Identification of aging-related genes in diagnosing osteoarthritis via integrating bioinformatics analysis and machine learning.

BACKGROUND: Osteoarthritis (OA) is one of the main causes of pain and disability in the world, it may be caused by many factors. Aging plays a significant role in the onset and progression of OA. However, the mechanisms underlying it remain unknown. Our research aimed to uncover the role of aging-related genes in the progression of OA. METHODS: In Human OA datasets and aging-related genes were obtained from the GEO database and the HAGR website, respectively. Bioinformatics methods including Gene Ontology (GO), Kyoto Encyclopedia of Genes Genomes (KEGG) pathway enrichment, and Protein-protein interaction (PPI) network analysis were used to analyze differentially expressed aging-related genes (DEARGs) in the normal control group and the OA group. And then weighted gene coexpression network analysis (WGCNA), the least absolute shrinkage and selection operator (LASSO) regression, and the Random Forest (RF) machine learning algorithms were used to find the hub genes. RESULTS: Four overlapping hub genes: HMGB2, CDKN1A, JUN, and DDIT3 were identified. According to the nomogram model and receiver operating characteristic (ROC) curve analysis, four hub DEARGs had good diagnostic value in distinguishing normal from OA. Furthermore, the qRT-PCR test demonstrated that HMGB2, CDKN1A, JUN, and DDIT3 mRNA expression levels were lower in OA group than in normal group. CONCLUSION: Finally, these four-hub aging-related genes may help us understand the underlying mechanism of aging in osteoarthritis and could be used as possible diagnostic and therapeutic targets.

Enhanced recovery after surgery combined with quantitative rehabilitation training in early rehabilitation after total knee replacement: a randomized controlled trial.

BACKGROUND: The number of patients undergoing total knee replacement (TKR) is increasing yearly; however, there is still a relative lack of specific, individualized, and standardized protocols for functional exercise after TKR. Quantitative rehabilitation training was developed to improve the recovery of postoperative joint function, increase patient satisfaction, shorten the length of the hospital stay, improve the quality of life, and promote rapid patient recovery. AIM: We aimed to compare the effectiveness of quantitative rehabilitation training based on the enhanced recovery after surgery (ERAS) concept with conventional rehabilitation training in the early rehabilitation of patients with TKR. DESIGN: This was a single-centre, prospective, randomized controlled trial. SETTING: Inpatient department. POPULATION: Participants were patients who underwent unilateral total knee replacement. METHODS: Based on the ERAS concept, a quantitative rehabilitation training program was developed for the quantitative group, and the control group underwent conventional rehabilitation training. Seventy-eight patients undergoing TKR were randomly divided into two blinded groups: the quantitative rehabilitation group and the conventional rehabilitation group. The analysis was performed according to per-protocol practice. The primary outcome metric was the Hospital for Special Surgery Knee Score (HSS Score), and secondary outcomes included patient satisfaction, Visual Analog Pain Score (VAS), time to get out of bed for the first time after surgery, 6-minute-walk test (6MWT), quality-of-life score (SF-36), and number of days in the hospital. The incidence of postoperative complications was also recorded. RESULTS: There was no significant difference in HSS scores between the two groups before surgery (P=0.967), but the quantitative rehabilitation training group had significantly higher scores at two weeks (P=0.031), 3 months (P<0.01), and 12 months (P<0.01) after surgery than did the conventional rehabilitation training group, and both groups had higher HSS scores than before surgery. The quantitative training group had significantly higher VAS scores at 24 hours and three days postoperatively than the conventional training group (P<0.01), while there was no statistical significance at any other time points. The quantitative rehabilitation group had an earlier time to get out of bed for the first time after surgery (P<0.01), a longer 6MWT distance (P=0.028), and higher patient satisfaction and quality of life scores (SF-36) (P<0.01) that did the control group. The number of days in the hospital was lower in the quantitative training group than in the control group (P<0.001). There was no significant difference in the incidence of postoperative complications between the two groups. CONCLUSIONS: Compared with conventional rehabilitation training, quantitative rehabilitation training based on the ERAS concept was found to be safe and effective and can accelerate the recovery of joint function after surgery, shorten hospitalization time, improve patient satisfaction, and promote rapid recovery. CLINICAL REHABILITATION IMPACT: The quantitative rehabilitation training based on the ERAS concept provides a new program for rehabilitation exercises after total knee arthroplasty, which is safe and reliable, accelerates the recovery of joint function, and should be considered for clinical promotion.

High-Intensity Progressive Rehabilitation Versus Routine Rehabilitation After Total Knee Arthroplasty: A Randomized Controlled Trial.

BACKGROUND: This study aimed to compare the effectiveness of high-intensity progressive rehabilitation training with routine training in the early treatment of patients undergoing total knee arthroplasty. METHODS: There were 78 patients who underwent total knee arthroplasty and were randomized into high-intensity progressive training and routine rehabilitation training groups (RRT). The primary outcome measures were the American Hospital for Special Surgery Knee Score (HSS), with secondary outcomes including patient satisfaction, visual analog pain score, first time of standing after surgery, 6-minute walk test, 36-Item Short Form Survey (SF-36), and length of hospital stay. The incidence of postoperative complications were recorded. RESULTS: The HSS scores were higher in the intervention group at 2 weeks, 3 months, and 12 months postoperatively (P < .001). The RRT group had higher visual analog pain scores than the intervention group at 24 hours, 3 days, and 2 weeks after surgery (P < .001). The intervention group had an earlier the first time of standing after surgery and a longer 6-minute walk test distance (P < .001, P = .028, P < .001, P < .001). Patient satisfaction was higher in the intervention group, with a higher quality of life rating at 3 months postoperatively (P < .001). However, 1 year after surgery, the 2 groups had no significant differences in mental component summaries. The length of hospital stay was shorter in the intervention group than in the RRT group. CONCLUSION: Compared to routine training, high-intensity progressive rehabilitation training is more effective. It reduces postoperative patient pain, accelerates recovery of joint function, increases patient satisfaction, improves quality of life, shortens hospital stays, and promotes rapid recovery.

Characteristics and time points to inhibit ferroptosis in human osteoarthritis.

Ferroptosis is a form of cell death that is triggered by iron-dependent lipid peroxidation and is closely associated with osteoarthritis. The primary interventions for inhibiting ferroptosis in osteoarthritis are anti-lipid peroxidation and iron chelation. The objective of our study is to investigate the characteristics of ferroptosis in osteoarthritis and identify the optimal time points for inhibiting ferroptosis to alleviate disease progression. Ferroptosis-related alterations and markers of OA were analyzed in paired intact and damaged cartilages from OA patients by immunofluorescence, qRT-PCR, mitochondrial membrane potential and immunohistochemistry. We also compared Ferroptosis-related alterations in cartilage of mild, moderate, and severe OA (according to the modified Mankin score). In addition, we compared the effect of Fer-1 on ferroptosis and the protection of chondrocytes by detecting markers of both ferroptosis and OA by immunofluorescence, CCK8 and qRT-PCR. Ferroptosis-related alterations (GPX4 downregulation, ACSL4 upregulation, MDA, LPO accumulation, Mitochondrial membrane potential decreased) in the damaged area cartilage were more severe than those in the intact area and increased with the progression of OA. Compared with mild OA group, the activity of chondrocytes treated with Fer-1 (a ferroptosis inhibitor) was increased, mitochondrial function was improved, and ferroptosis was reduced (GPX4 upregulation, SLC7A11 upregulation, ACSL4 downregulation,), and promoted the expression of COL2A1 and inhibited the expression of MMP13. However, these changes were not observed in moderate and severe OA chondrocytes. Ferroptosis occurs in a region-specific manner and is exacerbated with the progression of human OA cartilage degeneration. Inhibition of ferroptosis might had a therapeutic effect on chondrocytes with mild OA but had no significant therapeutic effect on chondrocytes with moderate to severe OA.

Pioglitazone-Loaded Cartilage-Targeted Nanomicelles (Pio@C-HA-DOs) for Osteoarthritis Treatment.

Background: Hyaluronic acid (HA) is a popular biological material for osteoarthritis (OA) treatment. Pioglitazone, a PPAR-γ agonist, has been found to inhibit OA, but its use is limited because achieving the desired local drug concentration after administration is challenging. Purpose: Herein, we constructed HA-based cartilage-targeted nanomicelles (C-HA-DOs) to deliver pioglitazone in a sustained manner and evaluated their efficacy in vitro and in vivo. Methods: C-HA-DOs were chemically synthesized with HA and the WYRGRL peptide and dodecylamine. The products were characterized by FT-IR, 1H NMR, zeta potential and TEM. The drug loading rate and cumulative, sustained drug release from Pio@C-HA-DOs were determined, and their biocompatibility and effect on oxidative stress in chondrocytes were evaluated. The uptake of C-HA-DOs by chondrocytes and their effect on OA-related genes were examined in vitro. The nanomicelle distribution in the joint cavity was observed by in vivo small animal fluorescence imaging (IVIS). The therapeutic effects of C-HA-DOs and Pio@C-HA-DOs in OA rats were analysed histologically. Results: The C-HA-DOs had a particle size of 198.4±2.431 nm, a surface charge of -8.290±0.308 mV, and a critical micelle concentration of 25.66 mg/Land were stable in solution. The cumulative drug release from the Pio@C-HA-DOs was approximately 40% at pH 7.4 over 24 hours and approximately 50% at pH 6.4 over 4 hours. Chondrocytes rapidly take up C-HA-DOs, and the uptake efficiency is higher under oxidative stress. In chondrocytes, C-HA-DOs, and Pio@C-HA-DOs inhibited H2O2-induced death, reduced intracellular ROS levels, and restored the mitochondrial membrane potential. The IVIS images confirmed that the micelles target cartilage. Pio@C-HA-DOs reduced the degradation of collagen II and proteoglycans by inhibiting the expression of MMP and ADAMTS, ultimately delaying OA progression in vitro and in vivo. Conclusion: Herein, C-HA-DOs provided targeted drug delivery to articular cartilage and improved the role of pioglitazone in the treatment of OA.

PPARγ activation suppresses chondrocyte ferroptosis through mitophagy in osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a prevalent disease plaguing the elderly. Recently, chondrocyte ferroptosis has been demonstrated to promote the progression of OA. Peroxisome proliferator-activated receptor-γ (PPARγ) is an important factor in maintaining cartilage health. However, the relationship between PPARγ and chondrocyte ferroptosis in OA and its mechanism is completely unclear. METHODS: We established a surgically induced knee OA rat model to investigate PPARγ and chondrocyte ferroptosis in OA. Rat knee specimens were collected for Safranin O/Fast Green staining and immunohistochemical staining after administered orally placebo or pioglitazone (PPARγ agonist) for 4 weeks. We used RSL3 to establish a chondrocyte ferroptosis model cultured in vitro to study the role of PPARγ activation toward ferroptosis, mitochondrial function, and PTEN-induced putative kinase 1 (Pink1)/Parkin-dependent mitophagy. GW9662 (PPARγ antagonist), Mdivi-1 (mitophagy inhibitor), and chloroquine (mitophagy inhibitor) were employed to investigate the mechanism of PPARγ-Pink1/Parkin-dependent mitophagy in the inhibition of ferroptosis. RESULTS: We found that PPARγ activation by pioglitazone attenuated not only OA but also inhibited the expression of the ferroptosis marker acyl-CoA synthetase long-chain family member 4 (ACSL4) at the same time in rats. Furthermore, in vivo and in vitro data indicated that PPARγ activation restored Pink1/Parkin-dependent mitophagy, improved mitochondrial function, inhibited chondrocyte ferroptosis, and delayed the progression of OA. CONCLUSIONS: The present study demonstrated that PPARγ activation attenuates OA by inhibiting chondrocyte ferroptosis, and this chondroprotective effect was achieved by promoting the Pink1/Parkin-dependent mitophagy pathway.

SCP2 mediates the transport of lipid hydroperoxides to mitochondria in chondrocyte ferroptosis.

Sterol carrier protein 2 (SCP2) is highly expressed in human osteoarthritis (OA) cartilage, accompanied by ferroptosis hallmarks, especially the accumulation of lipid hydroperoxides (LPO). However, the role of SCP2 in chondrocyte ferroptosis remains unexplored. Here, we identify that SCP2 transports cytoplasmic LPO to mitochondria in RSL3-induced chondrocyte ferroptosis, resulting in mitochondrial membrane damage and release of reactive oxygen species (ROS). The localization of SCP2 on mitochondria is associated with mitochondrial membrane potential, but independent of microtubules transport or voltage-dependent anion channel. Moreover, SCP2 promotes lysosomal LPO increase and lysosomal membrane damage through elevating ROS. However, SCP2 is not directly involved in the cell membrane rupture caused by RSL3. Inhibition of SCP2 markedly protects mitochondria and reduces LPO levels, attenuating chondrocyte ferroptosis in vitro and alleviating the progression of OA in rats. Our study demonstrates that SCP2 mediates the transport of cytoplasmic LPO to mitochondria and the spread of intracellular LPO, accelerating chondrocyte ferroptosis.

Eugenol suppresses the proliferation and invasion of TNF-α-induced fibroblast-like synoviocytes via regulating NF-κB and COX-2.

Eugenol (4-allyl -2- methoxyphenol), reportedly, a native compound that widely exists in a variety of plants, shows diverse biological activities such as anti-bacteria, anti-inflammation and anti-oxidation. This work was to delve into the effects and molecular mechanisms of eugenol on the phenotypes of fibroblast-like synovial cells from rheumatoid arthritis (RA). Fibroblast-like synovial cells treated with tumor necrosis factor-α (TNF-α) for 24 h received eugenol treatment. In this study, we found eugenol could inhibit TNF-α-induced proliferation, migration, invasion, angiogenesis and inflammatory response of fibroblast-like synovial cells, and promote apoptosis. Eugenol's target genes were significantly associated with vascular endothelial growth factor (VEGF) and NF-kappaB (NF-κB) signaling pathway. Eugenol reversed the promoting effect of TNF-α on the expression of NF-κB signaling pathway-related proteins as well as prostaglandin-endoperoxide synthase 2 (PTGS2, also known as COX-2) protein. In addition, the NF-κB pathway inhibitor Bay11-7082 markedly restrained the viability, migration, aggressiveness, and angiogenic and inflammatory responses of TNF-α-induced fibroblast-like synovial cells and promoted apoptosis. In conclusion, eugenol may represent a novel drug to suppress the progression of RA by inhibiting NF-κB signaling pathway and COX-2 expression in fibroblast-like synovial cells.

Regulatory effects of miR-28 on osteogenic differentiation of human bone marrow mesenchymal stem cells.

We aimed to assess the regulatory effects of miR-28 on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMMSCs). HBMMSCs isolated, cultured and induced (at P3) to undergo osteogenic induction. The expressions of miRNAs were detected by gene microarray, and differentially expressed miRNAs in hBMMSCs compared with induced cells were obtained by significance analysis of microarrays. The microarray findings were confirmed by RT-PCR. TargetScan showed that signal transducer and activator of transcription 1 (STAT1) was the downstream target gene of miR-28. The relationship between miR-28 and STAT1 was validated using dual-luciferase reporter gene assay. HBMMSCs were transfected with miR-28 mimics and STAT1 siRNA, respectively. Samples were collected on day 10 after osteogenic differentiation, and the alkaline phosphatase (AKP) activity, Runt-related transcription factor 2 (RUNX2, a key regulator of osteogenic differentiation) and STAT1 expressions were determined using kits, PCR and Western blotting, respectively. Cell proliferation and migration were detected through CCK-8 and Transwell assays, respectively. During the osteogenic differentiation of hBMMSCs, the expression level of miR-28 increased. MiR-28 specifically bound the 3'-untranslated region (3'UTR) of STAT1 mRNA. It inhibited STAT1 expression in a targeted manner during osteogenic differentiation. Interference with STAT1 partially mimicked the regulatory effects of miR-28 overexpression on the osteogenic differentiation of hBMMSCs. Interference with STAT1 or overexpression of miR-28 did not affect proliferation or migration. MiR-28 has gradually increased expression during the osteogenic differentiation of hBMMSCs, which can directly bind STAT1 3'UTR and inhibit its expression, thereby up-regulating AKP and RUNX2, and promoting osteogenic differentiation.

Influence of two-period cross-over design on the bioequivalence study of gefitinib tablets in beagle dogs.

Generally, two-period cross-over design is used in bioequivalence (BE) study. High intra-subject variability of gefitinib was reported in a clinical BE study, and significant changes in gefitinib exposures were observed among different periods in our previous BE study in dogs. Therefore, commercial gefitinib tablets from the same batch were used in the present study and assigned to two groups: the testing drug (GF1) group and the reference drug (GF2) group. A single-oral-dose, two-period cross-over study with a 7-day washout (approximately 24 half-lives) under fasting condition was conducted in 12 dogs to explore the factors. The results showed that the mean values of AUC(0-t), AUC(0-∞), Cmax and Tmax of these two GF1 and GF2 groups were similar. However, the GF1 and GF2 did not meet the acceptance criteria of bioequivalence with 90% confidence intervals, since the values obtained were 76.22%-117.43% for AUC(0-t) and 87.55%-131.59% for Cmax. ANOVA revealed a significant difference between the two periods (P < 0.05). Interestingly, the mean AUC(0-t) of gefitinib in the period 2 was 2.3-fold greater than that in the period 1, while Cmax in the period 2 was 1.7-fold higher than that in the period 1. However, the volume of distribution was significantly decreased, becoming 0.4-fold lower in the period 2. No statistically significant difference in the half-life and Tmax was observed between the two periods (P < 0.05). The pharmacokinetic alteration might come from the different physiological absorption and/or metabolism between periods. Since a 7-day washout interval was applied, DDI risk from P450s and/or P-gp would not play a significant role in the non-bioequivalence. As regard the variability, the intra-subject variation crossing the periods was much larger than the inter-subject variation within each period. The absorption and/or metabolism function of the gut bacteria might play an important role in the increasing exposure of gefitinib in the second period, especially with the comparison with the analysis from the high-fat-diet treatments in humans. Therefore, further studies might be needed to evaluate whether the assessment of bioequivalence could be facilitated by a much longer washout interval allowing the recovery of gut bacteria.

Inhibition effects of eight anti-coronavirus drugs on glycosides metabolism and glycosidases in human gut microflora.

The effects of eight oral anti-coronavirus drugs (lopinavir, ritonavir, chloroquine, darunavir, ribavirin, arbidol, favipiravir, oseltamivir) on the metabolism of four specific glycosides (polydatin, geniposide, quercitrin, glycyrrhizin) and on the activities of three major glycosidases (ß-glucosidase, α-rhamnosidase, ß-glucuronidase) from gut microflora were explored in vitro and determined by LC-MS/MS. The metabolism of polydatin, geniposide, quercitrin and glycyrrhizin was significantly inhibited by one or several anti-coronavirus drugs of 100 µM around 1 h and 4 h (P<0.05), among which darunavir could strongly reduce the production of genipin (70.6% reduction), quercitin (80.6% reduction) and glycyrrhetinic acid (37.9% reduction), which may cause a high risk of herb-drug interactions (HDI). Additionally, chloroquine reduced the production of genipin and quercitin by more than 75% (P<0.05), whereas arbidol had no significant influence on the metabolism of polydatin, quercitrin and glycyrrhizin (P>0.05) so that its risk may be lower. The inhibition of darunavir on ß-glucosidase was relatively strong (IC50 = 193±23 µM), and the inhibition became weaker on ß-glucuronidase and α-rhamnosidase (IC50>500 µM). The consistency between gut microflora and glycosidase system indicated that the inhibition of darunavir on the activity of ß-glucosidase and ß-glucuronidase may be the main reason for affecting the metabolism of geniposide, glycyrrhizin and polydatin in gut microflora. However, for the inhibition of darunavir and chloroquine on the metabolism of quercetrin, there was no correlation between gut microflora and α-rhamnosidase system. Assessing the risk of HDI mediated by glycosidases in gut microflora may be conducive to the safety and efficacy of combining traditional herbal and Western medicine for the treatment of patients with Covid-19.

Study on specific proteins involved in articular cartilage regeneration activity induced by decalcified bone transplantation.

BACKGROUND: Through a comprehensive analysis of the joint synovial fluid produced in the process of rabbit articular cartilage regeneration, the role and characteristics of knee synovial fluid in the process of decalcified bone transplantation-induced articular cartilage regeneration were explored. METHODS: Twenty New Zealand white rabbits (approximately 2.5 kg in weight) were selected, and bilateral distal femoral bones from two randomly selected rabbits were extracted. After decalcification, the bones were cut into 2 mm × 4 cm long decalcified bone strips. Meanwhile, the other 18 rabbits were randomly divided into three groups: the test group (8 rabbits), the positive control group (6 rabbits), and the blank group (4 rabbits). In the test group, the decalcified bone joint was transplanted into the rabbits at the articular cartilage defect; in the positive control group, the articular cartilage defect of the rabbits were treated and put aside; in the blank group, no rabbits were treated. On the day of transplantation, and on the 4th, 8th, 12th, and 16th weeks after transplantation, the joint synovial fluid of each group was taken for two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, and related database verification and identification, and compared with the positive control group and the blank group. RESULTS: Using 2D-PAGE to separate various proteins in the synovial fluid of the rabbit knee joints, it was found that there were differential protein spots in the test group compared with the blank group and the positive control group. After conducting a comparative search and query in the UniProt database, through comprehensive analysis, it was finally found that three proteins with molecular weights of 23,429.4, 57,431.4, and 26,071.1 that may be related to the regeneration of articular cartilage appeared in the test group. CONCLUSIONS: In the process of inducing the regeneration of articular cartilage using decalcified bone transplantation, knee joint synovial fluid produced specific proteins, which may play an important role in the regeneration of articular cartilage. These findings may offer novel ideas in laying a foundation for the in-depth study of articular cartilage regeneration.

Roles of diclofenac and its metabolites in immune activation associated with acute hepatotoxicity in TgCYP3A4/hPXR-humanized mice.

Diclofenac (DCF) is a widely used nonsteroidal anti-inflammatory drug, but it comes with a high risk of drug-induced liver injury (DILI). Despite the quinone-imine adduct pathways, the immunotoxicity is recently considered as another factor for DILI. However, such immune responses are still elusive. In the present study, investigation of the immune response in the acute hepatotoxicity model of TgCYP3A4/hPXR-humanized mice was conducted by administration of DCF and DCF metabolites, respectively. In a single dose intraperitoneal injection of 80 mg/kg DCF, the pharmacokinetic results showed the major DCF metabolites, including 4'-hydroxy-diclofenac (4'-OH-DCF), 5-hydroxy-diclofenac (5-OH-DCF) and diclofenac glucuronide (DCF-G) were generated after DCF treatment. Not only DCF, but those DCF metabolites could also directly cause different degrees of acute liver injury as significantly increased the serum ALT levels in a short time period in the TgCYP3A4/hPXR-humanized mice. Furthermore, the three DCF metabolites could directly stimulate the significant elevation of serum immune-related factors in varying degrees. Transcriptome analysis revealed the differentially expressed genes in the liver of DCF-G treated mice were mostly involved with the "immune system process" and "cell death" and related to "IL-17 signaling pathway" and "TNF-α signaling pathway", but 5-OH-DCF had little effect on the expressions of those genes. These results indicate that the metabolite DCF-G plays an important role in the activation of the hepatic immune system, which might be involved in the pathogenesis of DCF-induced acute liver injury.

Studies on oral bioavailability and first-pass metabolism of withaferin A in rats using LC-MS/MS and Q-TRAP.

Withaferin A (WA) is one of the major bioactive steroidal lactones with extensive pharmacological activities present in the plant Withania somnifera. The absolute oral bioavailability of WA remains unknown and human-related in vitro data are not available. Therefore, in the present study, the absolute oral bioavailability of WA in male rats and the in vitro screening of absorption factors by Q-trap and LC-MS/MS analysis were conducted to explore possible clinical properties of WA. The developed and validated analytical methods were successfully applied to the pharmacokinetic studies and in vitro measurement of WA. The oral bioavailability was determined to be 32.4 ± 4.8% based on intravenous (5 mg/kg) and oral (10 mg/kg) administrations of WA in male rats. The in vitro results showed that WA could be easily transported across Caco-2 cells and WA did not show as a substrate for P-glycoprotein. Moreover, the stability of WA was similar between male rat and human in simulated gastric fluid (stable), in intestinal microflora solution (slow decrease) and in liver microsomes (rapid depletion, with a half-life of 5.6 min). As such, the first-pass metabolism of WA was further verified by rat intestine-liver in situ perfusion, revealing that WA rapidly decreased and 27.1% remained within 1 h, while the content of three major metabolites (M1, M4, M5) identified by Q-trap increased. This perfusion result is consistent with the oral bioavailability results in vivo. The first-pass metabolism of WA might be the main barrier in achieving good oral bioavailability in male rats and it is predicted to be similar in humans. This study may hold clinical significance.

Studies of pharmacokinetics in beagle dogs and drug-drug interaction potential of a novel selective ZAK inhibitor 3h for hypertrophic cardiomyopathy treatment.

Overexpression of leucine-zipper and sterile-α motif kinase (ZAK) in heart has been closely associated with the development of hypertrophic cardiomyopathy (HCM). N-(3-(1H-pyrazolo[3,4-b]pyridin-5-yl)ethynyl) benzene-sulfonamides, novel highly selective ZAK inhibitors, had exhibited reasonable orally therapeutic effects on HCM in spontaneous hypertensive rat models. In the present study, a rapid and sensitive HPLC-MS/MS method for determining ZAK inhibitor 3h in beagle dog plasma was developed and validated. Meanwhile, the pharmacokinetics in beagle dog and drug-drug interaction potential of 3h had been conducted. The pharmacokinetic results showed that the absolute oral bioavailability for 3h in beagle dogs was determined to be 61.9%, which was significantly higher than that in the previous determination in Spragur-Dawley rats (F = 20%). The Cytochrome P450 enzymes and P-glycoprotein mediated drug-drug interactions by 3h were also investigated using dog and human liver microsomes and Caco-2 cells. The results demonstrated that only CYP2C9 was obviously inhibited (IC50 = 1.66 µM). Besides, 3h could significantly decrease digoxin efflux ratio in Caco-2 experiments in a dose-dependent manner (IC50 = 13.3 µM). Considering 3h strongly suppressed the ZAK kinase activity with an IC50 of 3.3 nM, there are significantly differences between this IC50 value for ZAK inhibition and the present determinations of IC50 values. In general, the clinical drug-drug interaction potential for 3h could be well monitored during the treatment of HCM.

Comparison of in vitro/in vivo blood distribution and pharmacokinetics of artemisinin, artemether and dihydroartemisinin in rats.

Artemisinin and its derivatives have been widely used for treatment of malaria and the therapeutic targets are considered within the red blood cells. In the recent studies on the erythrocytes' uptake of artemisinin-derivatives in vitro, applying the radioisotope-labeled technology, it was trying to predict the in vivo disposition properties, but different distribution results were revealed from a preliminary study in one human. The pharmacokinetic differences among blood cells and plasma still remain unclear. To explore the therapeutic related pharmacokinetics and compare the in vitro-in vivo blood distribution in rats, an improving blood sample preparation and LC-MS/MS detection method was developed and successfully validated. The lower limit of quantification was smaller than the previous studies. In the in vitro blood distribution studies, the content ratios from blood cells to plasma were compared in the concentrations from 20 ng/mL to 1000 ng/mL. Such ratios were determined to be 1.1-1.6 for artemisinin, 0.9-1.2 for artemether, and around 0.7 for dihydroartemisinin. In the oral administration pharmacokinetic studies in rats, the concentration ratios from blood cells to plasma were from high (2.6-3.6) to medium (1.3-2.5), and low (0.5-1.5) for artemisinin, artemether, and dihydroartemisinin respectively in all measuring time points, displaying the similar affinity order toward blood cells in artemisinin > artemether > dihydroartemisinin as the in vitro measurements. The dosages of 10 mg/kg for intravenous administrations of artemisinin and 200 mg/kg for oral administrations of artemisinin or artemether were used for the pharmacokinetic study in rats. The geometric mean exposures (AUC(0-t)) of artemisinin, artemether and dihydroartemisinin in blood cells were determined to be 2.6 folds, 1.7 folds, or 1.2 folds greater than those in plasma, respectively. Referring to the in vitro distribution, the AUC(0-t) ratios from the blood cells measurements to the plasma measurements of these three antimalarial drugs were also in a similar trend as the in vitro distribution measurements. Furthermore, the half-life (t1/2) of artemether in blood cells was even longer than that in plasma, while the clearance of artemisinin, artemether, or dihydroartemisinin in blood cells was slower than that in plasma. Particularly, it was found that the concentrations of artemisinin and artemether were presented in blood cells over longer time period than in plasma above their antimalarial IC50, which might result from both the affinity toward blood cells and the drugs clearance differences between blood cells and plasma. These results were indicated that the exposures and pharmacokinetic properties in the whole blood or the blood cells should be taken into account for the drug candidates with higher distribution affinity toward blood cells especially for the antimalarial drugs.

Development of an optimized cytotoxicity assay system for CYP3A4-mediated metabolic activation via modified piggyBac transposition.

Drug-induced hepatotoxicity is often caused by cytochrome P450 (CYP)-dependent metabolism of drugs into reactive metabolites. Assessment of hepatotoxicity induced by bioactive compounds is hampered by low CYP expression within in vitro cell lines. To overcome this limitation, piggyBac transposition and monoclonal expansion were used to generate a HepG2 cell line with stable and homogenously high expression of CYP3A4, a prominent CYP isoform. Our studies demonstrate the generated line's constant CYP3A4 expression and activity for over 40 cell passages; to date, it has been in subculture for more than a year without addition of Puromycin. This cell line was utilized to evaluate cytotoxicity of two bioactive (troglitazone and acetaminophen) and two non-bioactive (citrate and galactosamine) compounds by MTT assay. Cell viability significantly decreased upon treatment with bioactive drugs. Moreover, cell lines used in the present study were more sensitive to toxic effects of troglitazone than previously reported. Therefore, this HepG2 cell-based assay system may provide a suitable hepatic model for predicting CYP3A4-mediated hepatotoxicity during preclinical drug development.