Search

The influence of genetic polymorphism of metabolic enzymes on drug–drug interactions (DDI) should be thoroughly investigated owing to its remarkable effect on therapeutic treatments that are futile. Here, different from earlier articles focused on various CYP subfamilies and their isoforms, the effect of genetic polymorphs of a particular isoform, CYP2C9, was comprehensively studied. Using diclofenac as probe substrate, the influence of three CYP2C9 variants (CYP2C9*1, *2 and *3) on DDI was conducted from the measurement of inhibitory ability of six drugs towards three variants. A modified LC–MS/MS method according to former report was constructed for the determination of 4ʹ-hydroxydiclofenac in CYP2C9 enzyme incubation system and validated for accuracy, precision, linearity range within the acceptance criteria for regulatory guidelines. After selecting appropriate incubation time and enzyme concentration, the kinetic parameters of three CYP2C9 variants towards diclofenac were examined and half inhibitory concentrations (IC50) of six drugs towards three CYP2C9 variants were determined. IC50 of paroxetine and losartan towards CYP2C9*2 were almost threefold higher and 2.5-fold lower than that towards CYP2C9*1, respectively. IC50 of glibenclamide towards CYP2C9*3 was twofold more than that towards CYP2C9*1. Our results could offer effective guidelines for co-administration of these six drugs with various CYP2C9 substrates in individuals carrying variant CYP2C9 alleles, also broaden the application area of LC–MS/MS. [ABSTRACT FROM AUTHOR]

Huashanshen dripping pill (HSS), a commonly used traditional Chinese medicine, has been widely used in China due to its properties of antiasthma, expectorant, and antitussive. However, it was less in-depth understanding of the chemical composition, their absorption in plasma and potential biological mechanism. In this research, ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was developed to simultaneously identify the chemical constituents and absorption in rat plasma after oral administration of HSS. IgE-sensitized to ovalbumin was induced in BALB/c mice. ELISA and molecular docking analysis were carried out to identify their antiasthmatic mechanisms. As a result, 11 compounds were tentatively characterized by comparing the retention time, ion fragments, and the accurate mass measurement of [M + H]+ ions in the HSS. Two alkaloids including d-anisodamine and l-anisodamine were identified in the rat plasma after oral administration of HSS. The pills alleviated airway inflammation in lung tissues, and inhibited IgE, IL-4, and IL-5 in the serum. Furthermore, there were several amino acid residues involved in anisodamine-targeting receptors of hydrogen bonds and hydrophobic interactions via molecular docking indication. All in all, anisodamine would be the main component in the HSS whose antiasthmatic effects based on the inhibition of IgE, IL-4, and IL-5 production. [ABSTRACT FROM AUTHOR]

Host-gut microbiota metabolic interactions are closely associated with health and disease. A manifestation of such co-metabolism is the vast structural diversity of bile acids (BAs) involving both oxidative stereochemistry and conjugation. Herein, we describe the development and validation of a LC-MS-based method for the analysis of human C24 BA metabolome in serum and urine. The method has high throughput covering the discrimination of oxidative stereochemistry of unconjugated species in a 15-min analytical cycle. The validated quantitative performance provided an indirect way to ascertain the conjugation patterns of BAs via enzyme-digestion protocols that incorporated the enzymes, sulfatase, β-glucuronidase, and choloylglycine hydrolase. Application of the method has led to the detection of at least 70 unconjugated BAs including 27 known species and 43 newly found species in the post-prandial serum and urine samples from 7 nonalcoholic steatohepatitis patients and 13 healthy volunteers. Newly identified unconjugated BAs included 3α, 12β-dihydroxy-5β-cholan-24-oic acid, 12α-hydroxy-3-oxo-5β-cholan-24-oic acid, and 3α, 7α, 12β-trihydroxy-5β-cholan-24-oic acid. High-definition negative fragment spectra of the other major unknown species were acquired to facilitate future identification endeavors. An extensive conjugation pattern is the major reason for the “invisibility” of the newly found BAs to other common analytical methods. Metabolomic analysis of the total unconjugated BA profile in combination with analysis of their conjugation patterns and urinary excretion tendencies have provided substantial insights into the interconnected roles of host and gut microbiota in maintaining BA homeostasis. It was proposed that the urinary total BA profile may serve as an ideal footprint for the functional status of the host-gut microbial BA co-metabolism. In summary, this work provided a powerful tool for human C24 BA metabolome analysis that bridges the gap between GC-MS techniques in the past age and LC-MS techniques currently prevailing in biomedical researches. Further applications of the present method in clinical, translational research, and other biomedical explorations will continue to boost the construction of a host-gut microbial co-metabolism network of BAs and thus facilitate the decryption of BA-mediated host-gut microbiota crosstalk in health and diseases.ᅟ [ABSTRACT FROM AUTHOR]

Bile acids (BAs) are cholesterol metabolites with important biological functions. They undergo extensive host-gut microbial co-metabolisms during the enterohepatic circulation, creating a vast structural diversity and resulting in great challenges to separate and detect them. Based on the bioanalytical reports in the past decade, this work developed three chromatographic gradient methods to separate a total of 48 BA standards on an ethylene-bridged hybrid (BEH) C18 column and high-strength silica (HSS) T3 column and accordingly unraveled the factors affecting the separation and detection of them by liquid chromatography coupled with mass spectrometry (LC-MS). It was shown that both the acidity and ammonium levels in mobile phases reduced the electrospray ionization (ESI) of BAs as anions of [M−H], especially for those unconjugated ones without 12-hydroxylation. It was also found that the retention of taurine conjugates on the BEH C18 column was sensitive to the strength of formic acid and ammonium in mobile phases. By using the volatile buffers with an equivalent ammonium level as mobile phases, we comprehensively demonstrated the effects of the elution pH value on the retention behaviors of BAs on both the BEH C18 column and HSS T3 column. Based on the retention data acquired on a C18 column, we presented the ionization constants (p K ) of various BAs with the widest coverage beyond those of previous reports. When we made attempts to establish the structure-retention relationships (SRRs) of BAs, the lack of discriminative structural descriptors for BA stereoisomers emerged as the bottleneck problem. The methods and results presented in this work are especially useful for the development of reliable, sensitive, high-throughput, and robust LC-MS bioanalytical protocols for the quantitative metabolomic studies. [ABSTRACT FROM AUTHOR]

Cerebralcare granule (CG) is a preparation of Traditional Chinese Medicine that widely used in China. It was approved by the China State Food and Drug Administration for treatment of headache and dizziness associated with cerebrovascular diseases. In the present study, we aimed to investigate whether CG had protective effect against d-galactose (gal)-induced memory impairment and to explore the mechanism of its action. d-gal was administered (100 mg/kg, subcutaneously) once daily for 8 weeks to induced memory deficit and neurotoxicity in the brain of aging mouse and CG (7.5, 15, and 30 g/kg) were simultaneously administered orally. The present study demonstrates that CG can alleviate aging in the mouse brain induced by d-gal through improving behavioral performance and reducing brain cell damage in the hippocampus. CG prevents aging mainly via suppression of oxidative stress response, such as decreasing NO and MDA levels, renewing activities of SOD, CAT, and GPx, as well as decreasing AChE activity in the brain of d-gal-treated mice. In addition, CG prevents aging through inhibiting NF-κB-mediated inflammatory response and caspase-3-medicated neurodegeneration in the brain of d-gal treated mice. Taken together, these data clearly demonstrates that subcutaneous injection of d-gal produced memory deficit, meanwhile CG can protect neuron from d-gal insults and improve memory ability. [ABSTRACT FROM AUTHOR]

Nine novel process impurities of N-[ N-benzoyl- O-(2-dimethylaminoethyl)- l-tyrosyl]- l-phenylalaninol ( Y101) observed during the laboratory optimization and later during its bulk synthesis are described in this article. The impurities were monitored by HPLC, and their structures were tentatively assigned on the basis of fragmentation patterns in LC−MS/MS and NMR spectroscopies. All of the impurities were synthesized, and their assigned constitutions were confirmed by co-injection in HPLC. In addition to the formation, synthesis, and characterization, the strategy for minimizing these impurities to a level accepted by the International Conference on Harmonisation (ICH) was also described. [ABSTRACT FROM AUTHOR]

Purpose: To develop a new bionic system from an existing drug dissolution/absorption simulating system (DDASS) to simultaneously predict the release and absorption of enteric-coated formulations. Methods: In accordance with the pH-dependent characteristics of enteric-coated formulations, the modified DDASS was designed to effectively imitate the pH change process of the formulations' transfer from stomach to intestine in vivo. Omeprazole enteric-coated tablets were chosen as the model drug to verify the rationality and feasibility of the modified DDASS. The correlations between USP I system release and beagle dog absorption, as well as between modified DDASS elution/permeation and beagle dog absorption, were investigated by linear and nonlinear regression analyses, respectively. Results: In vitro- in vivo correlation between the modified DDASS elution/permeation method and beagle dog absorption was higher than between the USP I system release and beagle dog absorption in both analytical methods. The ratio of first-order permeation rate constant to first-order release rate constant was consistent with that from modified DDASS. Conclusions: The modified DDASS provided more information than the USP I system did in the evaluation of enteric-coated formulations. The proposed bionic system model could serve as a new method for improving drug effectiveness. [ABSTRACT FROM AUTHOR]

More and more herbal medicines are found to be the substrates of drug transporters. In this paper, chromatography/tandem mass spectrometry (LC/MS/MS) combined with radioisotope method was used for the quantification of catalpol, a traditional Chinese medicine, to study the affinity relationship between herbal medicines and transporters. Catalpol uptake experiment was carried out by using several transporters (OAT1, OCT2, OAT3, OATP1B1 and OATP2B1). And samples were precipitated with methanol and quantified with LC/MS/MS. The results show that catalpol has a good affinity with OCT2-transfected S2 cells. After studying drug-drug interaction between catalpol and C-tetraethylammonium (TEA), we found that catalpol is able to facilitate TEA transport mediated by OCT2, suggesting that catalpol could probably be a new promoter of OCT2. [ABSTRACT FROM AUTHOR]

rapid and sensitive liquid chromatography-tandem mass spectrometric method (LC-MS-MS) has been developed for simultaneous determination of 20( R)-25-methoxyl-dammarane-3,12,20-triol (25-OCH-PPD) and its active metabolite, 20( R)-dammarane-3,12,20,25-tetrol (25-OH-PPD) in beagle dog's plasma. Diazepam was employed as an internal standard. Sample preparation involved a liquid-liquid extraction with diethyl ether and dichloromethane (3:2, v/v). Chromatographic separation was achieved on a Phenomenex C material (50 × 2.0 mm i.d., 4 μm) using methanol-water-formic acid (124:26:0.3, v/v/v) as the mobile phase at a flow rate of 0.25 mL min. The present method exhibited good linearity over the concentration range of 2-500 ng mL for 25-OCH-PPD and 2-60 ng mL for 25-OH-PPD. The lower limit of quantification was 2 and 2 ng mL for 25-OCH-PPD and 25-OH-PPD, respectively. The intra- and inter-day precision values for 25-OCH-PPD and 25-OH-PPD met the requirements of the FDA guidance, and the accuracy (RE) ranged from −4.1 to 5.6% calculated from QC samples. No endogenous compound was found to interfere with the analysis. 25-OCH-PPD and 25-OH-PPD were stable during all storage, pretreatment and analytical periods. The validated method was successfully used to study the pharmacokinetics of 25-OCH-PPD in beagle dogs after oral administration at a dose of 12 mg kg. [ABSTRACT FROM AUTHOR]

Recent studies suggested that the histidine residues at 118 and 122 play an important role for the toxicity of staphylococcal enterotoxin C subtype 2 (SEC2), and the substitutions of both histidines with alanine can severely impair the fever activity of SEC2. We hypothesized that promising SEC2 antitumor agent with low toxicity and enhanced superantigen activity can be constructed by introducing related mutations at protein functional sites of SEC2. We showed that the SEC2 mutants H122A and H118A/H122A exhibited improved superantigen activity after introducing the point mutations at Thr20 and Gly22. A resultant mutant, named as SAM-3, has considerable abilities to inhibit the growth of H22 and Hepa1-6 tumor cells in vitro and colon 26 solid tumor in vivo. Furthermore, SAM-3 also exhibits significantly reduced toxicity compared with native SEC2. The study provides a novel strategy for designing promising superantigen immunotherapeutic agent. The constructed SEC2 mutant SAM-3 can be used as a powerful candidate for cancer immunotherapy and could compensate the deficiency caused by toxicity of native SEC2 in clinic. [ABSTRACT FROM AUTHOR]

5-Fluorouracil (5-FU) has a broad spectrum of anti-tumor activity, widely applied to the treatment of cancers. However, it is necessary to determine the plasma concentration of 5-FU in clinical practice due to its narrow therapeutic index. Therefore, a simple, economic and sensitive high-performance liquid chromatography (HPLC) method was developed and validated for the determination of 5-FU in human plasma. Ethyl acetate was chosen as extraction reagent. Chromatographic separation was performed on a Diamonsil C18 column (250 mm × 4.6 mm i.d., 5 μm) with the mobile phase consisting of methanol and 20 mmol/L ammonium formate using a linear gradient elution at a flow rate of 0.8 mL/min. 5-FU and 5-bromouracil (5-BU) were detected by UV detector at 265 nm. The calibration curve was linear over the concentration range of 5–500 ng/mL and the correlation coefficient was not less than 0.992 6 for all calibration curves. The intra- and inter-day precisions were less than 10.5% and 4.3%, respectively, and the accuracy was within ±3.7%. The recovery at all concentration levels was 80.1±8.6%. 5-FU was stable under possible conditions of storing and handling. This method is proved applicable to therapeutic drug monitoring and pharmacokinetic studies of 5-FU in human. [ABSTRACT FROM AUTHOR]

To compare the pharmacokinetics and pharmacodynamics of PEG30-rhG-CSF administered to beagle dogs at three diff erent dosages with PEG20-rhG-CSF administered at one dosage, and to provide an experimental basis for clinical trials. Beagle dogs received single, subcutaneous doses of PEG30-rhG-CSF at 100, 200 and 400 μg/kg or PEG20-rhG-CSF at 200 μg/kg. PEG30-rhG-CSF and PEG20-rhG-CSF concentrations in serum were analyzed using an enzyme-linked immunosorbent assay (ELISA). WBC, ANC and PLT counts of whole blood samples were measured using fully automated analytic instrumentation. Pharmacokinetic and pharmacodynamic parameters were calculated using DAS 2.0 statistical analysis so ware. The pharmacokinetic parameters of PEG30-rhG-CSF calculated from the serum concentration data determined by ELISA were as follows: the mean elimination half-life (t1/2ke) was 40.6 h (33.5∼45.4 h); the mean time to reach peak concentration (Tmax) was 19.2 h (11.7∼24.0 h); the drug clearance from the serum (CL) was decreased with increasing doses; the peak concentration (Cmax) and the area under the serum concentration-time curve (AUC) were increased with increasing doses. For PEG20-rhG-CSF, the half-life was shorter (12 h) and Tmax was achieved much earlier (10 h) relative to PEG30-rhG-CSF. The AUC of PEG30-rhG-CSF was much greater than that of PEG20-rhG-CSF, and the relative bioavailability with a subcutaneous injection was 158.7%. Administration of single doses of PEG30-rhG-CSF resulted in substantial increases in the absolute neutrophil count (ANC). The time to reach ANCmax (ANCTmax) was 72 h. The maximum observed absolute neutrophil counts (ANCmax) and the area over the baseline effect curve (AOBEC) was increased with increasing doses. The effect-elimination half-life (t1/2E) ranged from 60 h to 80 h after subcutaneous administration. The PLT count was slightly elevated 8∼12 h after s.c. injection, and declined after 24 h. The mean elimination half-life of PEG30-rhG-CSF was longer than that of PEG20-rhG-CSF at the same dose, and the other main pharmacokinetic and pharmacodynamic parameters of PEG30-rhG-CSF, including Cmax, ANCmax, AUC and AOBEC were much greater than those following PEG20-rhG-CSF injection. [ABSTRACT FROM AUTHOR]