High-performance liquid chromatographic procedure for the quantitative determination of paclitaxel (Taxol) in human plasma.

An isocratic high-performance liquid chromatographic method has been developed and validated for the quantitative determination of paclitaxel (Taxol), a novel antimitotic, anticancer agent, in human plasma. The analysis required 0.5 ml of plasma, and was accomplished by detection of the UV absorbance of paclitaxel at 227 nm following extraction and concentration. The method involved extraction of paclitaxel from plasma, buffered with 0.5 ml of 0.2 M ammonium acetate (pH 5.0), onto 1-ml cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the mobile phase, acetonitrile-methanol-water (4:1:5, v/v/v) containing 0.01 M ammonium acetate (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5 microns column. The retention time of paclitaxel was 10 min. The validated quantitation range of the method was 10-1000 ng/ml (0.012-1.17 microM) of paclitaxel in plasma. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of clinical study samples. The observed recovery for paclitaxel was 83%. Epitaxol, a biologically active stereoisomer, and baccatin III, a degradation product, were also chromatographically separated from taxol by this assay. The method was applied to samples from a clinical study of paclitaxel in cancer patients, providing a pharmacokinetic profiling of paclitaxel.

Phase I trial and clinical pharmacology of elsamitrucin.

Elsamitrucin (BMY-28090) is an antitumor antibiotic first described in 1985 that has significant oncolytic activity against a number of murine tumors including P388, L1210, B16 and M5076, as well as against MX1 and HCT116 xenografts. Preclinical toxicology studies of elsamitrucin revealed edema of multiple organs associated with hypoproteinemia and, at lethal doses, severe multiorgan toxicity. We conducted a phase I clinical trial (31 patients) of elsamitrucin administered as a 10-min i.v. infusion every 3 weeks. The starting dose (0.6 mg/m2) was 1/3 of the dog low toxic dose. The maximum tolerated dose was 30 mg/m2. Dose-limiting toxicity was reversible hepatic dysfunction manifested by elevated transaminase levels not associated with bilirubin, alkaline phosphatase, or lactate dehydrogenase elevations. Other toxicities included nausea, vomiting, malaise, and phlebitis. Because the hepatic toxicity was brief and reversible, a subsequent study (18 patients) was conducted with elsamitrucin administered every 2 weeks. Reversible grade 3 hepatotoxicity was again observed at 30 mg/m2. Plasma and urine samples from patients receiving doses of 0.6-36 mg/m2 were analyzed for drug content. The maximum plasma concentration and area under the plasma concentration versus time curve values increased linearly with doses up to 25 mg/m2 but not at higher doses. The terminal half-lives, total body clearances, and volume of distribution were 36-60 h, 10-19 liters/h/m2, and 400-1100 liters/m2, respectively. Less than 5% was excreted in the urine in 24 h as parent compound. Bile was collected from one patient with an indwelling biliary catheter. Approximately 22% of the dose was excreted in 48 h, suggesting that biliary excretion of elsamitrucin may be an important route of drug elimination. Based on reversible hepatic toxicity, the phase II recommended dose of elsamitrucin is 25 mg/m2 every 2 weeks.

Phase I trial of tallysomycin S10b, a bleomycin analogue.

Bleomycin 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 beta half-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, rectal cancer and lung cancer. 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.

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

The 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 0.2 M phosphate buffer (pH 8.0), 125 ng 1-naphthol (internal standard) in 25 microliters 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 microliters mobile phase and injected (200 microliters). To 1.0 ml urine were added 100 microliters MeOH and 1.0 ml 0.5 M 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 microliters). An IBM C-8 column 5-microns 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/l. 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 less than 3% for plasma and less than 5% for urine. Accuracies were within 6% of the nominal value and recoveries were 75% and 90% for plasma and urine, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The disposition of carboplatin in the beagle dog.

Carboplatin 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 non-renal 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.

The disposition of carboplatin in ovarian cancer patients.

Carboplatin 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 t1/2 alpha of 1.6 h and a t1/2 beta 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 +/- 3 l, 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 pharmacokinetics in patients with a Clcr of about 60 ml/min or greater, but dose reductions are necessary for patients with mild renal failure.

In vitro stability, plasma protein binding and blood cell partitioning of 14C-carboplatin.

Radiochemically pure 14C-labeled carboplatin, cis-diammine [1,1-cyclobutane (1-14C) dicarboxylato (2-)-0,0'] platinum (II), was added to fresh human, dog and rat plasma, at concentrations ranging from 1 to 100 micrograms 14C-carboplatin/ml. After 10 min incubation at ambient temperature, the plasma was ultrafiltered in Amicon Centrifree micropartition units to generate protein-free plasma ultrafiltrate (PU). Total radioactivity was determined by liquid scintillation counting. A mean (+/- SD) of 102% +/- 2.0%, 99.5% +/- 1.9%, and 99.0% +/- 1.0% of the 14C-carboplatin added to fresh human, dog, and rat plasma respectively was recovered in the PU. 14C-carboplatin was incubated at 37 degrees C with fresh plasma (60 micrograms/ml) and urine (200 micrograms/ml) from humans and dogs for 120 h, and samples were removed at appropriate times for analysis of carboplatin, 1,1-cyclobutane dicarboxylic acid and cyclobutane carboxylic acid. The latter were separated by HPLC on a C-18 column with a mobile phase of H2O/CH3CN/0.3 M tetrabutylammonium phosphate (880:50:20 v/v/v), and the column eluants at the retention time of each compound were collected and counted for total radioactivity. Carboplatin degraded in each of the matrices with a corresponding release of 1,1-cyclobutane dicarboxylic acid. 14C-carboplatin (50 micrograms/ml) was incubated at 37 degrees C with fresh human, dog and rat blood and the distribution of radioactivity into the cellular fraction was determined. Radioactivity did not distribute into the blood cells of humans or dogs, but after 5 h, 44% of the radioactivity in rat blood was associated with the cellular fraction. These results show that carboplatin, at physiological concentrations, does not bind instantaneously and reversibly to the plasma proteins of rat, dog or human, and that the molecule slowly degrades in plasma and urine in vitro with the release of 1,1-cyclobutane dicarboxylic acid. The remaining diammine platinum (II) portion of the molecule therefore accounts for the essentially irreversible protein binding of the platinum from carboplatin.

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

Specific, 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 Centrifree micropartition system, and samples were injected onto a LiChrosorb 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 micrograms/ml. The lower limit of quantification was 1.0 micrograms/ml plasma and 5.0 micrograms/ml urine. Constituents 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 micrograms/ml, respectively. Within-day accuracy was 96% or greater for both matrices. Carboplatin was not bound to the Centrifree 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 (less than or equal to 10%) over a range of 1-50 micrograms/ml. In human plasma at 37 degrees 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 degrees C. Biological samples, therefore, should be stored frozen and analyzed within a week of collection to obtain valid results.

Evaluation of two new megestrol acetate tablet formulations in humans.

The bioequivalence of two new investigational 160 mg tablets, one containing the regular form and the other a micronized form of megestrol acetate, was determined relative to a commercially available 40 mg tablet (Megace). The tablets were administered to 24 male subjects in a three-way cross-over study, balanced for sequence, with a week between administrations. The 40 mg tablets were administered q.i.d. at 08.00, 12.00, 18.00 and 22.00 h, while the 160 mg tablets were administered once at 08.00 h. Plasma samples were collected at appropriate times out to 96 h after administration and were analysed for megestrol acetate with a validated high performance liquid chromatographic procedure. Based on the times to maximum plasma concentrations (2.5 to 2.8 h), the absorption rate constant was the same for each of the tablets. Relative to the 40 mg q.i.d. dose, the 160 mg regular and the 160 mg micronized tablets had mean relative bioavailabilities of 97 per cent and 118 per cent, respectively.

A radioimmunoassay procedure for tallysomycin S10b in human plasma and urine.

A 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 degrees 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.

Liquid chromatographic procedure for the quantitative analysis of megestrol acetate in human plasma.

A simple, sensitive, and reproducible high-performance liquid chromatographic (HPLC) procedure was developed for the quantitative analysis of megestrol acetate in human plasma. An internal standard, 2,3-diphenyl-1-indenone, was added to 0.5 mL of plasma followed by extraction with hexane. The residue remaining after evaporation of hexane was reconstituted in methanol and injected onto a mu-Bondapak C18 column. The column was eluted with acetonitrile:methanol:water:acetic acid (41:23:36:1), and the eluant was monitored at 280 nm. Megestrol acetate and the internal standard eluted at 6-7 and 12-14 min, respectively. The peak height ratio (megestrol acetate/internal standard) versus plasma concentration was linear over a range of 10-600 ng of megestrol acetate/mL of plasma, and the limit of detection was 5 ng/mL. The mean intra- and interassay accuracies were within 3% of the actual values. The mean intra- and interassay precision, as estimated by RSD, were 4 and 6%, respectively. Constituents in human plasma and megestrol, a possible degradation product, did not interfere in the assay. The procedure was applied to the analysis of plasma samples from subjects receiving 40 mg of Megace q.i.d.

Bioequivalence evaluation of new megestrol acetate formulations in humans.

The bioequivalence of two new investigational 160 mg tablets, one containing the regular form and the other a micronized form of megestrol acetate, was determined relative to a commercially available 40 mg tablet. The tablets were administered to 24 male subjects in a three-way crossover study, balanced for sequence, with 1 week between administrations. The 40 mg tablets were administered qid at 8 AM, 12 PM, 6 PM, and 10 PM, while the 160 mg tablets were administered once at 8 AM. Plasma samples were collected at appropriate times up to 96 hours after administration and were analyzed for megestrol acetate with a validated high-performance liquid chromatographic procedure. Based on the times to maximum plasma concentrations (2.5 to 2.8 h) the rate of absorption was the same for each of the tablets. Relative to the 40 mg qid dose, the 160 mg regular and the 160 mg micronized tablets had mean relative bioavailabilities of 97% and 118%, respectively.

Human intravenous pharmacokinetics and absolute oral bioavailability of cefatrizine.

Cefatrizine was administered intravenously and orally at dose levels of 250, 500, and 1,000 mg to normal male volunteers in a crossover study. Intravenous pharmacokinetics were dose linear over this range; mean peak plasma concentrations at the end of 30-min infusions were, respectively, 18, 37, and 75 micrograms/ml, total body clearance was 218 ml/min per 1.73 m2, renal clearance was 176 ml/min per 1.73 m2, and mean retention time in the body was 1.11 h. Cumulative urinary excretion of intact cefatrizine was 80% of the dose, and half-lives ranged from 1 to 1.4 h. Steady-state volume of distribution was 0.22 liters/kg. On oral administration, the absolute bioavailabilities of cefatrizine were 75% at 250 and 500 mg and 50% at 1,000 mg. The mean peak plasma concentrations and peak times were, respectively, 4.9, 8.6, and 10.2 micrograms/ml at 1.4, 1.6, and 2.0 h, mean residence times were 2.4, 2.6, and 3.1 h, and mean absorption times were 1.3, 1.6, and 1.9 h. Oral renal clearance and half-life values corresponded well to the intravenous values. Cumulative urinary excretion of intact cefatrizine (as percentage of dose) was 60 at 250 mg, 56 at 500 mg, and 42 at 1,000 mg. It is hypothesized that the lack of oral dose linearity between the 500- and 1,000-mg doses is due to a component of cefatrizine absorption by a saturable transport process. Relative absorption at the high dose would be sufficiently slow that an absorption "window" would be passed before maximum bioavailability could be attained. It is not expected that the observed bioavailability decrease at doses exceeding 500 mg will have any therapeutic significance, since clinical studies are establishing efficacy for a recommended unit dosage regimen of 500 mg.

Disposition of orally administered 14C-prednimustine in cancer patients.

A 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 micrograms 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 micrograms/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.

Disposition of anagrelide, an inhibitor of platelet aggregation.

A single dose of 14C-anagrelide (6,7-dichloro-1,5-dihydroimidazo [2,1-b] quinazoline-2(3H)-one monohydrochloride) equivalent to 1 mg free base and containing 100 muCi 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 chromatography (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 recovered in the feces. Several urinary metabolites were detected by HPLC.

Disposition of parenteral butorphanol in man.

The metabolism and elimination of 3H-butorphanol (levo-3, 14-dihydroxy-N-(cyclobutylmethyl)[15-3H]morhinan) tartrate were determined in man after therapeutic im (2 mg) and iv (1 mg) doses. As judged from urinary excretion of radioactivity, the im dose was completely absorbed. Butorphanol was rapidly distributed to tissues, had a plasma half-life of about 3 hr, and was extensively metabolized prior to elimination. The major route of elimination was renal, with fecal excretion being a minor route. Hydroxybutorphanol [3, 14-dihydroxy-N-(trans-3'-hydroxycyclobutylmethyl)morphinan] was isolated and identified as a major urinary metabolite and was also present in the plasma. The disposition of butorphanol is compared and contrasted to the disposition of morphine and pentazocine.

Human pharmacokinetics of a new braod-spectrum parenteral cephalosporin antibiotic, ceforanide.

The pharmacokinetics of the l-lysine salt of ceforanide were studied after intravenous administration of 1132 and 2264 mg as 30-min constant-rate infusions and after intramuscular administration of 556 and 1132 mg. The peak intravenous plasma concentrations were 136 and 222 microgram/ml at termination of infusion, and 12-hr trough concentrations were 5.9 and 9.0 microgram/ml, respectively. The peak intramuscular plasma concentrations were 38 and 74 microgram/ml at 1.0-1.3 hr after dosing, and 12-hr trough concentrations were 3.9 and 6.7 microgram/ml, respectively. When 19 successive intravenous and intramuscular doses at these levels were administered at 12-hr intervals, there was no tendency toward drug accumulation. The major drug elimination route was urinary excretion; 85% of the dose was excreted unchanged in the urine within 12 hr, and no metabolites with antibiotic activity were observed in urine. The mean terminal plasma half-life was 2.98 hr, the mean plasma protein binding was 80.6%, the steady-state volume of distribution was 12 liters, the plasma clearance was 45.9 ml/min/1.73 m2, and the renal clearance was 34.9 ml/min/1.73 m2. The pharmacokinetic properties and antibacterial activity spectrum indicate that this antibiotic should be effective in treating human bacterial infections when administered at 12-hr intervals. It is presently under clinical investigation.