Topotecan Concentrations Research Articles

A Phase I Study to Characterize the Safety, Tolerability, and Pharmacokinetics of Topotecan at 4 mg/m2 Administered Weekly as a 30‐Minute Intravenous Infusion in Patients With Cancer

Topotecan pharmacokinetics at higher infusion rates (4 mg/m2 over 30 minutes) have not been studied. The authors report a pharmacokinetics and safety study of this dose in advanced cancer patients. Sixteen patients were given a 4-mg/m2 topotecan infusion intravenously (IV) over 30 minutes weekly for 3 weeks, repeated every 28 days. Pharmacokinetics were determined after the first dose. Plasma concentrations of total topotecan were measured to derive CL, V(ss), C(max), t(max), t(1/2), AUC(0-t), and AUC(0-infinity). Plasma total topotecan concentrations decreased biexponentially, with a mean CL value of 20.6 L/h, V(ss) value of 101 L, and t(1/2) value of 5.0 h. Nine significant adverse events (all hematologic) were topotecan related. Grade 3 or less adverse events included anemia, thrombocytopenia, leukopenia, and fatigue. Pharmacokinetics of the 4-mg/m2 infusion of topotecan over 30 minutes are comparable to findings from studies of lower and higher doses. Toxicities are similar to previous reports.

In-vitro cytotoxicity, in-vivo biodistribution and anti-tumour effect of PEGylated liposomal topotecan

In attempt to increase the accumulation of topotecan in tumours and improve its anti-cancer activity, PEGylated liposome (H-PEG) containing topotecan was prepared. The in-vitro cytotoxicity, in-vivo biodistribution pattern and anti-tumour effect of H-PEG were studied systemically. Compared with free topotecan or conventional liposome (H-Lip), H-PEG improved the cytotoxic effect of topotecan against human ovarian carcinoma A2780 and human colon carcinoma HCT-8 cells. The IC50 value (concentration leading to 50% cell-killing) of H-PEG decreased 5 fold (P<0.01) and 9 fold (P<0.01) against A2780 and HCT-8 cells compared with H-Lip, respectively. The results of biodistribution studies in sarcoma S(180) tumour-bearing mice showed that liposomal encapsulation increased the concentration of total topotecan and the ratio of lactone form in plasma. H-PEG resulted in a 70-fold and 3.7-fold increase in AUC(0-->24 h) compared with free topotecan and H-Lip, respectively. Moreover, H-PEG increased the accumulation of topotecan in tumours and the relative tumour uptake ratio compared with free topotecan was 5.2, and higher than that of H-Lip. The anti-cancer effect studies in murine heptocarcinoma H(22) tumour-bearing mice showed that H-PEG improved the therapeutic efficiency of topotecan and decreased the toxicity of topotecan to a certain extent compared with H-Lip. These results indicated that PEG-modified liposome might be an efficient carrier of topotecan.

Monitoring Drug-Induced γH2AX as a Pharmacodynamic Biomarker in Individual Circulating Tumor Cells

Circulating tumor cells (CTC) in peripheral blood of patients potentially represent a fraction of solid tumor cells available for more frequent pharmacodynamic assessment of drug action than is possible using tumor biopsy. However, currently available CTC assays are limited to cell membrane antigens. Here, we describe an assay that directly examines changes in levels of the nuclear DNA damage marker gammaH2AX in individual CTCs of patients treated with chemotherapeutic agents. An Alexa Fluor 488-conjugated monoclonal gammaH2AX antibody and epithelial cancer cell lines treated with topotecan and spiked into whole blood were used to measure DNA damage-dependent nuclear gammaH2AX signals in individual CTCs. Time-course changes in both CTC number and gammaH2AX levels in CTCs were also evaluated in blood samples from patients undergoing treatment. The percentage of gammaH2AX-positive CTCs increased in a concentration-dependent manner in cells treated with therapeutically relevant concentrations of topotecan ex vivo. In samples from five patients, percent gammaH2AX-positive cells increased post-treatment from a mean of 2% at baseline (range, 0-6%) to a mean of 38% (range, 22-64%) after a single day of drug administration; this increase was irrespective of increases or decreases in the total CTC count. These data show promise for monitoring dynamic changes in nuclear biomarkers in CTCs (in addition to CTC count) for rapidly assessing drug activity in clinical trials of molecularly targeted anticancer therapeutics as well as for translational research.

Episcleral Implants for Topotecan Delivery to the Posterior Segment of the Eye

Purpose. Intravenous or periocular topotecan has been proposed as new treatment modality for patients with advanced intraocular retinoblastoma, but systemic topotecan lactone exposure induced by both approaches may cause toxicity. The purpose of this study was to develop a topotecan-loaded ocular delivery system to minimize systemic exposure and achieve selective transscleral penetration. Methods. Biocompatible polymer implants containing low (0.3 mg) or high (2.3 mg) topotecan load were manufactured and characterized in vitro. Adrenaline (500 mug) was coloaded to induce local vasoconstriction in vivo in 2 of 4 animal groups. Implants were inserted into the episclera of rabbits, and topotecan (lactone and total) concentrations in ocular tissues and plasma were determined over a period of 48 hours. Results. In vitro, implants released 30% to 50% of the loaded drug within 48 hours and 45% to 70% by day 10. In vivo, topotecan lactone was highly accumulated in locally exposed ocular tissues (ranging from 10(5) to 10(6) ng/g in sclera and choroid and 10(2) to10(3) ng/g in retina) over 48 hours with all the formulations studied. Low vitreous topotecan lactone levels (approximately 5 ng/mL) were found in animals receiving concomitant local vasoconstriction and high load implants. Topotecan lactone concentrations in plasma and in contralateral eyes were minimal or undetectable as a marker of tissue selectivity of the proposed strategy. Conclusions. These studies may contribute to improving the efficacy and safety of chemotherapy treatments for retinoblastoma and may support the role of the local vasculature and tissues promoting drug clearance and local accumulation during transscleral drug delivery.

Use of an Anti-Vascular Endothelial Growth Factor Antibody in a Pharmacokinetic Strategy to Increase the Efficacy of Intraperitoneal Chemotherapy

The efficacy of intraperitoneal chemotherapy for ovarian cancers is limited by poor penetration of drug into peritoneal tumors. Based on pharmacokinetic theory that suggests that penetration depth is primarily determined by the rate of drug removal via tumor capillaries, we have hypothesized that co-administration of antiangiogenic therapy will allow for decreased drug removal, increased drug concentrations in tumor, and increased efficacy of intraperitoneal chemotherapy. Pharmacokinetic modeling was conducted to simulate the effect of tumor blood flow on tumor concentrations of topotecan. Simulations predicted that tumor blood flow reductions, as potentially achieved by antiangiogenic therapy, would lead to substantial increases in tumor concentrations after intraperitoneal chemotherapy but would lead to a slight decrease after systemic chemotherapy. Pharmacokinetic studies performed using the A2780 xenograft tumor model showed that animals receiving combined intraperitoneal topotecan and an anti-vascular endothelial growth factor (VEGF) monoclonal antibody had approximately 6.5-fold higher (p = 0.0015) tumor topotecan concentrations compared with animals receiving intraperitoneal topotecan alone, whereas there was no significant (p = 0.16) difference for systemic topotecan. Therapeutic studies conducted with two different drugs, topotecan and cisplatin, showed that animals receiving combined intraperitoneal chemotherapy and anti-VEGF therapy displayed superior survival relative to animals treated with chemotherapy alone (i.e., cisplatin or topotecan), anti-VEGF alone, or intravenous chemotherapy with concomitant anti-VEGF therapy. Combined intraperitoneal topotecan and anti-VEGF resulted in the complete cure of four of 11 mice. The proposed combination of antiangiogenic therapy and intraperitoneal chemotherapy, which was predicted to be beneficial by pharmacokinetic simulations, may provide substantial benefit to patients with peritoneal malignancies.

Sequential topoisomerase targeting and analysis of mechanisms of resistance to topotecan in patients with acute myelogenous leukemia

Resistance to topoisomerase I (TOP1)-targeting drugs such as topotecan often involves upregulation of topoisomerase II (TOP2), with accompanying increased sensitivity to TOP2-targeting drugs such as etoposide. This trial was designed to investigate sequential topoisomerase targeting in the treatment of patients with high-risk acute myelogenous leukemia. An initial cohort of patients received topotecan and cytosine arabinoside daily for 5 days. Serial samples of circulating mononuclear cells were examined to evaluate peak elevations of TOP2-alpha protein expression. In subsequent cohorts, etoposide was administered daily for 3 days, beginning 6 h after initiation of the topotecan infusion. The etoposide dose was escalated to determine a maximum-tolerated dose. Circulating mononuclear cells were analyzed for TOP1 mutations and ABCG2 protein expression. In addition, systemic and intracellular topotecan concentrations were measured. Thirty-one patients were enrolled. On the basis of TOP1-alpha protein levels in three patients with peripheral blast counts greater than 50%, etoposide administration began 6 h after initiation of the topotecan/cytosine arabinoside infusion. Using this schedule of administration, the maximum-tolerated dose of etoposide was 90 mg/m. No TOP1 mutations were identified, but increases in ABCG2 expression during the infusion were observed in mononuclear cells from two of four evaluable patients. Administration of etoposide 6 h after initiation of a topotecan/cytosine arabinoside infusion is feasible and is associated with clinical activity. Analysis of TOP2-alpha protein levels in this small number of patients indicated that peak increases occurred earlier than expected based on earlier publications. Upregulation of ABCG2 was detected in circulating cells and may represent an inducible form of drug resistance that should be investigated further.

The effect of PEG coating on in vitro cytotoxicity and in vivo disposition of topotecan loaded liposomes in rats

Amphoteric drugs encapsulated in PEGylated liposomes may not show superior therapeutic antitumor activity due to increased leakage rate of these drugs in presence of PEG-lipids. In order to investigate the effect of PEG coating on in vitro and in vivo characteristics of topotecan loaded liposomes, an amphoteric anticancer drug, PEGylated and conventional liposomes were prepared by lipid film hydration method. Various properties of the prepared nanoliposomes such as encapsulation efficiency, size, zeta potential, physical stability as well as the chemical stability of lactone form of topotecan, cytotoxicity and topotecan pharmacokinetics were evaluated. In vitro cytotoxic activity was evaluated on murine Lewis lung carcinoma (LLC) and human mammary adenocarcinoma (BT20) cells. Pharmacokinetic was evaluated in Wistar rats after i.v. injection of topotecan, formulated in PBS pH 7.4 or in conventional or in PEGylated liposomes. The conventional liposome (CL) formulation was composed of DSPC/cholesterol/DSPG (molar ratio; 7:7:3), while for PEGylated liposome the composition was DSPC/cholesterol/DSPG/DSPE-PEG 2000 (molar ratio; 7:7:3:1.28). The size of both liposomes was around 100 nm with polydispersity index of about 0.1. In comparison with free drug, liposomal topotecan showed more stability for topotecan lactone form in vitro. Compared to free topotecan, PEGylated and conventional liposomes improved cytotoxic effect of topotecan against the two cancer cell line studied. The results of pharmacokinetic studies in rats showed that both CL and PEGylated liposomal formulations increased the concentration of total topotecan in plasma, however, initial concentration and the values of AUC, MRT and t 1/2 β were much higher ( P < 0.001) for PEGylated liposomal drug than for conventional one or free drug. PEGylated liposome resulted in a 52-fold and 2-fold increases in AUC 0-infinity compared with that of free topotecan and CL, respectively. These results indicated that PEG modified liposome might be an effective carrier for topotecan.

Topotecan Vitreous Levels after Periocular or Intravenous Delivery in Rabbits: An Alternative for Retinoblastoma Chemotherapy

To determine the extent and the mechanism by which topotecan, a candidate agent for the treatment of retinoblastoma, gains access to the vitreous when administered by periocular injection or intravenous infusion. In vivo experiments were conducted in which albino rabbits received 1 mg topotecan by periocular injection (POI group; n = 30) or as a 30-minute intravenous infusion (IV group; n = 16). Plasma and vitreal topotecan concentrations were analyzed during the 10 hours after administration. A population pharmacokinetic model was fit to the data. Additionally, periocular injections were performed postmortem to study the effect of removing the blood vasculature barrier. Potentially active lactone topotecan levels were detected in the vitreous in the POI and IV groups. Both administration schedules induced high total topotecan plasma exposures because of absorption from the periocular depot, though plasma lactone area under the curve (AUC) was significantly higher in the IV group. Similar vitreal concentrations were found in treated and control eyes in the POI group. The transfer from the periocular compartment to the vitreous was negligible. The absence of drug levels in the control eye of the postmortem-injected rabbits confirmed the systemic delivery of topotecan. Local toxicity was not observed. As a consequence of a favored passage across the blood-retinal barrier, considerable topotecan vitreous levels were detected in a rabbit model after systemic or periocular administration. Transscleral entry in vivo was constrained by rapid clearance from the administration site.

Phenoxodiol-topotecan co-administration exhibit significant anti-tumor activity without major adverse side effects

Objective: We previously showed that Phenoxodiol is able to sensitize epithelial ovarian cancer cells to Paclitaxel, Carboplatin, Gemcitabine, and Docetaxel. The aim of this study was to determine the value of Phenoxodiol-Topotecan co-administration. Methods: Nine epithelial ovarian cancer cell lines isolated from ascites or ovarian tissue were treated with increasing concentrations of Topotecan (5-500 ng/ml) with or without Phenoxodiol pre-treatment (10 μg/ml) for 24h and cell viability was measured using CellTiter 96® AQueous One Solution Cell Proliferation Assay. The effect of Phenoxodiol-Topotecan combination therapy in vivo was determined using the topotecan resistant A2780 mouse xenograft model. Results: In vitro, pre-treatment with Phenoxodiol lowers the topotecan IC50 from >500 ng/ml to 2.5-100 ng/ml in 5 out of 9 cell lines tested. Results from animal experiments confirmed the advantage of Phenoxodiol-Topotecan combination therapy over monotherapy controls. Median tumour kinetics from animals receiving Phenoxodiol-Topotecan in combination was significantly slower compared to those animals receiving the respective monotherapies. In addition, co-administration with Phenoxodiol reversed Topotecan-induced myelosuppression. Conclusion: Phenoxodiol-Topotecan combination therapy allows the administration of both agents at lower doses while retaining significant anti-tumor activity compared to monotherapy. These findings suggest that the Phenoxodiol-Topotecan combination may represent an alternative treatment modality for the management of ovarian cancer and justifies further investigation in the clinical setting.

Replication-deficient Adenovirus Induces Host Topoisomerase I Activity: Implications for Adenovirus-mediated Gene Expression

Replication-deficient adenoviruses are useful vectors for the transfer of therapeutic transgenes to malignant and non-malignant tissues. Yet their clinical application is limited by the potential toxicity of viral infection and the transient nature of transgene expression. Although transgene expression from adenovirus vectors is initially higher than expression of transgenes transduced by other viral or non-viral vectors, it is often insufficient to generate a significant therapeutic effect. We addressed this issue by searching for DNA-targeted viral-induced host responses potentially restricting transgene expression. Nuclear protein extracts from livers of rats systemically infected with replication-deficient adenovirus exhibited enhanced topoisomerase I activity compared with extracts from uninfected animals. Consequently, the inhibition of topoisomerase I by the anti-cancer drug topotecan greatly enhanced transgene expression in adenovirus-infected hepatic cells, colon cancer and prostate cancer cell cultures, mouse liver, human ex vivo tumor specimens, and mouse tumor in vivo. The enhancement could not be ascribed to non-specific genotoxic stress, cell death, or cell-cycle perturbation. These findings are significant for gene therapy as they reveal novel aspects of the host anti-adenovirus response and set the stage for the development of a rational molecular-pharmacological approach to increase the effectiveness, and safety, of adenovirus-mediated cancer therapeutics.

The effect of Topotecan on oxidative stress in MCF-7 human breast cancer cell line.

Topotecan, a semisynthetic water-soluble derivative of camptothecin exerts its cytotoxic effect by inhibiting topoisomerase I and causes double-strand DNA breaks which inhibit DNA function and ultimately lead to cell death. In previous studies it was shown that camptothecin causes ROS formation. The aim of this study was to investigate if Topotecan like camptotecin causes oxidative stress in MCF-7 human breast cancer cell line. Determining the oxidant effect of Topotecan may elucidate a possible alternative mechanism for its cytotoxicity. MCF-7 cells were cultured and exposed to Topotecan for 24 h at 37 degrees C. The viability of the cells (% of control) was measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Lipid peroxidation (TBARS), protein oxidation (carbonyl content), sulfhydryl, glutathione (GSH) levels, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were determined in MCF-7 cells with and without Topotecan incubation. We found the IC(50) concentration of Topotecan as 0.218 microM in MCF-7 cells. This concentration of Topotecan was used in the incubations of the cells. Our data indicated increased oxidative status, as revealed by increased lipid peroxidation and protein oxidation, and decreased GSH and sulfhydryl levels in MCF-7 cells exposed to Topotecan compared to control cells. In contrast, there was a slight increase in SOD and a significant increase in GPx and catalase activity in MCF-7 cells incubated with Topotecan compared to the control. These results support our hypothesis that Topotecan increases oxidative stress in MCF-7 cells.

Pharmacokinetics, pharmacodynamics and adherence to oral topotecan in myelodysplastic syndromes: a Cancer and Leukemia Group B study

To evaluate medication adherence, pharmacokinetics and exposure versus response relationships in patients with myelodysplastic syndromes (MDS). Ninety adult patients with MDS received oral topotecan (1.2 mg/m2) either once a day for 10 days or twice a day for 5 days every 21 days for up to six cycles. Dosing histories were collected using electronic monitoring devices fitted to medication vials. Topotecan plasma concentrations were measured, and exposure was determined by a sparse sampling approach and Bayesian estimation methods. Relationships between exposure and clinical response and toxicity were evaluated using logistic regression. Overall adherence was excellent with 90% of patients taking the prescribed number of doses in cycle 1. Adherence did not differ between the two regimens. Topotecan pharmacokinetics were described using a one compartment open model with first order absorption and elimination. Pharmacokinetic parameter estimates did not differ between the once a day and twice a day dosing groups. While topotecan exposure was greater in the twice a day arm compared to the once a day arm due to drug accumulation, exposure did not correlate with clinical response. However, the probability of needing a platelet transfusion in the twice a day arm was significantly increased (by 35%) as a result of greater steady-state plasma topotecan concentrations. Adherence is high in patients with MDS receiving oral topotecan, whether the drug is prescribed once or twice daily. The optimal schedule cannot be determined from this study, as there was no evident relationship between any pharmacokinetic parameter and clinical response.

EFFECTS OF DIHYDROPYRIDINES AND PYRIDINES ON MULTIDRUG RESISTANCE MEDIATED BY BREAST CANCER RESISTANCE PROTEIN: IN VITRO AND IN VIVO STUDIES

Breast cancer resistance protein (BCRP, ABCG2) is a recently identified member of the ATP-binding cassette family of cell surface transport proteins. This study was conducted to investigate the effect of a series of newly synthesized 1,4-dihydropyridines and pyridines, designed as potent P-glycoprotein inhibitors, on BCRP-mediated drug efflux both in vitro and in vivo. The effects of 25 synthesized dihydropyridines and corresponding pyridines along with 4 commercially available dihydropyridines (niguldipine, nicardipine, nifedipine, and nitrendipine) on the intracellular accumulation of the BCRP substrate mitoxantrone were evaluated in BCRP-expressing human breast cancer MCF-7/MX100 and human non-small cell lung cancer H460/MX20 cells. At a 2.5 microM concentration, 24 of 25 newly synthesized dihydropyridines and pyridines produced a significant increase of mitoxantrone accumulation in both cell lines. The most potent compound was able to enhance mitoxantrone accumulation approximately 4.5-fold, greater than that obtained with 10 microM fumitremorgin C, which is a specific BCRP inhibitor. The results from the two cell lines showed good correlation (r(2) = 0.71, p < 0.01). Niguldipine, nicardipine, and nitrendipine also demonstrated potent BCRP inhibition, whereas nifedipine had no effect. The effects of the dihydropyridine and pyridine compounds on mitoxantrone cytotoxicity paralleled their effects on mitoxantrone accumulation. Coadministration of a selected dihydropyridine compound, I(m) [DHP-014; 3-(3-(4,4-diphenylpiperidin-1-yl)propyl) 5-methyl 4-(3,4-dimethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate)] with topotecan, a good BCRP substrate and a moderate to poor P-glycoprotein substrate, resulted in significant increases in the systemic exposure and peak concentration of topotecan in Sprague-Dawley rats when oral topotecan (2 mg/kg) was combined with 20 mg/kg DHP-014. The observed increase of topotecan exposure provides proof-of-concept for in vivo inhibition of BCRP by these agents.

In Vitro andIn Vivo Studies of Different Liposomes Containing Topotecan

Liposome as a carrier of topotecan (TPT), a promising anticancer drug, has been reported in attempt to improve the stability and antitumor activity of TPT. However, the biodistribution pattern of TPT liposome in vivo and PEG-modified liposome containing TPT have not been studied systemically. In this paper, the in vitro stability and in vivo biodistribution behavior of several liposomes containing TPT with different lipid compositions and PEG-modification were studied. Compared with the 'fluid' liposome (S-Lip) composed of soybean phosphatidylcholine (SPC), the 'solid' liposome (H-Lip) composed of hydrogenated soybean phosphatidylcholine HSPC decreased the leaking efficiency of TPT from liposome and enhanced the stability of liposome in fetal bovine serum (FBS) or human blood plasma (HBP). The results of biodistribution studies in S180 tumor-bearing mice showed that liposomal encapsulation increased the concentrations of total TPT and the ratio of lactone form in plasma. Compared with free TPT, S-Lip and H-Lip resulted in 5- and 19-fold increase in the area under the curve (AUC(0-->infinity)), respectively. PEG-modified H-Lip (H-PEG) showed 3.7-fold increase in AUC(0-->infinity) compared with H-Lip, but there was no significant increase in t(1/2) and AUC(0-->infinity) for PEG-modified S-Lip (S-PEG) compared with S-Lip. Moreover, the liposomal encapsulation changed the biodistribution behavior, and H-Lip and H-PEG dramatically increased the accumulation of TPT in tumor, and the relative tumor uptake ratios were 3.4 and 4.3 compared with free drug, respectively. There was also a marked increase in the distribution of TPT in lung when the drug was encapsulated into H-Lip and H-PEG. Moreover, H-PEG decreased the accumulation of TPT in bone marrow compared with unmodified H-Lip. All these results indicated that the membrane fluidity of liposome has an important effect on in vitro stability and in vivo biodistribution pattern of liposomes containing TPT, and PEG-modified 'solid' liposome may be an efficient carrier of TPT.

The Effect of Quercetin on Topotecan Cytotoxicity in MCF-7 and MDA-MB 231 Human Breast Cancer Cells 1

Topotecan, which is a Camptothecin derivative, shows a large spectrum in anti-tumor activity. Topotecan exerts its cytotoxic effect on tumor cells mainly by inhibition of topoisomerase I activity resulting in double-strand DNA breaks. In our study, we investigated the combined cytotoxic action of Topotecan and Quercetin in MCF-7 and MDA-MB 231 human breast cancer cells. To examine the possible relation between the cytotoxic activity of Topotecan and oxidative stress, we measured ROS and nitrite levels in both human breast cell lines. MCF-7 and MDA-MB 231 cells were exposed to Topotecan, Quercetin, or a combination of both agents for 24 h at 37 degrees C. The viability of the cells was measured using the colorimetric MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. We determined reactive oxygen species and nitrite levels as indicators of oxidative stress in both cell lines with and without Topotecan and/or Quercetin incubations using fluorometric dichlorofluorescin diacetate (DCFH-DA) and diaminonaphtalene (DAN) assay. The IC(50) concentration of Topotecan was 100 ng/ml in MCF-7 cell line and 160 ng/ml in MDA-MB231 cell line. Treatment with Quercetin enhanced cytotoxicity of Topotecan as 1.4-fold in MCF-7 and 1.3-fold in MDA-MB-231 cell line. A significant increment on ROS and nitrite levels was found in MCF-7 and MDA-MB-231 cells following Topotecan incubation. Our results suggest that Topotecan has cytotoxic activity against both of the breast cancer cell lines in vitro. A combination with Quercetin increases efficacy of Topotecan in the treatment of breast cancers. Our results indicate that increased oxidative stress plays a role in the cytotoxic action of Topotecan.

Simple and sensitive high performance liquid chromatographic method for the simultaneous quantitation of the lactone and carboxylate forms of topotecan in human plasma

A selective and highly sensitive isocratic high performance liquid chromatographic (HPLC) method is described for simultaneous determination of lactone and carboxylate species of topotecan, in plasma. The method utilizes a protein precipitation step with cold methanol (−20 °C) for sample preparation followed by separation on a Novapack C 18 column using ammonium acetate buffer, acetonitrile and triethylamine (84:16:1.5, v/v) containing tetrabutyl ammonium hydrogen sulfate (TBAHS) (2 mM) with a pH of 5 as the mobile phase. The eluted peaks were detected by a fluorescence detector was set at an excitation wavelength of 380 nm and an emission wavelength of 527 nm. The method was validated in the range of lactone and carboxylate forms of topotecan concentrations from 0.05 to 75 ng/ml. Intra- and inter-day precision expressed by the relative standard deviation was less than 8.50% and inaccuracy did not exceed 10% for lactone and carboxylate forms of topotecan. The limit of quantitation was 0.05 ng/ml using 0.50 ml plasma. Stability studies in plasma and plasma extract indicated that topotecan is stable for at least 2 weeks at −70 °C.

Compartmental modelling of the uptake kinetics of the anti‐cancer agent topotecan in human breast cancer cells

AbstractA compartmental model for the in vitro uptake kinetics of the anti‐cancer agent topotecan is proposed. This model provides a description of the activity of the drug, and subsequent delivery of active form to the nuclear DNA target. The unknown model parameters are estimated from two‐photon laser‐scanning microscopy data, which provide concentrations of topotecan (active plus inactive forms) in the extracellular region containing live human breast tumour cells (MCF‐7 cell line), the cytoplasm and the nucleus. This determines an output structure for which the model is uniquely identifiable, that is, the unknown parameters are uniquely determined from noise‐free, continuous and perfect data. The model allows in silico predictions of the dose dependence of target binding. Copyright © 2005 John Wiley &amp; Sons, Ltd.

Effect of ABCG2 genotype and mRNA expression on the bioavailability of topotecan

2015 Background: The ATP-binding cassette transporter ABCG2 (breast cancer resistance protein; BCRP) functions as an efflux transporter for many drugs and is expressed at high levels in the human small intestine. It has been demonstrated that some individuals possess a nonsynonymous single-nucleotide polymorphism at nucleotide 421, substituting lysine for glutamine on position 141 at exon 5, and express low amounts of ABCG2. Methods: In this prospective exploratory study, duodenal biopsies were obtained before oral administration of topotecan on day 1 (dose, 2.3 mg/m2) and i.v. infusion on day 2 (dose, 1.5 mg/m2) in 18 adult Caucasian cancer patients (10 female/8 male; median age, 54 years). Pharmacokinetic data were obtained on days 1 and 2. Topotecan concentrations in plasma were determined by liquid chromatography with fluorescence detection, and analyzed by noncompartmental methods. Enterocyte (villin)-corrected mRNA expression levels of ABCG2 and ABCB1 (P-glycoprotein) were measured by real-time TaqMan analysis, and all patients were genotyped by cycle sequencing for ABCG2 421C>A. Results: Complete topotecan pharmacokinetic data were available for 13 patients. The mean (± SD) area under the curves after oral and i.v. administration were 109 ± 35.7 ng×h/mL and 218 ± 62.3 ng×h/mL, respectively. Substantial intra- and interindividual variation in mRNA expression of ABCG2 and ABCB1 was observed in the intestinal samples (N = 12). Two patients were heterozygous variant for ABCG2421C>A (14%; allele frequency, 7.14%), and 12 patients carried the wild-type sequence; 4 samples showed no amplification. The heterozygous CA allele was associated with a 1.3-fold increased oral bioavailability of topotecan [31.4% (wild-type, N = 10) versus 42.0% (heterozygous, N = 2); P = 0.037]. The lowest combined mRNA expression of ABCG2 and ABCB1 was observed in one of the patients that was heterozygous variant for ABCG2 421C>A. Conclusions: This pilot study indicates that ABCG2 genotype is correlated with altered topotecan pharmacokin-etics. The current observation warrants confirmation and further investigation in a larger patient population involving different ethnic groups. No significant financial relationships to disclose.

Effect of ABCG2 genotype and mRNA expression on the bioavailability of topotecan

2015 Background: The ATP-binding cassette transporter ABCG2 (breast cancer resistance protein; BCRP) functions as an efflux transporter for many drugs and is expressed at high levels in the human small intestine. It has been demonstrated that some individuals possess a nonsynonymous single-nucleotide polymorphism at nucleotide 421, substituting lysine for glutamine on position 141 at exon 5, and express low amounts of ABCG2. Methods: In this prospective exploratory study, duodenal biopsies were obtained before oral administration of topotecan on day 1 (dose, 2.3 mg/m2) and i.v. infusion on day 2 (dose, 1.5 mg/m2) in 18 adult Caucasian cancer patients (10 female/8 male; median age, 54 years). Pharmacokinetic data were obtained on days 1 and 2. Topotecan concentrations in plasma were determined by liquid chromatography with fluorescence detection, and analyzed by noncompartmental methods. Enterocyte (villin)-corrected mRNA expression levels of ABCG2 and ABCB1 (P-glycoprotein) were measured by real-time TaqMan analysis, and all patients were genotyped by cycle sequencing for ABCG2 421C>A. Results: Complete topotecan pharmacokinetic data were available for 13 patients. The mean (± SD) area under the curves after oral and i.v. administration were 109 ± 35.7 ng×h/mL and 218 ± 62.3 ng×h/mL, respectively. Substantial intra- and interindividual variation in mRNA expression of ABCG2 and ABCB1 was observed in the intestinal samples (N = 12). Two patients were heterozygous variant for ABCG2421C>A (14%; allele frequency, 7.14%), and 12 patients carried the wild-type sequence; 4 samples showed no amplification. The heterozygous CA allele was associated with a 1.3-fold increased oral bioavailability of topotecan [31.4% (wild-type, N = 10) versus 42.0% (heterozygous, N = 2); P = 0.037]. The lowest combined mRNA expression of ABCG2 and ABCB1 was observed in one of the patients that was heterozygous variant for ABCG2 421C>A. Conclusions: This pilot study indicates that ABCG2 genotype is correlated with altered topotecan pharmacokin-etics. The current observation warrants confirmation and further investigation in a larger patient population involving different ethnic groups. No significant financial relationships to disclose.

The role of topotecan for extending the platinum-free interval in recurrent ovarian cancer: an in vitro model

Objective. Topotecan, a novel topoisomerase-I inhibitor, is an active agent of second-line chemotherapy for extending the platinum-free interval (PFI) and improving the chances of a response to platinum in recurrent ovarian cancer patients. The aim of this study was to understand the molecular mechanism of topotecan-based second-line chemotherapy through an in vitro cell culture model and to gain clinical insight into sequencing issues for second-line treatment with novel agents versus retreatment with platinum. Study design. The human ovarian cancer cell line A2780 and the cisplatin resistance cell line A2780-CR were separately seeded in 6-well cell culture plates and then exposed to multiple concentrations of cisplatin plus paclitaxel or topotecan for 7 days. Surviving cells were recovered and cultured in drug-free media for 3 weeks and then replated in a 96-well microtiter plate. The LD 50 for these cells was determined by a cytotoxic MTT assay after exposure to multiple clinically relevant concentrations of cisplatin or topotecan. Surviving cells were cultured in drug-free media for an additional 4 weeks at which time the LD 50 was reassessed for each cell population by a second MTT assay. Using RT-PCR and Northern blot hybridization to measure mRNA expression, the molecular profile of these cells in terms of resistance was evaluated for the multidrug-resistant gene (MDR-1), multidrug-resistant protein (MRP), Topoisomerase-I, and β-Actin. Results. The LD 50 to cisplatin was unchanged in A2780-CR cells treated by topotecan. Those A2780-CR cells originally exposed to higher concentrations of cisplatin became more resistant to cisplatin in the MTT assays, while those A2780-CR cell lines treated with a combination of lower cisplatin concentrations and paclitaxel became more sensitive to cisplatin in the MTT assay ( P < 0.01). The second MTT assay demonstrated that the LD 50 for cisplatin in every cell line decreased significantly after a 4-week drug-free interval ( P < 0.01). There was no difference in the mRNA expression for MRP or topoisomerase-I regardless of cell line, or type or concentration of chemotherapeutic exposure. The mRNA for MDR-1 was uniquely overexpressed in the cisplatin-resistant cell line A2780-CR9 initially treated with low doses of cisplatin and paclitaxel, but was not amplified in A2780 ( P < 0.01). Conclusions. The acquired resistance to cisplatin in A2780 is potentially due to P-glycoprotein-mediated multidrug resistance. This acquired resistance to cisplatin is an unstable phenotype in that some cell populations become sensitive after a drug-free interval and topotecan treatment. This reversal of resistance, however, does not appear to be simply due to loss of MDR-1 expression. While in vivo confirmation is required, agents with novel mechanisms of action offer a strategy to extend the platinum-free interval and thereby improve survival in patients with recurrent ovarian cancer.