Published papers

Acute kidney injury (AKI) often correlates with poor prognosis and is followed by various severe unfavorable systemic outcomes. It is important to understand the pathophysiology of AKI for the development of novel therapeutic approaches toward promoting renal regeneration after injury. Recent studies have indicated that AKI-induced tubular cell death plays an active role in the onset of tissue regeneration however, the mechanisms underlying renal tubular repair after injury have yet to be understood. In the present study, we explored molecules that might serve as “danger” signals in mediating tubular regeneration. Kidneys of rats systemically administered the nephrotoxicant cisplatin (to induce AKI) exhibited massive cell proliferation. The proportion of proliferating cells in the total cell distribution was highest in the outer stripe of the outer medulla coincided with where the tubular damage was the most severe in this study. This finding suggests that soluble factors may have been released from damaged cells to stimulate the proliferation of neighboring tubular epithelial cells. In elucidating the mechanism of dying cell-to-surviving cell communication using normal rat kidney NRK-52E epithelial cells, we found a significant increase in ATP levels in supernatants of these cells after the induction of cell death using ultraviolet irradiation. Furthermore, treatment of conditioned supernatants with apyrase or suramin, which inhibits purinergic signaling, resulted in significant decreases in cell proliferation and migration activities. These results demonstrate a novel role for extracellular nucleotides, probably as danger signals in aggravating tubular regeneration after AKI.

The principle of nano-surface and molecular-orientation limited (nSMOL) proteolysis has a unique characteristic Fab-selective proteolysis for antibody bioanalysis that is independent of a variety of monoclonal antibodies by the binding antibody Fc via Protein A/G in a pore with 100 nm diameter and modified trypsin immobilization on the surface of nanoparticles with 200 nm diameter. Since minimizing peptide complexity and protease contamination while maintaining antibody sequence specificity enables a rapid and broad development of optimized methods for liquid chromatography-mass spectrometry (LC-MS) bioanalysis, the application of regulatory LC-MS for monitoring antibody biopharmaceuticals is expected. nSMOL is theoretically anticipated to be applicable for representative Fc-fusion biopharmaceuticals, because Protein A/G-binding site Fc exists on the C-terminus, and its functional domain is available to orient and interact with the reaction solution. In this report, we describe the validated LC-MS bioanalysis for monitoring Ethanercept and Abatacept using nSMOL technology. The quantitation range of Ethanercept in human serum was from 0.195 to 100 μg/mL using the signature peptide VFCTK (aa.43-47), and that of Abatacept was from 0.391 to 100 μg/mL using the signature peptide MHVAQPAVVLASSR (aa.1-14). Both proteins fulfilled the guideline criteria for low-molecular-weight drug compounds. The results indicate that the clinical and therapeutic monitoring for antibody and Fc-fusion biopharmaceuticals are adequately applicable using nSMOL proteolysis coupled with LC-MS bioanalysis.

Many biosimilars have been launched recently, however, a precise analytical method to evaluate their characteristics has not been developed. In this study, we established a new qualitative analysis technology for biomedicines using liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (LC/QTOF-MS), and assessed the original product and 4 biosimilars of filgrastim. Three peaks (Peak 1-3) appeared in total ion chromatogram by LC/QTOF-MS in the original brand-named drug. Filgrastim appeared as the main peak (Peak 1), and its molecular weight was calculated to be 18798.96 ± 0.02. When the original product was reduced by dithiothreitol, the molecular weight was 18802.19 ± 0.47, which was identical to the theoretical value based on amino acid sequence. These results indicate that it is possible to measure and distinguish molecular weights of untreated and reduced forms of filgrastim by LC/QTOF-MS. In addition, main peaks from all products including biosimilars showed exactly the same mass spectra and calculated molecular weights, suggesting that the main components (filgrastim) in these 4 products were considered to be qualitatively equal. On the other hand, the area ratios of 2 secondary peaks (Peaks 2 and 3) against the main peak (Peak 1) in 2 biosimilars were higher than those in the original product. The calculated molecular weights in Peaks 2 and 3 corresponded to that of polysorbate 80, which was one of the additives in the formulation. In conclusion, LC/QTOF-MS is a useful technique for qualitative component analysis of biomedicines.

Background: Infliximab (IFX) is a chimeric therapeutic monoclonal antibody targeting tumor necrosis factor alpha (TNFα)-mediated inflammatory immune diseases. However, despite of an initial good clinical response, decrease in response to long-term treatment is a common observation. Objective: Recent studies suggest that IFX level in circulation has a correlation with clinical bioavailability. Therefore, the management of IFX dosage for individual manifestation by IFX monitoring may be valuable for the improvement of therapeutic response and outcomes. Method: In order to develop a broad IFX therapeutic monitoring in human serum, we have developed the validated IFX bioanalysis for Remicade™ and its biosimilar product using our nano-surface and molecular-orientation limited proteolysis (nSMOL) technology coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The nSMOL chemistry has a unique property of Fab-selective proteolysis, and makes it possible a global bioanalysis for many monoclonal antibodies. Results: The quantitation range of IFX in serum was from 0.293 to 300 μg/ml with good linearity. Quantitation verification at the concentrations of 0.293, 0.879, 14.1 and 240 μg/ml was within 1.56-7.53% of precision and 98.9-111% of accuracy using H-chain signature peptide SINSATHYAESVK. Moreover, cross-verified bioanalysis of Remicade quantitation using biosimilar standard, and its opposite combination, obtained an identical and inter-comparative results. Conclusion: The nSMOL strategy has the potential as a practical therapeutic monitoring technology in IFX therapeutic applications.

心臓血管外科病棟におけるバンコマイシン(VCM)治療に焦点を当てて、プロトコルの基づく薬物治療管理(PBPM)の臨床的アウトカムを定量化し、VCM治療における薬剤師介入の効果について検討した。VCMが開始され治療薬物モニタリング(TDM)を行った54例を対象とし、プロトコル導入以前にVCM治療を行った29例(男性21名、女性8名、70±18歳)(I群)、プロトコル導入後にVCM治療を行った25例(男性17名、女性8名、67±15歳)(II群)に分けた。急性腎障害発現はI群9例(31.0%)、II群2例(8.0%)であった。治療域維持率の中央値は、I群50%、II群80%、中毒域到達率の中央値は、I群17%、II群0%であった。治療域維持率及び中毒域到達率に関する2群間の差は、敗血症、肺炎、感染性心内膜炎の背景を有する場合において大きい値であった。VCM血中濃度の日内変動はI群で大きい傾向があった。

2016年度の診療報酬の改定に伴い、処方せんの様式に残薬に関する指示項目が追加された。京都大学医学部附属病院(以下、京大病院)では、1.「保健医療機関へ疑義照会した上で調剤」と2.「保険医療機関へ情報提供」の指定選択項目に、京大病院の処方医が保険薬局薬剤師に対する指示(選択可能)の位置づけで、3.「残薬調整し調剤後にFAXで情報提供」を追加した。この変更によって、院外処方せんにおける残薬調整実施率は変更前の2.8倍に増加し、残薬調整に有効な手段であることを示した。その結果、薬剤費の削減効果も顕著であった。一方で、病院薬剤師の負担は多大で、保険薬局から病院に送付されるトレーシングレポートを電子カルテに取り込み医師にフィードバックしなければならない。今後は、残薬の発生そのものの減少を目指して、服薬アドヒアランスを向上させるための処方提案や服薬指導のさらなる充実に取り組む必要がある。(著者抄録)

Antibody drugs are effective therapeutic agents and provide treatment for many types of diseases such as cancer and rheumatoid arthritis. Because many antibody drugs are developed and approved, quantification technologies of antibodies are required for drug development and individualized therapy. We recently reported a high reproducible and robust therapeutic drug monitoring method for antibody drugs. This method was developed to be applicable to all type of antibody drugs and to provide accurate quantification values. The method is named nano-surface and molecular-orientation limited (nSMOL) proteolysis. nSMOL limits the access of protease to immunoglobulin G molecules and aims to selectively proteolyze on Fab region of substrate. However, the bioanalysis of Tocilizumab using nSMOL has not been validated. We newly discovered that acidified reduction pretreatment of Tocilizumab promotes digestive efficiency by nSMOL proteolysis. Exposure of Tocilizumab to Tris(2-carboxyethyl)phosphine hydrochloride before nSMOL proteolysis significantly improved the recovery of peptides. Under this condition, the quantification range of Tocilizumab in human serum was from 0.781 to 200 μg/mL. The quantification values of quality control samples fulfilled all guideline criteria for bioanalytical validation. The signature peptide with the highest quantitative sensitivity was on H-chain VTMLR. From these results, it is expected that the pretreatment with TCEP will broaden the application of antibody drugs quantification by nSMOL proteolysis.

BACKGROUND: Cutaneous warts are caused by human papilloma virus (HPV) infection. FIT039, a specific inhibitor of CDK9, suppresses the proliferation of DNA viruses in vitro. PURPOSE: We evaluated the safety, plasma concentrations, and efficacy of FIT039 delivered by single application of an adhesive skin patch on normal back skin and cutaneous warts. PATIENTS AND METHODS: In this placebo-controlled, dose-escalation, open-label, two-cohort phase I/II clinical trial, after a single administration of a 1% FIT039 patch, 3% FIT039 patch, or placebo on back skin, patients with cutaneous warts were treated with cryotherapy followed by a 1% FIT039 patch for 24 h in the first cohort. In the second cohort, cutaneous warts were treated with cryotherapy followed by a 3% FIT039 patch for 24 h. Adverse events and adverse drug reactions, the concentrations of FIT039, and surface area of cutaneous warts were evaluated. RESULTS: Neither irritant reactions nor symptoms related to FIT039 occurred when the FIT039 patches were applied to patients' backs or on warts in ten patients. The concentrations of FIT039 were under 0.1 ng/ml at every time point. The median wart surface area at 1 week after application of the 1% FIT039 patch was similar to baseline, while that of the 3% FIT039 patch was smaller than baseline. CONCLUSION: The FIT039 patch showed no topical or systemic adverse reactions when applied on normal skin or cutaneous warts. The safety and good adherence of the FIT039 patch are encouraging and support further studies to evaluate the efficacy of FIT039 in patients with cutaneous warts.

Endoplasmic reticulum (ER) stress may play a role in the etiology of Parkinson's disease (PD). We have previously reported that ubiquitin ligase 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase degradation 1 (HRD1) involved in ER stress degrades unfolded protein that accumulates in the ER due to loss of function of Parkin, which is a causative factor in familial PD. We have also demonstrated that cell death is suppressed by the degradation of unfolded proteins. These findings indicate that HRD1 may serve as a compensatory mechanism for the loss of function of Parkin in familial PD patients. However, the role of HRD1 in sporadic PD has not yet been identified. This study aimed to reveal the roles of HRD1 and associated molecules in a cellular model of PD. We demonstrated that expressions of HRD1 and Suppressor/Enhancer Lin12 1-like (SEL1L: a HRD1 stabilizer) increased in SH-SY5Y human neuroblastoma cells upon exposure to 6-hydroxydopamine (6-OHDA). The 6-OHDA-induced cell death was suppressed in cells overexpressing wt-HRD1, whereas cell death was enhanced in cells with knockdown of HRD1 expression. These results suggest that HRD1 is a key molecule involved in 6-OHDA-induced cell death. By contrast, suppression of SEL1L expression decreased the amount of HRD1 protein. As a result, 6-OHDA-induced cell death was enhanced in cells suppressing SEL1L expression, and this cell death was much more evident than that in cells with suppression of HRD1 expression. These findings strongly indicate that SEL1L is necessary for maintaining and stabilizing the amount of HRD1 protein, and stabilizing the amount of HRD1 protein through SEL1L may serve to protect against 6-OHDA-induced cell death. Furthermore, the expression of Parkin was reinforced when HRD1 mRNA had been suppressed in cells, but was not observed when SEL1L mRNA had been restrained. It is possible that Parkin expression is induced as a compensatory mechanism when HRD1 mRNA decreases. This intracellular transduction may suppress the enhancement of 6-OHDA-induced cell death caused by the loss of HRD1. Taken together with these results, it is suggested that HRD1 and its stabilizer (SEL1L) are key molecules for elucidating the pathogenesis and treatment of PD.

薬物相互作用に関する実験研究として、以下について概説した。1)非臨床試験データ等に基づくペランパネルの薬物相互作用、2)エゼチミブによるビタミンKの吸収阻害を介したワルファリンの相互作用、3)医薬品と飲料・健康食品の薬物動態学的相互作用。次いで、医療現場における薬物相互作用に関する調査研究に関して、事例を示しつつ、調査研究を行う際の注意点やポイントについて述べた。

医療現場における薬物相互作用への対応事例とその問題点について、以下の項目に分けて述べた。1)薬剤師による薬物相互作用のマネジメント・介入の報告、2)システムの問題、3)薬剤師による介入に影響を与える要因や介入時の注意点。また、薬物動態が変動する特別な背景をもつ患者における相互作用の考え方について、ゲノム変異、腎機能低下、肝機能低下、心機能低下、高齢者、新生児・幼児・小児、妊娠に分けて解説した。

日本医療薬学会学術第一小委員会では、医療現場において薬物相互作用をどのように評価しマネジメントに取り組むか、薬物相互作用に関連する問題点をどのように抽出し研究を進めるか、などに関して手引きの作成を検討している。会員に対し実施した手引き作成に関するアンケート調査の結果を紹介するとともに、医療現場における薬物相互作用の評価とマネジメント、薬物相互作用のガイドライン改定について解説した。

PURPOSE: Warfarin shows large inter- and intra-individual variabilities in its pharmacokinetics and pharmacodynamics. Sufficient understanding of factors affecting the response to warfarin is necessary to achieve improved outcomes for warfarin therapy. In this study, we evaluated effects of fasting on the anticoagulant properties of warfarin. METHODS: We conducted a retrospective observational study involving a total of 58 patients, who received cardiovascular surgeries and subsequent warfarin therapy. The effect of dietary intake on the anticoagulant properties with warfarin was assessed by measurement of the international normalized ratio of prothrombin time (PT-INR): the anticoagulant activities of warfarin were expressed as the warfarin sensitivity index (WSI). Additionally, fluctuations in WSI during the study period were obtained as differences between the maximum and minimum WSI. RESULTS: The maximum PT-INR and WSI values were significantly higher for patients who were fasting for different reasons during the postoperative period than those in the group without reduced dietary intake. The differences between maximum and minimum WSI in the fasting group significantly increased compared with those in the groups with moderate or no reduced dietary intake. Meanwhile, effects of other markers of clinical conditions including the baseline Child-Pugh score and Charlson Comorbidity Index on WSI were not significant. CONCLUSIONS: Our results indicate that postoperative fasting was significantly associated with the anticoagulation activity of warfarin. In patients fasting for different reasons during the postoperative period, closer control of PT-INR values and warfarin adjustments may be required to avoid adverse effects such as bleeding in warfarin treatment.

OBJECTIVES: This study aimed to assess whether the combined use of proton pump inhibitors (PPIs) with non-steroidal anti-inflammatory drugs (NSAIDs) or antibiotics (penicillins, macrolides, cephalosporins or fluoroquinolones) was associated with an increased risk of acute kidney injury (AKI). DESIGN: A nested case-control study. SETTING: A health insurance claims database constructed by the Japan Medical Data Center. PARTICIPANTS: Patients were eligible if they were prescribed a PPI, NSAID and antibiotic at least once between January 2005 and June 2017. The patients who were new PPI users and did not have any history of renal diseases before cohort entry were included (n=219 082). The mean age was 45 and 44% were women. INTERVENTIONS: Current use of PPIs, NSAIDs, or antibiotics. PRIMARY OUTCOME MEASURES: Acute kidney injury. RESULTS: During a mean follow-up of 2.4 (SD, 1.7) years, 317 cases of AKI were identified (incidence rate of 6.1/10 000 person-years). The current use of PPIs was associated with a higher risk of AKI compared with past PPI use (unadjusted OR, 4.09; 95% CI, 3.09 to 5.44). The unadjusted ORs of AKI for the current use of PPIs with NSAIDs, cephalosporins and fluoroquinolones, compared with the current use of PPIs alone, were 3.92 (95% CI, 2.40 to 6.52), 2.57 (1.43 to 4.62) and 3.08 (1.50 to 6.38), respectively. The effects of concurrent use of PPIs with NSAIDs, cephalosporins or fluoroquinolones remain significant in the adjusted model. The analyses on absolute risk of AKI confirmed the results from the nested case-control study. CONCLUSIONS: Concomitant use of NSAIDs with PPIs significantly increased the risk for AKI. Moreover, the results suggested that concomitant use of cephalosporins or fluoroquinolones with PPIs was associated with increased risk of incident AKI.

Chemotherapy-induced peripheral neuropathy (CIPN) can lead to discontinuation of chemotherapy and is consequently a serious impediment to effective cancer treatment. Due to our limited understanding of mechanisms underlying the pathogenesis of CIPN, no causal therapy has been approved for relief of this condition. We previously demonstrated that taxanes (paclitaxel and docetaxel) induce Schwann cell dedifferentiation, characterized by increased expression of p75 and galectin-3, ultimately leading to demyelination. These changes appear to be responsible for CIPN pathogenesis. This study was designed to identify a novel candidate therapeutic for CIPN with the ability to suppress paclitaxel-induced Schwann cell dedifferentiation. Given that elevation of cyclic adenosine monophosphate (cAMP) signaling participates in Schwann cell differentiation, we performed immunocytochemical screening of phosphodiesterase (PDE) inhibitors. We found that the PDE3 inhibitor cilostazol strongly promoted differentiation of primary cultures of rat Schwann cells via a mechanism involving cAMP/exchange protein directly activated by cAMP (Epac) signaling. Co-treatment with cilostazol prevented paclitaxel-induced dedifferentiation of Schwann cell cultures and demyelination in a mixed culture of Schwann cells and dorsal root ganglia neurons. Notably, continuous oral administration of cilostazol suppressed Schwann cell dedifferentiation within the sciatic nerve and the development of mechanical hypersensitivity in a mouse model of paclitaxel-related CIPN. Importantly, cilostazol potentiated, rather than inhibited, the anti-cancer effect of paclitaxel on the human breast cancer cell line MDA-MB-231. These findings highlight the potential utility of cilostazol as a causal therapeutic that avoids the development of paclitaxel-related CIPN without compromising anti-cancer properties.

Somnolence during brain function mapping is one of the factors that inhibit the accomplishment of the goals of awake craniotomy. We examined the effect of anesthesia depth measured by bispectral index (BIS) during pre-awake phase on somnolence during brain function mapping and also explored the factors associated with somnolence. We examined the association between BIS values during pre-awake phase and somnolence during the first 30 min of brain function mapping in 55 patients who underwent awake craniotomy at Kyoto University Hospital from 2015 to 2018. The pre-awake BIS value was defined as the mean BIS value for 60 min before the removal of the airway. Somnolence during brain function mapping was the primary outcome, defined as either of the following conditions: inability to follow up, disorientation, or inability to assess speech function. Additionally, we compared patient or perioperative variables between patients with/without somnolence. Somnolence occurred in 14 patients (25.5%), of which 6 patients (10.9%) were unable to complete brain function mapping. There was no significant difference in the pre-awake BIS value between patients with/without somnolence (median: 46 vs. 49, P = 0.192). Somnolence was not significantly associated with age, gender, and the number of preoperative anticonvulsive drugs, but patients with somnolence had a significantly lower preoperative Western Aphasia Battery (WAB) aphasia quotient score (median 93.8 vs. 98.6, P = 0.011). We did not find an association between pre-awake BIS value and somnolence during brain function mapping. Somnolence likely occurs in patients with a low preoperative WAB aphasia quotient score.