Xiping Li

Protective Effect of Calculus Bovis Sativus on Dextran Sulphate Sodium-Induced Ulcerative Colitis in Mice.

Calculus Bovis Sativus (CBS) is a commonly used traditional Chinese medicine, which has been reported to exhibit antispasmodic, fever-reducing, anti-inflammatory, and gallbladder-repairing effects. The present study aims to investigate the protective effect of CBS on dextran sulphate sodium- (DSS-) induced ulcerative colitis (UC) in mice. C57BL/6 male mice were exposed to 5% DSS in drinking water. CBS was given orally at 50 and 150 mg/kg once per day for 7 days. Body weight, disease activity index (DAI), colon length, colonic myeloperoxidase (MPO) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) and nitric oxide (NO) levels were measured. Administration of CBS significantly reserved these changes, decreased the MPO activity and MDA and NO level, and increased the SOD activity in the colon tissue. Histological observation suggested that CBS alleviated edema, mucosal damage, and inflammatory cells infiltration induced by DSS in the colon. Moreover, CBS significantly downregulated the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin- (IL-) 1β and IL-6 in the colon tissue. Our data suggested that CBS exerted protective effect on DSS-induced UC partially through the antioxidant and anti-inflammatory activities.

Evaluation of intestinal absorption of amtolmetin guacyl in rats: breast cancer resistant protein as a primary barrier of oral bioavailability.

AIMS The purpose of the present study was to investigate the role of efflux transporters on the intestinal absorption of amtolmetin guacyl (MED-15). MAIN METHODS The effects of P-glycoprotein (P-gp), multiple resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) inhibitors on intestinal absorption amount of MED-5 (tolmetin-glycine amide derivative), the metabolite formed from MED-15 in the intestinal epithelial cells were studied in the in vitro everted gut sac experiments. Moreover, the in situ single-pass intestine perfusion was adopted to clarify the role of efflux transporters in excreting MED-5 in knockout mice. The plasma concentration of MED-5 and tolmetin, the metabolite formed from MED-5 was determined in Bcrp1 knockout mice and wild-type mice. KEY FINDINGS BCRP inhibitor Ko143 (50 μM and 100 μM) significantly increased the intestinal absorption amount in jejunum, ileum and colon (p<0.05). However, no effect was observed in the presence of P-gp inhibitor verapamil and MRP2 inhibitor MK571 in each intestinal segment. Furthermore, the plasma concentration MED-5 and tolmetin, metabolites of MED-15, increased 2-fold and 4-fold, respectively, in Bcrp1 knockout mice compared with wild-type mice after the single-pass perfusion of small intestine with MED-15. SIGNIFICANCE It may be concluded that BCRP plays an important role in the intestinal efflux of MED-5 and limits the bioavailability after oral administration of MED-15.

Beneficial effect of Calculus Bovis Sativus on 17α-ethynylestradiol-induced cholestasis in the rat.

AIMS Calculus Bovis Sativus (CBS) shares similar pharmacological effects with Calculus Bovis like relieving hepatobiliary diseases. This study aims to investigate the effect and mechanism of CBS on 17α-ethynylestradiol (EE)-induced cholestasis in the rat. MAIN METHODS CBS (50 and 150 mg/kg per day) was intragastrically (i. g.) given to experimental rats for 5 consecutive days in coadministration with EE. The levels of serum biomarkers, hepatic malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were determined by biochemical methods. The bile flow in 2h was measured. The histopathology of the liver tissue was evaluated. The expression of transporter was studied by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. KEY FINDINGS CBS treatment significantly prevented EE-induced increases in serum levels of biomarkers. Decreased bile flow by EE was restored with CBS treatment. The tissue lesions were also relieved with CBS treatment. Western blot studies indicated that EE significantly decreased the protein expression of multidrug resistance-associated protein 2 (Mrp2) and breast cancer resistance protein (Bcrp), but notably increased P-glycoprotein (P-gp) protein, compared with the control group. CBS treatment significantly increased the protein expression of P-gp, Mrp2 and Bcrp compared with the EE group. RT-qPCR studies indicated that EE down-regulated Bcrp at transcriptional level. CBS up-regulated the mRNA expression of P-gp, Mrp2 and Bcrp compared with the EE group. SIGNIFICANCE The present study indicated that CBS exerted a beneficial effect on EE-induced cholestasis in the rat, which may result from its induction of P-gp, Mrp2 and Bcrp expression.

In vitro evaluation of the inhibitory potential of pharmaceutical excipients on human carboxylesterase 1A and 2.

Two major forms of human carboxylesterase (CES), CES1A and CES2, dominate the pharmacokinetics of most prodrugs such as imidapril and irinotecan (CPT-11). Excipients, largely used as insert vehicles in formulation, have been recently reported to affect drug enzyme activity. The influence of excipients on the activity of CES remains undefined. In this study, the inhibitory effects of 25 excipients on the activities of CES1A1 and CES2 were evaluated. Imidapril and CPT-11 were used as substrates and cultured with liver microsomes in vitro. Imidapril hydrolase activities of recombinant CES1A1 and human liver microsomes (HLM) were strongly inhibited by sodium lauryl sulphate (SLS) and polyoxyl 40 hydrogenated castor oil (RH40) [Inhibition constant (Ki) = 0.04±0.01 μg/ml and 0.20±0.09 μg/ml for CES1A1, and 0.12±0.03 μg/ml and 0.76±0.33 μg/ml, respectively, for HLM]. The enzyme hydrolase activity of recombinant CES2 was substantially inhibited by Tween 20 and polyoxyl 35 castor oil (EL35) (Ki = 0.93±0.36 μg/ml and 4.4±1.24 μg/ml, respectively). Thus, these results demonstrate that surfactants such as SLS, RH40, Tween 20 and EL35 may attenuate the CES activity; such inhibition should be taken into consideration during drug administration.

Protective effect of Calculus Bovis Sativus on intrahepatic cholestasis in rats induced by α-naphthylisothiocyanate.

Calculus Bovis Sativus (also referred to as in vitro Cultured Calculus Bovis), an artificial substitute of natural Calculus Bovis (Niuhuang in Chinese, a traditional Chinese medicine), has been widely used to relieve fever, diminish inflammation and normalize gallbladder function in the last decade. This study aims to investigate the effects and possible mechanisms of Calculus Bovis Sativus on α-naphthylisothiocyanate (ANIT)-induced intrahepatic cholestasis in rats. Calculus Bovis Sativus (50, 100 and 200 mg/kg per day) was intragastrically (i.g.) given to experimental rats for seven consecutive days. A single dose of ANIT (100 mg/kg i.g.) was given to rats on the fifth day to induce intrahepatic cholestasis. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkalinephosphatase (ALP) and total bilirubin (TBIL) were determined through biochemical methods. The bile duct was cannulated with a PE 10 polyethylene tube to collect bile for two hours and bile flow was calculated by the weight of each specimen. Moreover, the mechanism of Calculus Bovis Sativus was elucidated by determining liver malondialdehyde (MDA) content and superoxide dismutase (SOD) activity. The biochemical observations were supplemented by histopathological examinations. Our results showed that Calculus Bovis Sativus (50, 100 and 200 mg/kg) significantly prevented ANIT-induced changes in bile flow and serum levels of ALT, AST, ALP and TBIL. Furthermore, Calculus Bovis Sativus (50, 100 and 200 mg/kg) significantly reduced the elevated hepatic MDA content induced by ANIT and increased the hepatic SOD activity suppressed by ANIT. Accordingly, histopathology of the liver tissue showed that pathological injuries were relieved after Calculus Bovis Sativus (50, 100 and 200 mg/kg) pretreatment. In conclusion, Calculus Bovis Sativus exerted a protective effect on ANIT-induced intrahepatic cholestasis in rats, which may result from the attenuated oxidative damage in liver tissues.

Effects of Andrographolide on the Pharmacokinetics of Aminophylline and Doxofylline in Rats

Andrographolide, which is one of the main pharmaceutical ingredients in traditional Chinese medicine Andrographis paniculata, can clear heat, detoxify human body, cool blood and reduce swelling, etc. Respiratory tract infectious diseases have been treated with the combination of andrographolide and theophyllines clinically. As andrographolide inhibits the CYP1A2 activity in vitro, it potentially interacts with theophyllines that are mainly metabolized by CYP1A2. Therefore, we herein studied the effects of andrographolide on the pharmacokinetics of aminophylline and doxofylline in rats. The blood drug concentrations of aminophylline, doxofylline and its metabolite theophylline were determined by HPLC. The theophylline AUC(0-t) was significantly elevated confronting the combination of andrographolide and aminophylline compared to that of the control group (P<0.05). Meanwhile, when only aminophylline was used, the theophylline clearance rate was significantly higher than those in the case of combination (P<0.05). The pharmacokinetics parameters of doxofylline and its metabolite theophylline in the individual administration group showed no significantly different from that combined with andrographolide. The results suggest that andrographolide and aminophylline should not be simultaneously administered because the former may raise the risks of side effects by inhibiting the clearance of the latter. In contrast, it is more secure to combine doxofylline with andrographolide owing to the almost intact pharmacokinetics.

Chronopharmacodynamics and chronopharmacokinetics of pethidine in mice.

Background Many studies have demonstrated that the pharmacokinetics and pharmacodynamics of analgesic drugs vary according to the circadian time of drug administration. This study aims at determining whether the analgesic effect and pharmacokinetics of pethidine in male BALB/c mice are influenced by administration time. Methods A hot-plate test was used to evaluate the analgesic effect after pethidine (20 mg/kg) or saline injection at different dosing times. Mouse blood samples were collected at different intervals after dosing at 9:00 am and 9:00 pm, and were determined via liquid chromatography–tandem mass spectrometry (LC–MS/MS). Results A significant 24-h rhythm was observed in the latency to thermal response at 30 min after dosing, with the peak during the dark phase and the nadir during the light phase. Tolerance to analgesic effect was produced after chronic pethidine injection at 9:00 am or 9:00 pm, and the recovery from tolerance was faster during the dark phase. The peak concentration (Cmax) and area under the concentration–time curve (AUC) of pethidine and its metabolite norpethidine were significantly higher during the dark phase than during the light phase, but the total serum clearance (CL/F) exhibited the opposite trend. The rhythm of drug plasma concentration was positively correlated with the analgesic effect. Conclusion These results suggest that the pharmacodynamics and pharmacokinetics of pethidine in mice vary significantly according to the dosing time, which implies that the time of administration should be considered in the rational clinical use of pethidine to maximise analgesia and minimise the adverse effects.

Down-regulation of carboxylesterases 1 and 2 plays an important role in prodrug metabolism in immunological liver injury rats.

Liver plays a central role in xenobiotics metabolism, thus affecting the in vivo disposition and therapeutic effects of drugs. Carboxylesterases (CESs), with the main isoforms CES1 and CES2, are important in the metabolism of ester-type prodrugs. However, influences of immunological liver injury on the activity of CES remain undefined. In the present study, we demonstrated treatment with lipopolysaccharide (LPS) suppressed the activities of CES1 and CES2. The decreased activities of CES1 and CES2 were preliminarily assessed by the hydrolysis assay for their common substrate p-nitrophenyl acetate (PNPA) with rat hepatic microsomal enzyme. Subsequently, RT-PCR results showed that the levels of CES1 mRNA and mRNA of CES2 (AB010635) and CES2 (AY034877) in the model group were significantly lower than those of the normal control group (P<0.05). Western blot results showed that the expressions of CES1 and CES2 proteins were decreased (P<0.05). To further clarify the effects of LPS on the metabolic activities of CESs, pharmacokinetic studies were performed in rats by utilizing imidapril and irinotecan (CPT-11) as the specific substrates for CES1 and CES2, respectively. After treatment with LPS, AUC0-∞ and Cmax of imidaprilat were decreased from 2084.86±340.66ng·h(-1)·mL(-1) and 234.66±68.85ng·mL(-1) to 983.87±315.34ng·h(-1)·mL(-1) and 113.1±19.69ng·mL(-1) (P<0.05), respectively. Moreover, AUC0-∞ and Cmax of SN-38 were decreased from 8100±918.6ng·h(-1)·mL(-1) and 144.67±20.28ng·mL(-1) to 3270±500.5ng·h(-1)·mL(-1) and 56.19±10.38ng·mL(-1) (P<0.05), respectively. In summary, immunological liver injury remarkably attenuated the expressions and metabolic activities of CES1 and CES2, which may be associated with the regulatory effects of cytokines under inflammation.

Upregulation of PDZK1 by Calculus Bovis Sativus May Play an Important Role in Restoring Biliary Transport Function in Intrahepatic Cholestasis

Intrahepatic cholestasis is a main cause of hepatic accumulation of bile acids leading to liver injury, fibrosis, and liver failure. Our previous studies proved that Calculus Bovis Sativus (CBS) can restore biliary transport function through upregulating the multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) in 17α-ethynylestradiol- (EE-) induced intrahepatic cholestasis rats. The regulation mechanism of CBS on these transporters, however, remains unclear. This study was designed to evaluate the possible relationship between the effect of CBS on transport activities and the regulation of CBS on the expression of PDZK1, a mainly scaffold protein which can regulate MRP2 and BCRP. Intrahepatic cholestasis model was induced in rats with injection of EE for five consecutive days and then the biliary excretion rates and cumulative biliary excretions were measured. The mRNA and protein expression levels of PDZK1 were detected by reverse transcription-quantitative real-time polymerase chain reaction, western blot, and immunohistochemical analysis. When treated with CBS, cumulative biliary excretions and mRNA and protein expressions of PDZK1 were significantly increased in intrahepatic cholestasis rats. This study demonstrated that CBS exerted a beneficial effect on EE-induced intrahepatic cholestasis rats by restoring biliary transport function, which may result from the upregulation of PDZK1 expression.

Development of a rapid and simple LC-MS/MS method for identification and quality control of natural Calculus bovis and Calculus bovis sativus

Calculus bovis sativus (C. bovis sativus, CBS), with the same pharmacodynamics activities as those of natural C. bovis (CB), has been approved as an ideal substitute in China. However, in the latest edition of the Chinese Pharmacopoeia, only cholic acid and bilirubin are used as the markers for quality control. To overcome current limitations and to further improve its quality control method, a rapid and reliable method was herein developed for simultaneous qualitative and quantitative analyses of taurine and twelve bile acids (BAs) in natural CB and CBS by liquid chromatography coupled with tandem mass spectrometry. Taurine and BAs were separated using a Diamonsil C18 column (150 mm × 2.1 mm i.d., 5 μm) with acetonitrile: water containing 10 mM ammonium acetate (pH 3) as mobile phase in a gradient elution program. Mass spectra were obtained in the negative ion mode through multiple reaction monitoring of respective mass transitions. This method had satisfactory separation efficiency, high sensitivity, little potential interference, and short running time. The average recoveries ranged from 93.0% to 107.3%, with relative standard deviations of less than 9.0% for all thirteen analytes of interest. Taurine and characteristic BAs, especially conjugated BAs, may partially reflect the internal quality of CBS and natural CB. Combining the ratios of cholic acid/deoxycholic acid and unconjugated BAs/conjugated BAs with quantitative data of single BA and chromatographic profiles may be able to control the quality of CBS. In conclusion, this method can be used for explicit identification and quality control of CBS and natural CB.