A novel automated multi-cycle magnetic solid-phase extraction coupled to LC-MS/MS to study the disorders of six functional B vitamins in patients with gastroenterology and hyperhomocysteinemia.

B vitamins are essential for human life and their disorders can cause a variety of diseases. Solid-phase extraction (SPE) coupled to LC-MS/MS is a preferred technique for determining multiple B vitamins, however, their complexity in real biological matrices makes it hard to achieve satisfactory recovery and accuracy when simultaneous detection. In this study, a novel automated multi-cycle magnetic SPE (MSPE) coupled to the LC-MS/MS method was established using a mixed-mode anion exchange magnetic adsorbent for the simultaneous extraction of six functional B vitamins, including methylmalonic acid, riboflavin, pantothenic acid, 4-pyridoxic acid, folic acid, and 5-methyltetrahydrofolate. After three consecutive MSPE cycles, the recoveries of all analytes were between 51.5% and 89.6%. The method exhibited excellent sensitivity and linearity, with a dynamic range of 200-fold (R > 0.99 for all analytes), exceptional accuracy (ranging between 95.4% and 105.6%) and precision (with RSDs ≤ 6.2%) without significant matrix effects or interferences. Compared to manual SPE method, the automated multi-cycle MSPE method has better feasibility and greater vitamin coverage. It shows a high correlation with the manual method for the detection of 5-methyltetrahydrofolate and folate (R > 0.99). A study of patients from the gastroenterology department showed that those undergoing surgery and those with malignancies may be at risk of folate deficiency. In addition, patients with hyperhomocystinemia had higher levels of methylmalonic acid and lower levels of 5-methyltetrahydrofolate, which correlated with homocysteine levels (R = 0.404 and -0.311, respectively) and showed dose-response relationships. This method is highly automated and cost-effective, with minimal systematic error, making it suitable for the analysis of clinical samples.

Effect of visceral fat area on the accuracy of preoperative CT-N staging of colorectal cancer.

OBJECTIVE: To investigate the effect of visceral fat area (VFA) on the accuracy of preoperative CT-N staging of colorectal cancer. METHODS: We retrospectively reviewed the clinical and imaging data of 385 CRC patients who underwent surgical resection for colorectal cancer between January 2018 and July 2021. Preoperative CT-N staging and imaging features were determined independently by two radiologists. Using postoperative pathology as the gold standard, patients were divided into accurately and incorrectly staged groups, and clinical and imaging characteristics were compared between the two groups. VFA and subcutaneous fat area (SFA) at the L3 vertebral level, sex, age, BMI, tumor location, size, and tumor circumference ratio (TCR) were included. Logistic regression analysis was used to evaluate the independent factors influencing the accuracy of preoperative N staging of colorectal cancer. RESULTS: Of the 385 patients enrolled, 259 (67.27%) were in the preoperative N-stage accurate staging group, and 126 (32.73%) were in the incorrectly staged group. Univariate analysis showed that there were significant differences in BMI, tumor location, VFA, SFA, size and TCR between the two groups (P<0.05). Logistic regression analysis showed that VFA (95% CI: 1.277, 3.813; P=0.005) and TCR (95% CI: 1.649, 17.545; P=0.005) were independent factors affecting the accuracy of N staging. The optimal cutoff points for VFA and TCR in predicting incorrect staging were 110 cm2 and 0.675, respectively. CONCLUSIONS: Colorectal cancer patients with lower VFA and higher TCR and preoperative CT-N staging had an increased risk for diagnostic errors.

Effects of Maternal Dietary Enteromorpha prolifera Polysaccharide Iron Supplement on Mineral Elements and Iron Level of Neonatal Piglets.

Iron plays a key role in maternal health during pregnancy and fetal growth. Enteromorpha polysaccharide-iron (EP-Fe) as an organic iron chelate may improve the iron transmission of mother and offspring, ameliorate the poor pregnancy outcomes of sows, and alleviate the growth restriction of piglets caused by iron deficiency. This study aimed to evaluate the effects of maternal dietary supplementation with EP-Fe on reproductive performance and placental iron transmission of sows, as well as growth performance of piglets. Sixty pregnant sows at the 95th day of gestation were randomly divided into control group and EP-Fe group (EP-Fe, 139 mg kg-1). Blood samples of sows and neonatal piglets, colostrum, and tissue samples were collected on the day of delivery. The animal experiment ended at the 21st day of post-delivery. Results showed that maternal dietary EP-Fe increased colostrum iron (P < 0.05) of sows, as well as final litter weight (P < 0.05) and average daily weight of piglets (P < 0.05) during days 1-21 of lactation, as well as iron and manganese content in umbilical cord blood (P < 0.05) and hepatic iron of neonatal piglets (P < 0.01), and decreased fecal iron (P < 0.001), serum calcium (P < 0.05), phosphorus (P < 0.05), and zinc (P < 0.01) in the parturient sow. RT-qPCR results showed that Fpn1 and Zip14 in placenta, as well as TfR1 and Zip14 in duodenum of neonatal piglets, were activated by maternal EP-Fe supplement. These findings suggest that maternal dietary EP-Fe could increase iron storage of neonatal piglets via improving placental iron transport and iron secretion in colostrum, thus enhancing the growth performance of sucking piglets.

Safety and immunogenicity of a modified COVID-19 mRNA vaccine, SW-BIC-213, as a heterologous booster in healthy adults: an open-labeled, two-centered and multi-arm randomised, phase 1 trial.

BACKGROUND: We assessed the safety and immunogenicity of a core-shell structured lipopolyplex (LPP) based COVID-19 mRNA vaccine, SW-BIC-213, as a heterologous booster in healthy adults. METHODS: We conducted an open-labeled, two-centered, and three-arm randomised phase 1 trial. Healthy adults, who had completed a two-dose of inactivated COVID-19 vaccine for more than six months, were enrolled and randomized to receive a booster dose of COVILO (inactivated vaccine) (n = 20) or SW-BIC-213-25µg (n = 20), or SW-BIC-213-45µg (n = 20). The primary study endpoint was adverse events within 30 days post-boosting. The secondary endpoint was the titers of binding antibodies and neutralizing antibodies against the wild-type (WT) of SARS-CoV-2 as well as variants of concern in serum. The exploratory endpoint was the cellular immune responses. This trial was registered with http://www.chictr.org.cn (ChiCTR2200060355). FINDINGS: Between Jun 6 and Jun 22, 2022, 60 participants were enrolled and randomized to receive a booster dose of SW-BIC-213 (25 µg, n = 20, or 45 µg, n = 20) or COVILO (n = 20). The baseline demographic characteristics of the participants at enrollment were similar among the treatment groups. For the primary outcome, injection site pain and fever were more common in the SW-BIC-213 groups (25 µg and 45 µg). Grade 3 fever was reported in 25% (5/20) of participants in the SW-BIC-213-45µg group but was resolved within 48 h after onset. No fatal events or adverse events leading to study discontinuation were observed. For secondary and exploratory outcomes, SW-BIC-213 elicited higher and longer humoral and cellular immune responses than that in the COVILO group. INTERPRETATION: SW-BIC-213, a core-shell structured lipopolyplex (LPP) based mRNA vaccine, was safe, tolerable, and immunogenic as a heterologous booster in healthy Chinese adults. FUNDING: Shanghai Municipal Government, the Science and Technology and Economic Commission of Shanghai Pudong New Area, and mRNA Innovation and Translation Center of Shanghai.

Dietary nucleotides influences intestinal barrier function, immune responses and microbiota in 3-day-old weaned piglets.

Nucleotides (NTs) play a pivotal role in the growth and development of the intestine. This study aimed to evaluate the effects of nucleotides supplementation on the intestinal barrier function, immune responses and microbiota in 3-day-old weaned piglets. Ninety-six piglets weaned at 3-days after birth were randomly assigned to 2 treatments (6 replicates/treatment, 8 piglets/replicate) according to the average body weight. The dietary treatments consisted of the control (CON; fed a basal artificial milk) and nucleotides groups (NT; fed a basal artificial milk with 0.035 % nucleotides, the contents of CMP, UMP, AMP, GMP, and IMP were 1:1:1:1:1, respectively). Diarrhea rates were recorded, and blood and intestinal samples were collected on day 35 of the piglets. The current study showed that NTs supplementation tended to decrease the diarrhea rate of weaned piglets (P < 0.10). NTs increased villus height and the villus height-to-crypt depth (V/C) ratio in the ileum (P < 0.05). Dietary NTs up-regulated protein expression of ZO-1 in ileal mucosa (P < 0.05), and the protein expression of Occludin tended to increase. Furthermore, NTs up-regulated the mRNA expression of Mucin (MUC)2, while the mRNA expression of MUC4 was down-regulated in the ileal mucosa (P < 0.05). Besides, supplementation with NTs increased the ileal mucosa genes expression of IL-21, INF-γ, IL-10, IL-4, IL-6 and TNF-α (P < 0.05). Furthermore, dietary NTs increased the protein expression of NF-κB, IL-6 and TNF-α (P < 0.05), and the proteins expression of Occludin and p-NF-κB tended to be up-regulated in the ileal mucosa (P < 0.10). Furthermore, NTs supplementation increased short chain fatty acid in the colonic (P < 0.05). And NTs supplementation reduced the Firmicutes/Bacteroidota ratio in the colon, at the genus level, NTs enriched the relative abundance of Prevotella, Faecalibacterium and Olsenella (P < 0.05). These data indicate that NTs could increase the villus height, increase the V/C, regulate the expression of tight junction protein and mucin, improve the intestinal barrier of piglets, regulate the secretion of cytokines, improve the biological immunity, increase the abundance of beneficial bacteria, and thus reduce the diarrhea of piglets.

Pharmacokinetics, mass balance, and metabolism of [14C]TPN171, a novel PDE5 inhibitor, in humans for the treatment of pulmonary arterial hypertension.

TPN171 is a novel phosphodiesterase-5 (PDE5) inhibitor used to treat pulmonary arterial hypertension (PAH) and erectile dysfunction (ED), which currently is undergoing phase II clinical trials in China. In this single-center, single-dose, nonrandomized, and open design study, radiolabeled [14C]TPN171 was used to investigate the metabolic mechanism, pharmacokinetic characteristics, and clearance pathways of TPN171 in 6 healthy Chinese male volunteers. Each volunteer was administered a single oral suspension of 10 mg (100 µCi) of [14C]TPN171. We found that TPN171 was absorbed rapidly in humans with a peak time (Tmax) of 0.667 h and a half-life (t1/2) of approximately 9.89 h in plasma. Excretion of radiopharmaceutical-related components was collected 216 h after administration, accounting for 95.21% of the dose (46.61% in urine and 48.60% in feces). TPN171 underwent extensive metabolism in humans. Twenty-two metabolites were detected in human plasma, urine, and feces using a radioactive detector combined with a high-resolution mass spectrometer. According to radiochromatograms, a glucuronide metabolite of O-dealkylated TPN171 exceeded 10% of the total drug-related components in human plasma. However, according to the Food and Drug Administration (FDA) guidelines, no further tests are needed to evaluate the safety of this metabolite because it is a phase II metabolite, but the compound is still worthy of attention. The main metabolic biotransformation of TPN171 was mono-oxidation (hydroxylation and N-oxidation), dehydrogenation, N-dealkylation, O-dealkylation, amide hydrolysis, glucuronidation, and acetylation. Cytochrome P450 3A4 (CYP3A4) mainly catalyzed the formation of metabolites, and CYP2E1 and CYP2D6 were involved in the oxidative metabolism of TPN171 to a lesser extent. According to the incubation data, M1 was mainly metabolized to M1G by UDP-glucuronosyltransferase 1A9 (UGT1A9), followed by UGT1A7 and UGT1A10.

Impact of dietary supplementation with N-carbamoyl-aspartic acid on serum metabolites and intestinal microflora of sows.

BACKGROUND: N-Carbamoyl-aspartic acid (NCA) is a critical precursor for de novo biosynthesis of pyrimidine nucleotides. To investigate the cumulative effects of maternal supplementation with NCA on the productive performance, serum metabolites and intestinal microbiota of sows, 40 pregnant sows (∼day 80) were assigned into two groups: (1) the control (CON) and (2) treatment (NCA, 50 g t-1 NCA). RESULTS: Results showed that piglets from the NCA group had heavier birth weight than those in the CON group (P < 0.05). In addition, maternal supplementation with NCA decreased the backfat loss of sows during lactation (P < 0.05). Furthermore,16S-rRNA sequencing results revealed that maternal NCA supplementation decreased the abundance of Cellulosilyticum, Fournierella, Anaerovibrio, and Oribacterium genera of sows during late pregnancy (P < 0.05). Similarly, on the 14th day of lactation, maternal supplementation with NCA reduced the diversity of fecal microbes of sows as evidenced by significantly lower observed species, Chao1, and Ace indexes, and decreased the abundance of Lachnospire, Faecalibacterium, and Anaerovorax genera, while enriched the abundance of Catenisphaera (P < 0.05). Untargeted metabolomics showed that a total of 48 differentially abundant biomarkers were identified, which were mainly involved in metabolic pathways of arginine/proline metabolism, phenylalanine/tyrosine metabolism, and fatty acid biosynthesis, etc. CONCLUSION: Overall, the results indicated that NCA supplementation regulated intestinal microbial composition of sows and serum differential metabolites related to arginine, proline, phenylalanine, tyrosine, and fatty acids metabolism that may contribute to regulating the backfat loss of sows, and the birth weight and diarrhea rate of piglets. © 2022 Society of Chemical Industry.

Simple and fast determination of tetrodotoxin in human plasma based on hydrophilic-interaction/ion-exchange mixed-mode solid phase extraction combined with liquid chromatography-tandem mass spectroscopy.

In this study, we developed and validated a simple, fast and sensitive LC-MS/MS method for the measurement of tetrodotoxin (TTX) in human plasma. Three HILIC-type solid phase extraction (SPE) carriers (PSA, silica, Siphila i HILIX) with different stationary phase functional groups were compared. The Siphila i HILIX SPE plate containing multi-carboxyl groups was finally selected due to obviously better extraction recovery of TTX (about 80% of recovery from plasma samples) than the other two and no significant matrix effects were observed, which was speculated to have mixed-mode synergistic effects of hydrophilic interaction and ion exchange. 100µL plasma sample was precipitated rapidly with acetonitrile containing 1% trichloroacetic acid, and filtrates were loaded onto Siphila i HILIX 96 well SPE plate. After washed with 95% acetonitrile, TTX was eluted with 200µL of 50% acetonitrile containing 1% trichloroacetic acid. 2µL of elution solution was directly injected into LC-MS/MS and the total run time on a BEH amide column was 4.5 min. The method avoids the evaporation and ultrafiltration processes which is simple and timesaving (<30 min). TTX and internal standard (arginine-15N4) were monitored in positive mode using m/z 320.3→162.2 (quantification transition for TTX), 320.3→284.1 (confirmation transition for TTX) and 179.2→63.0 (transition for IS), respectively. The method was linear in the range of 0.1-20 ng/mL for TTX with the low limit of quantification (S/N > 10) of 0.1 ng/mL; the intra- and inter-assay accuracies were in the range of 98.5%-99.8% (relative standard deviations, RSDs ≤ 5.92%) and 98.8-99.5% (RSDs ≤ 6.23%), respectively. Biases of spiking analysis were ranged from -7.00% to 7.43% for healthy human plasma samples (RSDs ≤ 8.83%) and from -5.00% to 3.93% for hemolytic, high triglyceride, high cholesterol and high bilirubin plasma samples (RSDs ≤ 6.40%), which proved the good anti-interference property of the method. The results showed that the method is sensitive, accurate, specific, reliable, and can be used to monitor the concentration of TTX in plasma to meet the needs of clinical research and poisoning screening.

Maternal pyrimidine nucleoside supplementation regulates fatty acid, amino acid and glucose metabolism of neonatal piglets.

Pyrimidine nucleosides (PN) are abundant in mammalian milk and mainly involved in glycogen deposition and lipid metabolism. To investigate the effects of maternal supplementation with pyrimidine nucleoside on glucose, fatty acids (FAs), and amino acids (AAs) metabolism in neonatal piglets. Forty pregnant sows were randomly assigned into the control (CON) group (fed a basal diet, n = 20) or the PN group (fed a basal diet supplemented with PN at 150 g/t, n = 20). Litter size, born alive and birth litter weight were recorded. The serum and placenta of sows, and jejunum and liver of neonatal piglets were sampled. The results indicated that supplementing sow diets with PN decreased birth mortality and increased the birth weight of piglets (P < 0.05). In addition, neonates from sows supplemented with PN had higher glucose levels in serum and liver compared with the CON group (P < 0.05). Moreover, maternal PN supplementation regulated the ratio of saturated FAs and polyunsaturated FAs, and AAs content in serum and liver of piglets (P < 0.05). Furthermore, an up-regulation of mRNA expression of genes related to glucose and AA transport were observed in the neonatal jejunum from the PN group (P < 0.05). Additionally, hepatic protein expressions of phosphorylated hormone-sensitive lipase (P-HSL), HSL, sterol regulatory element-binding transcription factor 1c (SREBP-1c), and phosphorylated protein kinase B (P-AKT) was higher in the piglets from the PN group than the CON group (P < 0.05). Together, maternal PN supplementation may regulate nutrient metabolism of neonatal piglets by modulating the gene expression of glucose and AA transporters in placenta and jejunum, and the gene and protein expression of key enzymes related to lipid metabolism in liver of neonatal piglets, which may improve the reproductive performance of sows.

A phase I study comparing the pharmacokinetics of the biosimilar (RD12014) with liraglutide (Victoza) in healthy Chinese male subjects.

This study aimed to evaluate the pharmacokinetics (PKs), safety, and immunogenicity of the biosimilar (RD12014) compared to reference liraglutide (Victoza) in healthy Chinese male subjects, so as to provide the basis for the similarity evaluation of the two drugs. Eligible subjects were randomized 1:1 to two sequences (RD12014-Victoza or Victoza-RD12014). Subjects received a single 0.6 mg dose of Victoza or RD12014 by abdominal subcutaneous injection during the first period. After a 7-day washout period, subjects received the alternative drug during the second period. Blood samples were collected at predefined timepoints for PKs and immunogenicity assessment. The primary PK end points were maximum plasma concentration (Cmax ) and area under the concentration-time curve from time zero to the time of the last quantifiable concentration (AUC0-last ). PK bioequivalence was achieved, if the 90% confidence intervals (CIs) of the geometric mean ratio (GMR) of Cmax and AUC0-last were within the range of 80.00-125.00%. Safety was assessed throughout the study. The 90% CIs of the GMR of RD12014 to Victoza for Cmax and AUC0-last were completely within the range of 80.00-125.00%. Thirteen treatment-related adverse events (TRAEs) were reported in 11 subjects (22.4%) in the RD12014 group, compared to 12 TRAEs reported in 12 subjects (24.5%) in the Victoza group. The blood samples of 49 subjects were negative for anti-drug antibody and the neutralizing antibody was not further detected. This study demonstrated PK similarity of RD12014 to Victoza in healthy Chinese male subjects. Safety and immunogenicity profiles were comparable between the two groups.

Effect of different dosages of sodium butyrate and niacin on growth, faecal microbiota and Vitamin B metabolism in weaned piglets.

AIMS: Our study aimed to evaluate the effects of different dosages of sodium butyrate and niacin on the growth performance, faecal Vitamin B and microbiota in weaned piglets. METHODS AND RESULTS: Seventy-two weaned piglets (Duroc × Landrace × Yorkshire, age of 21 days) were randomly assigned to one of six treatments (12 pigs/treatment); the control (CT) group was administered a basal diet. The groups in which concentration ratios of sodium butyrate to niacin were 100: 1, 100: 2, 100: 4, 100: 8 and 100: 16 (BN1, BN2, BN4, BN8 and BN16) were administered a basal diet supplemented with 2000 mg kg-1 sodium butyrate and 20, 40, 80, 160 or 320 mg·kg-1 niacin. After 14-day treatment, the samples were collected. The results showed that feed conversion rate (FCR) was reduced and average daily gain (ADG) was increased in BN2 (p < 0.05). The diarrhoea index of pigs decreased with the low supplement. Additionally, compared with the CT group, other groups significantly increased (p < 0.05) the abundance of Firmicutes (BN4, phylum), Lactobacillaceae (BN8, family), Megasphaera (BN8, genus) and Lactobacillus (BN8, genus). Furthermore, the sodium butyrate and niacin supplementation influence Vitamin B1, Vitamin B2, pyridoxine, niacin, nicotinamide and Vitamin B12 (p < 0.05). Correlation analysis of the association of micro-organisms with Vitamin B indicated that changes of Vitamin B metabolism have a potential correlation with alterations of faecal microbiota in weaned piglets. CONCLUSIONS: The results indicated that adding sodium butyrate and niacin in the diet could promote the performance and improve the faecal microbiota and Vitamin B metabolism in weaned piglets. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study might provide clues to the research of correlations between faecal bacteria and faecal Vitamin B, and these findings will contribute to the direction of future research in weaned piglets.

Safety, tolerability, and pharmacokinetics of VV116, an oral nucleoside analog against SARS-CoV-2, in Chinese healthy subjects.

VV116 (JT001) is an oral drug candidate of nucleoside analog against SARS-CoV-2. The purpose of the three phase I studies was to evaluate the safety, tolerability, and pharmacokinetics of single and multiple ascending oral doses of VV116 in healthy subjects, as well as the effect of food on the pharmacokinetics and safety of VV116. Three studies were launched sequentially: Study 1 (single ascending-dose study, SAD), Study 2 (multiple ascending-dose study, MAD), and Study 3 (food-effect study, FE). A total of 86 healthy subjects were enrolled in the studies. VV116 tablets or placebo were administered per protocol requirements. Blood samples were collected at the scheduled time points for pharmacokinetic analysis. 116-N1, the metabolite of VV116, was detected in plasma and calculated for the PK parameters. In SAD, AUC and Cmax increased in an approximately dose-proportional manner in the dose range of 25-800 mg. T1/2 was within 4.80-6.95 h. In MAD, the accumulation ratio for Cmax and AUC indicated a slight accumulation upon repeated dosing of VV116. In FE, the standard meal had no effect on Cmax and AUC of VV116. No serious adverse event occurred in the studies, and no subject withdrew from the studies due to adverse events. Thus, VV116 exhibited satisfactory safety and tolerability in healthy subjects, which supports the continued investigation of VV116 in patients with COVID-19.

Development and validation of a ligand-binding assay for quantification of the F(ab')2 antivenom of Daboia russelii siamensis in human serum and its application to a phase I clinical study.

Daboia russelii siamensis accounts for most of snakebite mortalities in China, yet, specific treatment against the venom toxins is absent in clinical practice. The F(ab')2 antivenom of Daboia russelii siamensis is manufactured and approved for the clinical trial in China. To satisfy the need for clinical pharmacokinetic research, this study aimed to develop a ligand binding assay (LBA) for the quantification of F(ab')2 antivenom of Daboia russelii siamensis in human serum. A diverse combination of conditions was optimized based on the fitness of the calibration curve and selectivity. The established LBA undergoes thorough method validation according to the guidelines of regulatory authorities. In the calibration range 1.0-64 µg/mL, the correlation coefficient r2 was from 0.9970 to 1.000, indicating good fitness. Accuracy and precision were within ± 20%. Dilution linearity was observed in the ultra-high quality-control (QC) samples (500 µg/mL). In addition, the assay was free from hook effect, the endogenous interferences and exogenous interferences. The QC samples were stable under different handling and storage conditions. The validated assay was successfully applied to a phase I clinical study of the F(ab')2 antivenom of Daboia russelii siamensis in Chinese healthy volunteers. The peak concentrations exhibited dose-proportionality. In conclusion, this study provides a novel and reliable LBA method for the clinical pharmacokinetic research of F(ab')2 antivenom of Daboia russelii siamensis. It will facilitate further clinical trials in treating the snakebite of Daboia russelii siamensis.

Liquid chromatography-tandem mass spectrometric assay for TPN171 in human plasma.

A simple, rapid and accurate method for quantitative analysis of a highly selective phosphodiesterase-5 inhibitor (PDE5), TPN171 by high performance liquid chromatography and tandem mass spectrometry in human plasma was proposed and validated successfully using D3-TPN171 as internal standards (ISTD). An aliquot of 100 µL of plasma was mixed with internal standard and was precipitated with acetonitrile. Gradient elution was performed on a ACQUITY HSS T3 column (50 × 2.1 mm, 1.8 µm) coupled with a ACQUITY column in-line filter at 40℃, by 5 mM ammonium acetate in water containing 0.1 % formic acid and 0.1 % formic acid in acetonitrile as the mobile phase. The total analytical run time was 3.5 min. The analyte was monitored using multiple reaction monitoring (MRM) scan in positive polarity mode. The ion transition was m/z 442.2→113.2 and 445.2→116.2 for TPN171 and D3-TPN171 respectively. The method was validated for specificity, sensitivity, precision, accuracy, and other analytical parameters. The results found were satisfactory over the linear calibration range of 1-500 ng/mL. Within-day precisions ranged from 1.8 to 7.3 %, and between-day precisions from 2.3 to 4.9 %, accuracies were 95.5-99.8 %.The validated method was successfully applied to determine the plasma concentration after oral administration of 10 mg TPN171 in six healthy volunteers.

Direct analysis in real time-mass spectrometry for rapid quantification of five anti-arrhythmic drugs in human serum: application to therapeutic drug monitoring.

Therapeutic drug monitoring (TDM) is necessary for the optimal administration of anti-arrhythmic drugs in the treatment of heart arrhythmia. The present study aimed to develop and validate a direct analysis in real time tandem mass spectrometry (DART-MS/MS) method for the rapid and simultaneous determination of five anti-arrhythmic drugs (metoprolol, diltiazem, amiodarone, propafenone, and verapamil) and one metabolite (5-hydroxy(OH)-propafenone) in human serum. After the addition of isotope-labeled internal standards and protein precipitation with acetonitrile, anti-arrhythmic drugs were ionized by DART in positive mode followed by multiple reaction monitoring (MRM) detection. The use of DART-MS/MS avoided the need for chromatographic separation and allowed rapid and ultrahigh throughput analysis of anti-arrhythmic drugs in a total run time of 30 s per sample. The DART-MS/MS method yielded satisfactory linearity (R2 ≥ 0.9906), accuracy (86.1-109.9%), and precision (≤ 14.3%) with minimal effect of biological matrixes. The method was successfully applied to analyzing 30 clinical TDM samples. The relative error (RE) of the concentrations obtained by DART-MS/MS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was within ± 13%. This work highlights the potential usefulness of DART for the rapid quantitative analysis of anti-arrhythmic drugs in human serum and gives rapid feedback in the clinical TDM practices.

Proteomics analysis reveals a potential new target protein for the lipid-lowering effect of Berberine8998.

Berberine8998 is a newly synthesized berberine derivative with better lipid-lowering activity and improved absorption. The objective of this study was to investigate the effects of berberine8998 on serum cholesterol and lipid levels in vivo and to examine the mechanisms involved. Hamsters on high-fat diet (HFD) were administered berberine or berberine8998 (50 mg·kg-1·d-1, ig) for 3 weeks. Berberine8998 administration significantly lowered the total cholesterol, triglycerides and LDL-C levels in HFD hamsters. Bioinformatics revealed that berberine and berberine8998 shared similar metabolic pathways and fatty acid metabolism was the predominant pathway. Western blot validation results showed that peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) and long-chain fatty acid-CoA ligase 1 (ACSL1), two proteins involved in fatty acid metabolism, were expressed differently in the berberine8998 group than in the untreated group and the berberine treatment group. Biochemistry results showed that berberine8998 significantly lowered the non-esterified fatty acid (NEFA) levels, which may lead to a reduction in TG levels in the berberine8998 treatment group and the differences observed in proteomics analyses. Pharmacokinetic analysis conducted in rats. After administration of berberine or berberine8998 (50 mg/kg, ig), berberine8998 exhibited a remarkably improved absorption with increasing bioavailability by 6.7 times compared with berberine. These findings suggest that berberine8998 lowers cholesterol and lipid levels via different mechanisms than berberine, and its improved absorption makes it a promising therapeutic candidate for the treatment of hypercholesterolemia and obesity.

Phase I dose escalation and pharmacokinetic study on the nanoparticle formulation of polymeric micellar paclitaxel for injection in patients with advanced solid malignancies.

Background Polymeric micellar paclitaxel (PM-paclitaxel) is a novel Cremophor EL-free, nanoparticle-encapsulated paclitaxel formulation administered through intravenous injection. The primary objective of this phase I trial was to determine the first cycle dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of PM-paclitaxel. Secondary objectives included the evaluation of the safety, antitumor activity, and pharmacokinetic (PK) profile of PM-paclitaxel in patients with advanced malignancies. Methods The PM-paclitaxel dose was escalated from 175 mg/m2 (level 1) to 435 mg/m2 (level 5). PM-paclitaxel was intravenously administered to patients for 3 h without premedication on day 1 of a 21-day cycle. Results Eighteen patients with confirmed advanced malignancies received PM-paclitaxel. DLT included grade 4 neutropenia (four patients) and grade 3 numbness (one patient), which occurred in one of the six patients who received 300 mg/m2 (level 3) PM-paclitaxel and all three patients who were treated with 435 mg/m2 PM-paclitaxel. Thus, the MTD of PM-paclitaxel was determined as 390 mg/m2 (level 4). Acute hypersensitive reactions were not observed. Partial response was observed in six of 18 patients (33.3%), three of whom had prior exposure to paclitaxel chemotherapy. The peak concentration and area under the curve values of paclitaxel increased with increasing dosage, indicating that PM-paclitaxel exhibits linear PKs. Conclusions PM-paclitaxel showed high MTD without additional toxicity, and exhibited desirable antitumor activity. The recommended dose of PM paclitaxel for phase II study is 300 mg/m2.

Oral Bioavailability and Mass Balance Studies of a Novel Anti-arrhythmic Agent Sulcardine Sulfate in Sprague-Dawley Rats and Beagle Dogs.

BACKGROUND AND OBJECTIVES: Sulcardine sulfate is a newly developed candidate drug used to control arrhythmias. The aim of this research was to investigate the pharmacokinetics, bioavailability and excretion characteristics of sulcardine in animals. METHODS: Sprague-Dawley rats were orally and intravenously given sulcardine at 20 and 40 mg/kg. Beagle dogs were also orally and intravenously dosed at 10 mg/kg. Both [3H]-labeled sulcardine and unlabeled sulcardine were given to rats. Feces, urine and bile were collected at 0-72 h for mass balance study. The contents of unlabeled sulcardine and radioactivity in samples were determined by a validated LC-MS/MS method and by liquid scintillation counting, separately. RESULTS: Sulcardine was rapidly eliminated in rats after dosing. The oral bioavailability was 34-35 % in rats, while a higher exposure was observed in dogs (bioavailability = 62.7 %). More than 90 % of dosed sulcardine was recovered, and approximately 20-40 % of the dose excreted into urine as the original form, and the remaining was found in feces and bile, most of which (about 40 %) was transformed into metabolites. No difference was observed between sexes. Metabolism may occur to a large extent after oral administration in rats but to a smaller extent in dogs. CONCLUSIONS: Sulcardine was extensively absorbed in both rats and dogs after oral administration. The mass balance data indicated that sulcardine was widely metabolized in rats after oral administration.

Pharmacokinetics, safety, and tolerability of sulcardine sulfate: an open-label, single-dose, randomized study in healthy Chinese subjects.

Sulcardine sulfate (Sul) is a novel anti-arrhythmic agent as a potential treatment for atrial fibrillation and ventricular arrhythmias. This study was conducted to investigate the pharmacokinetic profile, safety, and tolerability of Sul in healthy Chinese subjects. In this open-label, single-dose, randomized study, 10 healthy subjects were assigned to receive Sul doses of 200, 400, and 800 mg under fasting conditions (Cohorts A, B, and C, respectively) or 400 mg under fed conditions (Cohort D). The study incorporated a crossover design, separated by a seven-day washout period. Blood samples were collected before treatment and at successive time intervals up to 48 h after treatment. Sul concentrations in plasma samples were determined using a validated LC-MS/MS method. Tolerability was determined by clinical evaluation and adverse event (AE) monitoring. Pharmacokinetic results demonstrated that Cmax and AUC(0-t) of Sul increased with an increasing dose. The mean t1/2 values for Cohorts A, B, and C were 16.85, 17.66, and 11.87 h, respectively. No statistically significant differences were observed between men and women for the main pharmacokinetic parameters, with the exception of t1/2 in Cohorts B and C. No significant differences were observed in the absorption and bioavailability of Sul between the fed and fasted states (P > 0.05). Four subjects reported mild AEs during the study. No serious AEs were reported. Sul was shown to be safe and well tolerated in healthy Chinese subjects. Pharmacokinetics studies demonstrated that Sul has adequate oral absorption and bioavailability properties.