Takuya Iwamoto

DNA intrastrand cross‐link at the 5′‐GA‐3′ sequence formed by busulfan and its role in the cytotoxic effect

Busulfan (1,4‐butanediol dimethanesulfonate) has been used widely for the treatment of patients with chronic myelogenous leukemia. Busulfan is bifunctional and thus may effectively induce DNA damage, which may play an important role in the cytotoxicity. In this study, we compared the cytotoxicity of bifunctional busulfan with that of monofunctional ethyl methanesulfonate (EMS) in human promyelocytic leukemia HL‐60 cells. Busulfan showed a significant inhibitory effect on cell growth, whereas the cells grew in the presence of EMS. To clarify the mechanism of cytotoxicity of busulfan, we investigated DNA damage induced by busulfan using 32P‐5′‐end‐labeled DNA fragments obtained from the human p16 tumor suppressor gene. Busulfan induced DNA damage dose‐dependently, whereas EMS caused little DNA damage. DNA‐sequencing experiments using piperidine and 3‐methyladenine DNA glycosylase indicated that busulfan caused double‐base lesions mainly at 5′‐GA‐3′and, to a lesser extent, at 5′‐GG‐3’sequences. Time of flight mass spectrometry confirmed that busulfan forms an intrastrand cross‐link at the 5′‐GA‐3’sequence, in addition to mono‐alkylation. The mechanism and the role of cross‐linking at the 5′‐GA‐3’sequence are discussed in relation to the cytotoxicity induced by busulfan.

MicroRNA-145 Post-transcriptionally Regulates the Expression and Function of P-glycoprotein in Intestinal Epithelial Cells

P-glycoprotein (P-gp/MDR1) is a multispecific efflux transporter regulating the pharmacokinetics of various drugs. Although P-gp expression in the small intestine is elevated after liver ischemia-reperfusion (I/R) injury, the regulatory mechanism remains to be clarified. MicroRNAs (miRNAs) play an important role in the post-transcriptional regulation of the expression of drug transporters. Here, we investigated the intestinal expression profile of miRNAs after liver I/R and the role of miRNAs in the post-transcriptional regulation of P-gp in intestinal epithelial cells. Microarray analysis showed that microRNA-145 (miR-145) level was decreased in the small intestine of I/R rats. This downregulation of miR-145 was further confirmed by real-time polymerase chain reaction. In silico analysis revealed that 3′-untranslated regions (UTRs) of rat Mdr1a, mouse Mdr1a, and human MDR1 mRNA retain binding sites for miR-145. Luciferase assays using MDR1 3′-UTR reporter plasmid in HEK293 cells showed that luciferase activity was decreased by the overexpression of miR-145, and the deletion of miR-145 binding site within MDR1 3′-UTR abolished this decreased luciferase activity. The downregulation of miR-145 in Caco-2 cells, an epithelial cell line derived from human colon, increased P-gp expression and efflux activity of rhodamine 123, but not MDR1 mRNA level. These findings demonstrated that miR-145 negatively regulates the expression and function of P-gp through the repression of mRNA by direct interaction on the 3′-UTR of MDR1 mRNA. In addition, the downregulation of miR-145 should significantly contribute to the elevated intestinal P-gp expression after liver I/R. Our results provide new insight into the post-transcriptional regulation of intestinal P-gp.

Decreased Oral Absorption of Cyclosporine A after Liver Ischemia-Reperfusion Injury in Rats: The Contribution of CYP3A and P-Glycoprotein to the First-Pass Metabolism in Intestinal Epithelial Cells

The bioavailability of orally administrated cyclosporine A (CsA) is poor and variable in liver transplantation recipients. Little information is available about the effect of liver ischemia-reperfusion (I/R) injury, which is associated with liver transplantation, on the intestinal first-pass metabolism of CsA. In the present study, we investigated the pharmacokinetics of CsA after liver I/R and assessed the effect of liver I/R via CYP3A and P-glycoprotein (P-gp) on its intestinal first-pass metabolism. When CsA alone was administrated orally, the area under the concentration-time curve (AUC) in the I/R rats was significantly decreased compared with that in the sham rats. On the other hand, there were no significant differences in the AUC between I/R and sham rats when CsA was administrated intravenously or orally with ketoconazole. After intraloop administration of CsA to the small intestine (upper, middle, and lower portions) of the I/R and sham rats, the AUC0–15 min in the upper intestine was significantly lower in the I/R rats than in the sham rats. CYP3A activity and the expression levels of P-gp in the upper intestine of the I/R rats were significantly higher than those of the sham rats. Our study clearly demonstrates for the first time that liver I/R decreases the oral bioavailability of CsA and that this is attributable principally to increased first-pass metabolism mediated by CYP3A and P-gp in the upper small intestine. The present findings provide useful information for the etiology of liver I/R injury and appropriate use of CsA after liver transplantation.

MicroRNAs as regulators of drug transporters, drug-metabolizing enzymes, and tight junctions: implication for intestinal barrier function.

Drug transporters, drug-metabolizing enzymes, and tight junctions in the small intestine function as an absorption barrier and sometimes as a facilitator of orally administered drugs. The expression of these proteins often fluctuates and thereby causes individual pharmacokinetic variability. MicroRNAs (miRNAs), which are small non-coding RNAs, have recently emerged as a new class of gene regulator. MiRNAs post-transcriptionally regulate gene expression by binding to target mRNA to suppress its translation or regulate its degradation. They have been shown to be key regulators of proteins associated with pharmacokinetics. Moreover, the role of miRNAs on the expression of some proteins expressed in the small intestine has recently been clarified. In this review, we summarize current knowledge regarding the role of miRNAs in the regulation of drug transporters, drug-metabolizing enzymes, and tight junctions as well as its implication for intestinal barrier function. MiRNAs play vital roles in the differentiation, architecture, and barrier function of intestinal epithelial cells, and directly and/or indirectly regulate the expression and function of proteins associated with drug absorption in intestinal epithelial cells. Moreover, the variation of miRNA expression caused by pathological and physiological conditions as well as genetic factors should affect the expression of these proteins. Therefore, miRNAs could be significant factors affecting inter- and intra-individual variations in the pharmacokinetics and intestinal absorption of drugs. Overall, miRNAs could be promising targets for personalized pharmacotherapy or other attractive therapies through intestinal absorption of drugs.

Impact of closed-system drug transfer device on exposure of environment and healthcare provider to cyclophosphamide in Japanese hospital

In spite of current recommended safe handling procedures, the potential for the exposure of healthcare providers to hazardous drugs exists in the workplace. A reliance on biological safety cabinets to provide total protection against the exposure to hazardous drugs is insufficient. Preventing workplace contamination is the best strategy to minimize cytotoxic drug exposure in healthcare providers. This study was conducted to compare surface contamination and personnel exposure to cyclophosphamide before and after the implementation of a closed-system drug transfer device, PhaSeal, under the influence of cleaning according to the Japanese guidelines. Personnel exposure was evaluated by collecting 24 h urine samples from 4 pharmacists. Surface contamination was assessed by the wiping test. Four of 6 wipe samples collected before PhaSeal indicated a detectable level of cyclophosphamide. About 7 months after the initiation of PhaSeal, only one of 6 wipe samples indicated a detectable level of cyclophosphamide. Although all 4 employees who provided urine samples had positive results for the urinary excretion of cyclophosphamide before PhaSeal, these levels returned to minimal levels in 2 pharmacists after PhaSeal. In combination with the biological safety cabinet and cleaning according to the Japanese guidelines, PhaSeal further reduces surface contamination and healthcare providers exposure to cyclophosphamide to almost undetectable levels.

Romidepsin (FK228), a potent histone deacetylase inhibitor, induces apoptosis through the generation of hydrogen peroxide

Romidepsin (FK228) is a potent histone deacetylase (HDAC) inhibitor, which has a potent anticancer activity, but its molecular mechanism is unknown. We investigated the mechanism of FK228‐induced apoptosis in the human leukemia cell line HL‐60 and its hydrogen peroxide (H2O2)‐resistant sub‐clone, HP100, and the human colon cancer cell line Caco‐2. Cytotoxicity and DNA ladder formation induced by FK228 could be detected in HL‐60 cells after a 24‐h incubation, whereas they could not be detected in HP100 cells. Trichostatin A (TSA), an HDAC inhibitor, induced DNA ladder formation in both HL‐60 and HP100 cells. In contrast, FK228 inhibited HDAC activity in both HL‐60 and HP100 cells to a similar extent. These findings suggest that FK228‐induced apoptosis involves H2O2‐mediated pathways and that TSA‐induced apoptosis does not. Flow cytometry revealed H2O2 formation and a change in mitochondrial membrane potential (Δψm) in FK228‐treated cells. FK228 also induced apoptosis in Caco‐2 cells, which was prevented by N‐acetyl‐cysteine, suggesting that reactive oxygen species participate in apoptosis in various types of tumor cells. Interestingly, in a cell‐free system, FK228 generated superoxide (O2−) in the presence of glutathione, suggesting that H2O2 is derived from dismutation of O2− produced through redox‐cycle of FK228. Therefore, in addition to HDAC inhibition, H2O2 generated from FK228 may participate in its apoptotic effect. (Cancer Sci 2010;)

Altered functions and expressions of drug transporters in liver, kidney and intestine in disorders of local and remote organs: possible role of oxidative stress in the pathogenesis

Background: Oxidative stress is important in the pathogenesis of various diseases. Drug transporters expressed in the liver, kidney and intestine regulate the distribution, elimination and absorption of drugs. These procedures of drugs in diseases of these organs, mediated by drug transporters, often fluctuate and thereby cause inter- and intra-individual pharmacokinetic variability. Objective: In the present review article, we explore recent reports demonstrating the variance of expressions and/or functions of drug transporters in diseases of the liver, kidney and intestine, in which oxidative stress is involved at least in part in their etiology, and summarize perspectives for further study and the application to clinical situations. Results/conclusion: Various ATP binding cassette and solute carrier transporter expressions were up- or down-regulated in remote organs as well as in the local organ of diseases when these organs were injured, at least in part by oxidative stress, suggesting that oxidative stress is a factor affecting the expression and function of drug transporters. The present review provides useful information for further investigation on the roles of drug transporters in inter- and intra-individual pharmacokinetic variability and for refining dosing regimens in the state of organ diseases.

Mechanism of UVA-dependent DNA Damage Induced by An Antitumor Drug Dacarbazine in Relation to its Photogenotoxicity

PurposeIt has been reported that dacarbazine (DTIC) is photogenotoxic. The purpose of this study is to clarify the mechanism of photogenotoxicity induced by DTIC.Materials and methodsWe examined DNA damage induced by UVA-irradiated DTIC using 32P-5′-end-labeled DNA fragments obtained from human genes. Formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in calf thymus DNA was measured by high performance liquid chromatograph with an electrochemical detector. Electron spin resonance (ESR) spin-trapping experiments were performed to detect radical species generated from UVA-irradiated DTIC.ResultsUVA-irradiated DTIC caused DNA damage at guanine residues, especially at the 5′-GGT-3′ sequence in the presence of Cu(II) and also induced 8-oxodG generation in calf thymus DNA. DTIC-induced photodamage to DNA fragments was partially inhibited by catalase, whereas 8-oxodG formation was significantly increased by catalase. NaN3, a carbene scavenger, inhibited DNA damage and 8-oxodG formation in a dose-dependent manner, suggesting that carbene intermediates are involved. The ESR spin-trapping experiments demonstrated the generation of aryl radicals in the process of photodegradation of DTIC.ConclusionPhotoactivated DTIC generates the carbene and aryl radicals, which may induce both DNA adduct and 8-oxodG formation, resulting in photogenotoxicity. This study could provide an insight into the safe usage of DTIC.

Carboplatin‐induced severe hypersensitivity reaction: Role of IgE‐dependent basophil activation and FcεRI

Basophil activation was observed in patients with a history of carboplatin‐induced severe hypersensitivity reaction (HR). However, the precise mechanism by which carboplatin induces basophil activation and the associated surrogate markers remains to be elucidated. To investigate whether IgE‐dependent mechanisms, including the overexpression of FcεRI, participate in carboplatin‐induced basophil activation, 13 ovarian cancer patients were enrolled: 5 with a history of carboplatin‐induced severe hypersensitivity reaction within the past 2 years, and 8 with no such history. The expression levels of FcεRI, IgE, and CD203c on basophils were measured using a flow cytometer. Immunoglobulin E‐dependent basophil activation was evaluated by testing for IgE passive sensitization using lactic acid, and by testing for phosphatidylinositol 3‐kinase inhibition, using wortmannin. In three patients positive for carboplatin hypersensitivity, pretreatment with wortmannin almost completely inhibited carboplatin‐induced basophil activation (P < 0.05). In a healthy control subject, whose own IgE showed no response to carboplatin, acquired reactivity to carboplatin when exposed to plasma from patients positive for carboplatin hypersensitivity. This did not occur when the same experiment was carried out using plasma from the patients negative for carboplatin hypersensitivity. Moreover, pretreatment with omalizumab, a monoclonal anti‐IgE antibody, almost completely blocked carboplatin‐induced basophil activation in the plasma of patients positive for carboplatin hypersensitivity. On further investigation, the HR‐positive group had significantly higher levels of FcεRI compared with the negative group (P < 0.05). In conclusion, an IgE‐dependent mechanism incorporating FcεRI overexpression participates in carboplatin‐induced severe HR. These results establish the relevance of monitoring the pharmacodynamic changes of basophils to prevent carboplatin‐induced severe HR.

Drug Interaction Between Itraconazole and Bortezomib: Exacerbation of Peripheral Neuropathy and Thrombocytopenia Induced by Bortezomib

Study Objective. To investigate whether a drug interaction exists between bortezomib and the cytochrome P450 (CYP) 3A4 inhibitor itraconazole and/or the CYP2C19 inhibitor lansoprazole that results in increased severity of bortezomib‐induced peripheral neuropathy and thrombocytopenia.