Determination of Vitamin A Total Body Stores in Children from Dried Serum Spots: Application in a Low- and Middle-Income Country Community Setting.

BACKGROUND: The retinol isotope dilution (RID) method has been used to evaluate vitamin A (VA) status in healthy adults and children in low- and middle-income countries (LMIC) and to assess the efficacy of various VA interventions. OBJECTIVE: The study was designed to examine whether dried serum spots (DSS) can be applied to RID when conducting VA total body store (TBS) assessments in community settings. METHODS: Four days after an oral dose of 0.4 mg [13C10]retinyl acetate was administered to Filipino children (12-18 mo), a single blood draw was divided to isolate both serum and plasma. Serum (40 µL) was spotted and dried on Whatman 903 cards and shipped at ambient temperature whereas liquid plasma (LP) was frozen at -80°C and shipped on dry ice. The VA tracer to tracee ratio from DSS and LP was quantified by LC-MS/MS. Comparisons between DSS and LP paired samples (n = 72) were made for [13C10]retinol specific activity (SAp) by Pearson's correlation and for VA TBS by Bland-Altman analysis. RESULTS: The sum of 3 coextracted DSS were required to consistently detect [13C10]retinol above the LC-MS/MS limit of quantitation (LOQ). [13C10]retinol SAp from DSS was highly correlated with SAp from LP (r = 0.945; P < 0.01). A comparison of methods for TBS determination using Bland-Altman analysis indicated agreement with an intraindividual difference of 24.7 µmol (4.6%). Mean total liver reserve (TLR) values from DSS and LP were 1.7 µmol/g (± 0.6 SD) and 1.6 µmol/g (± 0.6 SD), respectively. CONCLUSIONS: VA TBS can be determined from DSS thereby reducing the logistics and cost of maintaining a cold chain by shipping samples at ambient temperature and, thus, making the RID technique more feasible in LMIC community settings. This trial was registered at https://clinicaltrials.gov as NCT03030339.

Physiological significance of link between polycyclic aromatic hydrocarbons and serum nutritional biomarkers.

It is well-known that exposure to polycyclic aromatic hydrocarbons (PAH) may cause adverse health impacts. However, there are few investigations assessing the association between PAH exposure and the nutritional status of the general population. Thus, the purpose of this investigation was to assess the correlation between PAH metabolites and nutritional biomarkers in the U.S. general population. From the 2003-2006 National Health and Nutrition Examination Survey, 4,545 eligible participants were included in this cross-sectional study. To assess PAH exposure, ten urinary PAH metabolites were measured. Eleven serum nutritional biomarkers including carotenoids and vitamins were measured. The association between PAH metabolites and serum nutritional biomarkers was investigated using multivariate linear regression models. Increased 2-hydroxyfluorene was inversely correlated with elven serum nutritional biomarkers: α-carotene (ß = -0.529, p < 0.001), ß-cryptoxanthin (ß = -0.968, p < 0.001), cis-ß carotene (ß = -0.149, p < 0.001), lutein and zeaxanthin (ß = -1.188, p < 0.001), retinyl palmitate (ß = -0.145, p < 0.001), retinyl stearate (ß = -0.025, p = 0.006), total lycopene (ß = -1.074, p < 0.001), trans-ß carotene (ß = -2.268, p < 0.001), trans-lycopene (ß = -0.466, p < 0.003), retinol (ß = -0.694, p = 0.004) and 25-hydroxyvitamin D (ß = -1.247, p = 0.007). Increased 3-hydroxyfluorene was inversely correlated with eleven serum nutritional biomarkers: α-carotene (ß = -0.740, p < 0.001), ß-cryptoxanthin (ß = -1.377, p < 0.001), cis-ß carotene (ß = -0.205, p < 0.001), lutein and zeaxanthin (ß = -1.521, p < 0.001), retinyl palmitate (ß = -0.209, p < 0.001), retinyl stearate (ß = -0.034, p = 0.014), total lycopene (ß = -1.20, p = 0.007), trans-ß carotene (ß = -3.185, p < 0.001), trans-lycopene (ß = -0.490, p = 0.039), retinol (ß = -1.366, p < 0.001) and 25-hydroxyvitamin D (ß = -2.483, p < 0.001). Increased 1-hydroxypyrene was inversely correlated with eight serum nutritional biomarkers: α-carotene (ß = -0.601, p = 0.001), ß-cryptoxanthin (ß = -1.071, p = 0.001), cis-ß carotene (ß = -0.170, p = 0.001), lutein and zeaxanthin (ß = -1.074, p < 0.001), retinyl palmitate (ß = -0.214, p = 0.005), retinyl stearate (ß = -0.041, p = 0.043), total lycopene (ß = -1.664, p = 0.011) and retinol (ß = -1.381, p = 0.011). These results demonstrate that PAH exposure is significantly correlated with decreased levels of serum nutritional biomarkers.

Toxicological and pharmacokinetic analysis at therapeutic dose of SRS27, an investigational anti-asthma agent.

SRS27, an andrographolide analogue, had been proven to have therapeutic properties at a dose of 3 mg/kg in both in vitro and in vivo asthma models of our previous study. The present study focuses on the pharmacokinetic and toxicity profile of this compound to provide further evidence for the development of this compound as an anti-asthma agent. A simple pharmacokinetic study was performed in female BALB/c mice to measure blood plasma concentration of the compound at therapeutic dose. At a single dose of 3 mg/kg, SRS27 had a relatively short half-life but was able to achieve a concentration range of 13-19 µM that is related to its in vitro bioactivities. With regard to toxicity profile, SRS27 appears to be safe, as no histopathological changes were observed in the liver, kidneys and ovaries of SRS27-treated female BALB/c mice. In addition, there was no significant change in the mean body weight and organ weight of the animals in the SRS27-treated groups compared with the vehicle-treated control group at the end of the treatment. This fully supports the absence of any significant changes in peripheral blood leukocyte counts of SRS27-treated mice. Rewardingly, this acute toxicity study also revealed that SRS27 has a wide therapeutic window as no toxicity symptoms were detected with a dose up to 60 mg/kg daily when tested for 14 days. These results provide strong justification for further investigation of SRS27 as a potential new anti-asthma agent.

Vitamin A and D Absorption in Adults with Metabolic Syndrome versus Healthy Controls: A Pilot Study Utilizing Targeted and Untargeted LC-MS Lipidomics.

SCOPE: Persons with metabolic syndrome (MetS) absorb less vitamin E than healthy controls. It is hypothesized that absorption of fat-soluble vitamins (FSV) A and D2 would also decrease with MetS status and that trends would be reflected in lipidomic responses between groups. METHODS AND RESULTS: Following soymilk consumption (501 IU vitamin A, 119 IU vitamin D2 ), the triglyceride-rich lipoprotein fractions (TRL) from MetS and healthy subjects (n = 10 age- and gender-matched subjects/group) are assessed using LC-MS/MS. Absorption is calculated using area under the time-concentration curves (AUC) from samples collected at 0, 3, and 6 h post-ingestion. MetS subjects have ≈6.4-fold higher median vitamin A AUC (retinyl palmitate) versus healthy controls (P = 0.07). Vitamin D2 AUC is unaffected by MetS status (P = 0.48). Untargeted LC-MS lipidomics reveals six phospholipids and one cholesterol ester with concentrations correlating (r = 0.53-0.68; P < 0.001) with vitamin A concentration. CONCLUSIONS: The vitamin A-phospholipid association suggests increased hydrolysis by PLB, PLRP2, and/or PLA2 IB may be involved in the trend in higher vitamin A bioavailability in MetS subjects. Previously observed differences in circulating levels of these vitamins are likely not due to absorption. Alternate strategies should be investigated to improve FSV status in MetS.

Quantitative analysis and pharmacokinetic comparison of multiple bioactive components in rat plasma after oral administration of Qi-Shen-Ke-Li formula and its single-herb extracts using ultra-high-performance liquid chromatography-tandem mass spectrometry.

Qi-Shen-Ke-Li (QSKL), a traditional Chinese formula prepared from six herbs, has long been used for the treatment of coronary heart disease and chronic heart failure. However, the herbal combination mechanism and underlying material basis of this multi-herbal formula are not clear. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to simultaneously determine multiple bioactive compounds in QSKL was established and validated. Using the developed method, 18 bioactive components in rat plasma after oral administration of QSKL formula and its single herb extracts were quantified. Based on these results, pharmacokinetic (PK) parameters (T1/2 , Tmax , Cmax , AUC0-48h , and AUC0-∞ ) of the 18 bioactive components were analyzed and compared using PKSlover 2.0 PK software. The experimental data suggested that significant changes in PK profiles were observed between the QSKL formula and its single-herb extracts. The herbal combination in QSKL significantly influences the system exposure and the PK behaviors of the 18 bioactive components, indicating multicomponent interactions among the herbs. This study provides insight into the herbal combination mechanism and underlying material basis of the QSKL formula.

Comparison of pulmonary availability and anti-inflammatory effect of dehydroandrographolide succinate via intratracheal and intravenous administration.

Dehydroandrographolide succinate (DAS) injection, which was approved in China for the treatment of viral pneumonia and upper respiratory tract infections, is often off-label used for nebulization therapy to avoid the adverse drug reactions associated with the injection. However, the aerodynamic properties and pulmonary fate of nebulized DAS was largely uninvestigated. In this study, the main objectives were to evaluate the in vitro aerodynamic deposition profiles of nebulizer generated aerosols and comparatively investigate the local drug availability and anti-inflammatory efficacy of DAS between intratracheal and intravenous dosing. The in vitro evaluation of aerodynamic characteristics and droplet size distribution showed more than 50% aerosol particles with size being <5 µm, allowing the aerosols to reach the lower respiratory tract. Following intratracheal administration, the drug underwent pulmonary absorption into the bloodstream, rendering an absolute bioavailability of 47.3%. Compared to the intravenous delivery, the intratracheal administration dramatically increased the drug availability in the lung tissue in rats by more than 80-fold, leading to an improved and prolonged local anti-inflammatory efficacy in a lipopolysaccharide induced lung injury model in mice. The present results demonstrated that inhalation delivery of DAS is a convenient and effective alternative to intravenous injections.

An HPLC Ultraviolet Method Using Low Sample Volume and Protein Precipitation for the Measurement of Retinol in Human Serum Suitable for Laboratories in Low- and Middle-Income Countries.

BACKGROUND: Assessing vitamin A status in populations remains a high public health priority for low- and middle-income countries. However, analytical difficulties with serum retinol measurements persist in international laboratories. Nearly all participants in a Centers for Disease Control and Prevention external quality assessment program use HPLC to measure serum retinol, but round-to-round results failing to meet acceptable criteria suggest the need to provide a straightforward stable HPLC ultraviolet (UV) method that can be adopted by these laboratories to improve performance. We present a protein precipitation HPLC-UV method that measures serum retinol below the deficiency cutoff value (<0.7 µmol/L or 20 µg/dL) that is suitable for low- and middle-income countries and uses commercially available materials. METHODS: Serum (25 µL) added to retinyl acetate was precipitated with acetonitrile (125 µL) to extract retinol. Solvent-based calibration solutions required no extraction. Calibration used either single-point (50 µg/dL) or multipoint solutions (0.52-100 µg/dL). C18 column (4.6 × 100 mm) and acetonitrile with 0.1% triethylamine/water (83/17, v/v) as isocratic mobile phase (1.1 mL/min), achieved baseline separation (7 minutes). RESULTS: With only 25 µL of serum, the limit of detection was 0.52 µg/dL. Single- and multipoint calibration generated equivalent results. Over several years, between-run imprecision was ≤7.1% in multiple quality-control materials. Overall mean (CV) method bias for NIST-certified reference materials (e-series) was -0.2% (5.8%). Maximally, 180 samples were processed within 24 h. CONCLUSIONS: This method was robust and stable over years and accurately measured serum retinol with low-volume samples. Thus, it may be of interest to low- and middle-income countries and to pediatric and finger stick applications.

Development and application of a rapid and sensitive LC-MS/MS method for simultaneous quantification of six diterpene lactones in rats after oral administration of Rhizoma Dioscoreae Bulbiferae extract.

Diterpene lactones have been considered as the main therapeutic and hepatotoxic constituents of Rhizoma Dioscoreae Bulbiferae in recent years. In this work, a simple, rapid and accurate LC-MS/MS method was established and validated to determine six diterpene lactones in rat plasma simultaneously, including Diosbulbin B (DIOB), Diosbulbin C (DIOC), Diosbulbin D (DIOD), Diosbulbin G (DIOG), Diosbulbin J (DIOJ) and Diosbulbin L (DIOL), after oral administration of Rhizoma Dioscoreae Bulbiferae extract. The six diterpene lactones, with the inclusion of two pairs of isomer (DIOB & D, DIOC & L), and Buspirone (internal standard, IS) were successfully separated using an XDB-C18 column with the gradient elution, consisting of water with 0.1% (v/v) FA and methanol with 0.1% (v/v) FA, under a flow rate of 0.50 mL/min in 5.8 min. Precursor-product ion transitions were optimized to be m/z 362.1 → 317.1, 363.1 → 207.1, 345.0 → 299.2, 364.3 → 347.0, 396.3 → 379.3, 363.2 → 345.1 and 386.3 → 122.2 for DIOB, DIOC, DIOD, DIOG, DIOJ, DIOL and buspirone at positive ion mode with an electrospray ionization source (ESI), respectively. The linearity ranges of this present method were 0.50 to 500 µg/L for DIOB, 20.0 to 20,000 µg/L for DIOC and 2.00 to 2000 µg/L for DIOD, DIOG, DIOJ and DIOL, respectively. And the LLOQs were as low as 0.20 µg/L for DIOB, 20.0 µg/L for DIOC and 2.00 µg/L for DIOB, D, G, J and L. The accuracy of each analyte was within the range of 95.8% to 101.0% and the precision was <11.3%. No matrix effect and carry over was observed, and the recovery of the six analytes ranged from 87.3% to 109% with the RSD <11.4% within the concentrations range. The validated method was further applied to the pharmacokinetics investigation of DIOB, DIOC, DIOD, DIOG, DIOJ and DIOL successfully after oral administration of Rhizoma Dioscoreae Bulbiferae extract at 1.53 g/kg in rats.

Therapeutic potential of andrographolide-loaded nanoparticles on a murine asthma model.

Corticosteroids commonly prescribed in asthma show several side-effects. Relatively non-toxic andrographolide (AG) has an anti-asthmatic potential. But its poor bioavailability and short plasma half-life constrain its efficacy. To overcome them, we encapsulated AG in nanoparticle (AGNP) and evaluated AGNP for anti-asthmatic efficacy on murine asthma model by oral/pulmonary delivery. AGNP had 5.47% drug loading with a sustained drug release in vitro. Plasma and lung pharmacokinetic data showed predominantly improved AG-bioavailability upon AGNP administered orally/by pulmonary route. Cell numbers, IL-4, IL-5, and IL-13 levels in broncho-alveolar lavage fluid and serum IgE content were reduced significantly after administration of AGNP compared to free-AG treatment. AGNP-mediated suppression of NF-κß was predominantly more compared to free-AG. Further, pulmonary route showed better therapeutic performance. In conclusion, AGNP effectively controlled mild and severe asthma and the pulmonary administration of AGNP was more efficacious than the oral route.

Pharmacokinetics and relative bioavailability of tiamulin in broiler chicken as influenced by different routes of administration.

The bioavailability and pharmacokinetic disposition of tiamulin in broiler chicken were investigated after administration through the crop, drinking water, and feed at 40 mg/kg body weight. Residues of tiamulin in tissues of broiler chicken were also assessed. Plasma and tissue concentrations of tiamulin were analyzed by reverse-phase high-performance liquid chromatography (HPLC) method. Plasma concentration-time data were described by the non-compartmental model for all three routes, and pharmacokinetic parameters were calculated. There were no significant differences (p > 0.05) in pharmacokinetic parameters and mean plasma concentrations of tiamulin between three routes tested (crop, water, and feed), indicating equal efficacy. Tiamulin residues in edible tissues (muscles, skin, and fat) were lower than the advocated maximum residue limit (MRL of 0.1 µg/g and that of liver was 1 µg/g) on the 3rd day. No traces were found on the 5th day after drug administration. This indicated that the withdrawal period (less than 5 days) is very short, which makes it safer. This study shows that tiamulin can be used with equal efficacy through all routes of administration in broiler chicken (crop, water, and feed).

Prediction of lefamulin epithelial lining fluid penetration after intravenous and oral administration using Phase 1 data and population pharmacokinetics methods.

OBJECTIVES: Lefamulin is a semi-synthetic intravenous and oral pleuromutilin antibiotic with activity against pathogens commonly associated with community-acquired bacterial pneumonia. Using data from two Phase 1 studies, a population pharmacokinetics (PPK) model for lefamulin in plasma and epithelial lining fluid (ELF) was constructed. METHODS: Plasma pharmacokinetic (PK) data from a crossover, bioavailability, food-effect study and plasma and ELF PK data from a tissue penetration study in normal healthy volunteers were used to construct a PPK model for lefamulin. Model development involved refinement of a previous PPK model for intravenous and oral administration, followed by application of the model to plasma and ELF data from the tissue penetration study. The ELF penetration ratio of lefamulin was determined using model-based simulations. RESULTS: The PPK analysis data set contained 1103 plasma and 12 ELF lefamulin concentrations from 32 subjects. A three-compartment model with non-linear protein binding and two parallel absorption processes provided precise and unbiased estimated plasma concentration-time profiles. The absorption rate was slower and bioavailability was decreased after a high-fat/high-calorie meal. ELF data were well described using first-order rate constants into and out of the ELF compartment. The median predicted lefamulin total-drug ELF AUC0-24/free-drug plasma AUC0-24 ratio was ∼5:1 after intravenous or oral administration. CONCLUSIONS: The final PPK model allowed precise characterization of plasma and ELF exposures after intravenous and oral administration. The high ELF penetration ratio suggests that the penetration of lefamulin into the effect site is rapid and extensive, irrespective of route of administration.

Bioavailability of the diterpenoid 14-deoxy-11,12-didehydroandrographolide in rats and up-regulation of hepatic drug-metabolizing enzyme and drug transporter expression.

BACKGROUND: 14-Deoxy-11,12-didehydroandrographolide (deAND) is the second most abundant diterpenoid in Andrographis paniculata (Burm. f.) Nees, a traditional medicine used in Asia. To date, the biological activity of deAND has not been clearly investigated. PURPOSE: In this study, we intended to examine the modulatory effect of deAND on hepatic drug metabolism as well as its bioavailability. STUDY DESIGN: deAND prepared from A. paniculata was orally given to Sprague-Dawley rats and changes in plasma deNAD were determined by HPLC-MS. Modulation of deAND on drug-metabolizing enzyme and drug transporter expression as well as the possible mechanism involved was examined in primary rat hepatocytes. RESULTS: After a single oral administration of 50 mg/kg deAND to rats, the maximum plasma concentration (Cmax), time to reach the Cmax, area under the curve (AUC0-24h), mean retention time, and half-life (t1/2) of deAND were 2.65 ± 0.68 µg/ml, 0.29 ± 0.15 h, 6.30 ± 1.66 µg/ml•h, 5.55 ± 2.52 h, and 3.56 ± 1.05 h, respectively. The oral bioavailability was 3.42%. In primary rat hepatocytes treated with up to 10 µM deAND, a dose-dependent increase was noted in the expression of cytochrome P450 (CYP) 1A1/2, CYP2C6, and CYP3A1/2; UDP-glucuronosyltransferase (UGT) 1A1, NAD(P)H:quinone oxidoreductase (NQO1), π form of GSH S-transferase (GSTP), multidrug resistance-associated protein 2, p-glycoprotein, and organic anion transporter protein 2B1. Immunoblotting assay and EMSA revealed that deAND increases the nuclear translocation and DNA binding activity of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), and nuclear factor erythroid-derived 2-related factor 2 (Nrf2). Knockdown of AhR and Nrf2 expression abolished deAND induction of CYP isozymes and UGT1A1, NQO1, and GSTP expression, respectively. CONCLUSION: These results indicate that deAND quickly passes through enterocytes in rats and effectively up-regulates hepatic drug-metabolizing enzyme and drug transporter expression in an AhR-, PXR-, and Nrf2-dependent manner.

Multiple modulatory activities of Andrographis paniculata on immune responses and xenograft growth in esophageal cancer preclinical models.

BACKGROUND: Esophageal cancer (EC) is a malignant gastrointestinal cancer with high morbidity worldwide and is the fourth leading cause of cancer-related deaths in China. Even though surgery and/or chemotherapy/chemoradiation might achieve good therapeutic response, recurrence rate is high due to cancer metastasis. Hence, the use of alternative adjuvant treatments, such as herbal medicines, for metastatic EC remains a great desire of the patients. Our previous studies have demonstrated the anti-metastatic efficacy of hot water extract of Andrographis paniculata (APW) in human esophageal cancer cells and tumor-bearing nude mice. PURPOSE: In the present study, the immunomodulatory activities of APW were further evaluated in human peripheral blood mononuclear cells (PBMCs) and in a carcinogen-induced esophageal tumorigenesis model using immune-competent C57BL/6 mice. Besides, the inhibitory effects of APW on esophageal cancer cell line-based xenografts and patient-derived xenografts (PDX) were examined so as to illustrate the potential multi-targeted efficacies of APW in esophageal cancer in pre-clinical models. RESULTS: In vitro results showed that APW could stimulate proliferation of PBMCs, as well as TNF-α and IFN-γproductions. In mice with 4-nitroquinoline 1-oxide-induced tumorigenesis, 21-day oral treatment with APW (1600 mg/kg) decreased the level of dysplasia in esophagus and significantly modulated the population of regulatory T cells. The cytokines productions by spleen lymphocytes of APW-treated mice were shifted towards normal resting state (i.e. unchallenged with carcinogen). Furthermore, APW treatment suppressed the growth of cell line-based xenografts by significantly increasing apoptosis in tumors, without causing severe body weight loss as chemotherapeutics did. Most importantly, the inhibitory effects of APW treatment on esophageal patient-derived xenografts growth were demonstrated for the first time. Besides, several diterpenes were detected in the plasma after oral administration of APW in mice, suggesting that multi-components of APW were bioavailable and might have contributed towards the varied pharmacological activities demonstrated in our studies. CONCLUSION: APW was shown to possess anti-tumor, anti-metastatic and immunomodulatory activities in esophageal cancer cell-based and animal models, including immunocompromised mice model and clinically relevant PDX model. Our findings illustrated the potential multi-targeted efficacies of APW in esophageal cancer management.

Sex-dependent differences for larkspur (Delphinium barbeyi) toxicosis in yearling Angus cattle1.

Larkspur (Delphinium spp.) poisoning is a long-term problem for cattle grazing on rangelands of western North America. Results from preliminary experiments have suggested that differences in larkspur toxicity may exist between heifers and bulls. The objective of this study was to compare the physiological responses of yearling Angus heifers, steers, and bulls with a standardized dose of Delphinium barbeyi and to test the hypothesis that the response is sex dependent. Clinical signs of intoxication, including muscle coordination and function, were measured 24 h after oral dosing with larkspur by walking the cattle at a pace of 5 to 6 km h-1 for up to 40 min on an oval dirt track. Due to the experimental methods used, the variation in susceptibility to larkspur was not quantifiable for walking times of 0 or 40 min or more. Larkspur susceptible animals that were not able to walk (0 min; 36% of the animals) or larkspur resistant animals that walked the entire test period of 40 min (9% of the animals) resulted in censored or truncated data. The statistical methods (censReg and lmec) were used to adjust for data truncation or censoring. The heifers were only able to walk -8.9 ± 3.9 min (65.5% censored on the left) compared with 13.2 ± 3.7 min for bulls and 15.9 ± 2.7 min for steers. When heifers were compared with bulls and steers together, heifers walked 23.4 ± 4.5 min less (P < 0.0001). Serum alkaloid concentrations were measured immediately before walking, and deltaline concentrations averaged 266 ± 28, 131 ± 20, and 219 ± 28 ng mL-1 for all heifers, steers, and bulls, respectively, and serum methyllycaconitine concentrations averaged 660 ± 46, 397 ± 32, and 612 ± 34 ng mL-1 for all heifers, steers, and bulls, respectively. The relative risk of a zero walk time for yearling heifers is 330% that of yearling bulls (P = 0.0008). These results suggest that yearling Angus heifers are more susceptible to larkspur intoxication and, when possible, heifers should be kept from grazing larkspur-infested rangelands as a simple management tool to reduce the risk of fatal poisoning.

Evaluation of interconversion pharmacokinetics of 16α-hydroxycleroda-3,13(14)Z-dien-15,16-olide - a novel HMG-CoA reductase inhibitor and its acid metabolite using multi-compartmental pharmacokinetic model in mice.

16α-Hydroxycleroda-3,13(14)Z-dien-15,16-olide (4655K-09 or K-09) is a novel clerodane diterpene lactone reported for its anti-hyperlipidemic efficacy. The objective of the present study was to investigate the probable reversible metabolism of 4655K-09 and evaluate its effects on pharmacokinetic (PK) properties. The PK studies were carried out through intravenous (IV) bolus administration of 4655K-09 and K-9T in mice at a dose of 3, 6 and 12 mg/kg separately. The oral PK study of 4655K-09 was carried out at therapeutic dose of 25 mg/kg. The % AUC of metabolite converted to parent upon its administration % AUCK-09K-9T was found to be 27.28 ± 2.67. The multi-compartmental interconversion model defined reversible and irreversible clearances along with volumes of distribution for parent and metabolite. The results emphasized that hydrolysis of lactone to acid was more efficient than back conversion to parent due to greater extent of irreversible elimination of acid. Further, the role of interconversion in pharmacokinetics of 4655K-09 was evaluated through secondary parameters like conversion coefficients of parent to metabolite ( KK-9TK-09:0.08 ± 0.02 ), metabolite to parent ( KK-09K-9T : 0.019 ± 0.001), exposure enhancement (EE: 1.04 ± 0.006), and recycled fraction (RF: 0.042 ± 0.007), highlighted the minimal role of interconversion. The estimation of oral bioavailability remains unaffected when calculated through considering reversible metabolism. The present model-based interconversion pharmacokinetics of 4655K-09 in mice could be further extended to other species to support its development as anti-hyperlipidemic agent.

A sensitive LC-MS/MS method for the determination of triptolide and its application to pharmacokinetic research in rats.

Triptolide is one of the main active ingredients of Tripterygium wilfordii Hook. F. In this study, a sensitive LC-MS/MS method was established and validated to determine the concentration of triptolide in rat plasma. Triptolide and an internal standard [(5R)-5-hydroxytriptolide] were extracted from 100 µL of rat plasma with acetonitrile, and the dried residue was then reconstituted and reacted with benzylamine to produce benzylamine triptolide and benzylamine (5R)-5-hydroxytriptolide. Derivatization increased the sensitivity of triptolide detection by ~100-fold. Quantification was performed using a QTRAP 5500 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode with an ion transition m/z 468.5 → 192.0 for benzylamine triptolide and m/z 484.3 → 192.1 for benzylamine (5R)-5-hydroxytriptolide. Good linearity was observed in the range of 0.030-100 ng/mL with a lower limit of quantitation of 0.030 ng/mL. The intra- and inter-day precision was <6.5%, and the accuracy ranged from -11.7 to -4.4%. The recovery remained consistent and was reproducible at different concentrations. This method was successfully applied to the study of triptolide drug-drug interactions in Sprague-Dawley rats. With the use of itraconazole (40 mg/kg, p.o.) as a CYP3A inhibitor, the plasma exposure of triptolide in rats was increased by 36%.

Determination of kaurenoic acid in rat plasma using UPLC-MS/MS and its application to a pharmacokinetic study.

Kaurenoic acid (KA), a kaurane diterpene found in several medicinal plants, is an active ingredient with potential anti-inflammatory, anticonvulsant, antibacterial and antitumor activities. In this work, an ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) was firstly developed and validated to quantify kaurenoic acid in rat plasma. Rhein was chosen as the internal standard (IS) and the plasma was processed with one-step acetonitrile protein precipitation; the chromatographic separation was achieved on a HSS T3 (2.1 × 50 mm, 1.8 µm) column with the mobile phase consisting of acetonitrile and water containing 0.1% formic acid via gradient elution. An electrospray ionization source was applied and operated in the negative ion and multiple reaction monitoring (MRM) modes. Kaurenoic acid and IS were quantified using the transitions of m/z 301.2→301.2 (pseudo MRM) and m/z 283.2 → 238.9, respectively. The calibration curves were linear over the range of 5∼ 100 ng/mL (R2 = 0.990). The lower limit of quantification (LLOQ) was 5 ng/mL. The intra- and inter- day precision (RSD) ranged from 3.0% to 11.4%. The matrix effect and extraction recovery were within acceptable limits. The validated method was successfully applied to the pharmacokinetic study of kaurenoic acid in rats after oral administration at three dosages.

Relationships between the Toxicities of Radix Aconiti Lateralis Preparata (Fuzi) and the Toxicokinetics of Its Main Diester-Diterpenoid Alkaloids.

The processed lateral root of Aconitum carmichaelii Deb (Aconiti Radix lateralis praeparata or Fuzi) is a potent traditional herbal medicine extensively used in treatment of cardiovascular diseases, rheumatism arthritis, and bronchitis in many Asian countries. Although Fuzi has promising therapeutic effects, its toxicities are frequently observed. Three main C19-diester-diterpenoid alkaloids (DDAs) are believed to be the principal toxins of the herb. Although toxicokinetic profiles of the toxic DDAs have already been examined in several studies, they have seldom been correlated with the toxicities of Fuzi. The current article aimed to investigate the relationship between the up-to-date toxicokinetic data of the toxic DDAs and the existing evidence of the toxic effects of Fuzi. Relationships between the cardiac toxicity and the plasma and heart concentration of DDAs in mice and rats were established. Based on our findings, clinical monitoring of the plasma concentrations of DDAs of Fuzi is recommended to prevent potential cardiac toxicities. Additionally, caution with respect to potential hepatic and renal toxicity induced by Fuzi should be exercised. In addition, further analyses focusing on the preclinical tissue distribution profile of DDAs and on the long-term toxicokinetic-toxicity correlation of DDAs are warranted for a better understanding of the toxic mechanisms and safer use of Fuzi.

Novel nanocrystal-based solid dispersion with high drug loading, enhanced dissolution, and bioavailability of andrographolide.

OBJECTIVE: The current study sought to design a quickly dissolving, high drug loading nanocrystal-based solid dispersion (NC-SD) in order to improve the dissolution of poorly soluble drugs. METHODS: The NC-SD was prepared by means of combination of homogenization and spray-drying. Polymer hydroxypropylmethylcellulose (HPMC) was used as baseline dispersant for NC-SD of the model drug - andrographolide (AG). Three superdisintegrants cohomogenized with HPMC were used as codispersant for AG-NC-SD and compared to common water-soluble dispersants - mannitol and lactose. The dissolution characterization and oral bioavailability of AG-NC-SD were evaluated. RESULTS: The AG-NC-SD with the higher concentration of HPMC exhibited fast dissolution due to the enhanced wettability of HPMC. The water-soluble codispersants (mannitol and lactose) did not completely prevent AG-NC from aggregation during spray-drying. To achieve much faster AG release, cohomogenized superdisintegrants at a level of 20% must be used along with 25% HPMC. Compared with water-soluble dispersants like mannitol and lactose, superdisintegrants with high swelling capacity were much more effective dispersants for enhancing fast redispersion/dissolution of AG-NC-SD via a swelling-triggered erosion/disintegration mechanism. Surfactant-free AG-NC-SD with 15% cohomogenized sodium carboxymethyl starch combined with 15% HPMC and 10% lactose enhanced the dissolution further, without comprising drug loading, exhibited a barely compromised dissolution rate compared to precursor NC suspensions (f2>50), and possessed drug loading up to 67.83%±1.26%. The pharmacokinetics results also demonstrated that the AG-NC-SD significantly improved the bioavailability in vivo of AG (P<0.05), compared with to the coarse AG. CONCLUSION: This study illustrates that a quickly dissolving, high drug load, surfactant-free NC-SD can be prepared by using a superdisintegrant as codispersant, and provides a feasible strategy to improve the oral bioavailability of poorly soluble drugs.

Investigation of potential toxic components based on the identification of Genkwa Flos chemical constituents and their metabolites by high-performance liquid chromatography coupled with a Q Exactive high-resolution benchtop quadrupole Orbitrap mass spectrometer.

Genkwa Flos, a famous traditional Chinese medicine has been reported to have significant hepatotoxicity. A high-throughput and reliable method was established to explore potential toxic components by high-performance liquid chromatography coupled with a Q Exactive high-performance benchtop quadrupole-Orbitrap mass spectrometer. A total of 68 compounds including 22 chemical components and 46 metabolites were tentatively identified based on the accurately measured mass value, retention time, and fragmentation pattern. Besides, the metabolic pathways of main components in Genkwa Flos were also illustrated. The results indicated that hydroxylation, demethylation, methylation, glucuronidation, sulfation, cysteine conjugation, and glutathione conjugation participated in the metabolic reactions of Genkwa Flos. Moreover, 12 Genkwa Flos chemical components and 26 metabolites were detected in cell lysate, which were considered as the bound components to HL-7702 cells. In view of cell affinity theory, these compounds were preliminarily deduced to be potential toxic ingredients for the hepatotoxicity induced by Genkwa Flos. The results demonstrated that the developed method was a very feasible and efficient approach for the components identification even in the complex matrix. In conclusion, this study will provide a deep insight into the toxic substances of Genkwa Flos and lay a chemical basis for in-depth toxic studies on Genkwa Flos hepatotoxicity.