Inhibitory effects of tanshinones towards the catalytic activity of UDP-glucuronosyltransferases (UGTs).

CONTENTS: Danshen is a popular herb employed to treat cardiovascular and cerebrovascular diseases worldwide. Danshen-drug interaction has not been well studied. OBJECTIVE: The inhibitory effects of four major tanshinones (tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I) on UDP-glucuronosyltransferases (UGTs) isoforms were determined to better understand the mechanism of danshen-prescription drugs interaction. MATERIALS AND METHODS: In vitro recombinant UGTs-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction was employed. Tanshinones (100 µM) was used to perform the initial screening of inhibition capability. High-performance liquid chromatography (HPLC) was used to separate 4-MU and its glucuronide. In vitro-in vivo extrapolation (IV-IVE) was employed to predict in vivo inhibition situation. RESULTS: Cryptotanshinone inhibited UGT1A7 and UGT1A9 with IC50 values of 1.91 ± 0.27 and 0.27 ± 0.03 µM, respectively. Dihydrotanshinone I inhibited UGT1A9-catalyzed 4-MU glucuronidation reaction with the IC50 value of 0.72 ± 0.04 µM. The inhibition of cryptotanshinone towards UGT1A7 and UGT1A9 was best fit to competitive inhibition type, and UGT1A9 was non-competitively inhibited by dihydrotanshinone I. Using in vitro inhibition kinetic parameters (Ki) and in vivo maximum plasma concentration (Cmax) of cryptotanshinone and dihydrotanshinone I, the change of area-under-the-concentration-time curve (AUC) was predicted to be 0.4-4.2%, 3.7-56.3%, and 0.6-6.4% induced by cryptotanshinone and dihydrotanshinone inhibition towards UGT1A7 and UGT1A9, respectively. DISCUSSION AND CONCLUSION: The inhibitory effects of tanshinones towards important UGT isoforms were evaluated in the present study, which provide helpful information for exploring the mechanism of danshen-clinical drugs interaction.

Inhibition of UDP-Glucuronosyltransferases (UGTs) Activity by constituents of Schisandra chinensis.

Structure-activity relationship for the inhibition of Schisandra chinensis's ingredients toward (Uridine-Diphosphate) UDP-glucuronosyltransferases (UGTs) activity was performed in the present study. In vitro incubation system was employed to screen the inhibition capability of S. chinensis's ingredients, and in silico molecular docking method was carried out to explain possible mechanisms. At 100 µM of compounds, the activity of UGTs was inhibited by less than 90% by schisandrol A, schisandrol B, schisandrin, schisandrin C, schisantherin A, gomisin D, and gomisin G. Schisandrin A exerted strong inhibition toward UGT1A1 and UGT1A3, with the residual activity to be 7.9% and 0% of control activity. Schisanhenol exhibited strong inhibition toward UGT2B7, with the residual activity to be 7.9% of control activity. Gomisin J of 100 µM inhibited 91.8% and 93.1% of activity of UGT1A1 and UGT1A9, respectively. Molecular docking prediction indicated different hydrogen bonds interaction resulted in the different inhibition potential induced by subtle structure alteration among schisandrin A, schisandrin, and schisandrin C toward UGT1A1 and UGT1A3: schisandrin A > schisandrin > schisandrin C. The detailed inhibition kinetic evaluation showed the strong inhibition of gomisin J toward UGT1A9 with the inhibition kinetic parameter (Ki ) to be 0.7 µM. Based on the concentrations of gomisin J in the plasma of the rats given with S. chinensis, high herb-drug interaction existed between S. chinensis and drugs mainly undergoing UGT1A9-mediated metabolism. In conclusion, in silico-in vitro method was used to give the inhibition information and possible inhibition mechanism for S. chinensis's components toward UGTs, which guide the clinical application of S. chinensis.

Chirality Influence of Zaltoprofen Towards UDP-Glucuronosyltransferases (UGTs) Inhibition Potential.

Zaltoprofen (ZLT) is a nonsteroidal antiinflammation drug, and has been clinically employed to treat rheumatoid arthritis, osteoarthritis, and other chronic inflammatory pain conditions. The present study aims to investigate the chirality influence of zaltoprofen towards the inhibition potential towards UDP-glucuronosyltransferases (UGTs) isoforms. In vitro a recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation incubation system was employed to investigate the inhibition of (R)-zaltoprofen and (S)-zaltoprofen towards UGT isoforms. The inhibition difference capability was observed for the inhibition of (R)-zaltoprofen and (S)-zaltoprofen towards UGT1A8 and UGT2B7, but not for other tested UGT isoforms. (R)-zaltoprofen exhibited noncompetitive inhibition towards UGT1A8 and competitive inhibition towards UGT2B7. The inhibition kinetic parameters were calculated to be 35.3 µM and 19.2 µM for UGT1A8 and UGT2B7. (R)-zaltoprofen and (S)-zaltoprofen exhibited a different inhibition type towards UGT1A7. Based on the reported maximum plasma concentration of (R)-zaltoprofen in vivo, a high drug-drug interaction between (R)-zaltoprofen and the drugs mainly undergoing UGT1A7, UGT1A8, and UGT2B7-catalyzed glucuronidation was indicated.

Enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7.

UDP-glucuronosyltransferases (UGTs)-catalyzed glucuronidation conjugation reaction plays an important role in the elimination of many important clinical drugs and endogenous substances. The present study aims to investigate the enantioselective inhibition of carprofen towards UGT isoforms. In vitro a recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation incubation mixture was used to screen the inhibition potential of (R)-carprofen and (S)-carprofen towards multiple UGT isoforms. The results showed that (S)-carprofen exhibited stronger inhibition potential than (R)-carprofen towards UGT2B7. However, no significant difference was observed for the inhibition of (R)-carprofen and (S)-carprofen towards other UGT isoforms. Furthermore, the inhibition kinetic behavior was compared for the inhibition of (S)-carprofen and (R)-carprofen towards UGT2B7. A Lineweaver-Burk plot showed that both (S)-carprofen and (R)-carprofen exhibited competitive inhibition towards UGT2B7-catalyzed 4-MU glucuronidation. The inhibition kinetic parameter (Ki ) was calculated to be 7.0 µM and 31.1 µM for (S)-carprofen and (R)-carprofen, respectively. Based on the standard for drug-drug interaction, the threshold for (S)-carprofen and (R)-carprofen to induce a drug-drug interaction is 0.7 µM and 3.1 µM, respectively. In conclusion, enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7 was demonstrated in the present study. Using the in vitro inhibition kinetic parameter, the concentration threshold of (S)-carprofen and (R)-carprofen to possibly induce the drug-drug interaction was obtained. Therefore, clinical monitoring of the plasma concentration of (S)-carprofen is more important than (R)-carprofen to avoid a possible drug-drug interaction between carprofen and the drugs mainly undergoing UGT2B7-catalyzed metabolism.

Ultrastructural observation on pulmonary fibrosis in E9 rats treated with compound Carapax trionycis formula.

OBJECTIVE: To investigate ultrastructural changes in pulmonary tissue of a rat model of pulmonary fibrosis following treatment with compound Carapax trionycis (C. trionycis; Biejia in Chinese) formula. METHODS: Sixty male Sprague-Dawley rats were randomly divided into four groups (n=15): compound C. trionycis formula high-, middle-, and low-dose groups as well as model group. Pulmonary fibrosis was induced by bleomycin. Five rats from each group were sacrificed on day 7, 14 and 28 of the drug treatment, respectively. The pulmonary tissue was harvested followed by hematoxylin-eosin staining and subsequent transmission electron microscopy. The Szapiel's method was used to assess the degree of alveolitis and pulmonary fibrosis. RESULTS: Compared with the model group, the compound C. trionycis formula groups had slighter pulmonary alveolitis after the 7-day treatment and also had alleviated alveolar inflammation and pulmonary fibrosis after the 14-day treatment. After the 28-day treatment, the compound C. trionycis formula groups showed deposition of a small amount of fibrous tissue and lesions occupying less than 21% of the whole lung area, while the model group showed focal or diffuse fibrous deposition, narrow alveolar cavity, disordered lung structure, and lesions in larger than 51% of the whole lung area. CONCLUSIONS: The compound C. trionycis formula can inhibit the proliferation of collagen fibers and resist pulmonary fibrosis.

[Exogenous Nkx2-5 gene expression induces the expression of cardiac markers during P19 cell differentiation in vitro].

OBJECTIVE: To investigate the role of homeobox gene Nkx2-5 in cardiac myogenesis. METHODS: Two P19 cell lines, namely cells transfected with exogenous expression of Nkx2-5 gene and non-transfected cells, were cultured in suspension for 4 days to induce cell aggregation, and the cell aggregates were transferred to the Petri dish for further adherent culture. On days 4, 8, 12 and 16 of adherent culture, the expressions of α-sarcomeric actin (α-SA) and cardiac troponin T (cTnT) protein were detected by immunocytochemistry, and the mRNA expressions of GATA-4, α-myosin heavy chain (α-MHC) and atrial natriuretic factor (ANF) genes by RT-PCR. RESULTS: In the transfected cells, α-SA and cTnT protein expressions were detected on days 8, 12 and 16 of adhere culture, and their expressions increased gradually with time. α-SA and cTnT expression was significantly higher on day 16 than on day 8 of culture (P<0.01). RT-PCR analysis of the transfected cell showed the presence of GATA-4 expression on day 4 of adherent culture, and the expression increased on days 8 and 12 but decreased on day 16. ANF and α-MHC expressions were found on days 8, 12, and 16, increasing gradually over time and showing significant differences from those on day 4 (P<0.05 or P<0.01). The expression of α-MHC was significantly higher on days 12 and 16 than on day 8 (P<0.05 or P<0.01), and ANF expression was significantly higher on day 16 than on days 8 and 12 (P<0.01). The non-transfected cells were negative for the expressions of all these genes. CONCLUSION: Exogenous expression of Nkx2-5 gene can induce P19 cells to express cardiac markers in vitro.

Genistein inhibits carotid sinus baroreceptor activity in anesthetized male rats.

AIM: To study the effect of genistein (GST) on carotid baroreceptor activity (CBA). METHODS: The functional curve of carotid baroreceptor (FCCB) was constructed and the functional parameters of carotid baroreceptor were measured by recording sinus nerve afferent discharge in anesthetized male rats with perfused isolated carotid sinus. RESULTS: GST at 50, 100, and 200 micromol/L inhibited the CBA, which shifted FCCB to the right and downward, with a marked decrease in peak slope and peak integral value of carotid sinus nerve discharge in a concentration-dependent manner. Pretreatment with 100 micromol/L NG-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, did not affect the effect of GST on CBA. Pretreatment with 500 nmol/L Bay K8644, an agonist of calcium channels, could completely abolish the effect of GST on CBA. A potent inhibitor of tyrosine phosphatase, sodium orthovanadate (1 mmol/L), could attenuate the inhibitory effect of GST. CONCLUSION: GST inhibits CBA, and the effect may be mediated by protein tyrosine kinase inhibition and a decrease in Ca2+ influx through the stretch-activated channels.