Interactions of oleanane pentacyclic triterpenoids with human organic anion transporting polypeptide 1B1 and 1B3.

Oleanane pentacyclic triterpenoids have been widely used in clinical practice. However, studies on their interactions with hepatic transporters remain limited. In this study, we systematically investigated the inhibitory effects of 14 oleanane pentacyclic triterpenoids on organic anion transporting polypeptide 1B1 and 1B3 (OATP1B1 and OATP1B3), two liver-specific uptake transporters. Through fluorescence-based cellular uptake assays, we identified three potent OATP1B1 inhibitors (saikosaponin B1, saikosaponin A and 18ß-glycyrrhetinic acid) and five potent OATP1B3 inhibitors (echinocystic acid, 3-oxo-16α-hydroxy-olean-12-en-28ß-oic acid, chikusetsu saponin IVa, saikosaponin B1 and 18ß-glycyrrhetinic acid). Structural analysis revealed that free oleanane triterpenoids inhibited OATP1B1/1B3 more potently than triterpene glycosides. Despite their similar structures, 18ß-glycyrrhetinic acid exhibited much stronger inhibition on OATP1B1/1B3 than 18α-glycyrrhetinic acid, while both were substrates of OATP1B3. Interestingly, OATP1B3 overexpression significantly increased reactive oxygen species (ROS) levels in HepG2 cells after treatment with 18ß-glycyrrhetinic acid. To conclude, this study highlights the potential interactions of oleanane pentacyclic triterpenoids with OATP1B1/1B3, and provides novel insights into the anti-cancer activity of 18ß-glycyrrhetinic acid.

Potential Involvement of Organic Anion Transporters in Drug Interactions with Shuganning Injection, a Traditional Chinese Patent Medicine.

Traditional Chinese medicine injections have been widely used in China for the treatment of various diseases. Transporter-mediated drug-drug interactions are a major contributor to adverse drug reactions. However, the research on transporter-mediated Traditional Chinese medicine injection-drug interactions is limited. Shuganning injection is a widely used Traditional Chinese medicine injection for treating various liver diseases. In this study, we investigated the inhibitory effect of Shuganning injection and its four main ingredients (baicalin, geniposide, chlorogenic acid, and oroxylin A) on 9 drug transporters. Shuganning injection strongly inhibited organic anion transporter 1 and organic anion transporter 3 with IC50 values < 0.1% (v/v), and moderately inhibited organic anion transporter 2, organic anion transporting-polypeptide 1B1, and organic anion transporting-polypeptide 1B3 with IC50 values < 1.0%. Baicalin, the most abundant bioactive ingredient in the Shuganning injection, was identified as both an inhibitor and substrate of organic anion transporter 1, organic anion transporter 3, and organic anion transporting-polypeptide 1B3. Oroxylin A had the potential to act as both an inhibitor and substrate of organic anion transporting-polypeptide 1B1 and organic anion transporting-polypeptide 1B3. In contrast, geniposide and chlorogenic acid had no significant inhibitory effect on drug transporters. Notably, Shuganning injection markedly altered the pharmacokinetics of furosemide and atorvastatin in rats. Using Shuganning injection as an example, our findings support the implementation of transporter-mediated Traditional Chinese medicine injection-drug interactions in the development of Traditional Chinese medicine injection standards.

Cytochrome P450-mediated herb-drug interaction (HDI) of Polygonum multiflorum Thunb. based on pharmacokinetic studies and in vitro inhibition assays.

BACKGROUND: Polygonum multiflorum Thunb. (PM) is well known both in China and other countries of the world for its tonic properties, however, it has lost its former glory due to liver toxicity incidents in recent years. PURPOSE: The purpose of this study is to determine whether the occurrence of herb-drug interaction (HDI) caused by PM is associated with cytochrome P450 (CYP450) based on pharmacokinetic studies and in vitro inhibition assays. The objective was to provide a reference for the rational and safe use of drugs in clinical practice. METHODS: In this study, raw PM (R), together with its two processed products which included PM by Chinese Pharmacopoeia (M) and PM by "nine cycles of steaming and sunning (NCSS)" ("9"), were prepared as the main research objects. A method based on fluorescence technology was used to evaluate the inhibition levels of raw and processed PMs, as well as corresponding characteristic compounds on seven recombinant human cytochrome P450s (rhCYP450s). The pharmacokinetics of sulindac (a representative of commonly used nonsteroidal anti-inflammatory drugs) and psoralen (a major compound of Psoralea in combination with PM) in rat plasma were studied when combined with raw and different processed products of PM. RESULTS: The inhibitory level order of the three extracts on major different subtypes of CYP450 (CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, and CYP3A4) was: R > M > "9". However, the inhibition level of R and "9" is higher than that of M on CYP2C9. Further studies showed that trans-THSG and emodin could selectively inhibit CYP3A4 and CYP1A2, respectively. Epicatechin gallate mainly inhibited CYP3A4 and CYP1A2, followed by CYP2C8 and CYP2C9. Genistein mainly inhibited CYP3A4, followed by CYP2C9 and CYP2C8. CYP3A4 and CYP2C9 were also inhibited by daidzein. The inhibitory effects of all the PM extracts were associated with their characteristic compounds. The results of HDI showed that R increased sulindac exposure to rat blood, and R and M increased psoralen exposure to rat blood, which were consistent with corresponding metabolic enzymes. Overall, the in vitro and in vivo results indicated that PM, especially R, would be at high risk to cause toxicity and drug interactions via CYP450 inhibition. CONCLUSION: This study not only elucidates the scientific connotation of "efficiency enhancement and toxicity reduction" of PM by NCSS from the perspective of metabolic inhibition but also contributes to HDI prediction and appropriate clinical medication of PM.

Shunaoxin dropping pill improves cognitive functions of rats with chronic cerebral hypoperfusion via the microbiota-gut-brain axis.

Chronic cerebral hypoperfusion (CCH) is a major risk factor for cognitive decline and degenerative processes. Shunaoxin dropping pill (SNX) has been clinically used to treat cerebrovascular diseases. However, the effect and mechanism of SNX in treating CCH-induced cognitive impairment remain unclear. In this study, CCH was induced in rats using permanent bilateral common carotid artery ligation (2-VO). CCH rats were characterized by impaired spatial learning and memory ability, as well as increased oxidative stress and inflammation in the hippocampus. Additionally, CCH rats had reduced richness and biodiversity of fecal microbiota, which showed a strong correlation with altered serum metabolites. SNX significantly improved the cognitive impairment and restored the dysbiosis of fecal microbiota and serum metabolites in CCH rats. Notably, SNX did not prevent cognitive impairment in antibiotics-treated CCH rats. Our findings suggest that the microbiota-gut-brain axis is a promising therapeutic target for the treatment of CCH-induced cognitive impairment.

Selective Inhibition of Organic Cation Transporter 1 by Benzoylpaeoniflorin Attenuates Hepatic Lipid Accumulation through AMPK Activation.

Organic cation transporter 1 (OCT1) is a liver-specific transporter and plays an essential role in drug disposition and hepatic lipid metabolism. Therefore, inhibition of OCT1 may not only lead to drug-drug interactions but also represent a potential therapy for fatty liver diseases. In this study, we systematically investigated the inhibitory effect of 200 natural products on OCT1-mediated uptake of 4,4-dimethylaminostyryl-N-methylpyridinium (ASP+) and identified 10 potent OCT1 inhibitors. The selectivity of these inhibitors over OCT2 was evaluated using both in vitro uptake assays and in silico molecular docking analyses. Importantly, benzoylpaeoniflorin was identified as the most potent OCT1 inhibitor with the highest selectivity over OCT2. Additionally, benzoylpaeoniflorin prevented lipid accumulation in hepatocytes, with concomitant activation of AMPK and down-regulation of lipogenic genes, such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). To conclude, our findings are of significant value in understanding OCT1-based natural product-drug interactions and provide a natural source of OCT1 inhibitors which may hold promise for treating fatty liver diseases.

Transporter-mediated Natural Product-Drug Interactions.

The increasing use of natural products in clinical practice has raised great concerns about the potential natural product-drug interactions (NDIs). Drug transporters mediate the transmembrane passage of a broad range of drugs, and thus are important determinants for drug pharmacokinetics and pharmacodynamics. Generally, transporters can be divided into ATP binding cassette (ABC) family and solute carrier (SLC) family. Numerous natural products have been identified as inhibitors, substrates, inducers, and/or activators of drug transporters. This review article aims to provide a comprehensive summary of the recent progress on the research of NDIs, focusing on the main drug transporters, such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion transporter 1 and 3 (OAT1/OAT3), organic anion-transporting polypeptide 1B1 and 1B3 (OATP1B1/OATP1B3), organic cation transporter 2 (OCT2), multidrug and toxin extrusion protein 1 and 2-K (MATE1/MATE2-K). Additionally, the challenges and strategies of studying NDIs are also discussed.

3D-QSAR analysis of the interactions of flavonoids with human organic cation transporter 2.

Flavonoids are a class of phenolic and polyphenolic compounds widely distributed in vegetables, fruits, grains and herbs. Organic cation transporter 2 (OCT2) mediates the renal secretion of organic cations and is a key site of drug-drug interactions (DDIs). In this study, we systematically investigated the inhibitory effect of 28 flavonoids on OCT2-mediated uptake of 4-4-dimethylaminostyryl-N-methylpyridinium (ASP+). Among them, scullcapflavone II demonstrated the strongest inhibitory effect on OCT2-mediated uptake of ASP+ (IC50 =11.2 µM) in a competitive manner. Next, 3D-QSAR analyses of flavonoid OCT2 inhibitors were performed using both CoMFA and CoMSIA models. The date revealed that bulky substituents at the C-3 and C-4 positions of ring C as well as the C-7 position of ring A could prevent the interactions of flavonoids with OCT2. In contrast, a hydrophilic and negatively charge substituent on ring A was favorable for the interactions of flavonoids with OCT2. Consequently, baicalin (IC50 =220.2 µM) with a uronic acid substituent on ring A exhibited a stronger inhibition than baicalein (IC50 =294.5 µM); quercetin-3-O-galactoside (IC50 =497.4 µM) was a stronger inhibitor of OCT2 than rhamnetin 3-galactoside (IC50 =1409.0 µM). Taken together, our findings could be valuable in elucidating and predicting the interactions of flavonoids with OCT2.

Wedelolactone protects against cisplatin-induced nephrotoxicity in mice via inhibition of organic cation transporter 2.

The balance of cisplatin uptake and efflux, mediated mainly by organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1), respectively, determines the renal accumulation and nephrotoxicity of cisplatin. Using transporter-mediated cellular uptake assay, we identified wedelolactone (WEL), a medicinal plant-derived natural compound, is a competitive inhibitor of OCT2 and a noncompetitive inhibitor of MATE1. Wedelolactone showed a selectivity to inhibit OCT2 rather than MATE1. Cytotoxicity studies revealed that wedelolactone alleviated cisplatin-induced cytotoxicity in OCT2-overexpressing HEK293 cells, whereas it did not alter the cytotoxicity of cisplatin in various cancer cell lines. Additionally, wedelolactone altered cisplatin pharmacokinetics, reduced kidney accumulation of cisplatin, and ameliorated cisplatin-induced acute kidney injury in the Institute of Cancer Research mice. In conclusion, these findings suggest a translational potential of WEL as a natural therapy for preventing cisplatin-induced nephrotoxicity and highlight the need for drug-drug interaction investigations of WEL with other treatments which are substrates of OCT2 and/or MATE1.

Salidroside Improves Chronic Stress Induced Depressive Symptoms Through Microglial Activation Suppression.

Depression is a severe neurological disorder highly associated with chronic mental stress stimulation, which involves chronic inflammation and microglial activation in the central nervous system (CNS). Salidroside (SLDS) has been reported to exhibit anti-neuroinflammatory and protective properties on neurological diseases. However, the mechanism underlying the effect of SLDS on depressive symptoms has not been well elaborated. In the present study, the effects of SLDS on depressive behaviors and microglia activation in mice CNS were investigated. Behavioral tests, including Forced swimming test (FST), Open field test (OFT) and Morris water maze (MWM) revealed that SLDS treatment attenuated the depressive behaviors in stress mice. SLDS treatment significantly reduced the microglial immunoreactivity for both Iba-1 and CD68, characteristic of deleterious M1 phenotype in hippocampus of stress mice. Additionally, SLDS inhibited microglial activation involving the suppression of ERK1/2, P38 MAPK and p65 NF-κB activation and thus reduced the expression and release of neuroinflammatory cytokines in stress mice as well as in lipopolysaccharide (LPS)-induced primary microglia. Also, SLDS changed microglial morphology, attachment and reduced the phagocytic ability in LPS-induced primary microglia. The results demonstrated that SLDS treatment could improve the depressive symptoms caused by unpredictable chronic stress, indicating a potential therapeutic application of SLDS in depression treatment by interfering microglia-mediated neuroinflammation.

Inhibitory effect of sixteen pharmaceutical excipients on six major organic cation and anion uptake transporters.

To date, relatively little is known about the interactions of pharmaceutical excipients with hepatic and renal drug uptake transporters. The present study was designed to systematically evaluate the effects of 16 commonly consumed excipients on human organic cation transporter 1 and 2 (hOCT1 and hOCT2), human organic anion transporter 1 and 3 (hOAT1 and hOAT3) and human organic anion transporting polypeptide 1B1 and 1B3 (hOATP1B1 and hOATP1B3). The inhibitory effects and mechanisms of excipients on transporters were investigated using in vitro uptake studies, cell viability assays, concentration-dependent studies, and the Lineweaver-Burk plot method. Triton X-100 is a non-competitive inhibitor for all six transporters. Tween 20 inhibits hOCT2, hOAT1, hOAT3, and hOATP1B3 in a mixed way, whereas it competitively inhibits hOATP1B1. The inhibition of Tween 80 is competitive for hOCT2, non-competitive for hOATP1B1 and hOATP1B3, and mixed for hOAT1 and hOAT3. Concentration-dependent studies identify Triton X-100 as a strong inhibitor of hOCT1 and hOCT2 with IC50 values of 20.1 and 4.54 µg/mL, respectively. Additionally, Triton X-100, Tween 20, and Tween 80 strongly inhibit hOAT3 with IC50 values ≤31.0 µg/mL. The present study is significant in understanding the excipient-drug interactions and provides valuable information for excipient selection in drug development.

Comparative toxicity analysis of corannulene and benzo[a]pyrene in mice.

Increasing production of corannulene (COR), a non-planar polycyclic aromatic hydrocarbon (PAH) with promising applications in many fields, has raised a concern about its potential toxic effects. However, no study has been undertaken to evaluate its metabolism and toxicity in mammals. In this study, the acute toxicities of COR in mice were compared with benzo[apyrene (BaP), a typical planar PAH with almost the same molecular weight. After 3-day exposures, the concentrations of COR in both plasma and tissues of mice were higher than that of BaP. However, blood chemistry and tissue weight monitoring showed no observable toxicities in COR-exposed mice. Compared to BaP, exposure to COR resulted in less activation of the aryl hydrocarbon receptor (AhR) and thus less induction of hepatic cytochrome P450 1A(CYP1A) enzymes, which play a critical role in metabolism of both COR and BaP. Additionally, COR also elicited less oxidative stress and microbiota alteration in the intestine than did BaP. RNA-seq analysis revealed that liver transcriptomes are responsive to COR and BaP, with less alterations observed in COR-exposed mice. Unlike BaP, exposure to COR had no effects on hepatic lipid and xenobiotic metabolism pathways. Nonetheless, COR appeared to alter the mRNA expressions of genes involved in carcinogenicity, oxidative stress, and immune-suppression. To conclude, this study for the first time unveils a comparative understanding of the acute toxic effects of COR to BaP in mice, and provides crucial insights into the future safety assessment of COR.

Potent Inhibitors of Organic Anion Transporters 1 and 3 From Natural Compounds and Their Protective Effect on Aristolochic Acid Nephropathy.

Organic anion transporters 1 and 3 (OAT1 and OAT3) play a critical role in renal drug-drug interactions and are involved in the nephrotoxicity of many anionic xenobiotics. To date, relatively little is known about the interaction of natural compounds with OAT1 and OAT3. Of the 270 natural compounds screened in the present study, 21 compounds inhibited OAT1 and 45 compounds inhibited OAT3. Further concentration-dependent studies identified 7 OAT1 inhibitors and 10 OAT3 inhibitors with IC50 values of <10 µM, and most of them were flavonoids, the most commonly ingested polyphenolic compounds in the diet and herbal products. Computational modeling of OAT1 and OAT3 revealed the important residues for the recognition of inhibitors. The two strong OAT inhibitors, namely wedelolactone and wogonin, were evaluated for their in vivo interactions with the OAT substrate aristolochic acid I (AAI), a natural compound causing aristolochic acid-induced nephropathy (AAN) in many species. The cytotoxicity of AAI increased in two OAT-overexpressing cell lines, with more cytotoxicity in OAT1-overexpressing cells, suggesting a more important role of OAT1 than OAT3 in AAN. Both wedelolactone and wogonin markedly increased serum AAI concentrations in AAI-treated rats and ameliorated kidney injuries in AAI-treated mice. To conclude, the present findings are of significant value in understanding natural compound-drug interactions and provide a natural source for developing treatments for AAN.

Involvement of aryl hydrocarbon receptor in the cytotoxicity of corannulene and its derivatives.

Despite numerous studies on the toxicities of planar polycyclic aromatic hydrocarbons (PAHs), very little is known about the toxicological profiles of non-planar PAHs. In the present study, the cytotoxicity of corannulene (COR), a typical bowl-shaped PAH with a myriad of applications in the area of material chemistry, and benzo[a]pyrene (BaP), a typical planar PAH with similar molecular weight, were systematically compared in various cell lines. Compared with BaP, exposure to COR resulted in less cytotoxic responses in both human (HepG2) and murine (Hepa1-6) hepatoma cells, which was characterized with a slower cellular accumulation as well as a weaker induction of cytochrome P450 1 (CYP1/Cyp1) isozymes. Knockdown of aryl hydrocarbon receptor (AhR) by siRNA attenuated the inductive effect of COR on CYP1A/Cyp1a mRNA levels in these two cell lines. Further analysis revealed that derivatization greatly influenced the cytotoxicity of COR, which was positively correlated with their binding affinities to the AhR, as demonstrated by in silico molecular docking. Overall, these results suggest that AhR appears to be involved in the cytotoxic responses of COR and its derivatives, providing a fundamental understanding of the biological effects of bowl-like PAHs.

Paradoxical Effects of Emodin on ANIT-Induced Intrahepatic Cholestasis and Herb-Induced Hepatotoxicity in Mice.

Emodin is an active ingredient in many herbal medicines and has a broad spectrum of pharmacological activities. The current data indicate that emodin exerts its beneficial effect on alpha-naphthylisothiocyanate (ANIT)-induced intrahepatic cholestasis through its anti-oxidant and anti-inflammatory activities. Emodin has little effect on the concentrations of bile acids (BAs) in livers of ANIT-treated mice. Instead, emodin shows a potential pro-cholestatic effect by interfering with the crosstalk between AMP-activated protein kinase (AMPK) and farnesoid X receptor (Fxr) in the liver, which leads to a suppression of bile salt export pump (Bsep). Two emodin-containing herbs, namely Polygonum multiflorum (PM) and Semen cassiae (SC), markedly aggravate the intrahepatic cholestasis in ANIT-treated mice. SC interferes with the AMPK-Fxr crosstalk and suppresses Bsep in livers of mice. ANIT markedly increases the hepatic retention of emodin in SC-treated mice. The major SC constituents, in particular three anthraquinones, are able to activate AMPK in HepG2 cells and inhibit Bsep in primary mouse hepatocytes, with emodin showing the strongest activities. Together, the present study identifies a potential pro-cholestatic role of emodin in the hepatotoxicity of herbs.