Toshiyuki Sakaeda

An Update on Overcoming MDR1-Mediated Multidrug Resistance in Cancer Chemotherapy

The intrinsic or acquired resistance to anticancer drugs remains one of the most significant factors impeding the progress of cancer chemotherapy. This phenomenon often involves simultaneous resistance to other anticancer drugs that differ in their chemical structure and mode of action and are not even used in chemotherapy. This phenotype has been called multidrug resistance (MDR). Although the cellular basis underlying MDR is not fully understood, several factors mediating therapy resistance in tumors have been proposed. One of the mechanisms leading to chemoresistance of tumor cells is the increased activity of transporter proteins. The best-characterized transporter protein is MDR1/P-glycoprotein, and a number of clinical investigations have suggested that its intrinsic or acquired overexpression resulted in a poor clinical outcome of chemotherapy. Various types of compounds and techniques for the reversal of MDR1/P-glycoprotein-mediated MDR have been developed, and efforts have concentrated on the inhibition of function and suppression of expression. This review summarizes the current state of knowledge of MDR1/P-glycoprotein and the modulation of MDR by targeting MDR1/P-glycoprotein.

Intravenous Infusion of an Antisense Oligonucleotide Results in Exon Skipping in Muscle Dystrophin mRNA of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease that is characterized by muscle dystrophin deficiency. We report that intravenous (IV) infusion of an antisense oligonucleotide created an in-frame dystrophin mRNA from an out-of-frame DMD mutation (via exon skipping) which led to muscle dystrophin expression. A 10-year-old DMD patient possessing an out-of-frame, exon 20 deletion of the dystrophin gene received a 0.5 mg/kg IV infusion of an antisense 31-mer phosphorothioate oligonucleotide against the splicing enhancer sequence of exon 19. This antisense construct was administered at one-week intervals for 4 wk. No side effects attributable to infusion were observed. Exon 19 skipping appeared in a portion of the dystrophin mRNA in peripheral lymphocytes after the infusion. In a muscle biopsy one week after the final infusion, the novel in-frame mRNA lacking both exons 19 and 20 was identified and found to represent approximately 6% of the total reverse transcription PCR product. Dystrophin was identified histochemically in the sarcolemma of muscle cells after oligonucleotide treatment. These findings demonstrate that phosphorothioate oligonucleotides may be administered safely to children with DMD, and that a simple IV infusion is an effective delivery mechanism for oligonucleotides that lead to exon skipping in DMD skeletal muscles.

Group IB secretory phospholipase A2 induces neuronal cell death via apoptosis

Group IB secretory phospholipase A2 (sPLA2‐IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high‐affinity binding sites of sPLA2‐IB have been documented. However, it remains obscure whether sPLA2‐IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2‐IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2‐IB induced neuronal cell death in a concentration‐dependent manner. This death was a delayed response requiring a latent time for 6u2003h; sPLA2‐IB‐induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2‐IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Δ12‐PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase‐2 (COX‐2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2‐IB‐induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2‐IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Δ12‐PGJ2, might mediate sPLA2‐IB‐induced apoptosis.

Impact of clarithromycin resistance and CYP2C19 genetic polymorphism on treatment efficacy of Helicobacter pylori infection with lansoprazole- or rabeprazole-based triple therapy in Japan.

Objective Helicobacter pylori treatment failure is thought to be due mainly to polymorphic cytochrome P450 2C19 (CYP2C19) genetic polymorphism, associated with proton pump inhibitor metabolism, and antimicrobial susceptibility. This report has ascertained which was more important, CYP2C19 polymorphism or antimicrobial susceptibility, when using 1-week lansoprazole-based or rabeprazole-based triple therapy in Japan. Design An open, randomized, parallel group study. Setting One hundred and forty-five subjects with H. pylori-positive gastritis or peptic ulcers were randomly assigned to receive 30 mg lansoprazole twice daily (LAC group), 10 mg rabeprazole twice daily (RAC20 group), or 20 mg rabeprazole twice daily (RAC40 group), with 1000 mg amoxicillin twice daily and 400 mg clarithromycin twice daily for 1 week. Antimicrobial resistance testing was performed by E-test. More than 4 weeks after completion of treatment, H. pylori status was assessed by 13C-urea breath test, histology, and culture. Results Cure rates expressed as intention-to-treat and per-protocol analyses, respectively, were 79.6 and 83.0% with LAC, 85.4 and 89.1% with RAC20, and 83.3 and 88.9% with RAC40. In the case of clarithromycin-sensitive strains, the cure rates were more than 97%, regardless of CYP2C19 polymorphism. However, treatment succeeded in only one out of 16 clarithromycin-resistant strains. Conclusions The key to successful eradication of H. pylori, using lansoprazole or rabeprazole with clarithromycin and amoxicillin, is clarithromycin susceptibility, not CYP2C19 polymorphism.

Hypersensitivity reactions to anticancer agents: Data mining of the public version of the FDA adverse event reporting system, AERS

BackgroundPreviously, adverse event reports (AERs) submitted to the US Food and Drug Administration (FDA) database were reviewed to confirm platinum agent-associated hypersensitivity reactions. The present study was performed to confirm whether the database could suggest the hypersensitivity reactions caused by anticancer agents, paclitaxel, docetaxel, procarbazine, asparaginase, teniposide, and etoposide.MethodsAfter a revision of arbitrary drug names and the deletion of duplicated submissions, AERs involving candidate agents were analyzed. The National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0 was applied to evaluate the susceptibility to hypersensitivity reactions, and standardized official pharmacovigilance tools were used for quantitative detection of signals, i.e., drug-associated adverse events, including the proportional reporting ratio, the reporting odds ratio, the information component given by a Bayesian confidence propagation neural network, and the empirical Bayes geometric mean.ResultsBased on 1,644,220 AERs from 2004 to 2009, the signals were detected for paclitaxel-associated mild, severe, and lethal hypersensitivity reactions, and docetaxel-associated lethal reactions. However, the total number of adverse events occurring with procarbazine, asparaginase, teniposide, or etoposide was not large enough to detect signals.ConclusionsThe FDAs adverse event reporting system, AERS, and the data mining methods used herein are useful for confirming drug-associated adverse events, but the number of co-occurrences is an important factor in signal detection.

Human group IIA secretory phospholipase A2 potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channels in cultured rat cortical neurons

Mammalian group IIA secretory phospholipase A2 (sPLA2‐IIA) generates prostaglandin D2 (PGD2) and triggers apoptosis in cortical neurons. However, mechanisms of PGD2 generation and apoptosis have not yet been established. Therefore, we examined how second messengers are involved in the sPLA2‐IIA‐induced neuronal apoptosis in primary cultures of rat cortical neurons. sPLA2‐IIA potentiated a marked influx of Ca2+ into neurons before apoptosis. A calcium chelator and a blocker of the L‐type voltage‐sensitive Ca2+ channel (L‐VSCC) prevented neurons from sPLA2‐IIA‐induced neuronal cell death in a concentration‐dependent manner. Furthermore, the L‐VSCC blocker ameliorated sPLA2‐IIA‐induced morphologic alterations and apoptotic features such as condensed chromatin and fragmented DNA. Other blockers of VSCCs such as N type and P/Q types did not affect the neurotoxicity of sPLA2‐IIA. Blockers of L‐VSCC significantly suppressed sPLA2‐IIA‐enhanced Ca2+ influx into neurons. Moreover, reactive oxygen species (ROS) were generated prior to apoptosis. Radical scavengers reduced not only ROS generation, but also the sPLA2‐IIA‐induced Ca2+ influx and apoptosis. In conclusion, we demonstrated that sPLA2‐IIA potentiates the influx of Ca2+ into neurons via L‐VSCC. Furthermore, the present study suggested that eicosanoids and ROS generated during arachidonic acid oxidative metabolism are involved in sPLA2‐IIA‐induced apoptosis in cooperation with Ca2+.

Pharmacokinetic characteristics and therapeutic effects of mitomycin C-dextran conjugates after intratumoural injection

The pharmacokinetics and therapeutic effects of macromolecular prodrugs of mitomycin C (MMC), MMC-dextran conjugates (MMC-D) were studied after intratumoural injection in rats bearing Walker 256 carcinosarcoma. As the first step, the intratumoural disposition characteristics of these drugs were delineated in perfusion experiments employing a tissue-isolated tumour preparation. While MMC immediately disappeared from the tumour preparation following direct intratumoural injection, cationic and anionic MMC-D were retained in the tumour longer, demonstrating that the intratumoural clearance of MMC can be greatly retarded by dextran conjugation. The effect was more pronounced in the case of the cationic conjugate. Venous outflow data in the perfusion experiments were analyzed based on a compartment model in which the tumour tissue was assumed to consist of two compartments, one well- and the other poorly-perfused. The pharmacokinetic analysis revealed that macromolecular conjugation reduced elimination of MMC from the poorly-perfused region rather than well-perfused region. Simulation of conjugated and free MMC levels in the tissue using the calculated parameters clearly showed that intratumoural injection of MMC-D, especially the cationic form, can maintain a certain level of active free MMC in the tissue for a much longer time period. The long retention of cationic MMC-D in tumour after intratumoural injection was also confirmed by an in vivo pharmacokinetic study and whole body autoradiography in rats bearing subcutaneous Walker 256 carcinosarcoma. In addition, superior antitumour activity of cationic MMC-D was observed against subcutaneous tumours after intratumoural injection. Together with the finding that MMC is selectively toxic to hypoxic tumour cells at low concentrations, these pharmacokinetic studies strongly support the therapeutic efficacy of the macromolecular prodrugs.

Effects of endothelin B receptor agonists on amyloid β protein (25-35)-induced neuronal cell death

Endothelin (ET), a vasoconstrictive peptide, acts as an anti-apoptotic factor, and endothelin receptor B (ET(B) receptor) is associated with neuronal survival in the brain. In the Alzheimers disease (AD) brain, accumulation of amyloid beta protein (Abeta) is thought to cause neuronal cell death via apoptosis. In the present study, we investigated effects of ET(B) receptor agonists on Abeta-induced neuronal cell death. In primary cultures of rat cortical neurons, Abeta(25-35) caused neuronal cell death in a concentration- and time-dependent manner. Abeta(25-35)-induced neuronal cell death was accompanied by chromatin condensation and DNA fragmentation, exhibiting apoptotic features. ET-3 and IRL-1620, ET(B) receptor agonists, significantly prevented neurons from undergoing Abeta(25-35)-induced cell death. Prior to cell death, Abeta increased concentration of intracellular Ca(2+) ([Ca(2+)](i)). Nimodipine, an L-type voltage-sensitive Ca(2+) channel (L-VSCC) blocker, suppressed the Abeta-induced Ca(2) influx, and attenuated Abeta-induced neuronal apoptosis. On the other hand, omega-conotoxin GIVA, an N-type VSCC blocker and omega-conotoxin MVIIC and omega-agatoxin IVA, P/Q-type VSCC blockers, had no effect. ET-3 and IRL-1620 significantly blocked Abeta(25-35)-induced Ca(2) influx. Furthermore, BQ788, an ET(B) receptor antagonist, inhibited both an anti-apoptotic effect and an L-VSCC-inactivating effect of ET(B) receptor agonists. In conclusion, ET(B) receptor agonists exhibit a protective effect against neurotoxicity of Abeta. Furthermore, these agonists appear to act as anti-apoptotic factors by blocking of L-VSCCs.

Effects of S-2474, a novel nonsteroidal anti-inflammatory drug, on amyloid β protein-induced neuronal cell death

The accumulation of amyloid β protein (Aβ) in the brain is a characteristic feature of Alzheimers disease (AD). Clinical trials of AD patients with nonsteroidal anti‐inflammatory drugs (NSAIDs) indicate a clinical benefit. NSAIDs are presumed to act by suppressing inhibiting chronic inflammation in the brain of AD patients. In the present study, we investigated effects of S‐2474 on Aβ‐induced cell death in primary cultures of rat cortical neurons. S‐2474 is a novel NSAID, which inhibits cyclo‐oxygenase‐2 (COX‐2) and contains the di‐tert‐butylphenol antioxidant moiety. S‐2474 significantly prevented neurons from Aβ(25u2003–u200335)‐ and Aβ(1u2003–u200340)‐induced cell death. S‐2474 ameliorated Aβ‐induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA completely. Prior to cell death, Aβ(25u2003–u200335) generated prostaglandin D2 (PGD2) and free radicals from neurons. PGD2 is a product of cyclo‐oxygenase (COX), and caused neuronal cell death. S‐2474 significantly inhibited the Aβ(25u2003–u200335)‐induced generation of PGD2 and free radicals. The present cortical cultures contained little non‐neuronal cells, indicating that S‐2474 affected neuronal survival directly, but not indirectly via non‐neuronal cells. Both an inhibitory effect of COX‐2 and an antioxidant effect might contribute to the neuroprotective effects of S‐2474. In conclusion, S‐2474 exhibits protective effects against neurotoxicity of Aβ. Furthermore, the present study suggests that S‐2474 may possess therapeutic potential for AD via ameliorating degeneration in neurons as well as suppressing chronic inflammation in non‐neuronal cells.

Statin-associated muscular and renal adverse events: data mining of the public version of the FDA adverse event reporting system.

Objective Adverse event reports (AERs) submitted to the US Food and Drug Administration (FDA) were reviewed to assess the muscular and renal adverse events induced by the administration of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) and to attempt to determine the rank-order of the association. Methods After a revision of arbitrary drug names and the deletion of duplicated submissions, AERs involving pravastatin, simvastatin, atorvastatin, or rosuvastatin were analyzed. Authorized pharmacovigilance tools were used for quantitative detection of signals, i.e., drug-associated adverse events, including the proportional reporting ratio, the reporting odds ratio, the information component given by a Bayesian confidence propagation neural network, and the empirical Bayes geometric mean. Myalgia, rhabdomyolysis and an increase in creatine phosphokinase level were focused on as the muscular adverse events, and acute renal failure, non-acute renal failure, and an increase in blood creatinine level as the renal adverse events. Results Based on 1,644,220 AERs from 2004 to 2009, signals were detected for 4 statins with respect to myalgia, rhabdomyolysis, and an increase in creatine phosphokinase level, but these signals were stronger for rosuvastatin than pravastatin and atorvastatin. Signals were also detected for acute renal failure, though in the case of atorvastatin, the association was marginal, and furthermore, a signal was not detected for non-acute renal failure or for an increase in blood creatinine level. Conclusions Data mining of the FDAs adverse event reporting system, AERS, is useful for examining statin-associated muscular and renal adverse events. The data strongly suggest the necessity of well-organized clinical studies with respect to statin-associated adverse events.