Risk factor evaluation of cuff pressure of >30 cmH2O to stop air leakage during mechanical ventilation: A prospective observational study.

AIM: The commonly recommended endotracheal tube cuff pressure is 20-30 cmH2O. However, some patients require a cuff pressure of >30 cmH2O to prevent air leakage. The study aims to determine the risk factors that contribute to the endotracheal tube cuff pressure of >30 cmH2O to prevent air leakage. DESIGN: A multi-centre prospective observational study. METHODS: Eligible patients undergoing mechanical ventilation in the intensive care unit of three hospitals between March 2020 and July 2022 were included. The endotracheal tube cuff pressure to prevent air leakage was determined using the minimal occlusive volume technique. The patient demographics and clinical information were collected. RESULTS: A total of 284 patients were included. Among these patients, 55 (19.37%) patients required a cuff pressure of >30 cmH2O to prevent air leakage. The multivariate logistic regression results revealed that the surgical operation (odds ratio [OR]: 8.485, 95% confidence interval [CI]: 1.066-67.525, p = 0.043) was inversely associated with the endotracheal tube cuff pressure of >30 cmH2O, while the oral intubation route (OR: 0.127, 95% CI: 0.022-0.750, p = 0.023) and cuff inner diameter minus tracheal area (OR: 0.949, 95% CI: 0.933-0.966, p < 0.001) were negatively associated with the endotracheal tube cuff pressure of >30 cmH2O. Therefore, a significant number of patients require an endotracheal tube cuff pressure of ＞30 cmH2O to prevent air leakage. Several factors, including the surgical operation, intubation route, and difference between the cuff inner diameter and tracheal area at the T3 vertebra, should be considered when determining the appropriate cuff pressure during mechanical ventilation.

Sigma-1 receptor knockout disturbs gut microbiota, remodels serum metabolome, and exacerbates isoprenaline-induced heart failure.

Introduction: Heart failure (HF) is usually the end stage of the continuum of various cardiovascular diseases. However, the mechanism underlying the progression and development of HF remains poorly understood. The sigma-1 receptor (Sigmar1) is a non-opioid transmembrane receptor implicated in many diseases, including HF. However, the role of Sigmar1 in HF has not been fully elucidated. Methods: In this study, we used isoproterenol (ISO) to induce HF in wild-type (WT) and Sigmar1 knockout (Sigmar1-/-) mice. Multi-omic analysis, including microbiomics, metabolomics and transcriptomics, was employed to comprehensively evaluate the role of Sigmar1 in HF. Results: Compared with the WT-ISO group, Sigmar1-/- aggravated ISO-induced HF, including left ventricular systolic dysfunction and ventricular remodeling. Moreover, Sigmar1-/- exacerbated ISO-induced gut microbiota dysbiosis, which was demonstrated by the lower abundance of probiotics g_Akkermansia and g_norank_f_Muribaculaceae, and higher abundance of pathogenic g_norank_f_Oscillospiraceae and Allobaculum. Furthermore, differential metabolites among WT-Control, WT-ISO and Sigmar-/--ISO groups were mainly enriched in bile secretion, tryptophan metabolism and phenylalanine metabolism, which presented a close association with microbial dysbiosis. Corresponding with the exacerbation of the microbiome, the inflammation-related NOD-like receptor signaling pathway, NF-kappa B signaling pathway and TNF signaling pathway were activated in the heart tissues. Conclusion: Taken together, this study provides evidence that a Sigmar1 knockout disturbs the gut microbiota and remodels the serum metabolome, which may exacerbate HF by stimulating heart inflammation.

The risk factors of early hemorrhage after emergency intravenous thrombolysis in patients with acute ischemic stroke.

BACKGROUND: At present, the treatment of acute ischaemic stroke (AIS) by aticepase (rt-PA) in emergency veins has become the main treatment mode in hospital, but the research on early hemorrhage complications in patients with emergency thrombolysis is rarely reported. This research aims to study the earlier warning index of early hemorrhage complications in patients with emergency thrombolysis. METHODS: A retrospective analysis was performed on the clinical data of rt-PA intravenous thrombolysis-treated AIS patients in the advanced stroke center of the emergency department of a tertiary grade hospital from January 2018 to May 2020. Patients were divided into a hemorrhage group and non-hemorrhage group according to the hemorrhage situation within 24 hours after thrombolytic therapy. The differences between the 2 groups in terms of pre-thrombolysis risk factors were analyzed. Logistic regression analysis was used to analyze the independent risk factors associated with post-thrombolysis hemorrhage. RESULTS: After intravenous thrombolysis, the hemorrhage group had 91 cases and the non-hemorrhage group had 146 cases. Logistic regression analysis showed that atrial fibrillation, systolic blood pressure before thrombolysis, platelet count, and antiplatelet drugs were independent risk factors for hemorrhage after intravenous thrombolysis (P<0.05). CONCLUSIONS: Patients with AIS have a higher incidence of hemorrhage after intravenous thrombolysis. Atrial fibrillation, systolic blood pressure before thrombolysis, platelet count, and antiplatelet drugs were independent risk factors for hemorrhage after intravenous thrombolysis. These independent risk factors can provide a basis for clinical nurses to evaluate hemorrhage risk in AIS patients after intravenous thrombolysis.

The Braden Scale for Predicting the Outcome and Prognosis of Pressure Injuries in Older Inpatients: A Multicenter, Retrospective Cohort Study.

INTRODUCTION: The Braden scale is used to assess the risk of patients with pressure injuries (PIs), but there are limitations to the prediction of PI healing. There is a lack of tools for evaluating PI healing and outcome in clinical practice. OBJECTIVE: The purpose of this study was to examine the ability of the Braden scale to predict the outcome and prognosis of PIs in older patients. MATERIALS AND METHODS: Outcome indicator was the wound healing rate of patients with PIs at discharge. The receiver operating characteristic (ROC) and Hosmer-Lemeshow goodness-of-fit test were used to evaluate the discrimination and calibration. RESULTS: Completed data were available for 309 patients, 181 of whom (58.6%) were male. The Braden scale had poor discrimination to predict the outcome and prognosis of PIs with an area under the curve (AUC) of 0.63 (95% CI, 0.56-0.70; P = .01). Subgroup analyses showed the Braden scale had low diagnostic value for patients aged over 90 years (AUCROC = 0.56; 95% CI, 0.17-0.96; P = .738), patients with respiratory diseases (AUCROC = 0.51; 95% CI, 0.37-0.65; P = .908), and digestive system diseases (AUCROC = 0.59; 95% CI, 0.42-0.75; P = .342). The level of calibration ability by Hosmer-Lemeshow goodness-of-fit test was acceptable, defined as P >.200 (χ2 = 6.59; P = .473). In patients aged more than 90 years (χ2 = 4.88; P = .431) and female patients (χ2 = 7.03; P = .425), the Braden scale was also fitting. It was not suitable for patients with respiratory diseases (χ2 = 11.35; P = .078). CONCLUSIONS: The Braden scale had low discrimination for predicting the outcome and prognosis of PIs in older inpatients. The development of a new tool is needed to predict healing in patients with preexisting PIs.

Negative-pressure wound therapy for III/IV pressure injuries: A meta-analysis.

This meta-analysis was conducted to identify the potential benefits and the efficacy of negative-pressure wound therapy (NPWT) for III/IV pressure injuries (PIs) compared with standard wound care (SWC). Sixteen RCTs with 629 patients were included in our analysis. The methodological quality was assessed by the Cochrane Collaboration Tool. The outcomes included complete ulcer healing rate, wound healing time, pain score, the frequency of dressing change, hospitalization cost, the condition of the exudate, and the wound improvement. The percentage of healing rate was 61.45% for the NPWT group and 36.90% for SWC (95% CI: 1.32-1.70). There were significant differences in wound healing time (WMD = -16.47 days, 95% [CI (-22.36, - 10.59) days, P ≤ .001]). The pain score and hospitalization cost in NPWT was lower compared with SWC group (WMD = -2.39, 95% CI [-3.47, -1.30], P ≤ .001); (SMD = -2.55, 95% CI [-4.07, -1.03], P < .01). The frequency of dressing change in both NPWT groups was greatly reduced (SMD = -3.61, 95% [CI (-4.57, - 2.66) times, P ≤ .001]). Our meta-analysis indicated that NPWT was associated with greater improvements in improving PIs and shorting healing time for III/IV PIs. However, this conclusion needs to be confirmed by high-quality multicenter RCTs.

Incidence of Pressure Injury in Individuals With Spinal Cord Injury: A Systematic Review and Meta-analysis.

PURPOSE: The purpose of this systematic review and quantitative analysis of pooled data was to assess the global incidence of pressure injury (PI), across time frames and countries, in individuals with spinal cord injury (SCI). DESIGN: Systematic review and meta-analysis. SEARCH STRATEGY: PubMed, Web of Science, and EMBASE databases were systematically searched for studies published from database inception to January 2019, with only English language studies that reported the incidence of PIs in individuals with SCI were included. Study quality was assessed by a 14-item standardized checklist. We calculated the incidence of PIs as the number of new PIs in individuals with SCI and the total number of individuals with SCI during the study period. Findings are presented as incidence rate with 95% confidence intervals (CIs). RESULTS: The search yielded 1652 studies; after studies were reviewed for inclusion criteria, 29 studies representing N = 82,722 patients were retained for data extraction. The global incidence of PIs was 0.23 (95% CI, 0.20-0.26). Data for regional distribution by country showed a pooled incidence of 0.43 (95% CI, 0.28-0.57) in individuals with SCI in South American countries, 0.36 (95% CI, 0.16-0.56) in African countries, 0.25 (95% CI, 0.14-0.37) in European countries, 0.23 (95% CI, 0.19-0.27) in North American countries, and 0.16 (95% CI, 0.06-0.25) in Asian countries. The incidence was 0.22 (95% CI, 0.19-0.26) in developing countries versus 0.27 (95% CI, 0.17-0.37) in developed countries. From 2000 to 2009, the incidence of PIs in individuals with SCI was 0.28 (95% CI, 0.09-0.47). The incidence rate of PIs before 2000 and after 2009 was 0.23. The hospital- and community-acquired PI incidence was 0.22 (95% CI, 0.19-0.26) and 0.26 (95% CI, 0.20-0.32), respectively. CONCLUSIONS: Study findings indicate that more than 1 in 5 individuals with SCI will develop a PI. Individuals with SCI are at high risk of developing PI, especially in community settings or low- and middle-income developing countries. The findings highlight the importance of further investigation of risk factors and prevention and management strategies for PIs in individuals with SCI.

Predicting the occurrence of venous thromboembolism: construction and verification of risk warning model.

BACKGROUND: The onset of venous thromboembolism is insidious and the prognosis is poor. In this study, we aimed to construct a VTE risk warning model and testified its clinical application value. METHODS: Preliminary construction of the VTE risk warning model was carried out according to the independent risk warning indicators of VTE screened by Logistic regression analysis. The truncated value of screening VTE was obtained and the model was evaluated. ROC curve analysis was used to compare the test of Caprini risk assessment scale and VTE risk warning model. The cut-off value of the VTE risk warning model was used to evaluate the test effectiveness of the model for VTE patients with validation data set. RESULTS: The VTE risk warning model is p = ex / (1+ ex), x = - 4.840 + 2.557 • X10(1) + 1.432 • X14(1) + 2.977 • X15(1) + 3.445 • X18(1) + 1.086 • X25(1) + 0.249 • X34 + 0.282 • X41. ROC curve results show that: AUC (95%CI), cutoff value, sensitivity, specificity, accuracy, Youden index, Caprini risk assessment scale is 0.596 (0.552, 0.638), 5, 26.07, 96.50, 61.3%, 0.226, VTE risk warning model is 0.960 (0.940, 0.976), 0.438, 92.61, 91.83, 92.2%, 0.844, respectively, with statistically significant differences (Z = 14.521, P < 0.0001). The accuracy and Youden index of VTE screening using VTE risk warning model were 81.8 and 62.5%, respectively. CONCLUSIONS: VTE risk warning model had high accuracy in predicting VTE occurrence in hospitalized patients. Its test performance was better than Caprini risk assessment scale. It also had high test performance in external population.

Zinc Therapy Is a Reasonable Choice for Patients With Pressure Injuries: A Systematic Review and Meta-Analysis.

The aim of this systematic review was to summarize the evidence on the efficacy of zinc supplementation in patients with pressure injuries (PIs). Electronic data bases (Embase, MEDLINE, and Web of Science) were searched from inception to 2019 for randomized controlled trials (RCTs) and non-RCTs addressing the efficacy of zinc supplementation compared with a control nutrition invention on PI outcomes. The primary study outcome was the healing rate of PIs during treatment; the secondary outcomes were the improvement of PI area and pressure ulcer scale for healing (PUSH) score. A total of 7 studies were included in this systematic review and meta-analysis. The intervention group significantly had improved healing vs that of the control group (relative risk, 1.44; 95% CI, 1.01-2.06; P = 0.043, I2 = 19.3%). There was no obvious asymmetry in the funnel plot and no strong evidence of publication bias. Sensitivity analysis showed that meta-analysis has good stability. Studies showed a greater mean reduction in PI area. All the studies we included had a significant improvement in the PUSH score of PIs. Our systematic review and meta-analysis from clinical research confirmed that zinc therapy can promote wound healing and suggest that medical staff should consider providing patients with zinc during PI treatment.

The relationship between pressure injury complication and mortality risk of older patients in follow-up: A systematic review and meta-analysis.

Pressure injuries (PIs) have now become a common complication of the elderly patients. Some studies have observed that pressure injuries may increase mortality, but this area of evidence has not been evaluated and summarised. The aim of this study was to compare the mortality of patients with pressure injuries and those without pressure injuries. A meta-analysis of observational studies was performed. PubMed, Cochrane Library, Embase, and Web of Science were searched up to April 2019. Studies about mortality among the elderly patients with and without pressure injuries were included. Methodological quality was assessed by the Newcastle-Ottawa Scale (NOS). The fixed effect or random effect model was determined by the test of heterogeneity. The subgroup analysis was performed based on the pressure injuries stages, the region, and the type of study design. The meta-regression analysis was performed to investigate the relationship between the mortality and patients' enrolled year, average age, the incidence of pressure injuries, and gender ratio. The sensitivity analysis was used to explore the impact of an individual study by excluding one at a time. The hazard ratio (HR) and 95% confidence intervals (CIs) in terms of the comparison of two groups were extracted for meta-analysis. A survival curve between two groups by individual patient-level was drew. Eight studies with 5523 elderly patients were included in the analysis. Follow-up periods for the included studies ranged from about 0.5 to 3 years. The elderly patients who complicated with pressure injuries had a higher risk of death. The pooled HR was 1.78 (95% CI 1.46-2.16). A funnel plot showed no publication bias. Further subgroup analysis showed that HR values for the patient stage 3 to 4 pressure injuries (HR:2.41; 95% CI:1.08-5.37) were higher than stage 1-4 and 2-4 pressure injuries (HR: 1.66; 95% CI: 1.35-2.05; HR: 1.74; 95% CI: 1.16-2.60). The meta-regression analysis found that patients' enrolled year, average age, the incidence of pressure injuries, and gender ratio were not the sources of heterogeneity. Sensitivity analyses showed that the outcomes of the study did not change after removing the Onder's article. The survival curve at the individual patient-level also indicated that patients complicated with pressure injuries significantly increased the risk of death (HR: 1.958; 95% CI: 1.79-2.14) in elderly patients. Our meta-analysis indicated that patients complicated with pressure injuries are estimated to have a two times higher risk on mortality compared with patients without pressure injuries during the 3 years follow-up period. Particular attention should be given to the elderly patients who are at higher risk for mortality.

Development of a mechanism-based pharmacokinetic/pharmacodynamic model to characterize the thermoregulatory effects of serotonergic drugs in mice.

We have shown recently that concurrent harmaline, a monoamine oxidase-A inhibitor (MAOI), potentiates serotonin (5-HT) receptor agonist 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT)-induced hyperthermia. The objective of this study was to develop an integrated pharmacokinetic/pharmacodynamic (PK/PD) model to characterize and predict the thermoregulatory effects of such serotonergic drugs in mice. Physiological thermoregulation was described by a mechanism-based indirect-response model with adaptive feedback control. Harmaline-induced hypothermia and 5-MeO-DMT-elicited hyperthermia were attributable to the loss of heat through the activation of 5-HT1A receptor and thermogenesis via the stimulation of 5-HT2A receptor, respectively. Thus serotonergic 5-MeO-DMT-induced hyperthermia was readily distinguished from handling/injection stress-provoked hyperthermic effects. This PK/PD model was able to simultaneously describe all experimental data including the impact of drug-metabolizing enzyme status on 5-MeO-DMT and harmaline PK properties, and drug- and stress-induced simple hypo/hyperthermic and complex biphasic effects. Furthermore, the modeling results revealed a 4-fold decrease of apparent SC50 value (1.88-0.496 µmol/L) for 5-MeO-DMT when harmaline was co-administered, providing a quantitative assessment for the impact of concurrent MAOI harmaline on 5-MeO-DMT-induced hyperthermia. In addition, the hyperpyrexia caused by toxic dose combinations of harmaline and 5-MeO-DMT were linked to the increased systemic exposure to harmaline rather than 5-MeO-DMT, although the body temperature profiles were mispredicted by the model. The results indicate that current PK/PD model may be used as a new conceptual framework to define the impact of serotonergic agents and stress factors on thermoregulation.

Modification of 5-methoxy-N,N-dimethyltryptamine-induced hyperactivity by monoamine oxidase A inhibitor harmaline in mice and the underlying serotonergic mechanisms.

BACKGROUND: 5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and harmaline are indolealkylamine (IAA) drugs often abused together. Our recent studies have revealed the significant effects of co-administered harmaline, a monoamine oxidase inhibitor (MAOI), on 5-MeO-DMT pharmacokinetics and thermoregulation. This study was to delineate the impact of harmaline and 5-MeO-DMT on home-cage activity in mouse models, as well as the contribution of serotonin (5-HT) receptors. METHODS: Home-cage activities of individual animals were monitored automatically in the home cages following implantation of telemetry transmitters and administration of various doses of IAA drugs and 5-HT receptor antagonists. Area under the effect curve (AUEC) of mouse activity values were calculated by trapezoidal rule. RESULTS: High dose of harmaline (15mg/kg, ip) alone caused an early-phase (0-45min) hypoactivity in mice that was fully attenuated by 5-HT1A receptor antagonist WAY-100635, whereas a late-phase (45-180min) hyperactivity that was reduced by 5-HT2A receptor antagonist MDL-100907. 5-MeO-DMT (10 and 20mg/kg, ip) alone induced biphasic effects, an early-phase (0-45min) hypoactivity that was completely attenuated by WAY-100635, and a late-phase (45-180min) hyperactivity that was fully suppressed by MDL-100907. Interestingly, co-administration of MAOI harmaline (2-15mg/kg) with a subthreshold dose of 5-MeO-DMT (2mg/kg) induced excessive hyperactivities at late phase (45-180min) that could be abolished by either WAY-100635 or MDL-100907. CONCLUSIONS: Co-administration of MAOI with 5-MeO-DMT provokes excessive late-phase hyperactivity, which involves the activation of both 5-HT1A and 5-HT2A receptors.

Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: A new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics.

To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages. HRMS-based single ion monitoring (SIM) on a Q Exactive™ mass spectrometer was employed to improve assay specificity and sensitivity for DCP(SS) due to its poor fragmentation and low sensitivity with SRM detection. The assay has been validated for the protein drug in monkey serum using both surrogate peptides with excellent accuracy (within ±4.4%Dev) and precision (within 7.5%CV) with a lower limit of quantitation (LLOQ) at 10 ng mL(-1). The protein concentrations in monkey serum obtained from the DCP(SS)-based assay not only provided important pharmacokinetic parameters, but also confirmed in vivo stability of the peptide regions of interest by comparing drug concentrations with those obtained from the CP-based assay or from a ligand-binding assay (LBA). Furthermore, UHPLC-HRMS allowed simultaneous monitoring of the oxidized forms of both surrogate peptides to evaluate potential ex vivo/in vivo oxidation of one methionine present in each of both surrogate peptides. To the best of our knowledge, this is the first report of using a surrogate disulfide-containing peptide for LC-MS bioanalysis of a therapeutic protein.

Potentiation of 5-methoxy-N,N-dimethyltryptamine-induced hyperthermia by harmaline and the involvement of activation of 5-HT1A and 5-HT2A receptors.

5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and harmaline are serotonin (5-HT) analogs often abused together, which alters thermoregulation that may indicate the severity of serotonin toxicity. Our recent studies have revealed that co-administration of monoamine oxidase inhibitor harmaline leads to greater and prolonged exposure to 5-HT agonist 5-MeO-DMT that might be influenced by cytochrome P450 2D6 (CYP2D6) status. This study was to define the effects of harmaline and 5-MeO-DMT on thermoregulation in wild-type and CYP2D6-humanized (Tg-CYP2D6) mice, as well as the involvement of 5-HT receptors. Animal core body temperatures were monitored noninvasively in the home cages after implantation of telemetry transmitters and administration of drugs. Harmaline (5 and 15 mg/kg, i.p.) alone was shown to induce hypothermia that was significantly affected by CYP2D6 status. In contrast, higher doses of 5-MeO-DMT (10 and 20 mg/kg) alone caused hyperthermia. Co-administration of harmaline (2, 5 or 15 mg/kg) remarkably potentiated the hyperthermia elicited by 5-MeO-DMT (2 or 10 mg/kg), which might be influenced by CYP2D6 status at certain dose combination. Interestingly, harmaline-induced hypothermia was only attenuated by 5-HT1A receptor antagonist WAY-100635, whereas 5-MeO-DMT- and harmaline-5-MeO-DMT-induced hyperthermia could be suppressed by either WAY-100635 or 5-HT2A receptor antagonists (MDL-100907 and ketanserin). Moreover, stress-induced hyperthermia under home cage conditions was not affected by WAY-100635 but surprisingly attenuated by MDL-100907 and ketanserin. Our results indicate that co-administration of monoamine oxidase inhibitor largely potentiates 5-MeO-DMT-induced hyperthermia that involves the activation of both 5-HT1A and 5-HT2A receptors. These findings shall provide insights into development of anxiolytic drugs and new strategies to relieve the lethal hyperthermia in serotonin toxicity.

Study of melanin bleaching after immunohistochemistry of melanin-containing tissues.

Melanin may interfere with immunohistochemical staining. The goal of this study was to investigate the effects of trichloroisocyanuric acid (TCCA) bleaching, potassium permanganate bleaching, and potassium dichromate bleaching on melanin, tissue antigen, and 3,3'-diaminobenzidine (DAB) using melanin-containing and melanin-free tissue samples. Our results demonstrated that all 3 bleaching methods efficiently bleached melanin and partially destroyed tissue antigen. In addition, potassium permanganate bleaching and potassium dichromate bleaching clearly destroyed DAB, whereas TCCA bleaching had no significant effect on DAB. Therefore, neither potassium permanganate nor potassium dichromate is an ideal solution, whereas TCCA might be an ideal solution for melanin bleaching after the immunohistochemical staining of melanin-containing tissues. After immunostaining followed by TCCA bleaching, the melanin could be completely removed in all 120 malignant melanoma tissue sections. Compared with the control, the DAB intensity was clear, and the tissue structure and cellular nuclei were well maintained. It is worth noting that TCCA should be freshly prepared before each experiment, and used within 2 hours of its preparation. In addition, sections should not be incubated with TCCA for over 30 minutes.

Bioanalysis of propylparaben and p-hydroxybenzoic acid, and their sulfate conjugates in rat plasma by liquid chromatography-tandem mass spectrometry.

Two rugged liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the determination of propylparaben, its major metabolite, p-hydroxybenzoic acid (pHBA), and their sulfate conjugates have been developed and validated in citric acid-treated rat plasma. To prevent propylparaben being hydrolyzed to pHBA ex vivo, rat plasma was first treated with citric acid; then collected and processed at a reduced temperature (ice bath). Stable isotope labeled internal standards, d4-propylparaben, (13)C6-pHBA, and the d4-labeled internal standards of their sulfate conjugates were used in the methods. The analytes were extracted from the matrix using protein precipitation, followed by chromatographic separation on a Waters ACQUITY UPLC HSS T3 column. Quantification using negative ion electrospray was performed on a Sciex API 4000 mass spectrometer. The analytical ranges were established from 2.00 to 200 ng/mL for propylparaben, 50.0-5000 ng/mL for pHBA, 50.0-10,000 ng/mL for the sulfate conjugate of propylparaben (SPP) and 200-40,000 ng/mL for the sulfate conjugate of pHBA (SHBA). Inter- and intra-run precision for the quality control samples were less than 5.3% and 4.4% for all analytes; and the overall accuracy was within ±5.7% of the nominal values. The validated bioanalytical methods demonstrated excellent sensitivity, specificity, accuracy and precision and were successfully applied to a rat toxicology study under the regulations of Good Laboratory Practices (GLP). Strategies have been developed and applied toward overcoming the challenges related to analyte stability, and environmental and endogenous background.

Altered expression of small heterodimer partner governs cytochrome P450 (CYP) 2D6 induction during pregnancy in CYP2D6-humanized mice.

Substrates of a major drug-metabolizing enzyme CYP2D6 display increased elimination during pregnancy, but the underlying mechanisms are unknown in part due to a lack of experimental models. Here, we introduce CYP2D6-humanized (Tg-CYP2D6) mice as an animal model where hepatic CYP2D6 expression is increased during pregnancy. In the mouse livers, expression of a known positive regulator of CYP2D6, hepatocyte nuclear factor 4α (HNF4α), did not change during pregnancy. However, HNF4α recruitment to CYP2D6 promoter increased at term pregnancy, accompanied by repressed expression of small heterodimer partner (SHP). In HepG2 cells, SHP repressed HNF4α transactivation of CYP2D6 promoter. In transgenic (Tg)-CYP2D6 mice, SHP knockdown led to a significant increase in CYP2D6 expression. Retinoic acid, an endogenous compound that induces SHP, exhibited decreased hepatic levels during pregnancy in Tg-CYP2D6 mice. Administration of all-trans-retinoic acid led to a significant decrease in the expression and activity of hepatic CYP2D6 in Tg-CYP2D6 mice. This study provides key insights into mechanisms underlying altered CYP2D6-mediated drug metabolism during pregnancy, laying a foundation for improved drug therapy in pregnant women.

Fully validated LC-MS/MS assay for the simultaneous quantitation of coadministered therapeutic antibodies in cynomolgus monkey serum.

An LC-MS/MS assay was developed and fully validated for the simultaneous quantitation of two coadministered human monoclonal antibodies (mAbs), mAb-A and mAb-B of IgG4 subclass, in monkey serum. The total serum proteins were digested with trypsin at 50 °C for 30 min after methanol denaturation and precipitation, dithiothreitol reduction, and iodoacetamide alkylation. The tryptic peptides were chromatographically separated with a C18 column (2.1 × 100 mm, 1.7 µm) with mobile phases of 0.1% formic acid in water and acetonitrile. Four peptides, a unique peptide for each mAb and two confirmatory peptides from different antibody domains, were simultaneously quantified by LC-MS/MS in the multiple reaction-monitoring mode. Stable isotopically labeled peptides with flanking amino acids on C- and N-terminals were used as internal standards to minimize the variability during sample processing and detection. The LC-MS/MS assay showed lower limit of quantitation (LLOQ) at 5 µg/mL for mAb-A and 25 µg/mL for mAb-B. The intra- and interassay precision (%CV) was within 10.0% and 8.1%, respectively, and the accuracy (%Dev) was within ±5.4% for all the peptides. Other validation parameters, including sensitivity, selectivity, dilution linearity, processing recovery and matrix effect, autosampler carryover, run size, stability, and data reproducibility, were all evaluated. The confirmatory peptides played a critical role in confirming quantitation accuracy and the integrity of the drugs in the study samples. The robustness of the LC-MS/MS assay and the data agreement with the ligand binding data demonstrated that LC-MS/MS is a reliable and complementary approach for the quantitation of coadministered antibody drugs.

Pharmacokinetic interactions between monoamine oxidase A inhibitor harmaline and 5-methoxy-N,N-dimethyltryptamine, and the impact of CYP2D6 status.

5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT or street name "5-MEO") is a newer designer drug belonging to a group of naturally occurring indolealkylamines. Our recent study has demonstrated that coadministration of monoamine oxidase A (MAO-A) inhibitor harmaline (5 mg/kg) increases systemic exposure to 5-MeO-DMT (2 mg/kg) and active metabolite bufotenine. This study is aimed at delineating harmaline and 5-MeO-DMT pharmacokinetic (PK) interactions at multiple dose levels, as well as the impact of CYP2D6 that affects harmaline PK and determines 5-MeO-DMT O-demethylation to produce bufotenine. Our data revealed that inhibition of MAO-A-mediated metabolic elimination by harmaline (2, 5, and 15 mg/kg) led to a sharp increase in systemic and cerebral exposure to 5-MeO-DMT (2 and 10 mg/kg) at all dose combinations. A more pronounced effect on 5-MeO-DMT PK was associated with greater exposure to harmaline in wild-type mice than CYP2D6-humanized (Tg-CYP2D6) mice. Harmaline (5 mg/kg) also increased blood and brain bufotenine concentrations that were generally higher in Tg-CYP2D6 mice. Surprisingly, greater harmaline dose (15 mg/kg) reduced bufotenine levels. The in vivo inhibitory effect of harmaline on CYP2D6-catalyzed bufotenine formation was confirmed by in vitro study using purified CYP2D6. Given these findings, a unified PK model including the inhibition of MAO-A- and CYP2D6-catalyzed 5-MeO-DMT metabolism by harmaline was developed to describe blood harmaline, 5-MeO-DMT, and bufotenine PK profiles in both wild-type and Tg-CYP2D6 mouse models. This PK model may be further employed to predict harmaline and 5-MeO-DMT PK interactions at various doses, define the impact of CYP2D6 status, and drive harmaline-5-MeO-DMT pharmacodynamics.

Humanized transgenic mouse models for drug metabolism and pharmacokinetic research.

Extrapolation of the metabolic, pharmacokinetic and toxicological data obtained from animals to humans is not always straightforward, given the remarkable species difference in drug metabolism that is due in large part to the differences in drug-metabolizing enzymes between animals and humans. Furthermore, genetic variations in drug-metabolizing enzymes may significantly alter pharmacokinetics, drug efficacy and safety. Thus, humanized transgenic mouse lines, in which the human drug-metabolizing enzymes are expressed in mouse tissues in the presence or absence of mouse orthologues, have been developed to address such challenges. These humanized transgenic mice are valuable animal models in understanding the significance of specific human drug-metabolizing enzymes in drug clearance and pharmacokinetics, as well as in predicting potential drug-drug interactions and chemical toxicity in humans. This review, therefore, aims to summarize the development and application of some humanized transgenic mouse models expressing human drug-metabolizing enzymes. The limitations of these genetically modified mouse models are also discussed.

The riboswitch regulates a thiamine pyrophosphate ABC transporter of the oral spirochete Treponema denticola.

Thiamine pyrophosphate (TPP), a biologically active form of thiamine (vitamin B1), is an essential cofactor in all living systems. Microorganisms either synthesize TPP via de novo biosynthesis pathways or uptake exogenous thiamine from the environment via specific transporters. The oral spirochete Treponema denticola is an important pathogen that is associated with human periodontal diseases. It lacks a de novo TPP biosynthesis pathway and needs exogenous TPP for growth, suggesting that it may obtain exogenous TPP via a thiamine transporter. In this study, we identified a gene cluster that encodes a TPP ABC transporter which consists of a TPP-binding protein (TDE0143), a transmembrane permease (TDE0144), and a cytosolic ATPase (TDE0145). Transcriptional and translational analyses showed that the genes encoding these three proteins are cotranscribed and form an operon (tbpABC(Td)) that is initiated by a σ7°-like promoter. The expression level of this operon is negatively regulated by exogenous TPP and is mediated by a TPP-sensing riboswitch (Td(thi-)(box)). Genetic and biochemical studies revealed that the TDE0143 deletion mutant (T. denticola ΔtbpA) had a decreased ability to transport exogenous TPP, and the mutant failed to grow when exogenous TPP was insufficient. These results taken together indicate that the tbpABC(Td) operon encodes an ABC transporter that is required for the uptake of exogenous TPP and that the expression of this operon is regulated by a TPP-binding riboswitch via a feedback inhibition mechanism.