Cost-Effectiveness Analysis of FOLFIRI-Based First-Line Regimens for Metastatic Colorectal Cancer Using Clinical Decision Analysis.

OBJECTIVE: Metastatic colorectal cancer with KRAS wild type is treated using a range of drug regimens, including fluorouracil, irinotecan, and Leucovorin(FOLFIRI)plus bevacizumab(Bmab), cetuximab(Cmab), or panitumumab(Pmab). The present study aimed to identify the optimal regimen using a decision analysis method, in combination with clinical and economic evidence. METHOD: A simple Markov model with a monthly cycle time was constructed. Probabilistic variables for input into the model were derived from randomized controlled trials. Direct costs for the drugs, laboratory analyses, and medical staff were calculated and used in the model. RESULTS: The expected survival times and costs of FOLFIRI alone and combination therapies were 20.9 months and 2,299,198 yen for FOLFIRI, 29.9 months and 8,929,888 yen for Bmab, 27.8 months and 11,811,849 yen for Cmab, and 22.6 months and 8,795,622 yen for Pmab. The incremental cost-effectiveness ratios to FOLFIRI were 736,743 yen/month for Bmab, 1,378,645 yen/month for Cmab, and 3,821,426 yen/month for Pmab. CONCLUSIONS: These findings suggested that these regimens were not sufficiently cost-effective, although they have excellent therapeutic efficacy. From the economic point of view, these combination regimens were inferior to FOLFIRI alone.

The Use of an Antioxidant Enables Accurate Evaluation of the Interaction of Curcumin on Organic Anion-Transporting Polypeptides 4C1 by Preventing Auto-Oxidation.

Flavonoids have garnered attention because of their beneficial bioactivities. However, some flavonoids reportedly interact with drugs via transporters and may induce adverse drug reactions. This study investigated the effects of food ingredients on organic anion-transporting polypeptide (OATP) 4C1, which handles uremic toxins and some drugs, to understand the safety profile of food ingredients in renal drug excretion. Twenty-eight food ingredients, including flavonoids, were screened. We used ascorbic acid (AA) to prevent curcumin oxidative degradation in our method. Twelve compounds, including apigenin, daidzein, fisetin, genistein, isorhamnetin, kaempferol, luteolin, morin, quercetin, curcumin, resveratrol, and ellagic acid, altered OATP4C1-mediated transport. Kaempferol and curcumin strongly inhibited OATP4C1, and the Ki values of kaempferol (AA(-)), curcumin (AA(-)), and curcumin (AA(+)) were 25.1, 52.2, and 23.5 µM, respectively. The kinetic analysis revealed that these compounds affected OATP4C1 transport in a competitive manner. Antioxidant supplementation was determined to benefit transporter interaction studies investigating the effects of curcumin because the concentration-dependent curve evidently shifted in the presence of AA. In this study, we elucidated the food-drug interaction via OATP4C1 and indicated the utility of antioxidant usage. Our findings will provide essential information regarding food-drug interactions for both clinical practice and the commercial development of supplements.

Cost-Effectiveness Analysis of Cyclin-Dependent Kinase 4/6 Inhibitor Palbociclib for Inoperable or Recurrent Breast Cancer.

BACKGROUND: Palbociclib and endocrine therapy has been approved to treat hormone receptor-positive/human epidermal growth factor receptor 2-negative inoperable or recurrent breast cancer in Japan. However, this cotherapy imposes an economic burden on both patients and society because of its high cost. In this study, we assessed the cost-effectiveness of cotherapy with palbociclib and fulvestrant compared to fulvestrant monotherapy for inoperable or recurrent breast cancer. METHODS: The three-state Markov model was built by taking into count health stats in inoperable or recurrent breast cancer. The clinical outcomes of the therapies were drawn from published randomized controlled trials. Total regimen cost was calculated from medical receipts of patients at the Yamagata University Hospital. The cost-effectiveness was evaluated by the incremental cost-effectiveness ratio(ICER), in case that it was below 400,000 Yen per month. Markov chain Monte Carlo simulation was performed to assess probability. RESULTS: Acquisition cost of palbociclib and fulvestrant and fulvestrant monotherapy was 6,209,554 JPY and 780,870 JPY, and 25.7 and 22.8 months were achieved, respectively. ICER for the cotherapy was 1,847,721 JPY/quality adjusted life month(QALM)gained. CONCLUSIONS: The palbociclib and fulvestrant therapy provided better health outcomes than conventional fulvestrant monotherapy, but were costly and suggested to be less cost-effective.

Glutathione depletion results in S-nitrosylation of protein disulfide isomerase in neuroblastoma cells.

AIMS: Protein disulfide isomerase (PDI) is an essential enzyme involved in oxidative protein folding. PDI is S-nitrosylated in the brains of Alzheimer's disease patients, and S-nitrosylated PDI is considered one of main causes of Alzheimer's disease. However, the mechanisms underlying PDI S-nitrosylation have not yet been elucidated. Because glutathione (GSH) depletion is a pathological feature of Alzheimer's disease, we investigated the effect of GSH depletion on the S-nitrosylation level of PDI. MAIN METHODS: SH-SY5Y cells, which is a human derived neuroblastoma cells, were used in this study. Glutamate and buthionine sulfoximine (BSO) were used as GSH depletors. S-nitrosylated PDI was detected by biotin-switch assay. KEY FINDINGS: GSH depletion by glutamate, a cystine/glutamate antiporter xCT inhibitor, increased S-nitrosylated PDI at C343 in SH-SY5Y cells, and induced IRE1α phosphorylation. BSO, a γ-glutamylcysteine synthetase inhibitor, also increased S-nitrosylated PDI and phosphorylated IRE1α upon GSH depletion. Because S-nitrosylated PDI at C343 is stable in neuro cells, S-nitrosylated PDI by GSH depletion progresses to neurodegeneration by the induction of endoplasmic reticulum stress via phosphorylated IRE1α signaling from the early to late stage. Furthermore, treatment with neohesperidin, but not N-acetylcysteine (NAC), improved PDI S-nitrosylation level in GSH-depleted SH-SY5Y cells because nitrosylated compound of NAC induces PDI S-nitrosylation. SIGNIFICANCE: The results of our study provide a new insight into the mechanisms of neurodegeneration, and may be useful for the development of drugs for Alzheimer's diseases.

A hydrolysate of poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) suppresses Cav3.2-dependent pain by sequestering exogenous and endogenous sulfide.

Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132), an organogermanium, is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP) in aqueous solutions, and reduces inflammation, pain and cancer, whereas the underlying mechanisms remain unknown. Sulfides including H2S, a gasotransmitter, generated from l-cysteine by some enzymes including cystathionine-γ-lyase (CSE), are pro-nociceptive, since they enhance Cav3.2 T-type Ca2+ channel activity expressed in the primary afferents, most probably by canceling the channel inhibition by Zn2+ linked via coordinate bonding to His191 of Cav3.2. Given that germanium is reactive to sulfur, we tested whether THGP would directly trap sulfide, and inhibit sulfide-induced enhancement of Cav3.2 activity and sulfide-dependent pain in mice. Using mass spectrometry and 1H NMR techniques, we demonstrated that THGP directly reacted with sulfides including Na2S and NaSH, and formed a sulfur-containing reaction product, which decreased in the presence of ZnCl2. In Cav3.2-transfected HEK293 cells, THGP inhibited the sulfide-induced enhancement of T-type Ca2+ channel-dependent membrane currents. In mice, THGP, administered systemically or locally, inhibited the mechanical allodynia caused by intraplantar Na2S. In the mice with cyclophosphamide-induced cystitis and cerulein-induced pancreatitis, which exhibited upregulation of CSE in the bladder and pancreas, respectively, systemic administration of THGP as well as a selective T-type Ca2+ channel inhibitor suppressed the cystitis-related and pancreatitis-related visceral pain. These data suggest that THGP traps sulfide and inhibits sulfide-induced enhancement of Cav3.2 activity, leading to suppression of Cav3.2-dependent pain caused by sulfide applied exogenously and generated endogenously.

Bile Acid-Drug Interaction via Organic Anion-Transporting Polypeptide 4C1 Is a Potential Mechanism of Altered Pharmacokinetics of Renally Excreted Drugs.

Patients with liver diseases not only experience the adverse effects of liver-metabolized drugs, but also the unexpected adverse effects of renally excreted drugs. Bile acids alter the expression of renal drug transporters, however, the direct effects of bile acids on drug transport remain unknown. Renal drug transporter organic anion-transporting polypeptide 4C1 (OATP4C1) was reported to be inhibited by chenodeoxycholic acid. Therefore, we predicted that the inhibition of OATP4C1-mediated transport by bile acids might be a potential mechanism for the altered pharmacokinetics of renally excreted drugs. We screened 45 types of bile acids and calculated the IC50, Ki values, and bile acid−drug interaction (BDI) indices of bile acids whose inhibitory effect on OATP4C1 was >50%. From the screening results, lithocholic acid (LCA), glycine-conjugated lithocholic acid (GLCA), and taurine-conjugated lithocholic acid (TLCA) were newly identified as inhibitors of OATP4C1. Since the BDI index of LCA was 0.278, LCA is likely to inhibit OATP4C1-mediated transport in clinical settings. Our findings suggest that dose adjustment of renally excreted drugs may be required in patients with renal failure as well as in patients with hepatic failure. We believe that our findings provide essential information for drug development and safe drug treatment in clinics.

The Effectiveness of Antidiabetic Drugs in Treating Dementia: A Peek into Pharmacological and Pharmacokinetic Properties.

Dementia dramatically affects the activities of daily living and quality of life; thus, many therapeutic approaches for overcoming dementia have been developed. However, an effective treatment regimen is yet to be developed. As diabetes is a well-known risk factor for dementia, drug repositioning and repurposing of antidiabetic drugs are expected to be effective dementia treatments. Several observational studies have been useful for understanding the effectiveness of antidiabetic drugs in treating dementia, but it is difficult to conclusively analyze the association between antidiabetic drug treatment and the risk of developing dementia after correcting for potential confounding factors. Mechanism-based approaches may provide a better understanding of the effectiveness of antidiabetic drugs for treating dementia. Since the peripheral circulation and the central nerve system are separated by the blood-brain barrier, it is important to understand the regulation of the central glucose metabolism. In this review, we discuss the pharmacological and pharmacokinetic properties of antidiabetic drugs in relation to treating dementia.

The safety of pranlukast and montelukast during the first trimester of pregnancy: A prospective, two-centered cohort study in Japan.

For leukotriene receptor antagonists (LTRAs), especially pranlukast, safety data during pregnancy is limited. Therefore, we conducted a prospective, two-centered cohort study using data from teratogen information services in Japan to clarify the effects of LTRA exposure during pregnancy on maternal and fetal outcomes. Pregnant women who being counseled on drug use during pregnancy at two facilities were enrolled. The primary outcome of this study was major congenital anomalies. The incidence of major congenital anomalies in women exposed to montelukast or pranlukast during the first trimester of pregnancy was compared with that of controls. Logistic regression analysis was performed to analyze the effects of maternal LTRA use during the first trimester of pregnancy on major congenital anomalies. The outcomes of 231 pregnant women exposed to LTRAs (montelukast n = 122; pranlukast n = 106; both n = 3) and 212 live births were compared with those of controls. The rate of major congenital anomalies in the LTRA group was 1.9%. Multivariable logistic regression analysis revealed that LTRA exposure was not a risk factor for major congenital anomalies (adjusted odds ratio, 0.78; 95% confidence interval, 0.23-2.05; p = 0.653). In addition, no significant difference was detected in stillbirth, spontaneous abortion, preterm birth, and low birth weight between the two groups. The present study revealed that montelukast and pranlukast were not associated with the risk of major congenital anomalies. Our findings suggest that LTRAs could be safely employed for asthma therapy during pregnancy.

KRP-203 Is a Desirable Immunomodulator for Islet Allotransplantation.

BACKGROUND: The current standard immunosuppressive regimens, calcineurin inhibitors, have diabetogenic and anti-vascularization effects on islet grafts. KRP-203, a sphingosine-1-phosphate functional antagonist, exerts its immunomodulatory function through lymphocyte sequestration. However, the effect of this antagonist on islets is unclear. We examined the effect of KRP-203 on the islet function and vascularization and sought a calcineurin-free regimen for islet allotransplantation. METHODS: KRP-203 was administered for 14 d to mice, then diabetogenic effect was evaluated by blood glucose levels and a glucose tolerance test. Static glucose stimulation, the breathing index, and insulin/DNA were examined using isolated islets. Islet neovascularization was evaluated using a multiphoton laser scanning microscope. After islet allotransplantation with either KRP-203 alone, sirolimus alone, or both in combination, the graft survival was evaluated by blood glucose levels and immunohistochemical analyses. A mixed lymphocyte reaction was also performed to investigate the immunologic characteristics of KRP-203 and sirolimus. RESULTS: No significant differences in the blood glucose levels or glucose tolerance were observed between the control and KRP-203 groups. Functional assays after islet isolation were also comparable. The multiphoton laser scanning microscope showed no inhibitory effect of KRP-203 on islet neovascularization. Although allogeneic rejection was effectively inhibited by KRP-203 monotherapy (44%), combination therapy prevented rejection in most transplanted mice (83%). CONCLUSIONS: KRP-203 is a desirable immunomodulator for islet transplantation because of the preservation of the endocrine function and lack of interference with islet neovascularization. The combination of KRP-203 with low-dose sirolimus may be promising as a calcineurin-free regimen for islet allotransplantation.

Taisho-Sanshoku koi have hardly faded skin and show attenuated melanophore sensitivity to adrenaline and melanin-concentrating hormone.

Introduction: Koi carp, an ornamental fish derived from the common carp Cyprinus carpio (CC), is characterized by beautiful skin color patterns. However, the mechanism that gives rise to the characteristic vivid skin coloration of koi carp has not been clarified. The skin coloration of many teleosts changes in response to differences in the background color. This change in skin coloration is caused by diffusion or aggregation of pigment granules in chromatophores and is regulated mainly by sympathetic nerves and hormones. We hypothesized that there would be some abnormality in the mechanism of skin color regulation in koi carp, which impairs skin color fading in response to background color. Methods: We compared the function of melanin-concentrating hormone (MCH), noradrenaline, and adrenaline in CC and Taisho-Sanshoku (TS), a variety of tri-colored koi. Results and Discussion: In CC acclimated to a white background, the skin color became paler and pigment granules aggregated in melanophores in the scales compared to that in black-acclimated CC. There were no clear differences in skin color or pigment granule aggregation in white- or black-acclimated TS. The expression of mch1 mRNA in the brain was higher in the white-acclimated CC than that in the black-acclimated CC. However, the expression of mch1 mRNA in the brain in the TS did not change in response to the background color. Additionally, plasma MCH levels did not differ between white- and black-acclimated fish in either CC or TS. In vitro experiments showed that noradrenaline induced pigment aggregation in scale melanophores in both CC and TS, whereas adrenaline induced pigment aggregation in the CC but not in the TS. In vitro administration of MCH induced pigment granule aggregation in the CC but not in the TS. However, intraperitoneal injection of MCH resulted in pigment granule aggregation in both CC and TS. Collectively, these results suggest that the weak sensitivity of scale melanophores to MCH and adrenaline might be responsible for the lack of skin color change in response to background color in the TS.

Clinical Importance of Plasma Drug Concentration of Oral Molecular Targeted Drugs for Renal Cell Carcinoma.

PURPOSE: Therapeutic drug monitoring (TDM) is widely used in clinical practice to maximize drug efficacy and minimize toxicities. Currently, it is also practiced in the use of oral molecular targeted drugs. The objective of this study was to assess the clinical importance of measuring the systemic concentration of oral molecular targeted drugs used to treat renal cell carcinoma (RCC). METHODS: The systemic concentrations of the oral molecular targeted drugs sorafenib, sunitinib, axitinib, pazopanib, and everolimus used for RCC were useful for therapeutic interventions, and clinical outcomes were evaluated retrospectively. RESULTS: The interventional use of systemic drug concentration was confirmed in 26 of 87, and their categories are presented. The systemic concentration of sunitinib was useful in dose reduction and/or discontinuation (n = 10), dose escalation (n = 3), and adherence monitoring (n = 2). Nine of the 10 patients whose dose was reduced showed reduced adverse event. Two patients who were intervened in adherence monitor showed improved adherence. For axitinib, dose reduction and/or discontinuation (n = 1) and dose escalation (n = 6) were confirmed. For pazopanib, dose reduction and/or discontinuation (n = 1) and drug interaction detection (n = 1) were confirmed, both of them were confirmed to have reduced adverse events. For everolimus, dose reduction and/or discontinuation (n = 1) and drug interaction detection (n = 1) were confirmed, a patient with reduced dose recovered from adverse events. Interventions for sorafenib were not identified. CONCLUSIONS: This study demonstrated that systemic concentrations of oral molecular targeted drugs for RCC were considered to be clinically useful for dose adjustment, monitoring of treatment adherence, and the detection of drug interactions. Moreover, this information could be successfully used to guide individualized therapy to maximize the antitumor effects of these drugs.

Glucose-induced oxidative stress leads to in S-nitrosylation of protein disulfide isomerase in neuroblastoma cells.

BACKGROUND: Dementia places a significant burden on both patients and caregivers. Since diabetes is a risk factor for dementia, it is imperative to identify the relationship between diabetes and cognitive disorders. Protein disulfide isomerase (PDI) is an enzyme for oxidative protein folding. PDI S-nitrosylation is observed in the brain tissues of Alzheimer's disease patients. The aim of this study is to clarify the relationship between PDI S-nitrosylation and diabetes. METHODS: We used SH-SY5Y cells cultured in high-glucose media. RESULTS: S-nitrosylated PDI level increased at 7 days and remained high till 28 days in SH-SY5Y cells cultured in high-glucose media. Using PDI wild-type- or PDI C343S-expressing SH-SY5Y cells, PDI C343 was identified as the site of glucose-induced S-nitrosylation. IRE1α and PERK were phosphorylated at day 14 in the SH-SY5Y cells cultured in high-glucose media, and the phosphorylated status was maintained to day 28. To determine the effect of S-nitrosylated PDI on endoplasmic reticulum stress signaling, SH-SY5Y cells were treated with S-nitrosocystein (SNOC) for 30 min, following which the medium was replaced with SNOC-free media and the cells were cultured for 24 h. Only phosphorylated IRE1α treated with SNOC was associated with PDI S-nitrosylation. Neohesperidin, a flavonoid in citrus fruits, is a natural antioxidant. The treatment with neohesperidin in the final 7 days of glucose loading reversed PDI S-nitrosylation and improved cell proliferation. CONCLUSION: Glucose loading leads to S-nitrosylation of PDI C343 and induces neurodegeneration via IRE1α phosphorylation. GENERAL SIGNIFICANCE: The results may be useful for designing curative treatment strategies for dementia.

Enhancement and evaluation of a prescription audit system for direct oral anticoagulants using a check sheet.

BACKGROUND: Renal function and use of concomitant medications should be carefully monitored in patients subjected to treatment with direct oral anticoagulants (DOACs); the dose should be individually designed for each patient. Owing to the complex therapeutic indications and dose reduction criteria, pharmacists exercise caution when determining the optimal dose for each patient. A DOAC check sheet has been developed that is automatically printed in the dispensing room at the same time as the prescription and can be used by pharmacists to dispense DOACs promptly and correctly. The purpose of this study was to evaluate the system for dispensing DOACs using a check sheet. METHODS: The study was conducted at Tohoku University Hospital in Japan; prescriptions containing DOACs dispensed by the hospital pharmacists were evaluated. The DOAC check sheet described indications, dosage regimens, dose reduction criteria, and contraindications for each drug and included the patient's information. The check sheet was set to print automatically in the dispensing room at the same time as the prescription when an inpatient was prescribed DOACs. This check sheet was evaluated using a prescription survey and a questionnaire for pharmacists. RESULTS: The usefulness of this check sheet for the correct use of DOACs was evaluated. There were four inquiries out of 642 (0.6%) prescriptions from pharmacists to physicians regarding DOAC prescriptions, such as the dose introduced before DOAC check sheet utilization, and there were 21 out of 905 (2.3%) prescriptions when the DOAC check sheet was used it, showing a significant increase (p = 0.0089). After the introduction of this sheet, overdoses of DOACs were identified at the time of dispensing. Of the 52 pharmacists who responded to the questionnaire, 51 (98%) stated that the check sheet was useful. CONCLUSION: The use of the DOAC check sheet is likely to render safety to DOAC drug therapy for individual patients.

Role of OATP4C1 in Renal Handling of Remdesivir and its Nucleoside Analog GS-441524: The First Approved Drug for Patients with COVID-19.

PURPOSE: Remdesivir and its active metabolite are predominantly eliminated via renal route; however, information regarding renal uptake transporters is limited. In the present study, the interaction of remdesivir and its nucleoside analog GS-441524 with OATP4C1 was evaluated to provide the detailed information about its renal handling. METHODS: We used HK-2 cells, a proximal tubular cell line derived from normal kidney, to confirm the transport of remdesivir and GS-441524. To assess the involvement of OATP4C1 in handling remdesivir and GS-441524, the uptake study of remdesivir and GS-441524 was performed by using OATP4C1-overexpressing Madin-Darby canine kidney II (MDCKII) cells. Moreover, we also evaluated the IC50 and Ki value of remdesivir. RESULTS: The time-dependent remdesivir uptake in HK-2 cells was observed. The results of inhibition study using OATs and OCT2 inhibitors and OATP4C1 knockdown suggested the involvement of renal drug transporter OATP4C1. Remdesivir was taken up by OATP4C1/MDCKII cells. OATP4C1-mediated uptake of remdesivir increased linearly up to 10 min and reached a steady state at 30 min. Remdesivir inhibited OATP4C1-mediated transport in a concentration-dependent manner with the IC50 and apparent Ki values of 42 ± 7.8 µM and 37 ± 6.9 µM, respectively. CONCLUSIONS: We have provided novel information about renal handling of remdesivir. Furthermore, we evaluated the potential drug interaction via OATP4C1 by calculating the Ki value of remdesivir. OATP4C1 may play a pivotal role in remdesivir therapy for COVID-19, particularly in patients with kidney injury.

Phase II study of trifluridine/tipiracil (TAS-102) therapy in elderly patients with colorectal cancer (T-CORE1401): geriatric assessment tools and plasma drug concentrations as possible predictive biomarkers.

PURPOSE: The current study aimed to determine the efficacy of trifluridine/tipiracil for elderly patients with advanced colorectal cancer. METHODS: This single-arm, open-label, multicenter, phase II study included elderly patients aged 65 years or more who had fluoropyrimidine-refractory advanced colorectal cancer and received trifluridine/tipiracil (70 mg/m2, days 1-5 and 8-12, every 4 weeks). The primary endpoint was progression-free survival (PFS), while secondary endpoints included overall survival (OS), overall response rate (ORR), toxicities, association between efficacy and geriatric assessment scores, and association between toxicity and plasma drug concentrations. RESULTS: A total of 30 patients with a mean age of 73 years were enrolled. Median PFS was 2.3 months (95% confidence interval, 1.9-4.3 months), while median OS was 5.7 months (95% confidence interval, 3.7-8.9 months). Patients had an ORR of 0%, with 57% having stable disease. Grade 4 neutropenia was observed in 13% of the patients. Patients with a higher G8 score (15 or more) showed longer PFS than those with a lower G8 score (median 4.6 vs. 2.0 months; p = 0.047). Moreover, patients with grade 3 or 4 neutropenia showed higher maximum trifluridine concentrations than those with grade 1 or 2 neutropenia (mean 2945 vs. 2107 ng/mL; p = 0.036). DISCUSSION: The current phase II trial demonstrated that trifluridine/tipiracil was an effective and well-tolerated option for elderly patients with advanced colorectal cancer. Moreover, geriatric assessment tools and/or plasma drug concentration monitoring might be helpful in predicting the efficacy and toxicities in elderly patients receiving this drug. TRIAL REGISTRATION NUMBER: UMIN000017589, 15/May/2015 (The University Hospital Medical Information Network).

Simultaneous analysis of drugs administered to lung-transplanted patients using liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring.

Several drugs are administered to lung-transplanted patients, which are monitored using therapeutic drug monitoring (TDM). Therefore, we developed and validated a liquid chromatography-tandem mass spectrometry method to simultaneously analyze immunosuppressive drugs such as mycophenolic acid, antifungal drugs such as voriconazole and itraconazole, and its metabolite hydroxyitraconazole. Chromatographic separation was achieved using a C18 column and gradient flow of mobile phase comprising 20 mM aqueous ammonium formate and 20 mM ammonium formate-methanol solution. A simple protein precipitation treatment was performed using acetonitrile/methanol and mycophenolic acid-2 H3 , voriconazole-2 H3 , itraconazole-2 H4 , and hydroxyitraconazole-2 H4 as internal standards. The linearity ranges of mycophenolic acid, voriconazole, itraconazole, and hydroxyitraconazole were 100-20,000, 50-10,000, 5-1000, and 5-1000 ng/mL, respectively. The retention time of each target was less than 2 min. The relative errors in intra- and inter-day were within ±7.6%, the coefficient of variation was 8.9% or less for quality control low, medium, and high, and it was 15.8% or less for lower limit of quantitation. Moreover, the patient samples were successfully quantified, and they were within the linear range of measurements. Therefore, our new method may be useful for TDM in lung-transplanted patients.

Azelnidipine treatment reduces the expression of Cav1.2 protein.

AIMS: Azelnidipine, a third-generation dihydropyridine calcium channel blocker (DHP CCB), has a characteristic hypotensive effect that persists even after it has disappeared from the plasma, which is thought to be due to its high hydrophobicity. However, because azelnidipine is unique, it might have other unknown effects on L-type Cav1.2 channels that result in the long-lasting decrease of blood pressure. The aim of this study was to investigate the potential quantitative modification of Cav1.2 by azelnidipine. MAIN METHODS: HEK293 cells were used to express Cav1.2 channels. Immunocytochemical analysis was performed to detect changes in the surface expression of the pore-forming subunit of the Cav1.2 channel, Cav1.2α1c. Western blotting analysis was performed to evaluate changes in expression levels of total Cav1.2α1c and Cavß2c. KEY FINDINGS: The surface expression of Cav1.2α1c was markedly reduced by treatment with azelnidipine, but not with other DHP CCBs (amlodipine and nicardipine). Results obtained with a dynamin inhibitor and an early endosome marker suggested that the reduction of surface Cav1.2α1c was not likely caused by internalization. Azelnidipine reduced the total amount of Cav1.2α1c protein in HEK293 cells and rat pulmonary artery smooth muscle cells. The reduction of Cav1.2α1c was rescued by inhibiting proteasome activity. In contrast, azelnidipine did not affect the amount of auxiliary Cavß2c subunits that function as a chaperone of Cav1.2. SIGNIFICANCE: This study is the first to demonstrate that azelnidipine reduces the expression of Cav1.2α1c, which might partly explain its long-lasting hypotensive effect.

Plasma mycophenolic acid concentration and the clinical outcome after lung transplantation.

BACKGROUND: The therapeutic drug monitoring of mycophenolic acid (MPA) has been investigated for renal and heart transplantations; however, its usefulness in lung transplantation is unclear. METHODS: The MPA area under the plasma concentration-time curve (AUC) was calculated in 59 adult lung transplant recipients. The MPA AUC0-12 s were compared among the three groups determined by the presence of adverse events (no events, infection, and chronic lung allograft dysfunction [CLAD]). Next, MPA AUC0-12 thresholds for the adverse events were identified by receiver operating characteristic analysis. Cumulative occurrence rate of the adverse events was compared between two groups (adequate and inadequate groups) according to the thresholds. RESULTS: The MPA AUC0-12 s in the no event, infection, and CLAD groups were 30.3 ± 6.5, 36.8 ± 10.7, and 20.6 ± 9.6 µg·h/mL, respectively (P = .0027), while the tacrolimus trough levels were similarly controlled in the groups. The thresholds of MPA AUC0-12 for the occurrence of infection and CLAD were 40.5 and 22.8 µg·h/mL, respectively. The cumulative occurrence rate of adverse events of adequate group (15.3%) was significantly lower than that of inadequate group (56.0%) (P = .0050). CONCLUSIONS: The MPA AUC0-12 may affect the occurrence of adverse events in lung transplant recipients.

Selective Chemonucleolysis With Condoliase in Cynomolgus Monkeys.

Selective chemonucleolytic effects of condoliase, a glycosaminoglycan degrading enzyme, was investigated histopathologically in cynomolgus monkeys. Condoliase was administered once into the lumber intervertebral disc (IVD), and as a comparative control, chymopapain, a proteolytic enzyme, was administered in a similar manner. Histopathological changes of the IVD and the adjacent vertebral body (VB) were examined at 1 to 26 weeks after administration. Major changes induced by condoliase in the IVD were degenerative and necrotic changes in the nucleus pulposus, annulus fibrosus, cartilaginous endplate (CEP), and epiphyseal growth plate (EGP); focal disappearance of the EGP; and neovascularization and ossification of the CEP. Decreased/necrosis of bone marrow cells with new bone formation was observed in the VB. Cellular regeneration in the IVD was observed as a recovery changes on and after week 4. The changes in the IVD and VB subsided at week 26. Chymopapain induced qualitatively similar but more widely extended changes. The degrees of the changes in the IVD and VB were more severe than those of condoliase, and the changes were exacerbated even at week 26. These results indicated that histopathological changes caused by condoliase were less severe and more selective than those by chymopapain.

ATP-Binding Cassette Transporter C4 is a Prostaglandin D2 Exporter in HMC-1 cells.

ATP-binding cassette transporter C4 (ABCC4) is associated with multidrug resistance and the regulation of cell signalling. Some prostaglandins (PGs), including: PGE2, PGF2α, PGE3, and PGF3α are known substrates of ABCC4, and are released from some types of cells to exert their biological effects. In the present study, we demonstrate that PGD2 is a novel substrate of ABCC4 using a transport assay based on inside-out membrane vesicles prepared from ABCC4-overexpressing cells. Then, we used two types of cell lines with confirmed ABCC4 mRNA and PGD2 release capacity (human mast cell lines HMC-1 cells and human rhabdomyosarcoma cell lines TE671 cells) to evaluate the contribution of ABCC4. The extracellular levels of PGD2 were unchanged following addition of a selective ABCC4 inhibitor in TE671 cells. Pharmacological inhibition and knockdown of ABCC4 significantly reduced the extracellular levels of PGD2 by at least 53% in HMC-1 cells. Moreover, the extracellular levels of PGD2 decreased by at least 20% using the selective ABCC4 inhibitor in the other mast cell line RBL-2H3 cells. Therefore, our results suggest that ABCC4 functions as a PGD2 exporter in HMC-1 cells.