Azilsartan ameliorates ventricular hypertrophy in rats suffering from pressure overload-induced cardiac hypertrophy by activating the Keap1-Nrf2 signalling pathway.

OBJECTIVES: Investigate if azilsartan protects against myocardial hypertrophy by upregulating nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated pathways. METHODS: Abdominal aortic constriction (AAC)-induced cardiac hypertrophy in rats was applied. Azilsartan or vehicle was administered daily for 6 weeks in sham or AAC rats. Cardiac morphology and ventricular function were determined. Azilsartan effects upon neonatal rat cardiomyocyte (NRCM) hypertrophy and molecular mechanisms were studied in angiotensin (Ang) II-stimulated NRCMs in vitro. Nrf2-small interfering RNA (siRNA) was used to knockdown Nrf2 expression. Messenger RNA (mRNA)/protein expression of Kelch-like erythroid cell-derived protein (Keap)1 and Nrf2 and its downstream antioxidant enzymes was determined by real-time reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. KEY FINDINGS: Azilsartan treatment ameliorated cardiac hypertrophy/fibrosis significantly in AAC rats. Azilsartan increased expression of Nrf2 protein but decreased expression of Keap1 protein. Upregulation of protein expression of Nrf2's downstream antioxidant enzymes by azilsartan treatment was observed. Azilsartan inhibited Ang II-induced NRCM hypertrophy significantly and similar effects on the Keap1-Nrf2 pathway were observed in vivo. Nrf2 knockdown markedly counteracted the beneficial effects of azilsartan on NRCM hypertrophy and the Keap1-Nrf2 pathway. CONCLUSIONS: Azilsartan restrained pressure overload-induced cardiac remodelling by activating the Keap1-Nrf2 pathway and increasing expression of downstream antioxidant enzymes to alleviate oxidative stress.

Puerarin ameliorated pressure overload-induced cardiac hypertrophy in ovariectomized rats through activation of the PPARα/PGC-1 pathway.

Estrogen deficiency induces cardiac dysfunction and increases the risk of cardiovascular disease in postmenopausal women and in those who underwent bilateral oophorectomy. Previous evidence suggests that puerarin, a phytoestrogen, exerts beneficial effects on cardiac function in patients with cardiac hypertrophy. In this study, we investigated whether puerarin could prevent cardiac hypertrophy and remodeling in ovariectomized, aortic-banded rats. Female SD rats subjected to bilateral ovariectomy (OVX) plus abdominal aortic constriction (AAC). The rats were treated with puerarin (50 mg·kg-1 ·d-1, ip) for 8 weeks. Then echocardiography was assessed, and the rats were sacrificed, their heart tissues were extracted and allocated for further experiments. We showed that puerarin administration significantly attenuated cardiac hypertrophy and remodeling in AAC-treated OVX rats, which could be attributed to activation of PPARα/PPARγ coactivator-1 (PGC-1) pathway. Puerarin administration significantly increased the expression of estrogen-related receptor α, nuclear respiratory factor 1, and mitochondrial transcription factor A in hearts. Moreover, puerarin administration regulated the expression of metabolic genes in AAC-treated OVX rats. Hypertrophic changes could be induced in neonatal rat cardiomyocytes (NRCM) in vitro by treatment with angiotensin II (Ang II, 1 µM), which was attenuated by co-treatemnt with puerarin (100 µM). We further showed that puerarin decreased Ang II-induced accumulation of non-esterified fatty acids (NEFAs) and deletion of ATP, attenuated the Ang II-induced dissipation of the mitochondrial membrane potential, and improved the mitochondrial dysfunction in NRCM. Furthermore, addition of PPARα antagonist GW6471 (10 µM) partially abolished the anti-hypertrophic effects and metabolic effects of puerarin in NRCM. In conclusion, puerarin prevents cardiac hypertrophy in AAC-treated OVX rats through activation of PPARα/PGC-1 pathway and regulation of energy metabolism remodeling. This may provide a new approach to prevent the development of heart failure in postmenopausal women.

Inhibitory effect of α1D/1A antagonist 2-(1H-indol-3-yl)-N-[3-(4-(2-methoxyphenyl) piperazinyl) propyl] acetamide on estrogen/androgen-induced rat benign prostatic hyperplasia model in vivo.

Benign prostatic hyperplasia (BPH) is a common disorder of the urinary system in aging men. 2-(1H-indol-3-yl)-N-[3-(4-(2-methoxyphenyl) piperazinyl) propyl] acetamide (HJZ-3), which is derived from naftopidil, exhibited 97.7- and 64.6-fold greater inhibitory effects for a1D adrenoceptor than for a1B- and a1A-adrenoceptors in vitro, respectively. To investigate the therapeutic potential for treating BPH, we evaluated the pharmacological activity of HJZ-3. Specifically, we evaluated through estrogen/androgen-induced rat benign prostatic hyperplasia model in vivo. HJZ-3 effectively prevented the progression of rat prostatic hyperplasia by suppressing the increase in prostate index and reducing the quantitative analysis of the relative acinus volume, relative stroma, epithelial volume and epithelial thickness and expression of proliferating cell nuclear antigen and α-smooth muscle actin. HJZ-3 decreased α1A- and α1D-adrenoceptor protein expressions in prostate tissue. HJZ-3 is a good alternative for α1A- and α1D-adrenoceptor blocker. It may relax smooth muscle tone and relieve symptoms of BPH.

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.

Novel naftopidil derivatives containing methyl phenylacetate and their blocking effects on α1D/1A-adrenoreceptor subtypes.

α1-Adrenoceptor (α1-AR) antagonists are considered to be the most effective monotherapy agents for lower urinary tract symptoms associated with benign prostatic hyperplasia (LUTS/BPH). In this study, we synthesized compounds 2-17, which are novel piperazine derivatives that contain methyl phenylacetate. We then evaluated the vasodilatory activities of these compounds. Among them, we found that compounds 2, 7, 12, which contain 2-OCH3, 2-CH3 or 2, 5-CH3, respectively, exhibited potent α1-blocking activity similar to protype drug naftopidil (1). The antagonistic effects of 2, 7, and 12 on the (-)-noradrenaline-induced contractile response of isolated rat prostatic vas deferens (α1A), spleen (α1B) and thoracic aorta (α1D) were further characterized to assess the sub receptor selectivity. Compared with naftopidil (1) and terazosin, compound 12 showed the most desirable α1D/1A subtype selectivity, especially improved α1A subtype selectivity, and the ratios pA2 (α1D)/pA2 (α1B) and pA2 (α1A)/pA2 (α1B) were 17.0- and 19.5-fold, respectively, indicating less cardiovascular side effects when used to treat LUTS/BPH. Finally, we investigated the chiral pharmacology of 12. We found, however, that the activity of enantiomers (R)-12 and (S)-12 are not significantly different from that of rac-12.

Puerarin-7-O-glucuronide, a water-soluble puerarin metabolite, prevents angiotensin II-induced cardiomyocyte hypertrophy by reducing oxidative stress.

This study aimed to investigate the anti-oxidant and anti-hypertrophic effects of puerarin-7-O-glucuronide, a water-soluble puerarin metabolite. The anti-oxidant effects of puerarin-7-O-glucuronide were assessed by measurement of intracellular superoxide levels, total superoxide dismutase (SOD) activity, total anti-oxidant capacity, and glutathione (GSH)/glutathione disulfide (GSSG) ratio in cultured neonatal rat cardiomyocytes (NRCMs) stimulated with the xanthine oxidase (XO)/xanthine (X) system or angiotensin II. The activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and expression of NADPH oxidase subunits p22phox and p47phox were determined. The anti-hypertrophic effects of puerarin-7-O-glucuronide in angiotensin II-challenged NRCMs were characterized by changes in cell morphology and expression of hypertrophic genes. In the pharmacokinetic study, the plasma concentration of puerarin-7-O-glucuronide was determined by rapid resolution-liquid chromatography-tandem mass spectrometry (RR-LC-MS/MS). Puerarin-7-O-glucuronide prevented XO/X-induced increase in intracellular superoxide production and decreases in total SOD activity, GSH/GSSG ratio, and total anti-oxidant capacity. Puerarin-7-O-glucuronide also reversed angiotensin II-induced increases in intracellular superoxide production and NADPH oxidase activity and decreases in total SOD activity. These anti-oxidant effects of puerarin-7-O-glucuronide were accompanied by downregulation of p22phox and p47phox. Furthermore, puerarin-7-O-glucuronide prevented angiotensin II-induced increases in cell surface area and perimeter, as well as changes in Nppa, Myh7, and Myh6. In the pharmacokinetic study, puerarin-7-O-glucuronide was cleared with a half-life of 0.94 h after intravenous administration. Puerarin could be detected in rat plasma, albeit in low concentration, as early as 5 min after intravenous administration of puerarin-7-O-glucuronide. These anti-oxidant and anti-hypertrophic properties of puerarin-7-O-glucuronide were similar to those of its parent compound puerarin. These results support the development of puerarin-7-O-glucuronide as a novel pharmaceutical agent for therapeutic application.

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.

Pharmaceutical evaluation of naftopidil enantiomers: Rat functional assays in vitro and estrogen/androgen induced rat benign prostatic hyperplasia model in vivo.

Naftopidil (NAF) is a α1D/1A adrenoceptor selective drug used for the treatment of both benign prostatic hyperplasia and lower urinary tract symptoms (BPH/LUTS). However, NAF is used as a racemate in clinic. To compare the differences and similarities among two enantiomers and racemate, pharmacological activities were evaluated through rat functional assays in vitro and estrogen/androgen (E/T) induced rat BPH model in vivo. NAF and the two enantiomers showed similar blocking activity on α1 receptor. S-NAF exhibited more α1D/1A adrenoceptor subtype selectivity than R-NAF and the racemate. The selectivity ratios pA2 (α1D)/pA2 (α1B) and pA2 (α1A)/pA2 (α1B) were 40.7- and 16.2-fold, respectively. NAF and its enantiomers effectively prevented the development of rat prostatic hyperplasia via suppressing the increase of the prostatic wet weight, visually. The quantitative analysis of the relative acinus volume, relative stroma volume, relative epithelial volume, epithelial height and expression of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were carried out. S-NAF showed an advantage on the effect of inhibiting prostate wet weight and stroma volume over R-NAF and racemate NAF (P<0.05). Nevertheless, no other significant difference was observed between these two enantiomers. In conclusion, both R-NAF and S-NAF not only relax prostate muscle but also inhibit the prostate growth, thus relieve BPH.

[Pharmacokinetic Effect of Acteoside in Blood Deficiency Rats].

Objective: To study the different pharmacokinetics effect of acteoside extracted from Rehmanniae Radix Preparata in normal and blood deficiency rats. Methods: Injected acetyl phenylhydrazine and cyclophosphamide to make blood deficiency rats models subcutaneously,and gave mice the ethand extracts of Rehmanniae Radix preparata by oral administration,the concentration of acteoside in rats at different time points were detected by HPLC method, pharmacokinetic parameters were calculated by 3p87 software. Results: The determination of acteoside in the linear range were 0. 2 ~ 80 µg / m L, the limit of detection and quantification was 0. 03 and 0. 12µg/m L,respectively. Compared with the normal group,the content of AUC0-tand AUC0-∞of corresponding dose in model group rats increased significantly, and the average dwell time and the elimination half-life prolong significantly. Conclusion: This method has high specificity,high sensitivity and simple operation, which can be used for the determination to pharmacokinetic process of acteoside in blood deficiency model.

[Pharmacokinetic Study on Brucine in Different Administration Methods of Liposome in Rats].

OBJECTIVE: To compare the pharmacokinetic differences of brucine in rats after different administration methods of brucine liposome. METHODS: To determine brucine in rat plasma at different points in time by HPLC after oral administration, intramuscular injection, subcutaneous injection and intravenous injection of brucine liposome, respectively. The pharmacokinetic parameters were calculated and analyzed by DAS 3.0. RESULTS: Compared with other groups, AUC(0 --> t) of subcutaneous injection were higher, C(max) were lower and MRT(0 --> 1), were significantly improved. The pharmacokinetics parameters and absolute bioavailability of brucine show that bioavailability in rats after different administration methods of brucine liposome is subcutaneous injection > intramuscular injection > oral administration.

Simultaneous stereoselective analysis of naftopidil and O-desmethyl naftopidil enantiomers in rat feces using an online column-switching high-performance liquid chromatography method.

A novel online column-switching chiral high-performance liquid chromatography method was developed and validated for the simultaneous determination of naftopidil (NAF) and its O-desmethyl metabolites (DMN) enantiomers in rat feces. Direct and multiple injections of supernatant from rat feces homogenate were allowed through the column-switching system. Analyte extraction was performed on the Capcell Pak mixed-functional column by acetonitrile-phosphate buffer (pH 7.4; 10 mm; 8:92, v/v) flowing at 1 mL/min. Separation of NAF and DMN enantiomers was achieved on the Chiralpak IA column by methanol-acetonitrile-acetate buffer (pH 5.3; 5 mm; 45:33:22, v/v/v) flowing at 0.5 mL/min. The analytes were measured with a fluorescence detector at 290 nm (λ(ex)) and 340 nm (λ(em)). The validated method showed a good linearity [22.5-15,000 ng/mL for (+)-/(-)-NAF; 35-25,000 ng/mL for (+)-/(-)-DMN] and the lowest limits of quantification for NAF and DMN enantiomers were 22.5 and 35 ng/mL, respectively. Both intra- and inter-day variations were <10%. The assay was successfully applied to the fecal excretion of NAF and DMN enantiomers in rat after single oral administration of (±)-NAF. Nonstereoselective excretion of (+)- and (-)-NAF was found in feces, while stereoselective excretion of (+)- and (-)-DMN was observed with higher excretion levels of (+)-DMN, indicating that there may exist stereoselective metabolism for NAF enantiomers.

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).

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.