Quantification of topotecan and its metabolite N-desmethyltopotecan in human plasma, urine and faeces by high-performance liquid chromatographic methods

doi:10.1016/S0378-4347(99)00078-X

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 727, Issues 1–2, 30 April 1999, Pages 191-203

https://doi.org/10.1016/S0378-4347(99)00078-X Get rights and content

Abstract

Sensitive high-performance liquid chromatographic (HPLC) methods have been developed and validated for the simultaneous determination of the antitumor drug topotecan and its metabolite N-desmethyltopotecan in human plasma, urine and faeces. Both compounds are reversibly hydrolysed to their hydroxycarboxylate forms at physiologic pH. Separate HPLC systems have been developed for the determination of lactone and total (lactone plus hydroxycarboxylate forms) concentrations in plasma. The instability of the analytes in plasma requires immediate protein precipitation with ice-cold methanol. The lactone forms of the analytes were stable in the methanol extracts for at least 15 months when stored at −70°C. For the determination of the total levels, the plasma extracts were acidified with 25 mM phosphoric acid to convert the compounds into their lactone forms quantitatively. The sample pretreatment procedure for urine included dilution in methanol while the faecal samples were homogenized in distilled water and then extracted twice with an acetonitrile–ammonium acetate mixture. Separation was achieved on reversed-phase columns (Zorbax SB-C18) and detection was performed fluorimetrically at 380/527 nm. Within-run and between-run precisions were less than 10% and average accuracies were between 90 and 110%. The methods were used in a mass balance study in patients with malignant solid tumors to determine the disposition and routes of elimination of topotecan and N-desmethyltopotecan.

Introduction

Topotecan (Hycamtin™, Fig. 1) is a water-soluble anticancer analogue of camptothecin. The drug inhibits DNA replication and RNA transcription by stabilizing the cleavable complexes formed between the nuclear enzyme topoisomerase I and DNA [1], [2]. The lactone ring undergoes reversible hydrolysis which is pH-dependent (Fig. 1) [3]. It has been demonstrated that stabilization of the DNA–topoisomerase I complex, which is considered to mediate the antitumor activity of topotecan, requires the drug to be present in its lactone form [4]. Topotecan has been registered for the treatment of patients with metastatic carcinoma of the ovary after failure of first line chemotherapy [5]. Additionally, topotecan has shown clinical antitumor activity in the treatment of small cell lung cancer [6], [7]. Demethylation to form N-desmethyltopotecan has been identified as a metabolic pathway for topotecan in humans [8]. This metabolite was found in plasma and urine of patients treated with topotecan. Although various papers have been published on the quantification of topotecan [9], [10], [11], [12], these methodologies are not able to quantify N-desmethyltopotecan. Furthermore, no bio-analytical assays have been described for the determination of topotecan in faeces. We have developed and validated high-performance liquid chromatographic (HPLC) methods for the determination of topotecan and N-desmethyltopotecan in human plasma, urine and faeces in support of clinical studies to determine the pharmacokinetics and mass balance of topotecan.

Camptothecin derivatives are reversibly hydrolysed to their hydroxycarboxylate forms at physiologic pH. In vitro conversion is rapid and it is important that blood samples are collected in an appropriate fashion (rapid cooling, centrifuged under cooled conditions and subsequent protein precipitation) to fix the equilibrium for adequate quantitation of the lactone forms [9]. The plasma samples can then be acidified for the determination of the total levels of topotecan and N-desmethyltopotecan. The hydroxycarboxylate concentrations are determined by calculating the difference between these concentrations. Separate measurements of lactone and total levels in urine and faeces were considered irrelevant due to rapid interconversion reactions of the compounds during collection of these matrices.

Section snippets

Chemicals and control matrices

Topotecan (hydrochloride salt, SKF 104864-A, lot MM-15906-194, purity 89.2%) and N-desmethyltopotecan reference standard (hydrochloride salt, SB 209780-A, lot JW-19178-221A1, purity 86.5%) originated from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). Acetonitrile and methanol (HPLC gradient grade) were obtained from Biosolve (Valkenswaard, The Netherlands). Potassium dihydrogen phosphate, disodium hydrogen phosphate di-hydrate, triethylamine, 37% (w/v) hydrochloric acid, citric acid

Optimization of the methods

The former assay for the analysis of topotecan in human plasma [10] could not be used for the quantitative determination of N-desmethyltopotecan due to insufficient resolution between both compounds. Like all known camptothecin derivatives, topotecan and N-desmethyltopotecan are reversibly hydrolysed to their carboxylate forms and it was the intention to develop a methodology enabling the quantification of both forms of the analytes in one analytical run. Using an end-capped Zorbax SB-C18

Conclusion

Accurate, precise and sensitive HPLC assays have been developed for the quantification of topotecan and N-desmethyltopotecan in several human matrices. These assays have been used to support a clinical study to determine the routes of elimination and disposition of topotecan in patients with malignant solid tumors. To minimize analyte degradation, blood samples taken from patients should be processed immediately and stored at −70°C. Under these conditions, stability data indicated that

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A urine sample was collected before the initial dose was administered and from cumulative urine collected from 0 to 24 hours and from 24 to 48 hours after the first dose during the first cycle, and 10 mL aliquots were stored at < −30°C until analyzed. The plasma concentration of the lactone form of topotecan; the total plasma concentration of topotecan (sum of the lactone form and carboxylate form); the plasma concentration of N-desmethyl topotecan, an active metabolite of topotecan20; the total urinary concentration of topotecan; and the total urinary concentration of N-desmethyl topotecan were determined by high-performance liquid chromatography (HPLC) with fluorescence detection according to the method described by Rosing et al.21 Briefly, separation was achieved on reversed-phase columns, and detection was performed fluorometrically with excitation of 380 nm and emission of 527 nm. The lower limit for quantitative determination of topotecan in plasma (lactone and total topotecan) and in urine was 0.22 nmol/L and 55 nmol/L, respectively, and the limit for N-desmethyl topotecan in plasma (lactone and total topotecan) and urine was 0.23 nmol/L and 5.6 nmol/L, respectively.
We conducted a phase I trial of the topoisomerase I inhibitor topotecan for the purpose of determining the maximum tolerated dose (MTD) and the dose-limiting toxicities (DLTs) of topotecan when administered weekly to patients with advanced non–small-cell lung cancer.
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The recommended dose of topotecan for phase II studies in previously untreated patients is 6 mg/m² on days 1, 8, and 15, every 28 days, and 4 mg/m² appears to be a suitable dose for use in previously treated patients with this schedule.
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Quantification of topotecan and its metabolite N-desmethyltopotecan in human plasma, urine and faeces by high-performance liquid chromatographic methods

Abstract

Introduction

Section snippets

Chemicals and control matrices

Optimization of the methods

Conclusion

J. Pharm. Biomed. Anal.

J. Pharm. Biomed. Anal.

J. Chromatogr. B

J. Chromatogr. B

J. Chromatogr. B

J. Pharm. Biomed. Anal.

J. Pharm. Sci.