Biotransformation and disposition characteristics of HSK7653, a novel long-acting dipeptidyl peptidase-4 inhibitor for the treatment of type 2 diabetes.

AIM: To investigate the metabolism and disposition characteristics of HSK7653 in healthy male Chinese participants. METHODS: A single oral dose of 80 µCi (25 mg) [14C]HSK7653 capsules was administered to six healthy participants, and blood, plasma, urine and faeces were collected. Quantitative and qualitative analysis was conducted to investigate the pharmacokinetics, blood-to-plasma ratio, mass balance and metabolism of HSK7653. RESULTS: The drug was well absorbed and reached a maximum concentration at 1.25 h. The drug-related components (HSK7653 and its metabolites) were eliminated slowly, with a half-life (t1/2) of 111 h. Unchanged HSK7653 contributed to more than 97% of the total radioactivity in all plasma samples. The blood-to-plasma ratio (0.573-0.845) indicated that HSK7653 did not tend to distribute into blood cells. At 504 h postdose, up to 95.9% of the dose was excreted, including 79.8% in urine and 16.1% in faeces. Most of the radioactivity (75.5% dose) in excreta was unchanged HSK7653. In addition, nine metabolites were detected in urine and faeces. The most abundant metabolite was M6-2, a dioxidation product of HSK7653, which accounted for 4.73% and 2.63% of the dose in urine and faeces, respectively. The main metabolic pathways of HSK7653 in vivo included oxidation, pyrrole ring opening and sulphonamide hydrolysation. CONCLUSION: HSK7653 was well absorbed, slightly metabolized and slowly excreted in humans. The high plasma exposure and long t1/2 of HSK7653 may contribute to its long-lasting efficacy as a long-acting dipeptidyl peptidase-4 inhibitor.

A Multiple-Target Simultaneous Detection Method for Immunosorbent Assay and Immunospot Assay.

Enzyme-linked immunosorbent assay (ELISA) is one of the most common methods in biological studies, and enzyme-linked immunospot (ELISpot) is a method to measure specific cell numbers by detecting protein secretion at a single-cell level. However, these two current methods can only detect one signal at one time and the sensitivity is not high enough to test low-concentration samples, which are major shortcomings in systematically analyzing the samples of interest. Herein, we demonstrated fluorescence-based oligo-linked immunosorbent assay (FOLISA) and fluorescence-based oligo-linked immunospot (FOLISPOT), which utilized DNA-barcoded antibodies to provide a highly multiplexed method with signal amplification. Signal amplification and simultaneous multiple-target detection were achieved by DNA complementary pairing and modular orthogonal DNA concatemers. By comparing FOLISA with traditional ELISA and comparing FOLISPOT with traditional ELISPOT, we found that the detection sensitivities of FOLISA and FOLISPOT are much higher than those of traditional ELISA and ELISPOT. The detection limit of ELISA is around 3 pg/mL, and the detection limit of FOLISA is below 0.06 pg/mL. FOLISPOT can detect more spots than ELISPOT and can detect targets that are undetectable for ELISPOT. Furthermore, FOLISA and FOLISPOT allowed sequential detection of multiple targets by using a single dye or multiple dyes in one round and sequential detection in multiple rounds. Thus, FOLISA and FOLISPOT enabled simultaneous detection of a large number of targets, significantly improved the detection sensitivity, and overcame the shortcomings of ELISA and ELISPOT. Overall, FOLISA and FOLISPOT presented effective and general platforms for rapid and multiplexed detection of antigens or antibodies with high sensitivity, either in laboratory tests or potentially in clinic tests.

Pilot study of a novel nanobody 68 Ga-NODAGA-SNA006 for instant PET imaging of CD8+ T cells.

PURPOSE: Positron emission tomography (PET) with specific diagnostic probes for quantifying CD8+ T cells has emerged as a powerful technique for monitoring the immune response. However, most CD8+ T cell radiotracers are based on antibodies or antibody fragments, which are slowly cleared from circulation. Herein, we aimed to develop and assess 68 Ga-NODAGA-SNA006 for instant PET (iPET) imaging of CD8+ T cells. METHODS: A novel nanobody without a hexahistidine (His6) tag, SNA006-GSC, was designed, site-specifically conjugated with NODAGA-maleimide and radiolabelled with 68 Ga. The PET imaging profiles of 68 Ga-NODAGA-SNA006 were evaluated in BALB/c MC38-CD8+/CD8- tumour models and cynomolgus monkeys. Three volunteers with lung cancer underwent whole-body PET/CT imaging after 68 Ga-NODAGA-SNA006 administration. The biodistribution, pharmacokinetics and dosimetry of patients were also investigated. In addition, combined with immunohistochemistry (IHC), the quantitative performance of the tracer for monitoring CD8 expression was evaluated in BALB/c MC38-CD8+/CD8- and human subjects. RESULTS: 68 Ga-NODAGA-SNA006 was prepared with RCP > 98% and SA > 100 GBq/µmol. 68 Ga-NODAGA-SNA006 exhibited specific uptake in MC38-CD8+ xenografts tumours, CD8-rich tissues (such as the spleen) in monkeys and CD8+ tumour lesions in patients within 1 h. Fast washout from circulation was observed in three volunteers (t1/2 < 20 min). A preliminary quantitative linear relationship (R2 = 0.9668, p < 0.0001 for xenografts and R2 = 0.7924, p = 0.0013 for lung patients) appeared between 68 Ga-NODAGA-SNA006 uptake and CD8 expression. 68 Ga-NODAGA-SNA006 was well tolerated by all patients. CONCLUSION: 68 Ga-NODAGA-SNA006 PET imaging can instantly quantify CD8 expression with an ideal safety profile and is expected to be important for dynamically tracking CD8+ T cells and monitoring immune responses for individualised cancer immunotherapy. TRIAL REGISTRATION: NCT05126927 (19 November 2021, retrospectively registered).

Metabolite profiles and mass balance of fuzuloparib, a novel poly (ADP-ribose) polymerase inhibitor, in subjects with advanced solid cancers.

AIM: This trial (NCT04013048) investigated the metabolite profiles, mass balance and pharmacokinetics of fuzuloparib, a novel poly (ADP-ribose) polymerase inhibitor, in subjects with advanced solid cancers. METHODS: A single dose of 150 mg [14 C]fuzuloparib was administered to five subjects with advanced solid cancers. Blood, urine and faecal samples were collected, analysed for radioactivity and unchanged fuzuloparib, and profiled for metabolites. The safety of the medicine was assessed during the study. RESULTS: The maximum concentrations (Cmax ) of the total radioactivity (TRA) and unchanged fuzuloparib in plasma were 5.39 µg eq./mL and 4.19 µg/mL, respectively, at approximately 4 hours post dose. The exposure (AUC0-t ) of fuzuloparib accounted for 70.7% of the TRA in plasma, and no single metabolite was observed accounting for more than 10% of the plasma TRA. The recovery of TRA in excreta was 103.3 ± 3.8% in 288 hours, including 59.1 ± 9.9% in urine and 44.2 ± 10.8% in faeces. Sixteen metabolites of fuzuloparib were identified, including mono-oxidation (M1), hydrogenation (M2), di-oxidation (M3), trioxidation (M4), glucuronidation (M5, M7, M8) and de-ethylation (M6) products, and there was no specific binding between these metabolites and blood cells. Aliphatic hydroxylated fuzuloparib (M1-1) was the primary metabolite in the excreta, accounting for more than 40% of the dose for subjects. There were no serious adverse events observed in the study. CONCLUSION: Fuzuloparib was widely metabolized and excreted completely through urine and faeces in subjects with advanced solid cancer. Unchanged fuzuloparib was indicated to be the primary drug-related compound in circulation. [14 C]fuzuloparib was well-tolerated at the study dose.

Development of Physiology Based Pharmacokinetic Model to Predict the Drug Interactions of Voriconazole and Venetoclax.

PURPOSE: Venetoclax (VEN), an anti-tumor drug that is a substrate of cytochrome P450 3A enzyme (CYP3A4), is used to treat leukemia. Voriconazole (VCZ) is an antifungal medication that inhibits CYP3A4. The goal of this study is to predict the effect of VCZ on VEN exposure. METHOD: Two physiological based pharmacokinetics (PBPK) models were developed for VCZ and VEN using the bottom-up and top-down method. VCZ model was also developed to describe the effect of CYP2C19 polymorphism on its pharmacokinetics (PK). The reversible inhibition constant (Ki) of VCZ for CYP3A4 was calibrated using drug-drug interaction (DDI) data of midazolam and VCZ. The clinical verified VCZ and VEN model were used to predict the DDI of VCZ and VEN at clinical dosing scenario. RESULT: VCZ model predicted VCZ exposure in the subjects of different CYP2C19 genotype and DDI related fold changes of sensitive CYP3A substrate with acceptable prediction error. VEN model can capture PK of VEN with acceptable prediction error. The DDI PBPK model predicted that VCZ increased the exposure of VEN by 4.5-9.6 fold. The increase in VEN exposure by VCZ was influenced by subject's CYP2C19 genotype. According to the therapeutic window, VEN dose should be reduced to 100 mg when co-administered with VCZ. CONCLUSION: The PBPK model developed here could support individual dose adjustment of VEN and DDI risk assessment. Predictions using the robust PBPK model confirmed that the 100 mg dose adjustment is still applicable in the presence of VCZ with high inter-individual viability.

Therapeutic drug monitoring status of four common antibiotics: vancomycin, meropenem, linezolid and teicoplanin.

Accurate therapeutic drug monitoring (TDM) of vancomycin, meropenem, linezolid and teicoplanin are conducive to developing optimal therapeutic regimes for patients. However, the measurement status of those drugs in different laboratories has not been reported. In this study, four samples including two frozen plasma samples and two lyophilized plasma samples were measured by over 35 laboratories across China. The inter- and intra-laboratory %CV, biases (%) of laboratories and intra- and inter-measurement-system %CV were calculated and analyzed. The short-term stability and homogeneity of those drugs in samples were studied. The results of frozen and lyophilized samples were also compared to determine whether there were significant differences in their matrix effects on various measurement systems. Results showed most laboratories' intra-laboratory %CVs were less than 9% for all drugs, and the mean inter-laboratory %CVs were 18.4%, 86.4%, 19.1% and 37.1% for vancomycin, meropenem, linezolid and teicoplanin measurements, respectively. For vancomycin, the intra-measurement %CV of commercial measurement systems was found to be smaller than that of other measurement systems. For meropenem, linezolid and teicoplanin, the agreement among laboratories using self-developed methods (Liquid chromatography-mass spectrometry [LC-MS] or high-performance liquid chromatography [HPLC]) was not satisfactory as most intra-measurement system CVs% were over 20%. Drugs in lyophilized samples were found to be more stable than in frozen samples, and no obvious differences in matrix effects were found for those two kinds of processed samples on most measurement systems. In conclusion, this study depicted the measurement status of those drugs in clinical laboratories, and found the lyophilized samples were more suitable EQA material for those drugs.

Tumor-selective blockade of CD47 signaling with a CD47/PD-L1 bispecific antibody for enhanced anti-tumor activity and limited toxicity.

CD47, an immune checkpoint receptor frequently unregulated in various blood and solid tumors, interacts with ligand SIPRα on innate immune cells, and conveys a "do not eat me" signal to inhibit macrophage-mediated tumor phagocytosis. This makes CD47 a valuable target for cancer immunotherapy. However, the therapeutic utility of CD47-SIRPα blockade monoclonal antibodies is largely compromised due to significant red blood cell (RBCs) toxicities and fast target-mediated clearance as a result of extensive expression of CD47 on normal cells. To overcome these limitations and further improve therapeutic efficacy, we designed IBI322, a CD47/PD-L1 bispecific antibody which attenuated CD47 activity in monovalent binding and blocked PD-L1 activity in bivalent binding. IBI322 selectively bound to CD47+PD-L1+ tumor cells, effectively inhibited CD47-SIRPα signal and triggered strong tumor cell phagocytosis in vitro, but only with minimal impact on CD47 single positive cells such as human RBCs. In addition, as a dual blocker of innate and adaptive immune checkpoints, IBI322 effectively accumulated in PD-L1-positive tumors and demonstrated synergistic activity in inducing complete tumor regression in vivo. Furthermore, IBI322 showed only marginal RBCs depletion and was well tolerated in non-human primates (NHP) after repeated weekly injections, suggesting a sufficient therapeutic window in future clinical development of IBI322 for cancer treatment.

Eosinophil-to-monocyte ratio is a potential biomarker in the prediction of functional outcome among patients with acute ischemic stroke.

BACKGROUND: It has been shown that eosinophils are decreased and monocytes are elevated in patients with acute ischemic stroke (AIS), but the impact of eosinophil-to-monocyte ratio (EMR) on clinical outcomes among AIS patients remains unclear. We aimed to determine the relationship between EMR on admission and 3-month poor functional outcome in AIS patients. METHODS: A total of 521 consecutive patients admitted to our hospital within 24 h after onset of AIS were prospectively enrolled and categorized in terms of quartiles of EMR on admission between August 2016 and September 2018. The endpoint was the poor outcome defined as modified Rankin Scale score of 3 to 6 at month 3 after admission. RESULTS: As EMR decreased, the risk of poor outcome increased (p < 0.001). Logistic regression analysis revealed that EMR was independently associated with poor outcome after adjusting potential confounders (odds ratio, 0.09; 95% CI 0.03-0.34; p = 0.0003), which is consistent with the result of EMR (quartile) as a categorical variable (odds ratio, 0.23; 95% CI 0.10-0.52; ptrend < 0.0001). A non-linear relationship was detected between EMR and poor outcome, whose point was 0.28. Subgroup analyses further confirmed these associations. The addition of EMR to conventional risk factors improved the predictive power for poor outcome (net reclassification improvement: 2.61%, p = 0.382; integrated discrimination improvement: 2.41%, p < 0.001). CONCLUSIONS: EMR on admission was independently correlated with poor outcome in AIS patients, suggesting that EMR may be a potential prognostic biomarker for AIS.

Mass balance, pharmacokinetics and pharmacodynamics of intravenous HSK3486, a novel anaesthetic, administered to healthy subjects.

AIMS: This trial (NCT03751956) investigated the mass balance, pharmacokinetics and pharmacodynamics of HSK3486, a novel anaesthetic, in healthy subjects. METHODS: A single dose of 0.4 mg/kg [14 C]HSK3486 was administered to six healthy subjects. Blood, urine and faecal samples were collected, analysed for radioactivity, unchanged HSK3486 and profiled for metabolites. The Modified Observer's Assessment of Alertness/Sedation (MOAA/S) scale and vital signs were closely monitored during the study. RESULTS: The mean recovery of total radioactivity in excreta was 87.3% in 240 h, including 84.6% in urine and 2.65% in faeces. The exposure (AUC0-t ) of total radioactivity was much higher than that of unchanged HSK3486 in plasma, indicating there were circulating metabolites in plasma. The glucuronide conjugate of HSK3486 (M4) was found as the only major circulating metabolite in plasma (79.3%), while unchanged HSK3486 accounted for only 3.97% of the total radiation exposure. M4 also resulted in a longer estimated elimination half-life (t1/2 ) of total radioactivity than that of unchanged HSK3486 in plasma. Fortunately, the metabolite was detected to be not specific to red blood cells and was suggested to be nonhypnotic and nontoxic. All the subjects were quickly anaesthetized (2 min) after drug administration and woke up smoothly after a short time (5.5-14.1 min) with few residual effects. The only adverse event in the study was mild (grade 1) and consisted of hypotension. CONCLUSION: HSK3486 is a promising anaesthetic candidate with rapid onset of action and clear absorption, distribution, metabolism, excretion (ADME) processes. HSK3486 showed favourable pharmacokinetic characteristics, pharmacodynamic responses and safety at the study dose.

The gut microbes, Enterococcus and Escherichia-Shigella, affect the responses of heart valve replacement patients to the anticoagulant warfarin.

Numerous algorithms based on patient genetic variants have been established with the aim of reducing the risk of GI bleeding and thromboembolism during warfarin administration. However, approximately 35 % of individual warfarin sensitivity still remains unexplained. Few of warfarin algorithms take into account gut microbiota profiles. The identification of certain microbiome will provide new targets and new strategies for reducing the risk of bleeding and thromboembolism during warfarin administration. In this study, we collected plasma and stool samples from 200 inpatients undergoing heart valve replacement (HVR), which were classified as low responder (LR), high responder (HR) and normal responder (NR). Significant differences were observed in the diversity and relative abundance of the gut microbiota among the three groups. The genus Escherichia-Shigella was enriched significantly in the LRs (P = 3.189e-11), while the genus Enterococcus was enriched significantly in the HRs (P = 1.249e-11). The amount of VK2 synthesized by gut microbiota in LR group was much higher than that in HR group (P = 0.005). Whole genome shotgun sequencing indicated that the relative abundance of enzymes and modules associated with VK biosynthesis was significantly higher in LRs than in HRs or NRs. The 12 microbial markers were identified through tenfold cross-validation with a random forest model. The results provided a new microbial diagnostic model that can be used to inform modulation of warfarin dosage on the basis of patient intestinal flora composition.

Ultrasensitive and quantitative detection of EGFR mutations in plasma samples from patients with non-small-cell lung cancer using a dual PNA clamping-mediated LNA-PNA PCR clamp.

Circulating tumour DNA (ctDNA) is a potential proxy for tumour tissues. However, the analysis of mutations and mutational abundance using ctDNA remains challenging because ctDNA is present at low levels. In addition, the concordance between plasma and tumour tissues requires further investigation by high-sensitivity techniques. Here, we established an ultrasensitive, quantitative method for detecting rare mutations in plasma samples based on a dual PNA clamping-mediated LNA-PNA PCR clamp (LNA-dPNA PCR clamp). The novelty of our method is the coupling of PNA clamping with one-tube nested PCR to dually block wild-type DNA amplification and efficiently amplify mutant DNA. Then, four hotspot EGFR mutations (EGFR L858R, EGFR Exon 19 deletion, EGFR T790M, and EGFR C797S) were detected by our proposed method. Finally, we evaluated the concordance between plasma and tumour tissues by simultaneously detecting EGFR L858R by ddPCR and LNA-dPNA PCR clamp in 132 tissues and matched plasma samples from patients with NSCLC. For the four EGFR mutations, the amplification sensitivity of the LNA-dPNA PCR clamp was 100 copies per reaction, and the linearity was from 100 to 106-107 copies per reaction. The limit of detection for the LNA-dPNA PCR clamp was 0.01%-0.1%. The LNA-dPNA PCR clamp was similarly consistent with ddPCR in quantifying mutational abundance (R2 = 0.9568) and exhibited similar limit of detection (0.01%-0.1% vs. 0.01%), sensitivity (19.6 vs. 21.7), specificity (94.2 vs. 91.9), and concordance (68.2 vs. 67.4) to those of ddPCR for ctDNA detection. In conclusion, the LNA-dPNA PCR clamp will provide a labour-saving, cost-saving, ultrasensitive tool for detecting and quantifying plasma EGFR mutations.

Predicting the dopamine D2 receptor occupancy of ropinirole in rats using positron emission tomography and pharmacokinetic-pharmacodynamic modeling.

1. The purpose of this study was to measure dopamine D2/3 receptor occupancy (RO) as a marker of the clinical efficacy of ropinirole in rats via positron emission tomography (PET) using 18F-fallypride as the radiotracer and to explore the relationship between dopamine RO and the plasma concentration of ropinirole via pharmacokinetic-pharmacodynamic modeling. 2. Plasma was collected from 16 rats treated with one of four doses of ropinirole. For the time-dependent study, the data of 16 rats in the 15 mg/kg dose group at four time points were averaged, and another 24 rats were divided into three dose groups (5 mg/kg, 30 mg/kg and 60 mg/kg) for the dose-dependent study; the animals were assessed via 18F-fallypride PET scans. The correlation between dopamine RO and the ropinirole plasma concentration was investigated, and a pharmacokinetic-pharmacodynamic (PK-PD) model was established with WinNonlin 6.3 software. Both the plasma concentration and the binding potential changed in a time- and dose-dependent manner, and the plasma concentration that induces 50% RO (EC50) as calculated by the PK-PD model was 1391 ng/mL. 3. 18F-fallypride appeared to be a suitable radiotracer for ropinirole imaging, and its binding to the dopamine D2 receptor has time- and concentration-dependent characteristics. A theory-based PK-PD model was developed to describe the relationship between the plasma ropinirole concentration and RO, providing a methodological foundation for noninvasive and in vivo clinical evaluations of ropinirole treatment.

First-line anti-tuberculosis drugs induce hepatotoxicity: A novel mechanism based on a urinary metabolomics platform.

Tuberculosis (TB) has become a global public health and social threat. As clinical first-line drugs, rifampicin and isoniazid used in combination with pyrazinamide and ethambutol (the HRZE regimen) usually induce hepatotoxicity. However, the mechanisms underlying this phenomenon remain unclear, and studying the metabolic impact of co-treating TB patients with the HRZE regimen can provide new hepatotoxicity evidence. In this study, urine metabolites from TB patients were profiled using a high-resolution ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) platform. The tricarboxylic acid circulation, arginine and proline metabolism and purine metabolic pathways were found to be affected by anti-TB drugs. The levels of pyroglutamate, isocitrate, citrate, and xanthine were significantly decreased after the administration of HRZE. The above mentioned pathways were also different between drug-induced liver injury (DILI) and non-DILI patients. Urate and cis-4-octenedioic acid levels in the DILI group were significantly increased compared to those in the non-DILI group, while the cis-aconitate and hypoxanthine levels were significantly decreased. These results highlight that superoxide generation can aggravate the hepatotoxic effects of the HRZE regimen. In addition, our metabolomic approach had the ability to predict hepatotoxicity for clinical applications.

GC-MS based metabolomics of CSF and blood serum: Metabolic phenotype for a rat model of cefoperazone-induced disulfiram-like reaction.

Cefoperazone is most popularly used in the treatment of complicated infections clinically. Concomitant ingestion of ethnaol and cefoperazone may cause a disulfiram-like reaction. However, very little is known about the possible interactions between cefoperazone treatment and an alcohol with regard to the induction of disulfiram-like reaction. Study of the metabolic impact of cotreatment with cefoperazone and alcohol on animals can facilitate the identification of markers relevant to disulfiram-like reaction. In this study, the serum and cerebrospinal fluid (CSF) metabolites from Sprague-Dawley rats were profiled using gas chromatography mass spectrometry. Serum levels of valine, leucine, glycine, palmitelaidic acid, and 2-hydroxyisobutyrate in combination application group were significantly higher than those in the control; while alanine and pyruvate deceased in cotreatment group. Most TCA intermediates, glutamate and aspartate had lower CSF level in combination application group, except citrate. In addition, most carbohydrates, ethylmalonate and N-acetylaspartate had higher level compared with control group. These results highlight concomitant ingestion of alcohol and cefoperazone generated disulfiram-like reaction by way of disrupting normal metabolic pathway. Cefoperazone magnifes ethanol-induced impairment of TCA cycle and aspartate metabolism, thereby affects energy metabolism and neural transmission.

Theory-based pharmacokinetics and pharmacodynamics of S- and R-warfarin and effects on international normalized ratio: influence of body size, composition and genotype in cardiac surgery patients.

AIMS: The aims of this study are to apply a theory-based mechanistic model to describe the pharmacokinetics (PK) and pharmacodynamics (PD) of S- and R-warfarin. METHODS: Clinical data were obtained from 264 patients. Total concentrations for S- and R-warfarin were measured by ultra-high performance liquid tandem mass spectrometry. Genotypes were measured using pyrosequencing. A sequential population PK parameter with data method was used to describe the international normalized ratio (INR) time course. Data were analyzed with NONMEM. Model evaluation was based on parameter plausibility and prediction-corrected visual predictive checks. RESULTS: Warfarin PK was described using a one-compartment model. CYP2C9 *1/*3 genotype had reduced clearance for S-warfarin, but increased clearance for R-warfarin. The in vitro parameters for the relationship between prothrombin complex activity (PCA) and INR were markedly different (A = 0.560, B = 0.386) from the theory-based values (A = 1, B = 0). There was a small difference between healthy subjects and patients. A sigmoid Emax PD model inhibiting PCA synthesis as a function of S-warfarin concentration predicted INR. Small R-warfarin effects was described by competitive antagonism of S-warfarin inhibition. Patients with VKORC1 AA and CYP4F2 CC or CT genotypes had lower C50 for S-warfarin. CONCLUSION: A theory-based PKPD model describes warfarin concentrations and clinical response. Expected PK and PD genotype effects were confirmed. The role of predicted fat free mass with theory-based allometric scaling of PK parameters was identified. R-warfarin had a minor effect compared with S-warfarin on PCA synthesis. INR is predictable from 1/PCA in vivo.

Simultaneous determination of ambroxol and salbutamol in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study.

A rapid, selective and sensitive liquid chromatography-tandem mass spectrometry assay method was developed for simultaneous determination of ambroxol and salbutamol in human plasma using citalopram hydrobromide as internal standard (IS). The sample was alkalinized with ammonia water (33:67, v/v) and extracted by single liquid-liquid extraction with ethyl acetate. Separation was achieved on Waters Acquity UPLC BEH C18 column using a gradient program at a flow rate of 0.2 mL/min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the ion transitions m/z 378.9 → 263.6 (ambroxol), m/z 240.2 → 147.7 (salbutamol) and m/z 325.0 → 261.7 (IS). The total analytical run time was relatively short (3 min). Calibration curves were linear in the concentration range of 0.5-100.0 ng/mL for ambroxol and 0.2-20.0 ng/mL for salbutamol, with intra- and inter-run precision (relative standard deviation) <15% and accuracy (relative error) ranging from 97.7 to 112.1% for ambroxol and from 94.5 to 104.1% for salbutamol. The method was successfully applied in a clinical pharmacokinetic study of the compound ambroxol and salbutamol tablets.

Pharmacokinetics, metabolism, excretion and safety of iruplinalkib (WX-0593), a novel ALK inhibitor, in healthy subjects: a phase I human radiolabeled mass balance study.

BACKGROUND: Iruplinalkib is a novel anaplastic lymphoma kinase (ALK) inhibitor for the treatment of ALK-positive crizotinib-resistant NSCLC. RESEARCH DESIGN AND METHODS: A single oral dose of 120 mg/3.7 MBq [14C]iruplinalkib was administered to healthy subjects. Blood, urine and fecal samples were collected and analyzed for iruplinalkib and its metabolites. The safety of iruplinalkib was also assessed. RESULTS: Iruplinalkib was absorbed quickly and eliminated slowly from plasma, with a Tmax of 1.5 h and t1/2 of 28.6 h. About 88.85% of iruplinalkib was excreted at 312 h, including 20.23% in urine and 68.63% in feces. Seventeen metabolites of iruplinalkib were identified, and M3b (demethylation), M7 (cysteine conjugation), M11 (oxidative dehydrogenation and cysteine conjugation of M3b) and M12 (oxidative dehydrogenation and cysteine conjugation) were considered the prominent metabolites in humans. Iruplinalkib-related compounds were found to be covalently bound to proteins, accounting for 7.70% in plasma and 17.96% in feces, which suggested chemically reactive metabolites were formed. There were no serious adverse events observed in the study. CONCLUSIONS: Iruplinalkib was widely metabolized and excreted mainly through feces in humans. Unchanged iruplinalkib, cysteine conjugates and covalent protein binding products were the main drug-related compounds in circulation. Iruplinalkib was well tolerated at the study dose. TRIAL REGISTRATION: The trial is registered at ClinicalTrials.gov (Identifier: Anonymized).

Assessment of risk factors for acute graft-versus-host disease post-hematopoietic stem cell transplantation: a retrospective study based on a proportional odds model using a nonlinear mixed-effects model.

Background: Acute graft-versus-host disease (aGVHD) is a major complication following hematopoietic stem cell transplantation (HSCT). Objective: This study aimed to explore the risk factors for the incidence of aGVHD in patients post-HSCT. Design: This was a retrospective study. Methods: A total of 407 patients were enrolled. The patients' data were recorded from the medical records. The exposure of cyclosporine was estimated based on a population pharmacokinetics model. The occurrence of aGVHD was clinically graded and staged in severity from grades I to IV. A proportional odds model that estimated the cumulative probabilities of aGVHD was used to analyze the data using a nonlinear mixed-effects model. Then, the model parameters and plausibility were evaluated by bootstrap and visual predictive checks. Results: The typical probabilities were 18.9% and 17.9% for grade II and grades III-IV, respectively. The incidence of grade II and grade III-IV aGVHD for human leukocyte antigen (HLA) haplo sibling donor patients was higher than that for HLA-matched donor patients. The incidence of grade II and grade III-IV aGVHD decreased with increasing early cyclosporine trough concentration; however, cyclosporine exposure was not associated with the incidence of aGVHD. Conclusion: HLA matching and early cyclosporine trough concentration were important factors for the occurrence of aGVHD.

Exploring the complex relationship between vitamin K, gut microbiota, and warfarin variability in cardiac surgery patients.

BACKGROUND AND OBJECTIVES: Due to the high individual variability of anticoagulant warfarin, this study aimed to investigate the effects of vitamin K concentration and gut microbiota on individual variability of warfarin in 246 cardiac surgery patients. METHODS: The pharmacokinetics and pharmacodynamics (PKPD) model predicted international normalized ratio (INR) and warfarin concentration. Serum and fecal samples were collected to detect warfarin and vitamin K [VK1 and menaquinone-4 (MK4)] concentrations and gut microbiota diversity, respectively. In addition, the patient's medical records were reviewed for demographic characteristics, drug history, and CYP2C9, VKORC1, and CYP4F2 genotypes. RESULTS: The PKPD model predicted ideal values of 62.7% for S-warfarin, 70.4% for R-warfarin, and 76.4% for INR. The normal VK1 level was 1.34±1.12 nmol/ml (95% CI: 0.33-4.08 nmol/ml), and the normal MK4 level was 0.22±0.18 nmol/ml (95% CI: 0.07-0.63 nmol/ml). The MK4 to total vitamin K ratio was 16.5±9.8% (95% CI: 4.3-41.5%). The S-warfarin concentration of producing 50% of maximum anticoagulation and the half-life of prothrombin complex activity tended to increase with vitamin K. Further, Prevotella and Eubacterium of gut microbiota identified as the main bacteria associated with individual variability of warfarin. The results suggest that an increase in vitamin K concentration can decrease anticoagulation, and gut microbiota may influence warfarin anticoagulation through vitamin K2 synthesis. CONCLUSION: This study highlights the importance of considering vitamin K concentration and gut microbiota when prescribing warfarin. The findings may have significant implications for the personalized use of warfarin. Further research is needed to understand better the role of vitamin K and gut microbiota in warfarin anticoagulation.

A fully validated flow cytometry method to quantitatively analyze active rATG in human serum and its application in pharmacokinetic study for therapeutic drug monitoring.

Rabbit anti-thymocyte globulin (rATG) has been widely used to prevent graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The therapeutic window of rATG is narrow, and it may increase the risk of relapse, viral reactivation, delayed immune reconstitution and GvHD when overexposed or underexposed. Therefore, a reliable method for detecting the rATG concentration in human serum by flow cytometry was established and fully validated for therapeutic drug monitoring. In this method, Jurkat T cells were used to capture active rATG in human serum, and PE-labeled donkey anti-rabbit IgG was used as a secondary antibody. The method showed good specificity, selectivity and excellent linearity at concentration of 0.00300-20.0 AU/mL. The intra- and interday precision values were all within 20% at four concentration levels for the analyte. The stock solutions of rATG showed no significant degradation after storage at ambient temperature for 8 h and at - 80 °C for 481 days. No significant degradation of rATG in serum was observed at ambient temperature for 6 h, during six freezethaw cycles and at - 80 °C for at least 373 days. This method was fully validated and successfully applied to monitor active rATG concentration in serum of patients with haploid-identical hematopoietic stem cell transplantation.