Calcitriol ameliorates motor deficits and prolongs survival of Chrne-deficient mouse, a model for congenital myasthenic syndrome, by inducing Rspo2.

Signal transduction at the neuromuscular junction (NMJ) is compromised in a diverse array of diseases including congenital myasthenic syndromes (CMS). Germline mutations in CHRNE encoding the acetylcholine receptor (AChR) ε subunit are the most common cause of CMS. An active form of vitamin D, calcitriol, binds to vitamin D receptor (VDR) and regulates gene expressions. We found that calcitriol enhanced MuSK phosphorylation, AChR clustering, and myotube twitching in co-cultured C2C12 myotubes and NSC34 motor neurons. RNA-seq analysis of co-cultured cells showed that calcitriol increased the expressions of Rspo2, Rapsn, and Dusp6. ChIP-seq of VDR revealed that VDR binds to a region approximately 15 â€‹kbp upstream to Rspo2. Biallelic deletion of the VDR-binding site of Rspo2 by CRISPR/Cas9 in C2C12 myoblasts/myotubes nullified the calcitriol-mediated induction of Rspo2 expression and MuSK phosphorylation. We generated Chrne knockout (Chrne KO) mouse by CRISPR/Cas9. Intraperitoneal administration of calcitriol markedly increased the number of AChR clusters, as well as the area, the intensity, and the number of synaptophysin-positive synaptic vesicles, in Chrne KO mice. In addition, calcitriol ameliorated motor deficits and prolonged survival of Chrne KO mice. In the skeletal muscle, calcitriol increased the gene expressions of Rspo2, Rapsn, and Dusp6. We propose that calcitriol is a potential therapeutic agent for CMS and other diseases with defective neuromuscular signal transmission.

Why does malaise/fatigue occur? Underlying mechanisms and potential relevance to treatments in rheumatoid arthritis.

INTRODUCTION: Fatigue and malaise are commonly associated with a wide range of medical conditions, including rheumatoid arthritis (RA). Evidence suggests that fatigue and malaise can be overwhelming for patients, yet these symptoms remain inadequately-managed, largely due to an incomplete elucidation of the underlying causes. AREAS COVERED: In this assessment of the published literature relating to the pathogenesis of fatigue or malaise in chronic conditions, four key mechanistic themes were identified. Each theme (inflammation, hypothalamic-pituitary-adrenal axis, dysautonomia, and monoamines) is discussed, as well as the complex network of interconnections between themes which suggests a key role for inflammatory cytokines in the development and persistence of fatigue. EXPERT OPINION: Fatigue is multifaceted, poorly defined, and imperfectly comprehended. Moreover, the cause and severity of fatigue may change over time, as a consequence of the natural disease course or pharmacologic treatment. This detailed synthesis of available evidence permits us to identify avenues for current treatment optimization and future research, to improve the management of fatigue and malaise in RA. Within the development pipeline, several new anti-inflammatory therapies are currently under investigation, and we anticipate that the next five years will herald much-needed progress to reduce the debilitating nature of fatigue in patients with RA.

Quantitative Analysis of mRNA and Protein Expression Levels of Aldo-Keto Reductase and Short-Chain Dehydrogenase/Reductase Isoforms in the Human Intestine.

Enzymes catalyzing the reduction reaction of xenobiotics are mainly members of the aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies. The intestine, together with the liver, is responsible for first-pass effects and is an organ that determines the bioavailability of orally administered drugs. In this study, we evaluated the mRNA and protein expression levels of 12 AKR isoforms (AKR1A1, AKR1B1, AKR1B10, AKR1B15, AKR1C1, AKR1C2, AKR1C3, AKR1C4, AKR1D1, AKR1E2, AKR7A2, and AKR7A3) and 7 SDR isoforms (CBR1, CBR3, CBR4, DCXR, DHRS4, HSD11B1, and HSD17B12) in each region of the human intestine using next-generation sequencing and data-independent acquisition proteomics. At both the mRNA and protein levels, most AKR isoforms were highly expressed in the upper regions of the intestine, namely the duodenum and jejunum, and then declined toward the rectum. Among the members in the SDR superfamily, CBR1 and DHRS4 were highly expressed in the upper regions, whereas the expression levels of the other isoforms were almost uniform in all regions. Significant positive correlations between mRNA and protein levels were observed in AKR1A1, AKR1B1, AKR1B10, AKR1C3, AKR7A2, AKR7A3, CBR1, and CBR3. The mRNA level of AKR1B10 was highest, followed by AKR7A3 and CBR1, each accounting for more than 10% of the sum of all AKR and SDR levels in the small intestine. This expression profile in the human intestine was greatly different from that in the human liver, where AKR1C isoforms are predominantly expressed. SIGNIFICANCE STATEMENT: In this study comprehensively determined the mRNA and protein expression profiles of aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase isoforms involved in xenobiotic metabolism in the human intestine and found that most of them are highly expressed in the upper region, where AKR1B10, AKR7A3, and CBR1 are predominantly expressed. Since the intestine is significantly involved in the metabolism of orally administered drugs, the information provided here is valuable for pharmacokinetic studies in drug development.

Utilization of OATP1B Biomarker Coproporphyrin-I to Guide Drug-Drug Interaction Risk Assessment: Evaluation by the Pharmaceutical Industry.

Drug-drug interactions (DDIs) involving hepatic organic anion transporting polypeptides 1B1/1B3 (OATP1B) can be substantial, however, challenges remain for predicting interaction risk. Emerging evidence suggests that endogenous biomarkers, particularly coproporphyrin-I (CP-I), can be used to assess in vivo OATP1B activity. The present work under the International Consortium for Innovation and Quality in Pharmaceutical Development was aimed primarily at assessing CP-I as a biomarker for informing OATP1B DDI risk. Literature and unpublished CP-I data along with pertinent in vitro and clinical DDI information were collected to identify DDIs primarily involving OATP1B inhibition and assess the relationship between OATP1B substrate drug and CP-I exposure changes. Static models to predict changes in exposure of CP-I, as a selective OATP1B substrate, were also evaluated. Significant correlations were observed between CP-I area under the curve ratio (AUCR) or maximum concentration ratio (Cmax R) and AUCR of substrate drugs. In general, the CP-I Cmax R was equal to or greater than the CP-I AUCR. CP-I Cmax R < 1.25 was associated with absence of OATP1B-mediated DDIs (AUCR < 1.25) with no false negative predictions. CP-I Cmax R < 2 was associated with weak OATP1B-mediated DDIs (AUCR < 2). A correlation was identified between CP-I exposure changes and OATP1B1 static DDI predictions. Recommendations for collecting and interpreting CP-I data are discussed, including a decision tree for guiding DDI risk assessment. In conclusion, measurement of CP-I is recommended to inform OATP1B inhibition potential. The current analysis identified changes in CP-I exposure that may be used to prioritize, delay, or replace clinical DDI studies.

Improvement of Protein Expression Profile in Three-Dimensional Renal Proximal Tubular Epithelial Cell Spheroids Selected Based on OAT1 Gene Expression: A Potential In Vitro Tool for Evaluating Human Renal Proximal Tubular Toxicity and Drug Disposition.

The proximal tubule plays an important role in the kidney and is a major site of drug interaction and toxicity. Analysis of kidney toxicity via in vitro assays is challenging, because only a few assays that reflect functions of drug transporters in renal proximal tubular epithelial cells (RPTECs) are available. In this study, we aimed to develop a simple and reproducible method for culturing RPTECs by monitoring organic anion transporter 1 (OAT1) as a selection marker. Culturing RPTECs in spherical cellular aggregates increased OAT1 protein expression, which was low in the conventional two-dimensional (2D) culture, to a level similar to that in human renal cortices. By proteome analysis, it was revealed that the expression of representative two proximal tubule markers was maintained and 3D spheroid culture improved the protein expression of approximately 7% of the 139 transporter proteins detected, and the expression of 2.3% of the 4,800 proteins detected increased by approximately fivefold that in human renal cortices. Furthermore, the expression levels of approximately 4,800 proteins in three-dimensional (3D) RPTEC spheroids (for 12 days) were maintained for over 20 days. Cisplatin and adefovir exhibited transporter-dependent ATP decreases in 3D RPTEC spheroids. These results indicate that the 3D RPTEC spheroids developed by monitoring OAT1 gene expression are a simple and reproducible in vitro experimental system with improved gene and protein expressions compared with 2D RPTECs and were more similar to that in human kidney cortices. Therefore, it can potentially be used for evaluating human renal proximal tubular toxicity and drug disposition. SIGNIFICANCE STATEMENT: This study developed a simple and reproducible spheroidal culture method with acceptable throughput using commercially available RPTECs by monitoring OAT1 gene expression. RPTECs cultured using this new method showed improved mRNA/protein expression profiles to those in 2D RPTECs and were more similar to those of human kidney cortices. This study provides a potential in vitro proximal tubule system for pharmacokinetic and toxicological evaluations during drug development.

Development and Validation of a Proteomic Correlation Profiling Technique to Detect and Identify Enzymes Involved in Metabolism of Drugs of Concern.

To predict the variation of pharmacological or toxicological effect caused by pharmacokinetic variance, it is important to be able to detect previously unknown and unsuspected enzymes involved in drug metabolism. We investigated the use of proteomic correlation profiling (PCP) as a technique to identify the enzymes involved in metabolism of drugs of concern. By evaluating the metabolic activities of each enzyme (including isoforms of cytochrome P450, uridine 5' diphospho-glucuronosyltransferase, and hydrolases, plus aldehyde oxidase and carbonyl reductase) on their typical substrates using a panel of human liver samples, we were able to show the validity of PCP for this purpose. R or Rs and P values were calculated for the association between the protein abundance profile of each protein and the metabolic rate profile of each typical substrate. For the 18 enzymatic activities examined, 13 of the enzymes reported to be responsible for the reactions had correlation coefficients higher than 0.7 and were ranked first to third. For the remaining five activities, the responsible enzymes had correlation coefficients lower than 0.7 and lower rankings. The reasons for this were diverse, including confounding resulting from low protein abundance ratios, artificially high correlations of other enzymes due to limited sample numbers, the presence of inactive enzyme forms, and genetic polymorphisms. Overall, PCP was able to identify the majority of responsible drug-metabolizing enzymes across several enzyme classes (oxidoreductase, transferase, hydrolase); use of this methodology could allow more timely and accurate identification of unknown drug-metabolizing enzymes. SIGNIFICANCE STATEMENT: Proteomic correlation profiling using samples from individual human donors was proven to be a useful methodology for the identification of enzymes responsible for drug-metabolism. This methodology could accelerate the identification of unknown drug-metabolizing enzymes in the future.

Characterization of Enzymes Involved in Nintedanib Metabolism in Humans.

Nintedanib, which is used to treat idiopathic pulmonary fibrosis and non-small cell lung cancer, is metabolized to a pharmacologically inactive carboxylate derivative, BIBF1202, via hydrolysis and subsequently by glucuronidation to BIBF1202 acyl-glucuronide (BIBF1202-G). Since BIBF1202-G contains an ester bond, it can be hydrolytically cleaved to BIBF1202. In this study, we sought to characterize these metabolic reactions in the human liver and intestine. Nintedanib hydrolysis was detected in human liver microsomes (HLMs) (Clearance [CL int]: 102.8 ± 18.9 µL/min per mg protein) but not in small intestinal preparations. CES1 was suggested to be responsible for nintedanib hydrolysis according to experiments using recombinant hydrolases and hydrolase inhibitors as well as proteomic correlation analysis using 25 individual HLM. BIBF1202 glucuronidation in HLM (3.6 ± 0.3 µL/min per mg protein) was higher than that in human intestinal microsomes (1.5 ± 0.06 µL/min per mg protein). UGT1A1 and gastrointestinal UGT1A7, UGT1A8, and UGT1A10 were able to mediate BIBF1202 glucuronidation. The impact of UGT1A1 on glucuronidation was supported by the finding that liver microsomes from subjects homozygous for the UGT1A1*28 allele showed significantly lower activity than those from subjects carrying the wild-type UGT1A1 allele. Interestingly, BIBF1202-G was converted to BIBF1202 in HLS9 at 70-fold higher rates than the rates of BIBF1202 glucuronidation. An inhibition study and proteomic correlation analysis suggested that ß-glucuronidase is responsible for hepatic BIBF1202-G deglucuronidation. In conclusion, the major metabolic reactions of nintedanib in the human liver and intestine were quantitatively and thoroughly elucidated. This information could be helpful to understand the inter- and intraindividual variability in the efficacy of nintedanib. SIGNIFICANCE STATEMENT: To our knowledge, this is the first study to characterize the enzymes responsible for each step of nintedanib metabolism in the human body. This study found that ß-glucuronidase may contribute to BIBF1202-G deglucuronidation.

Impact of P-glycoprotein on intracellular drug concentration in peripheral blood mononuclear cells and K562 cells.

P-glycoprotein (P-gp) expression in lymphocytes is variable and 2-fold higher in rheumatoid arthritis (RA) patients with treatment resistance than in healthy subjects. To date the information on P-gp-mediated drug interaction in lymphocyte is limited. We analyzed the importance on P-gp in lymphocytes using peripheral blood mononuclear cells (PBMCs) together with K562, K562/Adr, and K562/Vin cells, which have various P-gp levels, as cell models, and dexamethasone, nintedanib and apafant as weak to good P-gp substrates. P-gp levels in K562, K562/Adr, and K562/Vin cells were 0.3-, 20-, and 106-fold of healthy PBMCs, respectively. While cell accumulation of apafant and nintedanib decreased in all cells with increasing P-gp levels, dexamethasone accumulation in K562/Adr was comparable to that in healthy PBMCs and K562 cells. Cell accumulations of substrates in cells with low P-gp expression were not significantly changed by the P-gp inhibitors at therapeutic concentrations. However, accumulation increased to 1.4-fold at highest in K562/Adr cells with higher P-gp expression than in PBMCs of the RA patients. These results suggest P-gp controls the cellular concentration of P-gp substrates in PBMCs or K562 cells but cellular concentration of a weak P-gp substrate would not be apparently affected even in cells with a sufficient P-gp expression.

Identification of HSD17B12 as an enzyme catalyzing drug reduction reactions through investigation of nabumetone metabolism.

Nabumetone, a nonsteroidal anti-inflammatory prodrug, is converted to a pharmacologically active metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA); however, it is 11-fold more efficiently converted to 4-(6-methoxy-2-naphthyl)butan-2-ol (MNBO) via a reduction reaction in human hepatocytes. The goal of this study was to identify the enzyme(s) responsible for MNBO formation from nabumetone in the human liver. MNBO formation by human liver microsomes (HLM) was 5.7-fold higher than in the liver cytosol. In a panel of 24 individual HLM samples with quantitative proteomics data, the 17ß-hydroxysteroid dehydrogenase 12 (HSD17B12) protein level had the high correlation coefficient (r = 0.80, P < 0.001) among 4457 proteins quantified in microsomal fractions during MNBO formation. Recombinant HSD17B12 expressed in HEK293T cells exhibited prominent nabumetone reductase activity, and the contribution of HSD17B12 to the activity in the HLM was calculated as almost 100%. MNBO formation in HepG2 and Huh7 cells was significantly decreased by the knockdown of HSD17B12. We also examined the role of HSD17B12 in drug metabolism and found that recombinant HSD17B12 catalyzed the reduction reactions of pentoxifylline and S-warfarin, suggesting that HSD17B12 prefers compounds containing a methyl ketone group on the alkyl chain. In conclusion, our study demonstrated that HSD17B12 is responsible for the formation of MNBO from nabumetone. Together with the evidence for pentoxifylline and S-warfarin reduction, this is the first study to report that HSD17B12, which is known to metabolize endogenous compounds, such as estrone and 3-ketoacyl-CoA, plays a role as a drug-metabolizing enzyme.

Impact of Direction of Transport on the Evaluation of Substrate Recognition of Mouse Multidrug and Toxin Extrusion Protein 1.

Multidrug and toxin extrusion protein (MATE/SLC47A) secretes metabolites and xenobiotics into the urine in the proximal tubules of the kidney. Uptake assays have been commonly used for evaluating MATE-mediated transport of new chemical entities in drug development. The purpose of this study was to examine the relationship between in vitro uptake activities by MATEs and the impact of MATE-mediated transport in in vivo renal secretion. In vitro uptake in mouse Mate1 (mMate1)-expressing human embryonic kidney 293 (HEK293) cells and several in vivo parameters from mMate1 knockout and wild-type mice were compared using nine cationic compounds (almotriptan, naratriptan, talinolol, sumatriptan, alogliptin, sitagliptin, rivaroxaban, saxagliptin, and vildagliptin). Compounds that showed statistically significant decrease in secretory clearances with respect to kidney concentrations (CLR,kidney) in mMate1 knockout mice were categorized as in vivo substrates in this study. A good correlation (R2 = 0.637) was observed between the in vitro uptake ratio and the in vivo ratio of CLR,kidney of mMate1 knockout mice and wild-type mice. This study supported the rationale of using an uptake assay to determine whether investigational compounds are the substrate of MATEs and to predict drug-drug interaction risk via renal secretion by MATE from the viewpoint of drug development in pharmaceutical companies. SIGNIFICANCE STATEMENT: We revealed that substrates judged by in vitro experiments using mouse multidrug and toxin extrusion (mMate)1-expressing cells were excreted in urine via mMate1 in vivo, and a good correlation (R2 = 0.637) was observed between in vitro uptake ratio and in vivo ratio of secretory clearance with respect to the kidney concentrations (CLR,kidney) of mMate1 knockout and wild-type mice. This study supported the rationale of using an uptake assay to predict potential human MATE1-mediated drug-drug interaction as a victim.

Pharmacokinetic-Interactions of BI 425809, a Novel Glycine Transporter 1 Inhibitor, With Cytochrome P450 and P-Glycoprotein Substrates: Findings From In Vitro Analyses and an Open-Label, Single-Sequence Phase I Study.

PURPOSE/BACKGROUND: Glycine transporter-1 inhibitors may ameliorate cognitive deficits in schizophrenia. This study evaluated potential drug-drug interactions with the glycine transporter-1 inhibitor BI 425809. METHODS/PROCEDURES: Interactions with cytochromes P450 (CYP) and P-glycoprotein (P-gp) were assessed in in vitro assays using human hepatocytes and Caco-2 cells, respectively. Pharmacokinetic characteristics of probe drugs were subsequently assessed in a Phase I, open-label, single-sequence crossover study in healthy male participants. Participants received a probe-drug cocktail containing midazolam (CYP3A4), warfarin (CYP2C9), and omeprazole (CYP2C19) and a separate dose of digoxin (P-gp), alone and on a background of steady-state BI 425809 25 mg once daily in 2 treatment periods. Adverse events were monitored. FINDINGS/RESULTS: In vitro assays revealed concentration-dependent induction of CYP3A4 and inhibition of P-gp by BI 425809. In the clinical study, 12 of 13 participants completed both periods. With BI 425809, area under the plasma concentration curve from administration to the last measurement (AUC 0-tz ) and maximum plasma concentration ( Cmax ) for midazolam were lower than when administered alone. Adjusted geometric mean ratios (90% confidence interval) were 70.6% (63.9%-78.1%) for AUC 0-tz and 77.6% (67.3%-89.4%) for Cmax . For warfarin and digoxin, AUC 0-tz and Cmax were similar with and without BI 425809. For omeprazole, BI 425809 slightly reduced AUC 0-tz but not Cmax versus omeprazole alone. No new safety signals were identified. IMPLICATIONS/CONCLUSIONS: These findings indicate induction of CYP3A4 by once-daily BI 425809 25 mg (the assumed highest therapeutic dose) and no meaningful effects on CYP2C9, CYP2C19, or P-gp in vivo.

Safety and efficacy of filgotinib for Japanese patients with RA and inadequate response to MTX: FINCH 1 52-week results and FINCH 4 48-week results.

OBJECTIVES: To present safety and efficacy of the JAK1 preferential inhibitor filgotinib in Japanese patients with prior inadequate response (IR) to methotrexate (MTX) from a 52-week randomised controlled parent study (PS) and long-term extension (LTE) through June 2020. METHODS: The PS (NCT02889796) randomised MTX-IR patients to filgotinib 200 (FIL200) or 100 mg (FIL100), adalimumab (ADA) 40 mg, or placebo; all took stable background MTX. At week (W) 24, placebo patients were rerandomised to FIL200 or FIL100. The primary endpoint was W12 American College of Rheumatology 20% improvement; safety was assessed by adverse event (AE) reporting. For the LTE (NCT03025308), eligible filgotinib patients continued FIL200/FIL100; ADA patients were rerandomised (blinded) to FIL200 or FIL100; all continued MTX. RESULTS: In all, 114/147 Japanese patients completed the PS, 115 enrolled in LTE, and 103 remained on study in June 2020. In the PS, AEs were consistent with the overall population, and W24 efficacy was maintained or improved through W52, comparable with the overall population. LTE AE incidences were similar between doses; filgotinib efficacy was consistent from baseline to W48 and similar between PS ADA and filgotinib patients. CONCLUSIONS: Among MTX-IR Japanese patients, filgotinib maintained efficacy over 1 year; LTE safety was consistent with the PS.

Integrated safety analysis of filgotinib treatment for rheumatoid arthritis in patients from Japan over a median of 1.5 years.

OBJECTIVE: Characterize safety of the Janus kinase-1 preferential inhibitor filgotinib (FIL) in Japanese patients with moderately to severely active rheumatoid arthritis (RA). METHODS: Data from three Phase 3 trials (NCT02889796, NCT02873936, and NCT02886728) and a long-term extension (NCT03025308) through September 2019 were integrated; patients received ≥1 dose of FIL 200 (FIL200) or 100 mg (FIL100) daily, or placebo (PBO). We calculated exposure-adjusted incidence rates (EAIRs) per 100 patient-years FIL exposure (100PYE) for treatment-emergent adverse events (TEAEs) and adverse events of special interest. RESULTS: Among 3691 total patients and 6080.7 PYE, 229 Japanese patients received FIL for 311.4 PYE (median 1.5, maximum 2.5 years). During the 12-week PBO-controlled period, serious TEAEs and TEAEs leading to study drug disruption were comparable between FIL and PBO. Serious infection rates were 1.9%, 0%, and 2% for FIL200, FIL100, and PBO during the PBO-controlled period; long-term FIL200 and FIL100 EAIRs were 3.8 and 2.1/100PYE. No herpes zoster (HZ) or major adverse cardiovascular events (MACEs) occurred during the PBO-controlled period; long-term FIL200 and FIL100 EAIRs were 3.0 and 2.1/100PYE (HZ) and 0.6 and 0/100PYE (MACE). CONCLUSION: Long-term FIL treatment (median 1.5, maximum 2.5 years exposure) was well tolerated at 100- and 200-mg doses in Japanese patients with RA.

Pelvic incidence affects postoperative dislocation rate in total hip arthropalsty patients with spinal fusion.

PURPOSE: The purpose of this study was to compare the clinical outcome of total hip arthroplasty (THA) with and without spinal fusion (SF), and to evaluate the radiographic characteristics of patients with dislocation after THA. METHODS: A case-controlled study of 53 patients (67 hips) who underwent both THA and SF was performed. The control group was matched to the SF group by age, gender and body mass index, and 106 patients (134 hips) were selected. Hip function was evaluated using the Japanese Orthopaedic Association (JOA) hip score. In addition, the incidence rates of postoperative complications were determined. Radiograph evaluation included lateral inclination and anteversion of the acetabular component. We also analysed sacral slope (SS), pelvic tilt (PT), and pelvic incidence (PI). RESULTS: The JOA hip score at final follow up was significantly poorer in the SF group compared to the control group. There were 5 cases of dislocations in the SF group and none in the control group. The anteversion of acetabular cup, PT, and PI were significantly higher in the SF group compare to the control group. In particular, the PI of patients with anterior dislocation were significantly higher compare to those of patients without dislocation. CONCLUSIONS: Our study showed that SF is a risk factor for a poorer clinical outcome and postoperative dislocation of THA. In addition, the abnormal value of PI may be a risk factor to predict dislocation after THA with SF.

Long-term safety and efficacy of filgotinib treatment for rheumatoid arthritis in Japanese patients naïve to MTX treatment (FINCH 3).

OBJECTIVES: To evaluate the long-term safety and efficacy of filgotinib (FIL) for Japanese patients with rheumatoid arthritis (RA) and limited/no prior methotrexate (MTX) exposure. We present a Japanese population subanalysis of a global randomised-controlled trial at Week 52 and interim long-term extension (LTE) to Week 48 through June 2020. METHODS: Patients were randomised to FIL 200 mg plus MTX, FIL 100 mg plus MTX, FIL 200 mg, or MTX for 52 weeks. At completion, eligible patients could enrol in the LTE. Those receiving FIL continued; those receiving MTX were rerandomised (blinded) to FIL 200 or 100 mg upon discontinuation of MTX. After a 4-week washout period, MTX could be re-added. RESULTS: Adverse event rates at Week 52 and in the LTE to Week 48 were comparable across treatment groups. Week 52 American College of Rheumatology 20% improvement (ACR20) rates were 83% (19/23), 82% (9/11), 75% (9/12), and 76% (19/25) for FIL 200 mg plus MTX, FIL 100 mg plus MTX, FIL 200 mg, and MTX, respectively. Through LTE Week 48, ACR20 rates were maintained. CONCLUSIONS: In the 56 Japanese patients treated with FIL, efficacy was maintained through Week 52 and beyond, with no increases in the incidence of adverse events.

Functional coupling of organic anion transporter OAT10 (SLC22A13) and monocarboxylate transporter MCT1 (SLC16A1) influencing the transport function of OAT10.

OAT10 (SLC22A13) is a transporter highly expressed in renal tubules and transporting organic anions including nicotinate, ß-hydroxybutyrate, p-aminohippurate, and orotate. In transport assays using Xenopus oocytes and HEK293 cells, we found that apparent substrate selectivity of OAT10 was different between the expression systems, particularly less pronounced uptake of ß-hydroxybutyrate in HEK293 cells. Because functional coupling between transporters may interfere with functional properties of the transporter, we searched for endogenous transporters in HEK293 cells that could affect OAT10. By means of comprehensive approach with co-immunoprecipitation followed by LC-MS/MS analysis, we identified monocarboxylate transporter MCT1 (SLC16A1) as physically coupled with OAT10. The knockdown of MCT1 in OAT10-expressing HEK293 cells increased the uptake of ß-hydroxybutyrate and nicotinate, common substrates of OAT10 and MCT1, whereas the uptake of orotate, a substrate of only OAT10, was not affected. MCT1 is supposed to act as an escape route and mediate the efflux of nicotinate and ß-hydroxybutyrate taken up by OAT10 localized nearby MCT1, as suggested by co-immunoprecipitation. This functional coupling would explain altered apparent substrate selectivity in HEK293 cells compared with Xenopus oocytes. The findings in this study warn in transporter studies that the expression system can interfere with assessing correct transport properties due to unexpected interactions with endogenous transporters.

Changes in perioperative C-reactive protein levels in patients with rheumatoid arthritis undergoing total knee arthroplasty in the biologic era.

This study aimed to investigate changes in and factors associated with perioperative serum C-reactive protein (CRP) levels in rheumatoid arthritis (RA) patients undergoing total knee arthroplasty (TKA) in the biologic era. A total of 173 patients (228 knees) with RA underwent elective primary TKA at our institute between January 1, 2006 and December 31, 2018. Of these, 214 cases among 161 patients were examined in this retrospective study after excluding 3cases among 3 patients who developed postoperative complications and 11 cases among 9 patients who were treated with tocilizumab. Factors associated with changes in CRP levels between baseline (preoperative) and day 7 after TKA [ΔCRP (0-7days)] were assessed by multiple regression analysis. Median (interquartile range) CRP levels were 0.69 (0.21, 1.82) mg/dl preoperatively, 5.66 (4.21, 7.61) mg/dl on postoperative day 1, 12.75 (9.79, 16.74) mg/dl on postoperative days 3-4, 3.26 (2.21, 4.85) mg/dl on postoperative day 7, and 0.87 (0.45, 1.81) mg/dl on postoperative day 14. Multivariate regression analysis revealed that body mass index ≥25 [partial regression coefficient (B)=1.03, P=0.012] and use of glucocorticoids (B=-0.86, P=0.017) were independently associated with ΔCRP (0-7days), whereas use of methotrexate and targeted drug modifying antirheumatic drugs and preoperative CRP levels (an objective biomarker of RA activity) were not. In conclusion, serum CRP levels increased rapidly after TKA and peaked on postoperative days 3-4, followed by a return to preoperative levels by postoperative day 14 in patients with RA. Obesity and the use of glucocorticoids were independently associated with changes in CRP levels.

Regional Differences in the Absolute Abundance of Transporters, Receptors and Tight Junction Molecules at the Blood-Arachnoid Barrier and Blood-Spinal Cord Barrier among Cervical, Thoracic and Lumbar Spines in Dogs.

PURPOSE: The purpose of the present study was to quantitatively determine the expression of transporters, receptors and tight junction molecules at the blood-arachnoid barrier (BAB) and blood-spinal cord barrier (BSCB) in cervical, thoracic and lumbar spines from dogs. METHODS: The expression levels of 31 transporters, 3 receptors, 1 tight junction protein, and 3 marker proteins in leptomeninges and capillaries isolated from spines (3 male and 2 female dogs) were determined by quantitative Targeted Absolute Proteomics (qTAP). The units were converted from fmol/µg protein to pmol/cm (absolute abundance at the BAB and the BSCB in a 1 cm section of spine). RESULTS: The expression of MDR1 and BCRP were greater at the BSCB compared to the BAB (especially in the cervical cord), and the expressions at the lumbar BSCB were lower than that for the cervical BSCB. Among the organic anionic and cationic drug transporters, OAT1, OAT3, MRP1, OCT2 and MATE1/2 were detected only in the BAB, and not at the BSCB). The expression of these transporters was higher in the order: lumbar > thoracic > cervical BAB. The expressions of GLUT1, 4F2hc, EAAT1, 2, PEPT2, CTL1, and MCT1 at the BSCB of the cervical cord were higher than the corresponding values for the cervical BAB, and these values decreased in going down the spinal cord. CONCLUSION: These results provide a better understanding of the molecular mechanisms underlying the concentration gradients of drugs and endogenous substances in the cerebrospinal fluid and parenchyma of the spinal cord.

Promethazine Downregulates Wnt/ß-Catenin Signaling and Increases the Biomechanical Forces of the Injured Achilles Tendon in the Early Stage of Healing.

BACKGROUND: Wnt/ß-catenin signaling suppresses the differentiation of cultured tenocytes, but its roles in tendon repair remain mostly elusive. No chemical compounds are currently available to treat tendon injury. HYPOTHESIS: We hypothesized that the inhibition of Wnt/ß-catenin signaling would accelerate tendon healing. STUDY DESIGN: Controlled laboratory study. METHODS: Tendon-derived cells (TDCs) were isolated from rat Achilles tendons. The right Achilles tendon was injured via a dermal punch, while the left tendon was sham operated. A Wnt/ß-catenin inhibitor, IWR-1, and an antihistamine agent, promethazine (PH), were locally and intramuscularly injected, respectively, for 2 weeks after surgery. The healing tendons were histologically and biomechanically evaluated. RESULTS: The amount of ß-catenin protein was increased in the injured tendons from postoperative weeks 0.5 to 2. Inhibition of Wnt/ß-catenin signaling by IWR-1 in healing tendons improved the histological abnormalities and decreased ß-catenin, but it compromised the biomechanical properties. As we previously reported that antihistamine agents suppressed Wnt/ß-catenin signaling in human chondrosarcoma cells, we examined the effects of antihistamines on TDCs. We found that a first-generation antihistamine agent, PH, increased the expression of the tendon marker genes Mkx and Tnmd in TDCs. Intramuscular injection of PH did not improve histological abnormalities, but it decreased ß-catenin in healing tendons and increased the peak force and stiffness of the healing tendons on postoperative week 2. On postoperative week 8, however, the biomechanical properties of vehicle-treated tendons became similar to those of PH-treated tendons. CONCLUSION: IWR-1 and PH suppressed Wnt/ß-catenin signaling and improved the histological abnormalities of healing tendons. IWR-1, however, compromised the biomechanical properties of healing tendons, whereas PH improved them. CLINICAL RELEVANCE: PH is a candidate repositioned drug that potentially accelerates tendon repair.