacokinetic studies.