Monoclonal antibody 44-3A6 doxorubicin immunoconjugates: comparative in vitro anti-tumor efficacy of different conjugation methods.

Doxorubicin (Dox) was coupled by four published methods to a murine monoclonal antibody (MAb) developed against human pulmonary adenocarcinoma. Dox immunoconjugates made with this murine IgG1 MAb, 44-3A6, were evaluated for anti-tumor activity against the human lung cancer cell line, A549. Dox was attached to the MAb (1) by an oxidized dextran T40 intermediate, (2) using dilute glutaraldehyde cross-linking, (3) with an acid-sensitive linker using cis-aconitic anhydride and EDCI, a water-soluble carbodiimide, and (4) using EDCI alone. The biological activity of the different conjugates was compared in vitro by antigen binding (whole-cell RIA) versus serial dilutions of unconjugated MAb cytotoxicity (dye exclusion/viability) versus long dilutions of the various Dox conjugates, Dox and mixtures of Dox and MAb. Preincubation of antigen-expressing tumor cells (A549) for 2 h with excess (250 micrograms/ml) unconjugated MAb prior to conjugate exposure was attempted in order to block the cytotoxic effect. The number of Dox molecules conjugated to 44-3A6 (molar incorporation ratio or MIR) ranged from 3 to 10/1 for carbodiimide linkage and up to 63/1 using the dextran intermediate. Cis-aconityl-derivatized Dox conjugates contained an average of 22 mol Dox/mol immunoglobulin, but drug incorporation was quite variable from experiment to experiment. Dilute glutaraldehyde cross-linking produced an average MIR of 10/1. After repeated attempts to minimize drug and/or MAb precipitation, the percentage decrease (versus MAb) in immunoreactivity of the drug conjugates ranged from 2 to 42% and was dependent on the coupling method and extent of aggregate formation in the preparation. Loss of biological activity (antigen binding and cytotoxicity) was significant when aggregation and precipitation occurred. There were additional losses (17-25%) after sterile filtration through low protein-binding (0.22 microns) filters. Immunoconjugates produced by glutaraldehyde cross-linking were reproducibly 5-10 times more potent against antigen-bearing tumor cells than Dox, and showed selectivity for inhibiting the viability of antigen-positive A549 cell line. Noncovalent mixtures of 44-3A6 and Dox were slightly more potent than Dox. Immunoconjugates produced by the aconityl method and the dextran intermediates were less effective than Dox, the glutaraldehyde-mediated conjugates or Dox and 44-3A6 mixtures. The unconjugated MAb was not cytotoxic when tested at concentrations up to 500 micrograms/ml. Blocking studies using 'cold', unlabeled MAb were of limited success.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacokinetics and pharmacodynamics of long-term continuous-infusion doxorubicin.

Steady-state plasma levels of doxorubicin and doxorubicinol were analyzed in 32 patients with advanced cancer, each of whom was given doxorubicin by long-term continuous infusion at progressively increasing infusion rates. Patients received doxorubicin for 2 to 50 weeks at rates of 0.2 to 6.1 mg/m2/day. Dose-limiting stomatitis and leukopenia were observed. The mean maximum steady-state doxorubicin concentration was 6.04 ng/ml at a mean maximum infusion rate of 3.92 mg/m2/day. Clearance mechanisms did not appear to be saturated at the durations or infusion rates used in this study. The maximum steady-state doxorubicin level and the ln (initial WBC) were significant correlates of the ln (nadir WBC) (p = 0.002 and 0.02, respectively). A model was constructed according to these two parameters that significantly describes ln (nadir WBC) (p = 0.001). Neither age, infusion rate, nor doxorubicinol level correlated with nadir WBC. Stomatitis did not correlate with any of these parameters. The demonstration of this pharmacodynamic relationship highlights the potential importance of pharmacologic data collection in ongoing attempts to predict the clinical effects of anticancer drugs.

Human plasma pharmacokinetics of thiotepa following administration of high-dose thiotepa and cyclophosphamide.

Thiotepa is an established alkylating agent whose pharmacokinetics in standard doses are well defined. In order to ascertain whether dose-dependent variations in pharmacokinetics occur, we have undertaken an analysis of plasma thiotepa levels in 16 patients entered on a phase I-II study of bialkylator chemotherapy. High-dose thiotepa (1.8 to 7.0 mg/kg) and cyclophosphamide (2.5 g/m2) were administered intravenously (IV) on days -6, -4, and -2 followed by autologous marrow reinfusion on day 0. Plasma and urinary thiotepa was assayed by gas chromatography. Biexponential plasma decay curves were seen in ten patients, with a t 1/2 alpha of 10.0 +/- 6.4 minutes, a t 1/2 beta of 174 +/- 61 minutes and a total body clearance of 379 +/- 153 mL/h/kg (mean +/- SD). Six patients displayed monoexponential plasma decay curves with a terminal t 1/2 of 137 +/- 83 minutes and a total body clearance of 440 +/- 195 mL/h/kg. Although there was a trend toward reduced plasma clearance in the three patients treated at the highest dose level, the available data suggest that metabolic clearance mechanisms for thiotepa were not saturated with the doses used in this study. By stepwise regression analysis, linear functions using only 15-minute and four-hour postinfusion plasma levels were derived that correlated closely with area under the plasma concentration X time curves (AUC) (P less than .002). We conclude that high-dose thiotepa results in similar pharmacokinetic values to conventional doses with no apparent dose-dependent variation. The value of specific time points to predict AUC and clearance will require prospective evaluation.

Localization and imaging with radioiodine-labeled monoclonal antibodies in a xenogeneic tumor model for human B-cell lymphoma.

Two MoAbs directed towards human B-cell malignancies have been studied in a preclinical animal model to evaluate their potential for in vivo imaging and therapy of B-cell lymphomas. Anti-B1 reacts with virtually all immunoglobulin-bearing malignancies and non-T acute lymphoblastic leukemia. Anti-J5 reacts with the common acute lymphoblastic leukemia antigen found on non-T acute lymphoblastic leukemia and follicular lymphomas. Anti-T1 which recognizes the CD5 antigen on most T-cell leukemias and lymphomas was used as a control antibody. These monoclonal antibodies were radiolabeled with 125I or 131I by the ICl method. Namalwa (B-cell) and MOLT-4 (T-cell) tumors were grown s.c. in irradiated nude mice. The highest tissue concentration of 125I-labeled anti-J5 in Namalwa-bearing mice was in blood and tumor. The tumor/blood ratio ranged from 0.7-1.2, with the highest ratio 4 days after injection. Pharmacokinetic analysis indicated that the t1/2 beta of anti-J5 from blood and other tissues ranged from 40-50 h, while the t1/2 beta for tumor averaged 65 h. The area under the curve of tumor was 2- to 5-fold higher than the area under the curve of liver, kidney, skin, and muscle. The peak tissue levels of 125I-labeled anti-B1 in Namalwa-bearing mice were again in blood and tumor and 6 days following injection more than 5-fold greater activity was found in tumor compared to normal tissues other than blood. The tumor/blood ratio was 1.2 and 0.7 at 4 and 6 days after injection. 125I-labeled anti-B1 showed minimal uptake in antigen-negative MOLT-4 tumors and 125I-labeled anti-T1 showed little uptake in Namalwa tumors. Scintigraphic images were obtained following the injection of 131I-labeled anti-J5 and anti-B1 in nude mice bearing Namalwa tumors. These results indicate that radiolabeled anti-J5 and anti-B1 show promise as diagnostic and possibly therapeutic agents for human B-cell lymphoma, although there may be a limitation to clinical utility due to cross-reactivity with some normal cells.

High-performance liquid chromatographic assay for mitoxantrone in plasma using electrochemical detection.

A sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of mitoxantrone in plasma using electrochemical detection. Bisantrene was chosen as the internal standard. A reversed-phase, 10-microns muBondapak C18 analytical column (30 cm X 3.9 mm) with an isocratic mobile phase of 28% acetonitrile in 80 mM sodium formate buffer (pH 3.0) was used. The eluent was monitored by both electrochemical detection at an applied potential of +0.75 V vs. Ag/AgCl and visible absorbance at 660 nm. Only electrochemical detection was able to quantitate the internal standard and provided ten times higher sensitivity than visible absorbance for mitoxantrone with a detection limit as low as 0.1 ng/ml. Calibration curves in the range 0.1-1000 ng/ml showed good linearity (r = 0.998) and precision (coefficient of variation less than 10%). This HPLC method utilized a reproducible and inexpensive liquid-liquid extraction procedure. Using methylene chloride, the extraction efficacy of mitoxantrone from plasma was 85.3% with a coefficient of variation less than 2.1%. This new assay was then applied to measure mitoxantrone concentrations in plasma obtained from two leukemic patients receiving 12 mg/m2 mitoxantrone as a 1-h infusion.

Clinical pharmacodynamics of continuous infusion teniposide: systemic exposure as a determinant of response in a phase I trial.

Teniposide (VM-26) is an effective anticancer drug usually administered as a short infusion in doses of 150 to 165 mg/m2. The objectives of the trial reported here were to evaluate clinical responses and assess pharmacokinetic parameters as a determinant of outcome when VM-26 was administered as a 72-hour continuous infusion (CI) with doses escalated from 300 to 750 mg/m2 per course. Twenty-eight patients with recurrent leukemia, lymphoma, or neuroblastoma received 53 courses of CI VM-26 and 16 had measurable responses. There were two partial remissions and one stable disease among seven neuroblastoma patients and 13 of 21 leukemia/lymphoma patients had oncolytic responses (greater than or equal to 75% decrease in circulating blasts). Toxicity included moderate to severe mucositis and myelosuppression. Pharmacokinetic parameters determined during 35 courses administered to 23 patients were highly variable. Clearance (CI) ranged between 3.7 and 43.8 ml/min/m2, resulting in VM-26 plasma concentrations from 2.8 to 30.6 mg/L across all dose levels. The interpatient pharmacokinetic variability reflected in CI and VM-26 steady state concentrations (Css), obscured any dose-response relationship. However, when pharmacokinetic parameters for responding and nonresponding patients were compared, statistically significant relationships were observed. For responders, the mean Css was 15.2 mg/L and mean CI was 12.1 mL/min/m2; for nonresponders, mean Css was 6.2 mg/L (P less than .01) and mean CI was 21.3 mL/min/m2 (P less than .05). Thus, patients with higher CI and lower Css were less likely to respond. Clinical responses occurred in ten of ten patients with Css greater than 12 mg/L, and only five of 13 patients with Css less than 12 mg/L (P less than .01). In this study, interpatient pharmacokinetic variability yielded a four- to sixfold difference in intensity of systemic exposure (Css) within the same dose level, which was an important determinant of clinical response. These data indicate that achieving a VM-26 target concentration for individual patients could ensure an increased intensity of systemic exposure in patients with a high CI and improve the likelihood of effective therapy.

Phase I clinical and pharmacological study of 72-hour continuous infusion of etoposide in patients with advanced cancer.

Etoposide (VP-16) is a semisynthetic epipodophyllotoxin that exhibits cell cycle phase specific cytotoxicity and enhanced effectiveness with increasing duration of drug exposure. We have therefore conducted a Phase I trial to determine the side effects, tolerable doses, and pharmacokinetic parameters of VP-16 given by continuous i.v. infusion to patients with advanced cancer. Eighteen patients were treated with varying dosages of VP-16 infused continuously for 72 consecutive hours every 28 days. Using this schedule, the maximally tolerated dosage of VP-16 was 150 mg/m2/day for patients with good performance status and 125 mg/m2/day for more debilitated cancer patients. Hematological toxicity was dose limiting with median granulocyte and platelet nadirs of 700/mm3 and 116,000/mm3, respectively, at a dose of 150 mg/m2/day. Other toxicities included only mild nausea, vomiting, and alopecia. Plasma and urine VP-16 concentrations were determined using a high-performance liquid chromatography assay. At a VP-16 dosage of 150 mg/m2/day, steady-state VP-16 concentrations were in the range of 2.1 to 7.0 micrograms/ml in all patients. Further pharmacokinetic analysis revealed that the plasma clearance of VP-16 was consistently near 25 ml/min/m2 (independent of dosage) and that renal clearance accounted for only 15% of VP-16 total plasma clearance. Patient age was found to be the most important factor correlating with plasma clearance of VP-16. Linear regression analysis also revealed that both the plasma VP-16 concentration at steady state and the concentration of VP-16 in plasma at 24 h from the start of the infusion correlated with hematological toxicity; no other patient characteristics correlated with hematological toxicity. The recommended VP-16 dose for Phase II trials of 72-h continuous infusion VP-16 is 150 mg/m2/day in patients with good performance status.

A clinical and pharmacokinetic study of mitoxantrone in acute nonlymphocytic leukemia.

Twenty-two patients with relapsed or refractory acute leukemia received 31 treatment courses of mitoxantrone (10 to 12 mg/m2/d) as a one-hour infusion for five days. Seven of the 13 patients who had greater than or equal to 95% reduction in the leukemia cell mass, calculated using the bone marrow examination on day 6, achieved a complete remission (CR). These remissions lasted up to 14 months without additional therapy. There were no CRs among the 18 patients who had less than 95% cytoreduction by day 6. The sequential addition of 5-azacytidine (200 mg/m2/d) for three days in those patients with residual disease on day 6 provided little additional benefit. Nonhematological toxicity from mitoxantrone was mild, although fever and infection were common. A new high-performance liquid chromatography (HPLC) assay was used to describe the clinical pharmacokinetics of mitoxantrone. Neither clinical response nor toxicity was strongly correlated with the peak plasma mitoxantrone concentration on the first day (mean +/- SD, 510 +/- 206 ng/mL), nor the area under the concentration-time curve (484 +/- 229 ng X h/mL), nor the systemic clearance (405 +/- 124 mL/min/m2). Mitoxantrone causes rapid cytoreduction in acute nonlymphocytic leukemia (ANLL), but the optimal dose and schedule remain to be determined.

Interpatient and intrapatient variability in vinblastine pharmacokinetics.

We analyzed interpatient and intrapatient differences in vinblastine pharmacokinetics in 24 patients treated with a bolus dose of vinblastine followed by prolonged (2 to 36 weeks) continuous infusion via an implantable pump. The bolus vinblastine serum clearance was 552 +/- 182 ml/min/m2, with a terminal half-life of 29.2 +/- 11.2 hours. After steady state was achieved (2 weeks), the infusion clearance was 646 +/- 221 ml/min/m2. Interpatient differences in serum albumin levels were correlated with both the bolus clearance (r = 0.49) and the initial (first month) infusion clearance (r = 0.39). The infusion clearance decreased over the duration of the infusion (726 vs. 489 ml/min/m2; P = 0.001;months 1 vs. 4). Analysis of intrapatient changes in vinblastine clearance demonstrated a positive correlation with albumin (P less than 0.01) and negative correlation with dose (P less than 0.05). These results are consistent with a nonlinear pharmacokinetic model. We conclude that interpatient and intrapatient differences in vinblastine pharmacokinetics can be attributed partially to changes in hepatic function and nonlinear elimination at higher doses.

Phase I clinical and pharmacokinetic study of bisantrene in refractory pediatric solid tumors.

Fourteen patients with pediatric malignant solid tumors, median age 15 years, received 22 courses of bisantrene in a Phase I study. Dosage escalations ranged from 10 to 120 mg/m2 daily for 5 consecutive days. Toxicity included myelosuppression and phlebitis. A sensitive (detection limit of 2 ng/ml) and specific HPLC method was developed to quantitate bisantrene in patient's plasma and urine. Peak plasma concentrations at the end of 60 minute infusions ranged from 568 ng/ml at 10 mg/m2 to 6800 ng/ml at the 100 mg/m2 dosage. The elimination half life (T 1/2 beta) averaged about 10 hours but increased to 20 hours in a patient with liver disease. Only 2.4 - 10% of the bisantrene dose was eliminated in the urine suggesting that the liver may be the major route of elimination for this antineoplastic anthracene derivative.

Continuous infusion high-dose cytosine arabinoside in refractory childhood leukemia.

Ten pediatric patients with refractory leukemia received continuous infusion high-dose cytosine arabinoside (ara-C) according to one of two escalating dosage schedules: (1) a 500-mg/m2 rapid infusion loading dose followed by 3.5 g/m2 per day continuous infusion daily for four consecutive days, or (2) a 600-mg/m2 rapid infusion loading dose followed by 5.0 g/m2 per day continuous infusion daily for four consecutive days. Major toxicity at the lower dosage level was grade IV hematopoietic aplasia of three weeks' duration. At the higher dosage level, there was a prohibitive toxicity in multiple organ systems including transient noncardiogenic pulmonary edema, fungal infections, peritonitis, severe diarrhea, transaminase elevations, and one treatment-related death due to acute renal failure. In contrast to other methods of administration of high-dose ara-C, no CNS toxicity occurred. Oncolytic responses were seen in all patients and two achieved brief, partial remissions. Steady-state plasma ara-C concentrations were 13 to 40 mumol/L at the 3.5-g/m2 dosage level and 10 to 225 mumol/L at the 5-g/m2 dosage level; CSF concentrations at both dosages ranged from 2 to 5 mumol/L. Intracellular levels and ratios of 1-beta-D-arabinofuranosylcytidine-5' triphosphate and endogenous deoxycytidine 5' triphosphate in marrow blasts varied widely at steady state during infusion. No positive correlation existed between steady-state plasma ara-C levels, toxicity, oncolytic effect, or intracellular nucleotide concentration.

Pharmacokinetics of etoposide (VP16) in children and adolescents with refractory solid tumors.

The clinical pharmacokinetics of etoposide were studied in eight pediatric patients with refractory solid tumors. The alpha-phase half-life, beta-phase half-life, volume of distribution, and elimination rate constant averaged 0.82 hr, 6.5 hr, 4.0 liters/sq m, and 0.25 hr-1, respectively. Noncompartmental parameters such as systemic clearance, mean residence time, and volume of distribution at steady-state averaged 20.9 ml/min/sq m, 7.8 hr, and 7.2 liters/sq m, respectively. A significant relationship between serum glutamic pyruvic transaminase and systemic clearance was observed, with patients having elevated serum glutamic pyruvic transaminase showing slower systemic clearance of etoposide. Systemic clearance, mean residence time, and beta-phase half-life of etoposide were significantly lower in those patients who had received cisplatin prior to their Phase II etoposide trial. The average pharmacokinetic values derived from these eight pediatric patients with solid tumors did not differ significantly from those previously reported in children with leukemia administered similar dosages and in adults given radioactively labeled etoposide.

Etoposide: a semisynthetic epipodophyllotoxin. Chemistry, pharmacology, pharmacokinetics, adverse effects and use as an antineoplastic agent.

Etoposide (VP 16) is a semi-synthetic derivative of 4'- demethylepipodophyllotoxin , a naturally occurring compound synthesized by the North American May apple (Podophyllum peltatum ) and the Indian species Podophyllum emodi Wallich . Although podophyllotoxins are classical spindle poisons causing inhibition of mitosis by blocking mitrotubular assembly, etoposide inhibits cell cycle progression at a premitotic phase (late S and G2), probably via inhibition of DNA synthesis. There appears to be a selective antileukemic dose response relationship when compared to normal hematopoietic elements. Etoposide is effective when administered orally at about twice the recommended parenteral dosage. Schedule dependency in both animal models and clinical trials has been observed; multiple dosing over three to five consecutive days is superior to weekly single dose administration. Etoposide's dose-limiting toxicity is myelosuppression (leukopenia), which is quite predictable; alopecia and Gl toxicity (nausea, vomiting, stomatitis) occur in about 20-30% of patients given recommended dosages. Etoposide appears to be one of the most active drugs for small cell lung cancer, testicular carcinoma (the Food and Drug Administration approved indication), ANLL and malignant lymphoma. Etoposide also has demonstrated activity in refractory pediatric neoplasms, hepatocellular, esophageal, gastric and prostatic carcinoma, ovarian cancer, chronic and acute leukemias and non-small cell lung cancer, although additional single and combination drug studies are needed to substantiate these data. Its contribution in front-line combination chemotherapeutic regimens for these cancers will be better defined in the forthcoming years. Etoposide appears to have minimal activity in breast cancer and, based on current data, it is inactive against malignant melanoma, colorectal adenocarcinoma and cancer of the head and neck, although the dosage and schedules used in many of the Phase II studies may have been suboptimal.

Teniposide (VM26) disposition in children with leukemia.

The clinical pharmacokinetics of teniposide (VM-26, NSC 122819) has been studied in 21 children (median age, 4.7 years) with acute lymphocytic leukemia. Teniposide was administered at a dosage of 165 mg/sq m as a 30- to 60-min i.v. infusion. Patients were studied either on the first or second dosage of the drug. Plasma samples were assayed for teniposide and metabolites by high-performance liquid chromatography with electro-chemical detection. Both compartmental and noncompartmental pharmacokinetic analyses were performed. Systemic clearance and apparent volume of distribution of steady state averaged 13.82 +/- 6.0 ml/min/sq m (S.D.) and 7.9 +/- 4.0 liter/sq m, respectively. Univariate and multivariate stepwise regression analyses were used to construct mathematical models to describe the relationships between certain patient-specific demographic and laboratory values and the pharmacokinetic parameters, systemic clearance, elimination rate constant, and area under the concentration-time curve. A significant relationship between serum alkaline phosphatase and systemic clearance, elimination rate constant, and area under the concentration-time curve was found, suggesting that liver function influences the disposition of this anticancer drug in humans.

High-performance liquid chromatographic analysis of the semisynthetic epipodophyllotoxins teniposide and etoposide using electrochemical detection.

A high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of two structurally similar and highly active anticancer drugs, etoposide (I) and teniposide (II), and their potential metabolites (hydroxy acid, picrolactone, and aglycone). The assay utilizes electrochemical detection, which imparts specificity and sensitivity sufficient to detect greater than or equal to 20 ng/mL in plasma, urine, and CSF. The mean assay coefficients of variation were 5.1 and 8.1% for teniposide (10 micrograms/mL) and etoposide (5 micrograms/mL), respectively. The extraction efficiencies were 86% for etoposide, 70% for its hydroxy acid metabolite, 66% for teniposide, and 54% for the hydroxy acid of teniposide. The correlation coefficient of the multilevel standard curve was greater than or equal to 0.995 over the concentration range of 0.05-50 micrograms/mL for the parent drugs and metabolites extracted from plasma. This method has been used to determine the concentrations of the parent drugs and their metabolites in the plasma, urine, and CSF of patients with cancer.

Comparative cytotoxic and cytokinetic effects of the epipodophyllotoxins 4'-demethylepipodophyllotoxin-9-(4,6-O-2-ethylidene-beta-D-glucopyranoside) and 4'-demethylepipodophyllotoxin-9-(4,6-O-2-thenylidene-beta-D-glucopyranoside) and their metabolites on human leukemic lymphoblasts.

We compared the effects of the epipodophyllotoxins 4'-demethylepipodophyllotoxin-9-(4,6-O-2-ethylidene-beta-D-glucopyranoside) (VP-16-213) and 4'-demethylepipodophyllotoxin-9-(4,6-O-2-thenylidene-beta-D-glucopyranoside) (VM-26) and several of their derivatives on cell cycle progression and viability of human leukemic lymphoblasts (CCRF-CEM). The cis-(picro)-lactone derivatives, like both VP-16-213 and VM-26, produced G2-phase arrest and cytotoxicity, but only at concentrations 100 times greater than required with the parent compounds. The epiaglycone derivative showed potent cytotoxicity: at 100 to 250 nM, it reduced cloning efficiency by 50%, an effect requiring 25 to 40 nM VM-26 and 340 to 425 nM VP-16-213. In contrast to VM-26 and VP-16-213, the epiaglycone arrested cells in M, consistent with evidence that it, like podophyllotoxin, is an inhibitor of microtubule assembly. At concentrations resulting in 50% cell kill and an increase of cells in M, however, the epiaglycone produced little change in the proportion of cells in G1 or early S phase, while podophyllotoxin produced a shift of cells to mid- and late S. The hydroxy acid derivatives, although found in detectable quantities in patients' urine and serum, were inactive in vitro. Structural differences among the compounds account for their different biochemical and cell kinetic effects and, hence, different cytotoxic activities. Because the epiaglycone is a potent compound and combines activities of both the podophyllotoxins and 4'-demethyl derivatives, further studies of its cytotoxicity are indicated.

High-performance liquid chromatographic assay for cytosine arabinoside, uracil arabinoside and some related nucleosides.

A novel, dual-column high-performance liquid chromatographic method for determination of the anti-cancer drug cytosine arabinoside (Ara-C) and its major metabolite uracil arabinoside (Ara-U) has been developed. The analytical procedure is sensitive (25 ng/ml) and specific for Ara-C, Ara-U and the endogenous nucleosides that may influence response to Ara-C therapy, cytidine and deoxycytidine. Conventional and high dose calibration curves were linear and the method precise with the assay coefficient of variation for Ara-C and Ara-U not greater than 9.1% over the range of 0.1-10 micrograms/ml. Accuracy was determined to be within +/- 3 to 9% over this concentration range. Using this method, patient plasma samples from both conventional dose (100-200 mg/m2 per day) and high dose (3500-6500 mg/m2 per day) Ara-C can be simultaneously analyzed for Ara-C, Ara-U and nucleosides so that comparative pharmacokinetic and pharmacodynamic studies can be conducted.

Quantitation of daunorubicin and its metabolites by high-performance liquid chromatography with electrochemical detection.

A selective and sensitive high-performance liquid chromatographic method was developed for the separation and quantitation of daunorubicin and its metabolites in serum, plasma, and other biological fluids. Daunorubicin and metabolites in human plasma were injected directly into the high-performance liquid chromatography system via a loop-column to pre-extract the drugs from the plasma, and quantitated against a multilevel calibration curve with adriamycin as the internal standard. The column effluent was monitored with an electrochemical detector at an applied oxidative potential of 0.65 V and by fluorescence. Daunorubicin and four metabolites were separated and characterized by this method. In a blinded evaluation of accuracy and precision, the mean coefficients of variation were 3.8, 3.6 and 9.8% at concentrations of 150, 75 and 15 ng/ml, respectively, and blank samples gave negligible readings. The amperometric sensitivity was greater than achieved by fluorescence detection, and offers an alternative method for quantitation of these compounds. The new method has a limit of detection of less than 2 ng on column, allowing quantitation of less than 10 ng/ml in plasma samples without organic extraction prior to chromatographic analysis.