Cheng-Feng Luo

Pharmacokinetics, tissue distribution and relative bioavailability of puerarin solid lipid nanoparticles following oral administration

Puerarin has various pharmacological effects; however, poor water-solubility and low oral bioavailability limit its clinical utility. A delivery system of solid lipid nanoparticles could enhance its oral absorption. The objective of this study was to investigate the pharmacokinetics, tissue distribution and relative bioavailability of puerarin in rats after a single dose intragastric administration of puerarin solid lipid nanoparticles (Pue-SLNs). The puerarin concentrations in plasma and tissues were determined by rapid resolution liquid chromatography electrospray ionization-tandem mass spectrometry. The C(max) value of puerarin after the administration of Pue-SLNs was significantly higher than that obtained with puerarin suspension (0.33±0.05 μg/mL vs. 0.16±0.06 μg/mL, P<0.01). The T(max) value after the administration of the Pue-SLNs was significantly shorter than that after puerarin suspension administration (40±0 min vs. 110±15.49 min, P<0.01). The AUC(0→t) values of puerarin were 0.80±0.23 mg h/L, and 2.48±0.30 mg h/L after administration of the puerarin suspension and Pue-SLNs, respectively. Following administration of the Pue-SLNs, tissue concentrations of puerarin also increased, especially in the target organs such as the heart and brain. These data suggest that SLNs are a promising delivery system to enhance the oral bioavailability of puerarin.

MicroRNA-26 was decreased in rat cardiac hypertrophy model and may be a promising therapeutic target.

Abstract: MicroRNA (miR)-26 was found to be downregulated in cardiac diseases. In this study, the critical role of miR-26 in myocardial hypertrophy in both in vivo and in vitro was investigated. Sixteen male Wistar rats that underwent sham or transverse abdominal aortic constriction (TAAC) surgery were divided into control or TAAC group. Cardiomyocytes were isolated from neonatal Sprague-Dawley rats. Our study demonstrated that miR-26a/b was downregulated in both TAAC rat model and cardiomyocytes. The results of luciferase assays also suggested that glycogen synthase kinase 3&bgr; (GSK3&bgr;) may be a direct target of miR-26. The overexpression of miR-26 attenuated GSK3&bgr; expression and inhibited myocardial hypertrophy. The downregulation of miR-26 reversed these effects. Furthermore, silence of GSK3&bgr; gene phenocopied the anti-hypertrophy effects of miR-26, whereas overexpression of this protein attenuated the effects of miR-26. Taken together, these data suggest that miR-26 regulates pathological structural changes in the rat heart, which may be associated with suppression of the GSK3&bgr; signaling pathway, and implicate the potential application of miR-26 in diagnosis and therapy of cardiac hypertrophy.

Determination of puerarin in rat plasma by rapid resolution liquid chromatography tandem mass spectrometry in positive ionization mode

A highly sensitive and specific method of rapid resolution liquid chromatography tandem mass spectrometry (RRLC-MS/MS) in positive ionization mode has been developed and validated for pharmacokinetic study of puerarin in rat plasma. Chromatography was carried out on a Zorbax XDB C18 reversed-phase column using a mobile phase comprising a mixture of methanol and 0.05% acetic acid in water (35:65, v/v) with a flow rate of 0.3 mL/min from 0 min to 5.4 min and then 0.6 mL/min from 5.41 min to 12 min. The mass spectrometer operated in ESI positive ionization mode. Multiple reaction monitoring (MRM) was used to measure puerarin and tectoridin (internal standard). The method was sensitive with a detection limit of 0.33 ng/mL. A good linear response was observed over a range of 10-2000 ng/mL in rat plasma. The inter- and intra-day precision ranged from 2.97% to 7.52% and accuracy from 93.70% to 101.60%. This validated method was applied successfully to a pharmacokinetic study in rat plasma after intravenous administration of puerarin. The main pharmacokinetic parameters were as follows: AUC(0→t) 45.37±13.19 (mgh/L), AUC(0→∞) 47.03±14.78 (mgh/L), MRT 1.03±0.46 (h), T(1/2) 1.31±0.31 (h), V(ss) 0.09±0.02 (L), V(z) 0.17±0.04 (L), Cl 0.10±0.04 (L/h).

Metabolic profile of puerarin in rats after intragastric administration of puerarin solid lipid nanoparticles

Puerarin has multiple pharmacological effects and is widely prescribed for patients with cardiovascular diseases including hypertension, cerebral ischemia, myocardial ischemia, diabetes mellitus, and arteriosclerosis. We have successfully prepared puerarin-loaded solid lipid nanoparticles (Pue-SLNs) for oral administration. Pue-SLNs are prepared using monostearin, soya lecithin, and poloxamer 188. SLNs may alter the course of puerarin absorption predominantly to and through lymphatic routes and regions, presumably following a transcellular path of lipid absorption, especially by enterocytes and polar epithelial cells of the intestine. The alteration of absorption might influence the metabolic profile of puerarin when incorporated into SLNs. In the present study, we investigated the metabolic profile of puerarin in rat plasma and urine using rapid resolution liquid chromatography–tandem mass spectrometry after a single-dose intragastric administration of Pue-SLNs in comparison with puerarin suspension. Two glucuronidated metabolites of puerarin, puerarin-4′-O-glucuronide and puerarin-7-O-glucuronide, were detected in rat plasma and urine after intragastric administration of Pue-SLNs, with the latter acting as the major metabolite. Similar results were found in rat plasma and urine after intragastric administration of puerarin suspension. The results suggest that incorporation of puerarin into SLNs does not change either the position of glucuronidation or the metabolic pathway of puerarin in rats.

Enantiospecific determination of naftopidil by RRLC–MS/MS reveals stereoselective pharmacokinetics and tissue distributions in rats

Naftopidil (NAF) is used as a racemate to treat benign prostatic hyperplasia (BPH) and to prevent prostate cancer. However, racemic NAF has low bioavailability; therefore, it is commonly administered at higher clinical dosages compared to other therapeutic BPH drugs. Differences in interactions between individual enantiomers and biological macromolecules may result in variations in pharmacokinetics and dispositions. This study aimed to investigate the pharmacokinetics, bioavailability and tissue distributions of NAF enantiomers in rats after intragastric administration of the individual enantiomers. A rapid and sensitive liquid chromatography coupled with triple-quadrupole mass spectrometric method (RRLC-MS/MS) was developed and validated for determination of NAF enantiomers in rat plasma, tissues, urine and feces. After intragastric administration, S(-)-NAF in plasma [maximum concentration (C(max)) = 186.4 ng/mL, area under the curve from 0 h to 24h (AUC(0-24 h)) = 877.9 ng h/mL] was significantly higher than that of R(+)-NAF (C(max) = 133.2 ng/mL, AUC(0-24 h) = 602.1 ng h/mL). Moreover, S(-)-NAF bioavailability was twice that of R(+)-NAF. R(+)-NAF distributions in the prostate, liver, and kidney were significantly higher than S(-)-NAF distributions (R/S ratios of 3.16, 1.33, and 2.90, respectively). These data reveal the stereoselective pharmacokinetic profiles of the two enantiomers in rats.

Contributions of Nrf2 to Puerarin Prevention of Cardiac Hypertrophy and its Metabolic Enzymes Expression in Rats

Previous evidence has suggested that puerarin may attenuate cardiac hypertrophy; however, the potential mechanisms have not been determined. Moreover, the use of puerarin is limited by severe adverse events, including intravascular hemolysis. This study used a rat model of abdominal aortic constriction (AAC)-induced cardiac hypertrophy to evaluate the potential mechanisms underlying the attenuating efficacy of puerarin on cardiac hypertrophy, as well as the metabolic mechanisms of puerarin involved. We confirmed that puerarin (50 mg/kg per day) significantly attenuated cardiac hypertrophy, upregulated Nrf2, and decreased Keap1 in the myocardium. Moreover, puerarin significantly promoted Nrf2 nuclear accumulation in parallel with the upregulated downstream proteins, including heme oxygenase 1, glutathione transferase P1, and NAD(P)H:quinone oxidoreductase 1. Similar results were obtained in neonatal rat cardiomyocytes (NRCMs) treated with angiotensin II (Ang II; 1 μM) and puerarin (100 μM), whereas the silencing of Nrf2 abolished the antihypertrophic effects of puerarin. The mRNA and protein levels of UGT1A1 and UGT1A9, enzymes for puerarin metabolism, were significantly increased in the liver and heart tissues of AAC rats and Ang II–treated NRCMs. Interestingly, the silencing of Nrf2 attenuated the puerarin-induced upregulation of UGT1A1 and UGT1A9. The results of chromatin immunoprecipitation-quantitative polymerase chain reaction indicated that the binding of Nrf2 to the promoter region of Ugt1a1 or Ugt1a9 was significantly enhanced in puerarin-treated cardiomyocytes. These results suggest that Nrf2 is the key regulator of antihypertrophic effects and upregulation of the metabolic enzymes UGT1A1 and UGT1A9 of puerarin. The autoregulatory circuits between puerarin and Nrf2-induced UGT1A1/1A9 are beneficial to attenuate adverse effects and maintain the pharmacologic effects of puerarin.

Nrf2 Is a Key Regulator on Puerarin Preventing Cardiac Fibrosis and Upregulating Metabolic Enzymes UGT1A1 in Rats

Puerarin is an isoflavone isolated from Radix puerariae. Emerging evidence shown that puerarin possesses therapeutic benefits that aid in the prevention of cardiovascular diseases. In this study, we evaluated the effects of puerarin on oxidative stress and cardiac fibrosis induced by abdominal aortic banding (AB) and angiotensin II (AngII). We also investigated the mechanisms underlying this phenomenon. The results of histopathological analysis, as well as measurements of collagen expression and cardiac fibroblast proliferation indicated that puerarin administration significantly inhibited cardiac fibrosis induced by AB and AngII. These effects of puerarin may reflect activation of Nrf2/ROS pathway. This hypothesis is supported by observed decreases of reactive oxygen species (ROS), decreases Keap 1, increases Nrf2 expression and nuclear translocation, and decreases of collagen expressions in cardiac fibroblasts treated with a combination of puerarin and AngII. Inhibition of Nrf2 with specific Nrf2 siRNA or Nrf2 inhibitor brusatol attenuated anti-fibrotic and anti-oxidant effects of puerarin. The metabolic effects of puerarin were mediated by Nrf2 through upregulation of UDP-glucuronosyltransferase (UGT) 1A1. The Nrf2 agonist tBHQ upregulated protein expression of UGT1A1 over time in cardiac fibroblasts. Treatment with Nrf2 siRNA or brusatol dramatically decreased UGT1A1 expression in puerarin-treated fibroblasts. The results of chromatin immunoprecipitation–qPCR further confirmed that puerarin significantly increased binding of Nrf2 to the promoter region of Ugt1a1. Western blot analysis showed that puerarin significantly inhibited AngII-induced phosphorylation of p38-MAPK. A specific inhibitor of p38-MAPK, SB203580, decreased collagen expression, and ROS generation induced by AngII in cardiac fibroblast. Together, these results suggest that puerarin prevents cardiac fibrosis via activation of Nrf2 and inactivation of p38-MAPK. Nrf2 is the key regulator of anti-fibrotic effects and upregulates metabolic enzymes UGT1A1. Autoregulatory circuits between puerarin and Nrf2-regulated UGT1A1 attenuates side effects associated with treatment, but it does not weaken puerarin’s pharmacological effects.

Human UDP-Glucuronosyltransferase 2B4 and 2B7 Are Responsible for Naftopidil Glucuronidation in Vitro

Naftopidil (NAF) is widely used for the treatment of benign prostatic hyperplasia and prevention of prostate cancer in elderly men. These patients receive a combination of drugs, which involves high risk for drug–drug interaction. NAF exhibits superior efficacy but must be administered at a much higher dosage than other therapeutic drugs. We previously showed that extensive glucuronidation of NAF enantiomers caused poor bioavailability. However, the metabolic pathway and mechanism of action of NAF enantiomer remain to be elucidated. The present study was performed to identify the human UDP-glucuronosyltransferases (UGTs) responsible for the glucuronidation of NAF enantiomers and to investigate the potential inhibition of UGT activity by NAF. The major metabolic sites examined were liver and kidney, which were compared with intestine. Screening of 12 recombinant UGTs showed that UGT2B7 primarily contributed to the metabolism of both enantiomers. Moreover, enzyme kinetics for R(+)-NAF, UGT2B7 (mean Km, 21 μM; mean Vmax, 1043 pmol/min/mg) showed significantly higher activity than observed for UGT2B4 and UGT1A9. UGT2B4 (mean Km, 55 μM; mean Vmax, 1976 pmol/min/mg) and UGT2B7 (mean Km, 38 μM; mean Vmax, 1331 pmol/min/mg) showed significantly higher catalysis of glucuronidation of S(-)-NAF than UGT1A9. In human liver microsomes, R(+)-NAF and S(-)-NAF also inhibited UGT1A9: mean Ki values for R(+)-NAF and S(-)-NAF were 10.0 μM and 11.5 μM, respectively. These data indicate that UGT2B7 was the principal enzyme mediating glucuronidation of R(+)-NAF and S(-)-NAF. UGT2B4 plays the key role in the stereoselective metabolism of NAF enantiomers. R(+)-NAF and S(-)-NAF may inhibit UGT1A9. Understanding the metabolism of NAF enantiomers, especially their interactions with metabolic enzymes, will help to elucidate potential drug–drug interactions and to optimize the administration of this medicine.

MICRORNA-26 REGULATES RAT CARDIAC REMODELLING BY SUPPRESSING GLYCOGEN SYNTHASE KINASE 3β

Objectives MicroRNA-26 (miR-26) was found to be down-regulated in the myocardium in cardiac remodelling animal models. Here we investigated the critical role of miR-26a/b on cardiac remodelling in vivo and in vitro. Methods Rats which underwent sham or transverse abdominal aortic constriction (TAAC) surgery were divided into control and TAAC group. Cardiomyocytes (CMs) and cardiac fibroblasts (CFs) were isolated from neonatal Sprague-Dawley rats. QPCR assay was applied to detect the expression levels of miR-26 a/b in the myocardial tissue and plasma of TAAC rats, and in CMs and CFs treated with angiotesinII(AngII). Gain- and loss-of-function studies were applied through overexpressing or inhibiting miR26a/b or Glycogen Synthase Kinase 3β (GSK3β) by liposomes transfecting. Results The data demonstrated that the expressive levels of miR-26a/b were down-regulated in cardiac tissues and plasma in TAAC rats, moreover in CMs and CFs treated with AngII. Furthermore, overexpression miR26a/b by transfected miR-26a/b mimics in CM or CF inhibited CM hypertrophy or CF collagen synthesis significantly, and down-regulating the expressive levels of miR-26a/b by transfected miR-26a/b inhibitors in CM or CF led to opposite effects, suggesting that miR-26 was an anti-hypertrophy and anti-fibrosis gene. Through luciferase assay our study suggested that Glycogen Synthase Kinase 3β (GSK3β) gene that was negatively regulated by miR-26 in CM and CF may be a direct target of miR-26. Overexpression of miR-26 attenuates the endogenous GSK3β mRNA and protein levels followed by the inhibition of CM hypertrophy and CF collagen synthesis. Down-regulation of miR-26 reversed these effects. Furthermore, silence of GSK3β gene phenocopied the anti-hypertrophy and anti-fibrosis effects of miR-26, whereas overexpression of this protein attenuated the effects of miR-26. Conclusions Our data highlight an important role of miR-26 in the control of pathological structural changes in rat heart, which may associated with suppressing the GSK3β signalling pathway, and implicate the potential application of miR-26 in diagnosis and therapy of cardiac remodelling.