Unveiling the microbiota of sauce-flavor Daqu and its relationships with flavors and color during maturation.

This study investigated the microbial community in three-color sauce-flavor Daqu (black, yellow, and white) throughout their maturation processes, together with their physicochemical factors, culturable microbes, flavor components, and fermenting vitalities. Results from high-throughput sequencing revealed distinct microbial diversity, with more pronounced variations in bacterial community than in fungal community. Firmicutes and Ascomycota emerged as the most dominant bacterial and fungal phyla, respectively, during maturation. Genus-level analysis identified Kroppenstedia, Virgibacillus, and Bacillus as dominant bacteria in black Daqu, yellow Daqu, and white Daqu, severally, while Thermoascus was shared as the core dominant fungi for these Daqu. Physicochemical factors, particularly acidity, were found to exert a significant impact on microbial community. Kroppenstedtia was the key bacteria influencing the color formation of these Daqu. Furthermore, correlations between dominant microbes and flavor compounds highlighted their role in Daqu quality. Molds (Aspergillus, Rhizomucor, and Rhizopus), excepting Bacillus, played a crucial role in the formation of pyrazine compounds. Consequently, this study offers innovative insights into the microbial perspectives on color and pyrazine formation, establishing a groundwork for future mechanized Daqu production and quality control of sauce-flavor baijiu.

Pharmacokinetics of Dacarbazine and Unesbulin and CYP1A2-Mediated Drug Interactions in Patients With Leiomyosarcoma.

Unesbulin is being investigated in combination with dacarbazine (DTIC) as a potential therapeutic agent in patients with advanced leiomyosarcoma (LMS). This paper reports the pharmacokinetics (PK) of unesbulin, DTIC, and its unreactive surrogate metabolite 5-aminoimidazole-4-carboxamide (AIC) in 29 patients with advanced LMS. Drug interactions between DTIC (and AIC) and unesbulin were evaluated. DTIC (1000 mg/m2 ) was administered to patients with LMS via 1-hour intravenous (IV) infusion on Day 1 of every 21-day (q21d) cycle. Unesbulin dispersible tablets were administered orally twice weekly (BIW), starting on Day 2 of every cycle, except for Cycle 2 (C2), where unesbulin was dosed either on Day 1 together with DTIC or on Day 2, 1 day after DTIC administration. The PK of DTIC, AIC, and unesbulin in Cycle 1 (C1) and C2 were estimated using noncompartmental analysis. DTIC and AIC were measurable immediately after the start of infusion and reached Cmax immediately or shortly after end of infusion at 1.0 and 1.4 hours (Tmax ), respectively. Coadministration of unesbulin orally at 200 mg or above with DTIC inhibited cytochrome P450 (CYP)1A2-mediated DTIC metabolism, resulting in 66.7% reduction of AIC exposures. Such inhibition could be mitigated when unesbulin was dosed the day following DTIC infusion. Repeated unesbulin dosing demonstrated evidence of clinical CYP1A2 induction and increased AIC Cmax by 69.4% and AUCinf by 57.9%. No meaningful difference in unesbulin PK was observed between C2 and C1. The combination therapy of 1000 mg/m2 IV DTIC q21d and 300 mg unesbulin BIW in a staggered regimen is well tolerated in patients with LMS.

Absorption, metabolism and excretion of 14C-emvododstat following repeat daily oral dose administration in human volunteers using a combination of microtracer radioactivity and high radioactivity doses.

Emvododstat is a potent inhibitor of dihydroorotate dehydrogenase and is now in clinical development for the treatment of COVID-19 and acute myeloid leukemia. Since the metabolism and pharmacokinetics of emvododstat in humans is time­dependent, a repeat dose study design using a combination of microtracer radioactivity and high radioactivity doses was employed to evaluate the metabolism and excretion of emvododstat near steady state. Seven healthy male subjects each received 16 mg/0.3 µCi 14C-emvododstat daily oral doses for 6 days followed by a 16 mg/100 µCi high radioactivity oral dose on Day 7. Following the last 16 mg/0.3 µCi 14C­emvododstat dose on Day 6, total radioactivity in plasma peaked at 6 h post-dose. Following a high radioactivity oral dose (16 mg/100 µCi) of 14C-emvododstat on Day 7, both whole blood and plasma radioactivity peaked at 6 h, rapidly declined from 6 h to 36 h post-dose, and decreased slowly thereafter with measurable radioactivity at 240 h post-dose. The mean cumulative recovery of the administered dose was 6.0% in urine and 19.9% in feces by 240 h post-dose, and the mean extrapolated recovery to infinity was 37.3% in urine and 56.6% in feces. Similar metabolite profiles were observed after repeat daily microtracer radioactivity oral dosing on Day 6 and after a high radioactivity oral dose on Day 7. Emvododstat was the most abundant circulating component, M443 and O-desmethyl emvododstat glucuronide were the major circulating metabolites; M474 was the most abundant metabolite in urine, while O­desmethyl emvododstat was the most abundant metabolite in feces. Significance Statement This study provides a complete set of the absorption, metabolism and excretion data of emvododstat, a potent inhibitor of dihydroorotate dehydrogenase, at close to steady state in healthy human subjects. Resolution of challenges due to slow metabolism and elimination of a lipophilic compound highlighted in this study can be achieved by repeat daily microtracer radioactivity oral dosing followed by a high radioactivity oral dosing at therapeutically relevant doses.

Absorption, distribution, metabolism and excretion of 14C-vatiquinone in rats, dogs, and human subjects.

Vatiquinone is a potent inhibitor of 15-lipoxygenase and is in clinical development for the treatment of mitochondrial diseases and other disorders characterised by high levels of oxidative stress and dysregulation of energy metabolism.In rats, 14C-vatiquinone-derived radioactivity was quickly and widely distributed throughout the body and cleared from most tissues by 24 h post-dose following a single oral dose of 14C-vatiquinone.Following oral administration, 94% of dose was recovered within seven days in rats, approximately 61% of dose was recovered within seven days in dogs and approximately 93% of dose was recovered within nine days in human subjects (IND 119220). Faecal excretion was the major route (>56% dose) in all species; urinary excretion was minimal in rats and dogs (<3% dose) but was higher in humans (∼ 22% dose).Following oral administration, vatiquinone was the dominant circulating component in rats and dogs but was minor in human subjects. There were no plasma metabolites that were more than 10% of total drug related exposures in all species.Following oral administration, vatiquinone was not detectable in urine but was the most prominent component in faeces in rats, dogs, and humans.

A clinical pharmacokinetic drug-drug interaction study between dextromethorphan and emvododstat, a potent anti-SARS-CoV-2 dihydroorotate dehydrogenase inhibitor.

PURPOSE: A therapeutic agent that targets both viral replication and the hyper-reactive immune response would offer a highly desirable treatment for severe acute respiratory syndrome corona virus 2 (SARS-CoV-2, coronavirus disease 2019, COVID-19) management. Emvododstat (PTC299; 4-chlorophenyl 6-chloro-1-[4-methoxyphenyl]-1,3, 4,9-tetrahydro-2H-pyrido[3,4-b]indole-2-carboxylate) was found to be a potent inhibitor of immunomodulatory and inflammation-related processes by inhibition of dihydroorotate dehydrogenase to reduce the severity of SARS-CoV-2 infections This drug interaction study was performed to determine if emvododstat was an inhibitor of CYP2D6. METHODS: Potential drug-drug interactions between emvododstat and a CYP2D6 probe substrate (dextromethorphan) were investigated by measuring plasma dextromethorphan and metabolite (dextrorphan) concentrations before and after emvododstat administration. On day 1, 18 healthy subjects received an oral dose of 30 mg dextromethorphan followed by a 4-day washout period. On day 5, subjects received an oral dose of 250 mg emvododstat with food. Two hours later, 30 mg dextromethorphan was administered. RESULTS: When given with emvododstat, plasma dextromethorphan concentrations increased substantially, while metabolite levels (dextrorphan) remained essentially the same. Maximum plasma dextromethorphan concentration (Cmax) increased from 2006 to 5847 pg/mL. Dextromethorphan exposure (AUC) increased from 18,829 to 157,400 h·pg/mL for AUC0-last and from 21,585 to 362,107 h·pg/mL for AUC0-inf following administration of emvododstat. When dextromethorphan parameters were compared before and after emvododstat, least squares mean ratios (90% confidence interval) were found to be 2.9 (2.2, 3.8), 8.4 (6.1, 11.5), and 14.9 (10.0, 22.1) for Cmax, AUC0-last, and AUC0-inf, respectively. CONCLUSION: Emvododstat appears to be a strong CYP2D6 inhibitor. No drug-related treatment emergent adverse effects (TEAEs) were considered to be severe or serious. TRIAL REGISTRATION: EudraCT 2021-004626-29, 11 May 2021.

A pharmacokinetic drug-drug interaction study between rosuvastatin and emvododstat, a potent anti-SARS-CoV-2 (COVID-19) DHODH (dihydroorotate dehydrogenase) inhibitor.

A therapeutic agent that targets both viral replication and the hyper-reactive immune response would offer a highly desirable treatment for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; COVID-19) management. Emvododstat (PTC299) was found to be a potent inhibitor of immunomodulatory and inflammation-related processes by the inhibition of dihydroorotate dehydrogenase (DHODH) to reduce SARS-CoV-2 replication. DHODH is the rate-limiting enzyme of the de novo pyrimidine nucleotide biosynthesis pathway. This drug interaction study was performed to determine whether emvododstat was an inhibitor of breast cancer resistance protein (BCRP) transporters in humans. Potential drug-drug interactions (DDIs) between emvododstat and a BCRP transporter substrate (rosuvastatin) were investigated by measuring plasma rosuvastatin concentrations before and after emvododstat administration. There was no apparent difference in rosuvastatin plasma exposure. The geometric means of maximum plasma rosuvastatin concentrations (Cmax ) were 4369 (rosuvastatin) and 5141 pg/mL (rosuvastatin + emvododstat) at 4 h postdose. Geometric mean rosuvastatin area under the concentration-time curve (AUC) from time 0 to the last measurable plasma concentration was 45 616 and 48 975 h·pg/mL when administered alone and after 7 days of b.i.d. emvododstat dosing, respectively. Geometric least squares mean ratios for Cmax and AUC were approximately equal to 1. Overall, administration of multiple doses of 100 mg emvododstat b.i.d. for 7 days in combination with a single dose of rosuvastatin was safe and well tolerated. Emvododstat can be safely administered with other BCRP substrate drugs. Hence, pharmacokinetic DDI mediated via BCRP inhibition is not expected when emvododstat and BCRP substrates are coadministered.

Clinical Drug-Drug Interaction Between Vatiquinone, a 15-Lipoxygenase Inhibitor, and Rosuvastatin, a Breast Cancer Resistance Protein Substrate.

Vatiquinone is a small-molecule inhibitor of 15-lipoxygenase in phase 3 development for patients with mitochondrial disease and Friedreich ataxia. The objective of this analysis was to determine the effect of vatiquinone on the pharmacokinetic profile of rosuvastatin, a breast cancer resistance protein substrate. In vitro investigations demonstrated potential inhibition of BCRP by vatiquinone (half maximal inhibitory concentration, 3.8 µM). An open-label, fixed-sequence drug-drug interaction study in healthy volunteers was conducted to determine the clinical relevance of this finding. Subjects received a single dose of 20-mg rosuvastatin followed by a 7-day washout. On days 8 through 14, subjects received 400 mg of vatiquinone 3 times daily. On day 12, subjects concomitantly received a single dose of 20-mg rosuvastatin. The geometric mean ratio for maximum plasma concentration was 77.8%; however, the rosuvastatin disposition phase appeared unaffected. The geometric mean ratios for the area under the plasma concentration-time curve from time 0 to time t and from time 0 to infinity were 103.2% and 99.9%, respectively. Mean rosuvastatin apparent elimination half-life was similar between treatment groups. These results demonstrate that vatiquinone has no clinically relevant effect on the pharmacokinetics of rosuvastatin.

First-in-Human Studies of Pharmacokinetics and Safety of Utreloxastat (PTC857), a Novel 15-Lipooxygenase Inhibitor for the Treatment of Amyotrophic Lateral Sclerosis.

Utreloxastat (PTC857) is a 15-lipoxygenase inhibitor being developed to treat amyotrophic lateral sclerosis. This first-in-human study investigated the safety and pharmacokinetics of utreloxastat in healthy volunteers (N = 82) in a double-blind, placebo-controlled trial. The effects of a single ascending dose (100-1000 mg), multiple ascending doses (150-500 mg), and food (500 mg) on the pharmacokinetics and safety of utreloxastat were evaluated. Following single doses, the time to maximum plasma concentration (Cmax ) was observed ≈4 hours after dosing and the terminal half-life ranged from 20 to 25.3 hours. The Cmax and area under the concentration-time curve (AUC) increased slightly over dose proportionally. Following multiple doses (once daily/twice daily), the apparent clearance reduced and terminal half-life was ≥33 hours. There was no apparent difference of exposure following morning or evening doses. Varying diets increased the Cmax and AUCs of utreloxastat but did not alter time to Cmax . There were no gender-based differences in exposure. Utreloxastat showed no marked safety signal following single doses up to 1000 mg and multiple doses over 14 days of 500 mg once daily or 250 mg twice daily. The results support further development of utreloxastat for the treatment of patients with amyotrophic lateral sclerosis at a 250-mg twice-daily dose administered with food.

Evaluation of vatiquinone drug-drug interaction potential in vitro and in a phase 1 clinical study with tolbutamide, a CYP2C9 substrate, and omeprazole, a CYP2C19 substrate, in healthy subjects.

PURPOSE: In this study, the drug-drug interaction potential of vatiquinone with cytochrome P450 (CYP) substrates was investigated in both in vitro and clinical studies. METHODS: The inhibitory potential of vatiquinone on the activity of CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5 was assessed in vitro. In an open-label, drug-drug interaction study in 18 healthy human subjects, a single oral dose of 500 mg tolbutamide and 40 mg omeprazole was administered on day 1, followed by a washout of 7 days. Multiple oral doses of 400 mg vatiquinone (three times a day [TID]) were administered from day 8 to day 13 with coadministration of a single oral dose of 500 mg tolbutamide and 40 mg omeprazole on day 12. RESULTS: In vitro, vatiquinone inhibited CYP2C9 (IC50 = 3.7 µM) and CYP2C19 (IC50 = 5.4 µM). In the clinical study, coadministration of vatiquinone did not affect the pharmacokinetic (PK) profile of tolbutamide and omeprazole. The 90% confidence intervals (CIs) of geometric least-square mean ratios for maximum plasma concentration (Cmax), areas under the plasma concentration-time curve (AUC0-t), and AUC0-inf of tolbutamide and omeprazole were entirely contained within the 80 to 125% no effect limit, except a minor excursion observed for Cmax of omeprazole (geometric mean ratio [GMR], 94.09; 90% CI, 78.70-112.50). Vatiquinone was generally well tolerated, and no clinically significant findings were reported. CONCLUSION: The in vitro and clinical studies demonstrated vatiquinone has a low potential to affect the pharmacokinetics of concomitantly administered medications that are metabolized by CYP enzymes.

The interaction between autophagy and the epithelial-mesenchymal transition mediated by NICD/ULK1 is involved in the formation of diabetic cataracts.

BACKGROUND: Cataracts are the leading cause of blindness and a common ocular complication of diabetes. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) and altered autophagic activity occur during the development of diabetic cataracts. The disturbed interaction of autophagy with EMT in LECs stimulated by high glucose levels may participate in cataract formation. METHODS: A rat diabetic cataract model induced by streptozotocin (STZ) and human lens epithelial cells (HLE-B3) stimulated with a high glucose concentration were employed in the study. These models were treated with rapamycin (an inhibitor of mammalian target of rapamycin (mTOR)), and N-(N-[3,5-difluorophenacetyl]-1-alanyl)-S-phenylglycine t-butyl ester (DAPT, an inhibitor of γ-secretase) alone or in combination. Lens opacity was observed and photographed under a slit-lamp microscope. Histological changes in paraffin sections of lenses were detected under a light microscope after hematoxylin and eosin staining. Alterations of autophagosomes in LECs were counted and evaluated under a transmission electron microscope. The expression levels of proteins involved in the EMT, autophagy, and the signaling pathways in LECs were measured using Western blotting and immunofluorescence staining. Cell migration was determined by performing transwell and scratch wound assays. Coimmunoprecipitation (Co-IP) was performed to verify protein-protein interactions. Proteins were overexpressed in transfected cells to confirm their roles in the signaling pathways of interest. RESULTS: In LECs, a high glucose concentration induces the EMT by activating Jagged1/Notch1/Notch intracellular domain (NICD)/Snail signaling and inhibits autophagy through the AKT/mTOR/unc 51-like kinase 1 (ULK1) signaling pathway in vivo and in vitro, resulting in diabetic cataracts. Enhanced autophagic activity induced by rapamycin suppressed the EMT by inducing Notch1 degradation by SQSTM1/p62 and microtubule-associated protein light chain 3 (LC3) in LECs, while inhibition of the Notch signaling pathway with DAPT not only prevented the EMT but also activated autophagy by decreasing the levels of NICD, which bound to ULK1, phosphorylated it, and then inhibited the initiation of autophagy. CONCLUSIONS: We describe a new interaction of autophagy and the EMT involving NICD/ULK1 signaling, which mediates crosstalk between these two important events in the formation of diabetic cataracts. Activating autophagy and suppressing the EMT mutually promote each other, revealing a potential target and strategy for the prevention of diabetic cataracts.

A Novel Tongue Squamous Cell Carcinoma Cell Line Escapes from Immune Recognition due to Genetic Alterations in HLA Class I Complex.

Immune checkpoint inhibitors (ICI) have made progress in the field of anticancer treatment, but a certain number of PD-L1 negative OSCC patients still have limited benefits from ICI immuno-therapy because of primary immune evasion due to immunodeficiency. However, in existing human OSCC cell lines, cell models that can be used to study immunodeficiency have not been reported. The objective of this study was to establish a PD-L1 negative OSCC cell line, profile whether the presence of mutated genes is associated with immune deficiency, and explore its influence on the immune recognition of CD8+ T cells in vitro. Here, we established a novel tongue SCC cell line (WU-TSC-1), which escapes from immune recognition by antigen presentation defects. This cell line was from a female patient who lacked typical causative factors. The expression of PD-L1 was negative in the WU-TSC-1 primary tumor, transplanted tumor, cultured cells and lipopolysaccharide stimulation. Whole exome sequencing (WES) revealed that WU-TSC-1 harbored missense mutations, loss of copy number and structural variations in human leukocyte antigen (HLA) class I/II genes. The tumor mutation burden (TMB) score was high at 292.28. In addition, loss of heterozygosity at beta-2-microglobulin (B2M)-a component of all HLA class I complex allotypes-was detected. Compared with the commonly used OSCC cell lines, genetic alterations in HLA class I and B2M impeded the proteins' translation and inhibited the activation and killing effect of CD8+ T cells. In all, the WU-TSC-1 cell line is characterized by genetic variations and functional defects of the HLA class I complex, leading to escape from recognition by CD8+ T cells.

Absorption, distribution, metabolism and excretion of 14C-Emvododstat following a single oral dose in rats and dogs.

Emvododstat is a potent inhibitor of dihydroorotate dehydrogenase and is now in clinical development for the treatment of acute myeloid leukaemia and COVID-19.Following an oral dose administration in Long-Evans rats, 14C-emvododstat-derived radioactivity was widely distributed throughout the body, with the highest distribution in the endocrine, fatty, and secretory tissues and the lowest in central nervous system.Following a single oral dose of 14C-emvododstat in rats, 54.7% of the dose was recovered in faeces while less than 0.4% of dose was recovered in urine 7 days post-dose. Emvododstat was the dominant radioactive component in plasma and faeces.Following a single oral dose of 14C-emvododstat in dogs, 75.2% of the dose was recovered in faeces while 0.5% of dose was recovered in urine 8 days post-dose. Emvododstat was the dominant radioactive component in faeces, while emvododstat and its two metabolites (O-desmethyl emvododstat and emvododstat amide bond hydrolysis product) were the major circulating radioactivity in dog plasma.

In vitro metabolism, pharmacokinetics and drug interaction potentials of emvododstat, a DHODH inhibitor.

Emvododstat was identified as a potent inhibitor of dihydroorotate dehydrogenase and is now in clinical development for the treatment of acute myeloid leukaemia and COVID-19. The objective of this paper is to evaluate the metabolism, pharmacokinetics, and drug interaction potentials of emvododstat.Emvododstat showed high binding to plasma protein with minimal distribution into blood cells in mouse, rat, dog, monkey, and human whole blood.O-Demethylation followed by glucuronidation appeared to be the major metabolic pathway in rat, dog, monkey, and human hepatocytes. CYP2C8, 2C19, 2D6, and 3A4 were involved in O-desmethyl emvododstat metabolite formation. Both emvododstat and O-desmethyl emvododstat inhibited CYP2D6 activity and induced CYP expression to different extents in vitro.Emvododstat and O-desmethyl emvododstat inhibited BCRP transporter activity but did not inhibit bile salt transporters and other efflux or uptake transporters. Neither emvododstat nor O-desmethyl emvododstat was a substrate for common efflux or uptake transporters investigated.Emvododstat is bioavailable in mice, rats, dogs, and monkeys following a single oral dose. The absorption was generally slow with the mean plasma Tmax ranging from 2 to 5 h; plasma exposure of O-desmethyl emvododstat was lower in rodents, but relatively higher in dogs and monkeys.

Correlation between neutrophil-to-lymphocyte ratio and clinical manifestations and complications of retinitis pigmentosa.

PURPOSE: The role of inflammation in retinitis pigmentosa (RP) has been receiving additional attention. However, the association between inflammation and the clinical manifestations and complications of RP is still unclear. This study aimed to evaluate the neutrophil-to-lymphocyte ratio (NLR) of RP complicated with cataract and explore the correlations between the NLR and specific clinical features of RP. METHODS: This retrospective study included 79 RP patients complicated with cataract (125 eyes) and 63 age- and sex-matched patients (63 eyes) with age-related cataract (ARC). Patients' ocular examination results were collected and complete blood count results were used to calculate NLRs. The correlations between the NLR of RP patients and the parameters of ocular examinations were analysed. RESULTS: The NLRs of RP patients with cataracts were significantly higher than those of ARC (1.93 ± 0.83 versus 1.65 ± 0.59, p = 0.029). The NLRs increased with the severity of posterior subcapsular cataract (PSC), zonular deficiency, poor preoperative best-corrected visual acuity (LogMAR>1), and visual field defects. Analysis of receiver operating characteristic curves suggested that NLR > 1.36 could predict higher degrees (PSC area >3%, >P1) of PSC (p = 0.002, 95% CI, 0.672-0.934), and that NLR > 2.12 could predict zonular weakness (p = 0.002, 95% CI, 0.665-0.928) in RP. CONCLUSION: The NLRs in RP patients with cataract are not only higher but also associated with several clinical manifestations of RP. The NLR can be a predictive biomarker of higher degrees of PSC (>P1) and zonular weakness in RP before cataract surgery. These results suggest that systemic inflammation may play a role in the pathogenesis of RP.

Ataluren metabolism: Ataluren-O-1ß-acyl glucuronide is a stable circulating metabolite in mouse, rat, dog and human.

Ataluren is an aromatic acid derivative with a 1,2,4-oxodiazole moiety. Ataluren-O-1ß-acyl glucuronide is a prominent circulatory metabolite in mice, rats, dogs, and humans following oral administration of ataluren. The objective of this paper was to evaluate the stability in vitro and in vivo of ataluren-O-1ß-acyl glucuronide metabolite. Ultrahigh performance liquid chromatography-mass spectrometry methods were developed to separate and monitor ataluren-O-1ß-acyl glucuronide and its possible migration isomers. In vitro stability was assessed in phosphate buffered saline as well as in control rat and human plasma. The disappearance of ataluren-O-1ß-acyl glucuronide and the formation of migration isomers were monitored by the ultrahigh performance liquid chromatography-mass spectrometry methods. In vitro, ataluren-O-1ß-acyl glucuronide underwent isomerization with an estimated half-life of approximately 1 h. However, ataluren-O-1ß-acyl glucuronide was stable and was the only detectable acyl glucuronide following oral administration of ataluren in mice, rats, dogs, and humans using the same analytical methods. Ataluren acyl glucuronide in mouse, rat, dog, and human plasma could be hydrolyzed by ß-glucuronidase, further confirming the structure of O-1ß-acyl glucuronide. These results demonstrated that ataluren-O-1ß-acyl glucuronide did not undergo migration in vivo. No clinical safety concern related to ataluren-O-1ß-acyl glucuronide migration has been detected.

In vitro cytochrome P450- and transporter-mediated drug interaction potential of 6ß-hydroxy-21-desacetyl deflazacort-A major human metabolite of deflazacort.

6ß-Hydroxy-21-desacetyl deflazacort (6ß-OH-21-desDFZ) is a major circulating but not biologically active metabolite of deflazacort (DFZ). In vitro studies were performed to evaluate cytochrome P450 (CYP)- and transporter-mediated drug interaction potentials of 6ß-OH-21-desDFZ. Up to 50 µM, the highest soluble concentration in the test system, 6ß-OH-21-desDFZ weakly inhibited (IC50  > 50 µM) the enzyme activity of CYPs 1A2, 2B6, 2C8, 2C9, and 2D6, while moderately inhibiting CYP2C19 and CYP3A4 with IC50 values of approximately 50 and 35 µM, respectively. The inhibition was neither time-dependent nor metabolism-based. Incubation of up to 50 µM 6ß-OH-21-desDFZ with plated cryopreserved human hepatocytes for 48 h resulted in no meaningful concentration-dependent induction of either mRNA levels or enzyme activity of CYP1A2, CYP2B6, or CYP3A4. In transporter inhibition assays, 6ß-OH-21-desDFZ, up to 50 µM, did not show interaction with human OAT1, OAT3, and OCT2 transporters. It weakly inhibited (IC50  > 50 µM) human MATE1, MATE2-K, and OCT1 transporter activity, and moderately inhibited human MDR1, OATP1B1, and OATP1B3 transporter activity with IC50 values of 19.81 µM, 37.62 µM, and 42.22 µM, respectively. 14 C-6ß-OH-21-desDFZ was biosynthesized using bacterial biotransformation and the subsequent study showed that 6ß-OH-21-desDFZ was not a substrate for human BCRP, MDR1, MATE1, MATE2-K, OAT1, OATP1B1, OATP1B3, and OCT2 transporters, but appeared to be an in vitro substrate for the human OAT3 uptake transporter. At plasma concentrations of 6ß-OH-21-desDFZ seen in the clinic, CYP- and transporter-mediated drug-drug interactions are not expected following administration of a therapeutic dose of DFZ in Duchenne muscular dystrophy (DMD) patients.

LncRNA MALAT1 Regulates miR-144-3p to Facilitate Epithelial-Mesenchymal Transition of Lens Epithelial Cells via the ROS/NRF2/Notch1/Snail Pathway.

Diabetic cataract is a common complication of diabetes. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a key event in the development of diabetic cataracts. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been reported to be highly expressed in different tissues of diabetic patients. This study is aimed at investigating the function and mechanism of MALAT1 in the regulation of EMT in human LECs under high glucose conditions. MALAT1, α-smooth muscle actin (α-SMA), fibronectin (FN), and nuclear factor erythroid-derived 2-like 2 (NRF2) were highly expressed in the LECs of diabetic cataract patients and in the human LECs under high glucose conditions; meanwhile, the decreased expressions of E-cadherin and zonula occludens 1 (ZO-1) were detected. Knockdown of MALAT1 could significantly reduce ROS, prevent EMT, arrest S phase cell cycle, and suppress the expression of total NRF2 and its nucleus translocation in LECs. Furthermore, after NRF2 was knocked down, total NRF2, α-SMA, and FN in cells, and NRF2, Notch intracellular domain (NICD), and Snail were decreased in the nucleus. Using bioinformatics methods, we predicted that MALAT1 and NRF2 shared the same microRNA-144-3p (miR-144-3p) combining site. Luciferase reporter coupled with qRT-PCR assays revealed that miR-144-3p was a target of MALAT1, which was confirmed to downregulate miR-144-3p in the LECs. In addition, after transfection of miR-144-3p mimics or inhibitor, western blot assay demonstrated that miR-144-3p negatively regulated the expression of total NRF2, α-SMA, and FN in cells, and NRF2, NICD, and Snail in the nucleus without affecting Kelch-like ECH-associated protein 1 (KEAP1). Finally, we confirmed that transfection of shMALAT1 inhibited NRF2 expression, and its mediated EMT could be rescued by miR-144-3p inhibitor; transfection of pcDNA3.1-MALAT1 promoted NRF2 expression, and its mediated EMT could be reversed by miR-144-3p inhibitor. In summary, we demonstrate that MALAT1 regulates miR-144-3p to facilitate EMT of LECs via the ROS/NRF2/Notch1/Snail pathway.

Metabolite V, an epoxide species is a minor circulating metabolite in humans following a single oral dose of deflazacort.

Deflazacort (Emflaza) was approved in the United States in 2017 for the treatment of the Duchenne muscular dystrophy in patients aged 2 years and older. Several deflazacort metabolites were isolated and identified from rats, dogs, monkeys, and humans. Among them, 1ß,2ß-epoxy-3ß-hydroxy-21-desacetyl deflazacort, referred to as Metabolite V, was reported to be one of the major circulating metabolites in humans. However, its quantitative distribution in plasma was not fully characterized. The objective of this study was to determine deflazacort plasma pharmacokinetics, metabolite profiles and their quantitative exposures in humans following a single oral dose. Six healthy male subjects were each administered a single oral dose of 60 mg [14 C]-deflazacort. Plasma and urine were collected and deflazacort metabolites in plasma were quantified by high performance liquid chromatography radio-profiling followed by liquid chromatography-mass spectrometry characterization. Metabolite V was isolated from urine and its structure was further confirmed by nuclear magnetic resonance analysis. These analyses demonstrated that deflazacort was not detectable in plasma; of the eight circulating deflazacort metabolites identified or characterized, the pharmacologically active metabolite 21-desacetyl deflazacort and inactive metabolite 6ß-hydroxy-21-desacetyl deflazacort accounted for 25.0% and 32.9% of the 0-24 hours plasma total radioactivity, respectively, while Metabolite V, an epoxide species, was a minor circulating metabolite, representing only about 4.7% of the total plasma radioactivity.

A novel mutation of LIM2 causes autosomal dominant membranous cataract in a Chinese family.

AIM: To identify mutations in the genes of a four-generation Chinese family with congenital membranous cataracts and investigate the morphologic changes and possible functional damage underlying the role of the mutant gene. METHODS: Whole exome analysis of thirteen members of a four-generation pedigree affected with congenital membranous cataracts was performed; co-segregation analysis of identified variants was validated by Sanger sequencing. All members underwent detailed physical and complete eye examinations. The physical changes caused by the mutation were analyzed in silico through homology modeling. The lens fiber block from a patient was observed under a scanning electron microscope (SEM). Cell membrane proteins and cytoplasmic proteins from the human lenses donated by one patient with cataract in this family and from the dislocated lens resulted from the penetrating ocular trauma of a patient unrelated with this family were extracted, and the expression and localization of MP20 and Cx46 were detected by Western blot (WB) assay in these proteins. RESULTS: A novel LIM2 heterozygous mutation (c.388C>T, p.R130C) was identified with congenital membranous cataracts inherited by an autosomal dominant (AD) pattern. Nystagmus and amblyopia were observed in all patients of this family, and exotropia and long axial length were observed in most patients. A/B ultrasound scan and ultrasound biomicroscopy revealed obvious thin crystalline lenses from 1.7 to 2.7 mm in central thickness in all cataract eyes. The bioinformatic analysis showed that the mutation was deleterious to the physiological function of LIM2-encoded MP20. Furthermore, by SEM, ultrastructure of the cataract nucleus showed that lens fiber cells (LFCs) remained morphologic characteristics of immature fiber cells, including flap cell surface with straight edges and lacking normal ball-and-socket joint boundaries, which implied that the differentiation of LFCs might be inhibited. Accumulation of MP20 and Cx46 in the cytoplasm was observed in the cytoplasm of the LFCs in human cataract lens. CONCLUSION: We identify a novel heterozygous LIM2 (c.388C>T, p.R130C) mutation inherited by an AD pattern. This LIM2 mutation causes the abnormal sub-localization of MP20 and Cx46 in LFCs resulting in membranous cataracts.

LC-MS/MS quantification of ataluren and ataluren acyl glucuronide in human plasma/urine: application in clinical studies.

Background: This paper describes for the first-time analytical procedures established to resolve the challenges associated with simultaneous and direct quantification of ataluren and ataluren-O-1ß-acyl glucuronide (AAG) by LC-MS/MS in human plasma and urine matrices. Methodology/results: The plasma quantification method was validated for calibration range of 12.5-12500 ng/ml for ataluren and 6.25-2500 ng/ml for AAG. The urine quantification method was validated for calibration range of 0.01-10 and 1-1000 µg/ml for ataluren and AAG, respectively. Plasma and urine samples were stabilized upon collection and through storage to prevent hydrolysis and acyl migration of AAG. Conclusion: Methods described in this paper enabled successful completion of ataluren clinical pharmacology studies for simultaneous pharmacokinetic assessment of ataluren and AAG.