Pharmaceutical and Biomedical Aspects of Topoisomerase I Inhibitors

Pre-clinical and clinical phannacokinetics plays an important rote in the development of new anticancer agents and in the refinement of already existing therapies. In clinical studies, phannacokinetic parameters, including area under the plasma concentration-time curve and/or time above a certain threshold concentration, have previously been shown to be related to the phannacodynamic outcomes, such as myelosuppression or anti-tumor response. In order to obtain reliable phannacokinetic parameters, analytical methodologies have to be developed and validated, enabling accurate detennination of concentrations of anticancer dmgs in biological matrices. These methodologies have to be validated in terms of selectivity, precision, accuracy and sensitivity, to obtain meaningful phannacokinetic results. During the last decade several analogues of the topoisomerase I inhibitor camptothecin have entered clinical practice. Topoisomerase I is a nuclear enzyme involved in the replication of DNA, by fonning a cleavable complex, i.e. the covalent interaction between DNA and the enzyme. The cleavable complex results in a single strand break of the DNA, resulting in relaxation, followed by replication and resealing of the break. The camptothecin topoisomerase I inhibitors reversibly stabilize the cleavable complex, resulting in single·strand DNA breaks and thus termination of DNA replication, subsequently followed by cell death. The camptothecin analogues share a pH-dependent reversible conversion between their pharmacologically active lactone form, which is able to diffuse across cell membranes, and their inactive ring·opened carboxylate form. The existence of the two forms of the camptothecin analogues requires quick sample handling at the site of the patient in order to acquire real·time pharmacokinetic data. In chapter 1, an overview of methodologies for the determination of camptothecin analogues is described and their applicability for pharmacokinetic analysis is discussed. In this thesis, methodologies for the quantitative determination of the topoisomerase I inhibitors topotecan, 9-aminocamptothecin and lurtotecan are described. The applicability of these assays is shown in both clinical pharmacokinetic and in vitro studies

Sensitive high-performance liquid chromatographic fluorescence assay for the quantitation of topotecan (SKF 104864-A) and its lactone ring-opened product (hydroxy acid) in human plasma and urine

A sensitive reversed-phase high-performance liquid chromatographic fluorescence method is described for the simultaneous determination of topotecan (I) and the hydrolysed lactone ring-opened product hydroxy acid (II) in plasma and for the determination of I in urine. To 250 μl of plasma, a 750-μl volume of cold methanol was added to stabilize the pH-dependent conversion of I into II. In plasma, the lower limit of quantitation (LLQ) for both compounds was 0.10 ng/ml. The between-day variation for I at the LLQ was 7.1% and for II was 5.5%. Prior to injection, urine samples were acidified with orthophosphoric acid and diluted with phosphate-buffered saline (PBS). In urine, the calibration curve for I was linear in the range of 10 to 250 ng/ml and the LLQ was 10 ng/ml. The assay was developed to enable pharmacological analysis of I, in on-going phase I and II studies, in patients with solid tumors

Topotecan lacks third space sequestration

The objective of this study was to determine the influence of pleural and ascitic fluid on the pharmacokinetics of the antitumor camptothecin derivative topotecan. Four patients with histological proof of malignant solid tumor received topotecan (0.45 or 1.5 mg/m2) p.o. on several occasions in both the presence and absence of third space volumes. Serial plasma and pleural or ascitic fluid samples were collected during each dosing and analyzed by high-performance liquid chromatography for both the intact lactone form of topotecan and its ring-opened carboxylate form. The apparent topotecan clearance demonstrated substantial interpatient variability but remained unchanged within the same patient in the presence [110 +/- 55.6 liters/ h/m2 (mean +/- SD of eight courses)] or absence of pleural and ascitic fluid [118 +/- 31.1 liters/h/m2 (mean +/- SD of seven courses)]. Similarly, terminal half-lives and area under the concentration-time curve ratios of lactone:total drug in plasma were similar between courses within each patient. Topotecan penetration into pleural and ascitic fluid demonstrated a mean lag time of 1.61 h (range, 1.37-1.86 h), and ratios with plasma concentration increased with time after dosing in all patients. The mean ratio of third space topotecan total drug area under the concentration-time curve to that in plasma was 0.55 (range, 0.26-0.87). These data indicate that topotecan can be safely administered to patients with pleural effusions or ascites and that there is substantial penetration of topotecan into these third spaces, which may prove beneficial for local antitumor effects

Effects of St. John's wort on irinotecan metabolism

St. John's wort (SJW), a widely used herbal product, has been implicated in drug interactions resulting from the induced expression of the cytochrome P450 CYP3A4 isoform. In this study, we determined the effect of SJW on the metabolism of irinotecan, a pro-drug of SN-38 and a known substrate for CYP3A4. Five cancer patients were treated with irinotecan (350 mg/m(2), intravenously) in the presence and absence of SJW (900 mg daily, orally for 18 days) in an unblinded, randomized crossover study design. The plasma levels of the active metabolite SN-38 decreased by 42% (95% confidence interval [CI] = 14% to 70%) following SJW cotreatment with 1.0 micro M x h (95% CI = 0.34 micro M x h to 1.7 micro M x h) versus 1.7 micro M x h (95% CI = 0.83 micro M x h to 2.6 micro M x h) (P =.033, two-sided paired Student's t test). Consequently, the degree of myelosuppression was substantially worse in the absence of SJW. These findings indicate that patients on irinotecan treatment should refrain from taking SJW because plasma levels of SN-38 were dramatically reduced, which may have a deleterious impact on treatment outcome

Inter- and intrapatient variability in oral topotecan pharmacokinetics: implications for body-surface area dosage regimens

Anticancer drugs still are dosed based on the body-surface area (BSA) of the individual patient, although the BSA is not the main predictor of the clearance for the majority of drugs. The relevance of BSA-based dosing has not been evaluated for topotecan yet. A retrospective pharmacological analysis was performed of kinetic data from four clinical Phase I studies in which topotecan was administered p.o. as a single agent combined with data from a combination study of topotecan and cisplatin. A strong correlation (r = 0.91) was found between the area under the plasma concentration time curve of the lactone and carboxylate forms of topotecan by plotting 326 data sets obtained from 112 patients receiving oral topotecan at dose levels ranging from 0.15-2.70 mg/m2. The intrapatient variability, studied in 47 patients sampled for 3 or more days, for the apparent lactone clearance, ranged from 7.4-69% (mean, 24 +/- 13%; median, 20%). The interpatient variabilities in the lactone clearance, calculated with the data of all studied patients, expressed in liter/h/m2 and in liter/h were 38% and 42%, respectively. In view of the relatively high inter- and intrapatient variabilities in topotecan clearance, in contrast to a variability of only 12% in the BSA of the studied patients, no advantage of BSA-based dosing was found over fixed dose regimens

Determination of the lactone and lactone plus carboxylate forms of 9-aminocamptothecin in human plasma by sensitive high-performance liquid chromatography with fluorescence detection

Two sensitive reversed-phase high-performance liquid chromatographic fluorescence methods, with simple sample handling at the site of the patient, are described for the determination of the lactone and lactone plus carboxylate forms of g-aminocamptothecin (9AC). For 9AC lactone, the sample preparation was a liquid-liquid extraction with acetonitrile-n-butyl chloride (1:4, v/v), whereas the sample preparation for 9AC total (lactone plus carboxylate) was a simple deproteinization with 5% perchloric acid-methanol (1:1, v/v), which results in the conversion of the carboxylate into the lactone form. The lower limits of quantitation were 50 pg/ml and 100 pg/ml for 9AC lactone and 9AC total, respectively. The within-run precisions at four tested concentrations were ≤6.3% for 9AC lactone and ≤5.3% for 9AC total. The between-run precisions were ≤8.9% and ≤5.6%, respectively. The assays were developed to enable pharmacological analysis of 9AC in a bioavailability and oral phase I study in patients with solid tumors

Determination of irinotecan (CPT-11) and its active metabolite SN-38 in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection

Sensitive high-performance liquid chromatographic assays have been developed to determine the levels of the lactone and lactone plus carboxylate (total) forms of the antitumor agent irinotecan (CPT-11) and its active metabolite SN-38, in human plasma. The related compound camptothecin was used as the internal standard. The selective sample pretreatment for the lactone forms involved a single solvent extraction with acetonitrile-n-butyl chloride (1:4, v/v), whereas the sample clean-up for the total forms was a simple protein precipitation with aqueous perchloric acid-methanol (1:1, v/v), which results in the conversion of the carboxylate to the lactone forms. Chromatography was carried out on a Hypersil ODS column, with detection performed fluorimetrically. The methods have been validated, and stability tests under various conditions have been performed. The lower limits of quantitation are 0.5 and 2.0 ng/ml for the lactone and total forms, respectively. The assays have been used in a single pharmacokinetic experiment in a patient to investigate the applicability of the method in vivo