The Impact of Breast Cancer Resistance Protein (BCRP/ABCG2) on Drug Transport Across Caco-2 Cell Monolayers.

Breast cancer resistance protein (BCRP) is expressed on the apical membrane of small intestinal epithelial cells and functions as an efflux pump with broad substrate recognition. Therefore, quantitative evaluation of the contribution of BCRP to the intestinal permeability of new chemical entities is very important in drug research and development. In this study, we assessed the BCRP-mediated efflux of several model drugs in Caco-2 cells using WK-X-34 as a dual inhibitor of P-glycoprotein (P-gp) and BCRP and LY335979 as a selective inhibitor of P-gp. The permeability of daidzein was high with an apparent permeability coefficient for apical-to-basal transport (P AB) of 20.3 × 10-6 cm/s. In addition, its efflux ratio (ER) was 1.55, indicating that the contribution of BCRP to its transport is minimal. Estrone-3-sulfate and ciprofloxacin showed relatively higher ER values (>2.0), whereas their BCRP-related absorptive quotient (AQ BCRP) was 0.21 and 0.3, respectively. These results indicate that BCRP does not play a major role in regulating the permeability of estrone-3-sulfate and ciprofloxacin in Caco-2 cells. Nitrofurantoin showed a P AB of 1.8 × 10-6 cm/s, and its ER was 7.6. However, the AQ BCRP was 0.37, suggesting minimal contribution of BCRP to nitrofurantoin transport in Caco-2 cells. In contrast, topotecan, SN-38, and sulfasalazine had low P AB values (0.81, 1.13, and 0.19 × 10-6 cm/s, respectively), and each AQ BCRP was above 0.6, indicating that BCRP significantly contributes to the transport of these compounds in Caco-2 cells. In conclusion, Caco-2 cells are useful to accurately estimate the contribution of BCRP to intestinal drug absorption. SIGNIFICANCE STATEMENT: We performed an in vitro assessment of the contribution of breast cancer resistance protein (BCRP) to the transport of BCRP and/or P-glycoprotein (P-gp) substrates across Caco-2 cell monolayers using absorptive quotient, which has been proposed to represent the contribution of drug efflux transporters to the net efflux. The present study demonstrates that the combined use of a BCRP/P-gp dual inhibitor and a P-gp selective inhibitor is useful to estimate the impact of BCRP and P-gp on the permeability of tested compounds in Caco-2 cells.

Image-guided thermosensitive liposomes for focused ultrasound drug delivery: Using NIRF-labelled lipids and topotecan to visualise the effects of hyperthermia in tumours.

Image guided drug delivery using imageable thermosensitive liposomes (iTSLs) and high intensity focused ultrasound (FUS or HIFU) has attracted interest as a novel and non-invasive route to targeted delivery of anti-cancer therapeutics. FUS-induced hyperthermia is used as an externally applied "trigger" for the release of a drug cargo from within thermosensitive drug carriers. It is suggested that sub-ablative hyperthermia significantly modifies the permeability of tumour vasculature and enhances nanoparticle uptake. Here we describe the preparation and use of magnetic resonance imaging (MRI) and near infrared fluorescence (NIRF) labelled thermosensitive liposomes for imaging and tracking of biodistribution and drug release in a murine cancer model. We prepared iTSLs to encapsulate topotecan (Hycamtin®), a chemotherapeutic agent which when released in tumours can be monitored by an increase in its intrinsic drug fluorescence. FUS was applied using feedback via subcutaneously placed fine-wire thermocouples to maintain and monitor hyperthermic temperatures. iTSL accumulation was detected within tumours using NIRF imaging immediately after liposome administration. Mild FUS-induced hyperthermia (3 min at 42 °C, 30 min post i.v. administration) greatly enhanced iTSLs uptake. A co-localised enhancement of topotecan fluorescence emission was also observed immediately after application of FUS indicating rapid triggered drug release. The phenomena of increased iTSL accumulation and concomitant topotecan release appeared to be amplified by a second mild hyperthermia treatment applied one hour after the first. MRI in vivo also confirmed enhanced iTSLs uptake due to the FUS treatments. Our imaging results indicate the effects of hyperthermia on the uptake of carriers and drug. FUS-induced hyperthermia combined with real time imaging could be used as a tool for tumour targeted drug delivery.

Optimization of liposomal topotecan for use in treating neuroblastoma.

The purpose of this work was to develop an optimized liposomal formulation of topotecan for use in the treatment of patients with neuroblastoma. Drug exposure time studies were used to determine that topotecan (Hycamtin) exhibited great cytotoxic activity against SK-N-SH, IMR-32 and LAN-1 neuroblastoma human cell lines. Sphingomyelin (SM)/cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Chol liposomes were prepared using extrusion methods and then loaded with topotecan by pH gradient and copper-drug complexation. In vitro studies showed that SM/Chol liposomes retained topotecan significantly better than DSPC/Chol liposomes. Decreasing the drug-to-lipid ratio engendered significant increases in drug retention. Dose-range finding studies on NRG mice indicated that an optimized SM/Chol liposomal formulation of topotecan prepared with a final drug-to-lipid ratio of 0.025 (mol: mol) was better tolerated than the previously described DSPC/Chol topotecan formulation. Pharmacokinetic studies showed that the optimized SM/Chol liposomal topotecan exhibited a 10-fold increase in plasma half-life and a 1000-fold increase in AUC0-24 h when compared with Hycamtin administered at equivalent doses (5 mg/kg). In contrast to the great extension in exposure time, SM/Chol liposomal topotecan increased the life span of mice with established LAN-1 neuroblastoma tumors only modestly in a subcutaneous and systemic model. The extension in exposure time may still not be sufficient and the formulation may require further optimization. In the future, liposomal topotecan will be assessed in combination with high-dose radiotherapy such as 131 I-metaiodobenzylguanidine, and immunotherapy treatment modalities currently used in neuroblastoma therapy.

OCULAR PHARMACOLOGY OF CHEMOTHERAPY FOR RETINOBLASTOMA.

PURPOSE: To review preclinical and clinical pharmacokinetic studies of the three most important chemotherapy drugs used for intraocular retinoblastoma and the contribution of the reported results to optimize treatment. METHODS: Systemic review of pharmacokinetic studies identified by a literature search at Pubmed using the keywords carboplatin, melphalan, topotecan, intravitreal, ophthalmic artery chemosurgery, pharmacokinetics, and retinoblastoma. RESULTS: A total of 21 studies were reviewed for assessing the preclinical and clinical pharmacokinetics of carboplatin, topotecan, and melphalan delivered by intravenous, periocular, ophthalmic artery, and intravitreal routes. Some preclinical studies were done before translation to the clinics. Others, despite encouraging preclinical data as reported for periocular topotecan did not correlate with clinical use. In addition, as was the case for melphalan after ophthalmic artery chemosurgery and despite nonfavorable preclinical information, some routes of drug delivery are clinically effective. Besides topotecan, complete knowledge of the pharmacokinetics of melphalan and carboplatin is still lacking. CONCLUSION: Pharmacokinetic knowledge of chemotherapy may aid to guide retinoblastoma treatment in favor of safety and efficacy. Nonetheless, results obtained in preclinical models should be translated with care to the clinics.

A pharmacokinetic evaluation of topotecan as a cervical cancer therapy.

INTRODUCTION: Topotecan was initially approved for the treatment of recurrent ovarian cancer. In cervical cancer, it has been investigated for its potential as part of systemic therapy for advanced and/or recurrent disease and in combination with cisplatin and radiation as a first-line treatment for advanced disease. As a topoisomerase I (topo I) inhibitor, its activity is predicted to be schedule-dependent and potentiated in a schedule-dependent manner by its interaction with DNA damaging agents. AREAS COVERED: The cytotoxicity of topotecan is believed to be due to double-stranded DNA damage produced when the DNA replication 'fork' on the opposing DNA strand is interrupted by the ternary complex formed by topotecan, topoisomerase I and DNA. This review focuses on: i) combination studies of cisplatin + topotecan both as neoadjuvant and with concomitant radiation; ii) adding this drug as a radiosensitizer in pilot studies for locally advanced disease and iii) topotecan as part of non-cisplatin combinations in metastatic disease. EXPERT OPINION: Cervical cancer continues to claim many victims among parts of the world where early detection and/or vaccination programs are not systemically applied. Topotecan is an attractive building block for improving therapy against advanced disease.

Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons.

Angelman syndrome is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele of the ubiquitin protein ligase E3A (UBE3A). In neurons, the paternal allele of UBE3A is intact but epigenetically silenced, raising the possibility that Angelman syndrome could be treated by activating this silenced allele to restore functional UBE3A protein. Using an unbiased, high-content screen in primary cortical neurons from mice, we identify twelve topoisomerase I inhibitors and four topoisomerase II inhibitors that unsilence the paternal Ube3a allele. These drugs included topotecan, irinotecan, etoposide and dexrazoxane (ICRF-187). At nanomolar concentrations, topotecan upregulated catalytically active UBE3A in neurons from maternal Ube3a-null mice. Topotecan concomitantly downregulated expression of the Ube3a antisense transcript that overlaps the paternal copy of Ube3a. These results indicate that topotecan unsilences Ube3a in cis by reducing transcription of an imprinted antisense RNA. When administered in vivo, topotecan unsilenced the paternal Ube3a allele in several regions of the nervous system, including neurons in the hippocampus, neocortex, striatum, cerebellum and spinal cord. Paternal expression of Ube3a remained elevated in a subset of spinal cord neurons for at least 12 weeks after cessation of topotecan treatment, indicating that transient topoisomerase inhibition can have enduring effects on gene expression. Although potential off-target effects remain to be investigated, our findings suggest a therapeutic strategy for reactivating the functional but dormant allele of Ube3a in patients with Angelman syndrome.

Mechanism of the pharmacokinetic interaction between methotrexate and benzimidazoles: potential role for breast cancer resistance protein in clinical drug-drug interactions.

The antifolate drug methotrexate (MTX) is transported by breast cancer resistance protein (BCRP; ABCG2) and multidrug resistance-associated protein1-4 (MRP1-4; ABCC1-4). In cancer patients, coadministration of benzimidazoles and MTX can result in profound MTX-induced toxicity coinciding with an increase in the serum concentrations of MTX and its main metabolite 7-hydroxymethotrexate. We hypothesized that benzimidazoles interfere with the clearance of MTX and/or 7-hydroxymethotrexate by inhibition of the ATP-binding cassette drug transporters BCRP and/or MRP2, two transporters known to transport MTX and located in apical membranes of epithelia involved in drug disposition. First, we investigated the mechanism of interaction between benzimidazoles (pantoprazole and omeprazole) and MTX in vitro in membrane vesicles from Sf9 cells infected with a baculovirus containing human BCRP or human MRP2 cDNA. In Sf9-BCRP vesicles, pantoprazole and omeprazole inhibited MTX transport (IC50 13 microm and 36 microm, respectively). In Sf9-MRP2 vesicles, pantoprazole did not inhibit MTX transport and at high concentrations (1 mm), it even stimulated MTX transport 1.6-fold. Secondly, we studied the transport of pantoprazole in MDCKII monolayers transfected with mouse Bcrp1 or human MRP2. Pantoprazole was actively transported by Bcrp1 but not by MRP2. Finally, the mechanism of the interaction was studied in vivo using Bcrp1-/- mice and wild-type mice. Both in wild-type mice pretreated with pantoprazole to inhibit Bcrp1 and in Bcrp1-/- mice that lack Bcrp1, the clearance of i.v. MTX was decreased significantly 1.8- to 1.9-fold compared with the clearance of i.v. MTX in wild-type mice. The conclusion is as follows: benzimidazoles differentially affect transport of MTX mediated by BCRP and MRP2. Competition for BCRP may explain the clinical interaction between MTX and benzimidazoles.

Phase I studies of nogitecan hydrochloride for Japanese.

BACKGROUND: SmithKline Beecham synthesized camptothecin analogs and identified nogitecan hydrochloride (topotecan) with a broad spectrum of antitumor activity and less toxicity than camptothecin. Because preclinical and overseas clinical data indicated the antitumor effect of nogitecan hydrochloride with a 5-day repeat-dose schedule, we carried out phase I studies in Japan to determine the maximum tolerated dose (MTD), pharmacokinetics, and antitumor effect of nogitecan hydrochloride. METHODS: Phase I studies of nogitecan hydrochloride given by single and 5-day repeat dosing were carried out in patients with various solid tumors at 15 medical institutions in Japan. Pharmacokinetic evaluations were performed for both single and 5-day repeated dosing. RESULTS: The dose-limiting factor (DLF) was reversible leucopenia, and the maximum tolerated dose (MTD) was higher than 22.5 mg/m2 in the single-dose study. In the 5-day repeat-dose study, the DLF was also reversible leucopenia, and the MTD was estimated to be 1.5 mg/m2 per day. The plasma concentration of nogitecan hydrochloride increased with increasing dose, and the half-life after single dosing ranged from 3 to 5h. There was no evidence of accumulation or delayed excretion during 5-day repeat dosing. CONCLUSION: Based on these results and the finding that there were responders among patients treated at 1.5 mg/m2 per day by 5-day repeat dosing in overseas studies, 5-day repeat dosing of 1.2mg/m2 per day, one dose level lower than the MTD, was selected for phase II studies in Japan.