Robert C. Gaver

High-performance liquid chromatographic procedures for the analysis of carboplatin in human plasma and urine

SummarySpecific, sensitive and reproducible high-performance liquid chromatographic procedures were developed for the quantitative analysis of carboplatin in human plasma and urine. Plasma and urine were ultrafiltered with an Amicon CentrifreeTM micropartition system, and samples were injected onto a LiChrosorbTM diol column. The mobile phase was CH3CN/H2O (92:8, v/v) for plasma and CH3CN/0.015% H3PO4 (89:11, v/v) for urine. The effluents were monitored at 229 nm. Carboplatin eluted by 10 min. The detector response was linear from 0.5 (plasma) or 5 (urine) to 500 μg/ml. The lower limit of quantification was 1.0 μg/ml plasma and 5.0 μg/ml urine. Constitutents in plasma and urine, and possible degradation products (cyclobutane mono- and dicarboxylic acids) did not interfere. Within-day precision was less than 4% for plasma and 9% and 4% for urine concentrations of 40 and 401 μg/ml, respectively. Within-day accuracy was 96% or greater for both matrices. Carboplatin was not bound to the CentrifreeTM membrane and recovery was 94% for plasma and 96% for urine. The storage stability of carboplatin in water, plasma, plasma ultrafiltrate, and urine and the extent of binding to human plasma proteins were evaluated. The percentage of carboplatin reversibly bound to plasma proteins was minimal (≤10%) over a range of 1–50 μg/ml. In human plasma at 37 °C the drug was stable for about 2 h, but then degraded with a half-life of 32 h. Carboplatin had limited stability in water, plasma, and urine stored at-25 °C. Biological samples, therefore, should be stored frozen and analyzed within a week of collection to obtain valid results.

The disposition of carboplatin in ovarian cancer patients

SummaryCarboplatin was given as a 30-min infusion to 11 ovarian cancer patients at doses of 170–500 mg/m2. The ages, weights, and creatinine clearances (Clcr) ranged from 44 to 75 years, from 44 to 74 kg, and from 32 to 101 ml/min, respectively. Plasma, plasma ultrafiltrate (PU), and urine samples were obtained at appropriate times for 96 h and were analyzed for platinum. The PU and urine were also analyzed for the parent compound by HPLC. In patients with a Clcr of about 60 ml/min or greater, carboplatin decayed biexponentially with a mean t1/2α of 1.6 h and a t1/2β of 3.0 h. The mean (±SD) residence time, total body clearance, and apparent volume of distribution were 3.5±0.4 h, 4.4±0.85 l/h, and 16±31l, respectively. Cmax and AUCinf values increased linearly with dose, and the latter values correlated better with the dose in mg than in mg/m2. No significant quantities of free, ultrafilterable, platinum-containing species other than the parent compound were found in plasma, but platinum from carboplatin became protein-bound and was slowly eliminated with a minimal t1/2 of 5 days. The major route of elimination was excretion via the kidneys. Patients with a Clcr of 60 ml/min or greater excreted 70% of the dose as the parent compound in the urine, with most of this occurring within 12–16 h. All of the platinum in 24-h urine was carboplatin, and only 2%–3% of the dosed platinum was excreted from 48 to 96 h. Patients with a Clcr of less than about 60 ml/min exhibited dose-disproportional increases in AUCinf and MRT values. The latter were inversely related to Clcr (r=-0.98). Over a dose range of 300–500 mg/m2, carboplatin exhibited linear, dose-independent pharmaco-kinetics in patients with a Clcr of about 60 ml/min or greater, but dose reductions are necessary for patients with mild renal failure.

The disposition of carboplatin in the beagle dog

SummaryCarboplatin was administered i.v. to four groups of three male beagle dogs at doses of 3, 6, 12, and 24 mg/kg (60–580 mg/m2). Plasma samples were obtained at appropriate times and protein-free plasma ultrafiltrates (PU) were generated with Amicon Centrifree micropartition systems. Urine was collected at 24-h intervals for 96 h. PU and urine samples were analyzed for carboplatin by HPLC and for total platinum by atomic absorption spectrophotometry. Carboplatin accounted for about 90% of the free platinum in plasma. The Cmax and AUCinf values for carboplatin and for free platinum increased linearly with dose. The terminal elimination half-life and mean residence times for carboplatin and free platinum were each about 1 h. Total-body clearances for carboplatin (5.6 l/h per m2) and free platinum (5.1 l/h per m2) were constant over the dose range studied, as were the respective volumes of distribution (5.7 and 5.0 l/m2). A mean of 46% of the dose was excreted as carboplatin in 24-h urine; and by 72 h, 70% of the platinum administered was excreted in the urine. Free platinum was cleared by both renal and nonrenal processes. These results show that a dose of carboplatin is rapidly excreted in the urine and that carboplatin and plasma-free platinum exhibit linear pharmacokinetics in the beagle dog.

Disposition of anagrelide, an inhibitor of platelet aggregation

A single dose of 14C‐anagrelide (6,7‐dichloro‐1,5‐dihydroimidazo [2,1‐b] quinazolin‐2(3H)‐one monohydrochloride) equivalent to 1 mg free base and containing 100 µCi radioactivity, was taken by five healthy, fasting men. Blood, plasma, urine, and feces were analyzed for total radioactivity. Plasma and urine concentrations of anagrelide were determined, and the urinary metabolite profile was established by high‐performance liquid chromotography (HPLC). The drug was rapidly absorbed with peak plasma levels of radioactivity equivalent to 50 ng anagrelide per milliliter at about 1 hr. These levels decreased to less than 10% of peak in 24 hr. Plasma levels of anagrelide peaked at 5 ng/ml at about 1 hr, decreased rapidly during the first 6 to 8 hr, and then declined more slowly, with an estimated terminal elimination half‐life of about 3 days. No significant quantities of radioactivity were associated with the cellular elements of blood. Anagrelide was extensively metabolized before elimination in urine. Means of 68% and 72% of the dose were excreted in urine as metabolites in 24 and 144 hr, and 10% of the dose was recovered in the feces. Several urinary metabolites were detected by HPLC.

Validated HPLC procedures for the analysis of BMY-28090 in human plasma and urine

SummaryThe compound BMY-28090 (elsamicin A) is a new fermentation product with antitumor properties, which has the same aglycone as chartreusin but contains two novel sugars. To define the disposition of BMY-28090 during phase I trials, HPLC procedures were developed and validated for the quantitation of the drug in human plasma and urine. To 1.0 ml plasma were added 0.5 ml 0.2M phosphate buffer (pH 8.0), 125 ng 1-naphthol (internal standard) in 25 μl MeOH and 5 ml ethyl acetate. After mixing and centrifugation, 4 ml ethyl acetate layer was removed, evaporated to dryness, and the residue was dissolved in 250 μl mobile phase and injected (200 μl). To 1.0 ml urine were added 100 μl MeOH and 1.0 ml 0.5M succinate buffer (pH 4.0). After mixing (30 s) and sonication (1 min), the solution was filtered in an Amicon Centrifree micropartition unit and injected (30 μl). An IBM C-8 column 5-μm and fluorescence detection (excitation at 254 mm, 418 nm emission filter) were used for both analyses. The mobile phases for plasma (2 ml/min) and urine (1.3 ml/min) were H2O/CH3CN (7:3 v/v) and H2O/CH3CN/MeOH (6:3:1 (v/v), respectively, with 1.5 ml 85% H3PO4 and 1.5 ml triethylamine/1. BMY-28090 eluted at 8–10 min and 1-naphthol, at 10–11 min. The standard curves were linear from 1 to 50 ng/ml plasma and from 10 to 1000 ng/ml urine. The within- and between-day precision was <3% for plasma and <5% for urine. Accuracies were within 6% of the nominal value and recoveries were 75% and 90% for plasma and urine, respectively. At 37°C, BMY-28090 was stable in plasma for at least 8 h but had a half-life of 36 h in urine. The drug was stable in plasma and urine for 30 days at −20°C.

Disposition of orally administered 14C-prednimustine in cancer patients

SummaryA single oral solution dose (40 mg/m2) of 14C-prednimustine was administered to each of four cancer patients. Plasma, urine, and feces were collected at appropriate times and analyzed for total radioactivity. Plasma samples were analyzed for prednimustine. Peak plasma levels of radioactivity (1–3 μg 14C-prednimustine equivalents) occurred at 1.5–3 h in three patients and at 5–6 h in one patient. No intact prednimustine was detected in the plasma; this means that if present, it would be at a concentration of 0.02 μg/ml or less and would account for less than 1% of the total drug-related material at the time of peak plasma levels. Solvent-extractable metabolites had a plasma half-life of about 8 h or less. By 24 h essentially all the plasma radioactivity appeared to be covalently bound, and it was eliminated slowly with an estimated terminal elimination half-life of about 10 days. Rapid urinary excretion occurred in the first 24 h, and 40%–60% of the dose was recovered in the urine in 72 h. Although prednimustine was well absorbed, the ester was subject to extensive presystemic metabolism and was not present in the systemic circulation after oral administration.

Evaluation of the availability of drugs incorporated in rat laboratory diet.

The extent of absorption of drugs incorporated in rat diet was determined with three chemical classes of test substances (acidic, basic, and neutral compounds). Radiolabeled drugs were administered to conditioned, fasted rats by oral gavage of a solution dosage form and by feeding the test compound incorporated in pulverized rat diet. The availability of the drug from the diet was determined by a comparison of the excretion of radioactivity in the urine after administration of the solution or the diet. The extent of absorption of water-soluble salts of acidic (3,4-dihydroxy-N-cyclobutylmethyl morphinan tartrate) and basic (9-methyl-3-(1H-tetrazol-5-yl) 4H-pyrido-[1,2a]-pyrimidin-4-one) compounds was equivalent from the diet and solution. The availability of the water-insoluble, neutral compound (8-methoxypsoralen) from the diet was 70% relative to an oral solution. The significance of these findings in relation to dissolution of drugs from diet mixtures and their use in the interpretation of toxicologic studies is discussed.

Phase I ― preliminary phase II trial of iproplatin, a cisplatin analogue

SummaryIproplatin was administered intravenously over 30 min daily for 5 consecutive days every 3 weeks to 80 evaluable patients with a variety of refractory solid tumor malignancies. Thrombocytopenia was the dose-limiting toxicity. Reversible drug-induced renal dysfunction was observed in 3 patients. One patient sustained mild ototoxicity but neurotoxicity was not encountered. Transient neutropenia, anemia, nausea, vomiting, diarrhea, elevations of liver enzymes, alopecia, and skin rash also occurred. The spectrum and severity of toxicity of iproplatin were found to differ from those of cisplatin. The maximally tolerated dose (MTD) was 45 mg/m2/day in patients who received prior chemotherapy and 65 mg/m2/day in those who did not. No complete responses occurred. Partial responses were obtained in 2/15 patients with colon cancer, 3/18 with breast cancer, 2/4 with carcinoma of unknown primary site and 1/2 with pancreatic cancer. Thirteen patients with lung (5), breast (4), colon (2), head and neck (1) and cervical (1) cancers had stable disease. Based on the different toxicity profiles between iproplatin and cisplatin and the possible antitumor efficacy of the former, phase II investigation of iproplatin has been initiated.

Phase I trial of tallysomycin S10b, a bleomycin analogue

SummaryBleomycin is an agent with significant antitumor efficacy whose major dose limiting toxicity is pulmonary fibrosis. Attempts have thus been made to identify congeners with reduced toxicity and with comparable or greater antitumor activity. Tallysomycin S10b is a bleomycin analogue possessing significantly greater potency, equal or reduced lung toxicity, and slightly greater antineoplastic activity when compared to the parent compound in preclinical studies. This report describes our experience with tallysomycin S10b in 30 patients with a variety of non-hematologic neoplasms. Pulmonary toxicity, occurring in 4 patients, was the major toxicity. The recommended cumulative dose of tallysomycin S10b was difficult to establish from the results of this study, as pulmonary toxicity appeared to be more idiosyncratic than dose-or schedule-dependent. The employment of more sensitive methods for detecting pulmonary toxicity in this study suggest that tallysomycin S10b may have reduced pulmonary toxicity compared to the parent compound.Both bleomycin and tallysomycin S10b have similar t1/2 βhalf-lives of 2–4 h. Six patients had prolonged terminal elimination half-lives of tallysomycin S10b, but no clear relationship between this phenomenon and efficacy or toxicity was evident.No complete or partial responses occurred. Disease stabilization occurred in 4 of 15 patients with diagnoses of renal cell carcinoma [2], rectal cancer [1] and lung cancer [1]. Five of eight patients with non-measurable disease had stable disease, including one with mesothelioma, one with carcinoma of the head and neck, two with renal cell cancer and one with colon carcinoma.

A radioimmunoassay procedure for tallysomycin S10b in human plasma and urine

SummaryA simple and sensitive radioimmunoassay (RIA) procedure was developed and validated for the analysis of tallysomycin S10b in human plasma and urine. The assay utilized antisera developed in rabbits, 125I-tallysomycin as the radioligand, and dextran-coated charcoal to separate free and bound antigen. The antibody was specific in that it did not cross-react with either tallysomycin A or bleomycin. The lower limit of quantification was 5 ng per ml plasma or urine, and the linear range of the assay was 5–320 ng tallysomycin S10b base per ml plasma or urine. The within-day assay variability (%RSD) for plasma and urine was 11% at a concentration of 50 ng per ml, and 3% and 7% for plasma and urine, respectively at 200 ng per ml. Within-day accuracy ranged from 100% to 108% of the theoretical value. Tallysomycin S10b was stable in human plasma and urine at concentrations of 20 and 160 ng per ml for at least 7 months when stored at-20°C. The method was applied to the analysis of plasma and urine samples from a patient given tallysomycin S10b as part of a phase I study.