Juanhong Zhang

Study on detection of mutation DNA fragment in gastric cancer by restriction endonuclease fingerprinting with capillary electrophoresis.

The DNA fragment detection focusing technique has further enhanced the sensitivity and information of DNA targets. The DNA fragment detection method was established by capillary electrophoresis with laser-induced fluorescence detection and restriction endonuclease chromatographic fingerprinting (CE-LIF-REF) in our experiment. The silica capillary column was coated with short linear polyarclarylamide (SLPA) using nongel sieving technology. The excision product of various restricted enzymes of DNA fragments was obtained by REF with the molecular biology software Primer Premier 5. The PBR322/BsuRI DNA marker was used to establish the optimization method. The markers were focused electrophoretically and detected by CE-LIF. The results demonstrate that the CE-LIF-REF with SLPA can improve separation, sensitivity and speed of analysis. This technique may be applied to analysis of the excision product of various restricted enzymes of prokaryotic plasmid (pIRES2), eukaryote plasmid (pcDNA3.1) and the PCR product of codon 248 region of gastric cancer tissue. The results suggest that this method could very sensitively separate the excision products of various restricted enzymes at a much better resolution than the traditional agarose electrophoresis.

Changes of pathological and physiological indicators affecting drug metabolism in rats after acute exposure to high altitude.

High altitude environments cause the human body to undergo a series of pathological, physiological and biochemical changes, which have a certain effect on drug pharmacokinetics. The objective of the present study was to observe changes in factors affecting pharmacokinetics in rats following acute exposure to high altitude and return to low altitude. A total of 21 male Wistar rats were randomly assigned to three groups. The rats in group A were maintained at low altitude in Shanghai, 55 m above sea level; those in group B were acutely exposed to high altitude in Maqu, Gansu, 4,010 m above sea level; and those in group C were acutely exposed to high altitude and then returned to low altitude. Blood was collected from the orbit for the analysis of significant biochemical indicators and from the abdominal aorta for blood gas analysis. Brain, lung and kidney tissues were removed to observe pathological changes. In group B, the pH, buffer base (BB), base excess (BE), total carbon dioxide content (ctCO2), oxygen saturation of arterial blood (sO2), oxygen tension of arterial blood (pO2), serum sodium (Na+) concentration, lactate dehydrogenase (LDH) activity and total protein (TP) level were significantly reduced, and the carbon dioxide tension of arterial blood (pCO2), serum chloride (Cl−) concentration, serum total bilirubin (TBIL) level and alkaline phosphatase (ALP) activity were significantly increased compared with those in group A (P<0.05). In group C, the pH, BB, BE, sO2, pO2, hemoglobin (Hb) level, serum Na+ concentration, LDH activity and TP level were significantly reduced, and the pCO2, serum Cl− concentration, alanine transaminase activity, TBIL and urea levels were significantly increased (P<0.05) compared with those in group A. The Hb and ALP levels in group C were significantly lower than those in group B (P<0.05); and the TP, TBIL and urea levels in group C were significantly higher than those in group B (P<0.05). Pathological observation revealed that the alveolar wall was hyperemic, edematous and incrassate, the alveolar epithelium was hyperplastic and infiltrated with neutrophilic granulocytes and the alveolar septum was widened; brain neurons were edematous with enlarged perivascular spaces, and hippocampal neurons were metamorphic and karyopyknotic; and kidney mesangial cells were hyperplastic, both following acute exposure to high altitude and after returning to low altitude. In conclusion, blood gas indices, biochemical indicators and functions of the heart, liver, kidney were significantly changed, and marked pathological changes occurred in the brain, liver and kidney following acute exposure to high altitude and also after returning to low altitude. These changes are likely to seriously affect the pharmacokinetics of drugs.

The Pharmacokinetics Evaluation and Bioequivalence of new Docetaxel Injections and Taxotere using Healthy Rats

The docetaxel of sterile freeze-dried powder inject ions was new developed injections due to overcome some drawb acks of Taxotere. We has been evaluated the pharmacokine tic properties and bioequivalence of the docetaxel of s terile freeze-dried powder injections and Taxotere by high ly selective and accurate LC-MS/MS method in healthy r ats. The pharmacokinetic parameters and bioequivalence o f two injections were obtained by the profession software (DAS, version 2.0). The 90% CIs for the In-transformed ra tios of C max : AUC 0-t and AUC 0~8 were 101.3%-104.1%, 99.8%- 100.8% and 99.4-100.6%, respectively (all, p < 0.001). In this study, we attained the pharmacokinetic paramet ers of the two injections’, meanwhile docetaxel of sterile freeze- dried powder injections appeared to be bioequivalen t to Taxotere in healthy rats. The result was beneficial ly to further study the pharmacokinetics and bioequivalen ce of the human in the future research.

Determination of rifampicin in rat plasma by modified large-volume direct injection RAM-HPLC and its application to a pharmcokinetic study.

A direct large volume injection high-performance liquid chromatography (HPLC) method with homemade restricted-access media (RAM) pre-column and combined with a column-switching valve was established and developed for determination rifampicin (RIP) in rat plasma. The rat plasma samples (100 μL) were injected directly onto pre-column, where RIP was retained and pre-concentrated, while proteins were washed to waste using a methanol-water (5:95) as the mobile phase at a flow rate of 1 mL/min. Then, by rotation of the switching valve at 5 min, the RIP were eluted from the pre-column and transferred to an Luna C18 analytical column by the chromatographic mobile phase consisting of methanol-acetonitrile-10 mm ammonium format (60:5:35) at a flow rate of 1 mL/min. The total analytical run time was 15 min with UV detection wavelength at 254 nm. Carbamazepine was used as the internal standard. Excellent linear correlation (r = 0.9993) was obtained in the range of 0.25-8 µg/mL for rat plasma. The intra-day and inter-day precisions of RIP were all <5.0%. The recoveries were in the range of from 99.98-113.66% for plasma. This on-line RAM-HPLC method was successfully applied to the pharmacokinetic study of RIP in rat plasma.

[Enantiomeric separation of propranolol by normal phase chiral liquid chromatography coupled, with tandem mass spectrometry].

A rapid and sensitive method was developed and validated using a normal phase liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for determination of propranolol enantiomers in pharmaceuticals. Sample preparation involved a single extraction step by the addition of methanol. Separation of propranolol enantiomers was achieved on a Chiralcel OD-H chiral column using a mobile phase consisting of n-hexane-ethanol-ammonia (70:30:0.4, v/v/v), and the flow rate was 0.40 mL/min for 20 min. The analyte was monitored by tandem mass spectrometry with electrospray positive ionization in multiple reaction monitoring (MRM) mode, using the transitions of m/z 260.2 --> 116.0. Propranolol enantiomers can be completely separated. The linear range was 2.5-1000 microg/L, and the limit of quantification (LOQ) was 2.5 microg/L. The values for within day and between day precisions and accuracies were well within the generally accepted criteria for analytical methods. The relative standard deviations (RSDs) were less than 2.64%, and the recoveries of the two enantiomers were 99.08%-102.58% and 100.21%-103.16%, respectively. The separation method is accurate, convenient, reliable, efficient, and can be subsequently used for quality control of propranolol enantiomers in pharmaceuticals.

Plateau hypoxia attenuates the metabolic activity of intestinal flora to enhance the bioavailability of nifedipine

Abstract Nifedipine is completely absorbed by the gastrointestinal tract and its pharmacokinetics and metabolism may be influenced by microorganisms. If gut microbes are involved in the metabolism of nifedipine, plateau hypoxia may regulate the bioavailability and the therapeutic effect of nifedipine by altering the metabolic activity of the gut microbiota. We herein demonstrated for the first time that gut flora is involved in the metabolism of nifedipine by in vitro experiments. In addition, based on the results of 16S rRNA analysis of feces in rats after acute plateau, we first confirmed that the plateau environment could cause changes in the number and composition of intestinal microbes. More importantly, these changes in flora could lead to a slower metabolic activity of nifedipine in the body after an acute plateau, resulting in increased bioavailability and therapeutic efficacy of nifedipine. Our research will provide basis and new ideas for changes in the fecal flora of human acutely entering the plateau, and contribute to rational drug use of nifedipine.

Pharmacokinetic changes of norfloxacin based on expression of MRP2 after acute exposure to high altitude at 4300 m

BACKGROUND This study was to investigate the influence of physiological changes and the expression of MRP2 efflux transporter on the pharmacokinetics of norfloxacin after acute exposure to high altitude 4300m. METHODS AND RESULTS The rats were randomly divided into high altitude group and plain group. Blood gas and biochemical analysis showed that the physiological parameters significantly changed at high altitude. The mRNA and protein expression of MRP2 in high altitude group were higher than plain group in rat small intestine and kidney, while was reduced in rat liver. The AUC, Ka and Cmax of norfloxacin were significantly reduced in high altitude group (p<0.05). However, the MRT, CL, t1/2 and Vd were significantly increased (p<0.05). CONCLUSIONS These results indicate that physiological indicators and expression levels of drug transporters MRP2 are changed in responded to high altitude, to severely affect norfloxacin pharmacokinetics. These changes may provide basis and new ideas to adjust the dosage and administration, so as to promote rational drug use in the high altitude.

[Online enrichment ability of restricted-access column coupled with high performance liquid chromatography by column switching technique for benazepril hydrochloride].

The online enrichment ability of the restricted-access media (RAM) column coupled with high performance liquid chromatography by column switching technique for benazepril hydrochloride in plasma was studied. The RAM-HPLC system consisted of an RAM column as enrichment column and a C18 column as analytical column coupled via the column switching technique. The effects of the injection volume on the peak area and the systematic pressure were studied. When the injection volume was less than 100 microL, the peak area increased with the increase of the injection volume. However, when the injection volume was more than 80 microL, the pressure of whole system increased obviously. In order to protect the whole system, 80 microL was chosen as the maximum injection volume. The peak areas of ordinary injection and the large volume injection showed a good linear relationship. The enrichment ability of RAM-HPLC system was satisfactory. The system was successfully used for the separation and detection of the trace benazepril hydrochloride in rat plasma after its administration. The sensitivity of HPLC can be improved by RAM pre-enrichment. It is a simple and economic measurement method.

Gut microbiota modulates drug pharmacokinetics

Abstract Gut microbiota, one of the determinants of pharmacokinetics, has long been underestimated. It is now generally accepted that the gut microbiota plays an important role in drug metabolism during enterohepatic circulation either before drug absorption or through various microbial enzymatic reactions in the gut. In addition, some drugs are metabolized by the intestinal microbiota to specific metabolites that cannot be formed in the liver. More importantly, metabolizing drugs through the gut microbiota prior to absorption can alter the systemic bioavailability of certain drugs. Therefore, understanding intestinal flora-mediated drug metabolism is critical to interpreting changes in drug pharmacokinetics. Here, we summarize the effects of gut microbiota on drug pharmacokinetics, and propose that the influence of intestinal flora on pharmacokinetics should be organically related to the therapeutic effects and side effects of drugs. More importantly, we could rationally perform the strategy of intestinal microflora-mediated metabolism to design drugs.

Effects of Gut Microbiota on Drug Metabolism and Guidance for Rational Drug Use under Hypoxic Conditions at High Altitudes

BACKGROUND Modern features of drug development such as low permeability, low solubility, and improved release affect the interplay of the gut microbiota and drug metabolism. In recent years, studies have established the impact of plateau hypoxia on gut microbiota, where drug use by plateau populations is affected by hypoxia- induced changes in intestinal microflora-mediated drug metabolism. METHODS In this review, we summarized the effects of gut microbiota on drug metabolism, and of plateau hypoxia on the intestinal flora, with the aim of providing guidance for the rational use of drugs in high-altitude populations. RESULTS The evidence clearly shows that alterations in gut microbiota can affect pro-drug activation, drug inactivation, and the biotransformation of xenobiotics. Additionally, plateau hypoxia alters drug metabolism by affecting intestinal flora. CONCLUSION This review provides an overview of the effects of gut microbiota on drug metabolism and provides guidance for rational drug use under hypoxic conditions at high altitudes.