Intestinal absorption mechanism of rotundic acid: Involvement of P-gp and OATP2B1.

ETHNOPHARMACOLOGICAL RELEVANCE: Ilicis Rotundae Cortex (IRC), the dried barks of Ilex rotunda Thunb. (Aquifoliaceae), has been used for the prevention or treatment of colds, tonsillitis, dysentery, and gastrointestinal diseases in folk medicine due to its antibacterial and anti-inflammatory effects. However, there is no report about the intestinal absorption of major compounds that support traditional usage. AIM OF STUDY: Considering the potential of rotundic acid (RA) - major biologically active pentacyclic triterpenes found in the IRC, this study was purposed to uncover the oral absorption mechanism of RA using in situ single-pass intestinal perfusion (SPIP) model, in vitro cell models (Caco-2, MDCKII-WT, MDCKII-MDR1, MDCKII-BCRP, and HEK293-OATP2B1 cells) and in vivo pharmacokinetics studies in rats. MATERIALS AND METHODS: The molecular properties (solubility, lipophilicity, and chemical stability) and the effects of principal parameters (time, compound concentrations, pH, paracellular pathway, and the different intestinal segments) were analyzed by liquid chromatography-tandem mass spectrometry. The susceptibility of RA to various inhibitors, such as P-gp inhibitor verapamil, BCRP inhibitor Ko143, OATP 2B1 inhibitor rifampicin, and absorption enhancer EGTA were assessed. RESULTS: RA was a compound with low water solubility (12.89 µg/mL) and strong lipophilicity (LogP = 4.1). RA was considered stable in all media during the SPIP and transport studies. The SPIP and cell experiments showed RA was moderate absorbed in the intestines and exhibited time, concentration, pH, and segment-dependent permeability. In addition, results from the cell model, in situ SPIP model as well as the in vivo pharmacokinetics studies consistently showed that verapamil, rifampicin, and EGTA might have significant effect on the intestinal absorption of RA. CONCLUSION: The mechanisms of intestinal absorption of RA might involve multiple transport pathways, including passive diffusion, the participation of efflux (i.e., P-gp) and influx (i.e., OATP2B1) transporters, and paracellular pathways.

Influence of verapamil on the pharmacokinetics of rotundic acid in rats and its potential mechanism.

CONTEXT: Rotundic acid (RA), a plant-derived pentacyclic triterpene acid, has been reported to possess extensive pharmacological activities. The poor bioavailability limits its further development and potential clinic application. OBJECTIVE: To clarify the potential mechanism for poor oral bioavailability. MATERIALS AND METHODS: The single-dose pharmacokinetics of orally administered RA (10 mg/kg) in Sprague-Dawley rats without or with verapamil (25 or 50 mg/kg) were investigated. Additionally, MDCKII-MDR1 and Caco-2 cell monolayers, five recombinant human cytochrome P450 (rhCYP) enzymes (1A2, 2C8, 2C9, 2D6 and 3A4), and rat liver microsomes were also conducted to investigate its potential mechanism. RESULTS: Verapamil could significantly affect the plasma concentration of RA. Co-administered verapamil at 25 and 50 mg/kg, the AUC0-∞ increased from 432 ± 64.2 to 539 ± 53.6 and 836 ± 116 ng × h/mL, respectively, and the oral clearance decreased from 23.6 ± 3.50 to 18.7 ± 1.85 and 12.2 ± 1.85 L/h/kg, respectively. The MDCKII-MDR1 cell assay showed that RA might be a P-gp substrate. The rhCYPs experiments indicated that RA was mainly metabolized by CYP3A4. Additionally, verapamil could increase the absorption of RA by inhibiting the activity of P-gp, and slow down the intrinsic clearance of RA from 48.5 ± 3.18 to 12.0 ± 1.06 µL/min/mg protein. DISCUSSION AND CONCLUSIONS: These findings indicated that verapamil could significantly affect the pharmacokinetic profiles of RA in rats. It was demonstrated that P-gp and CYP3A were involved in the transport and metabolism of RA, which might contribute to the low oral bioavailability of RA.

Transporters (OATs and OATPs) contribute to illustrate the mechanism of medicinal compatibility of ingredients with different properties in yuanhuzhitong prescription.

Various medicinal ingredients with different tastes are combined according to the theory of compatibility in Chinese materia medica to achieve a better efficacy, while the mechanism was not very clear. Here, the authors studied the interaction between ingredients and human transporters such as the kidney transporters OAT1 and OAT3, the liver transporters OATP1B1 and OATP1B3, and the intestine transporter OATP2B1 to discern the compatibility mechanism of ingredients with different tastes in the Yuanhuzhitong preparation (YHP) comprising Corydalis yanhusuo (CYH) and Angelica dahurica (AD), which could relieve pain by restraining the central system. The results show that tetrahydropalmatine (TDE), the major component of CYH, could be transported by OAT3 into kidney, OATP1B1 and OATP1B3 into liver, while imperatorin (IPT) and isoimperatorin (ISP), the two key components of AD, and AD extract showed strong inhibition to OAT1 and OAT3. What's more, AD extract also exerted strongly inhibition to human transporters OATP1B1 and OATP1B3. It was also detected that IPT, ISP, and AD extract significantly downregulated the expression of Oatp1a1, Oatp1a4, and Oatp1b2 of liver in mice. The in vivo results show that the concentration of TDE in liver and kidney significantly decreased, while the TDE concentration in blood and brain were both significantly enhanced in the presence of IPT, ISP, and AD extract. These results suggest that the ingredients in AD with pungent taste could enhance the exposure of TDE in blood and brain by inhibiting the uptake of TDE in liver and kidney. That is to say, TDE with bitter taste could "flood up" into the central nervous system to play its therapeutic effect by the cut-off of that into liver and kidney in the presence of ingredients within AD. This paper not only proves the meridian distribution of CYH in liver and kidney with the role of OAT3, OATP1B1, and OATP1B3, but also illustrates how to improve the efficacy of CYH by reasonable compatibility with AD. This study may offer a valuable clue to illustrate the mechanism of compatibility theory.

Effects of liquorice on pharmacokinetics of aconitine in rats.

Aconite alkaloids are the main bioactive ingredients existing in Aconitum, for instance aconitine (AC), which exhibit potent analgesic, antirheumatic and other pharmacological effects. In this study, effects of long-term treatment with liquorice on pharmacokinetics of AC in rats were investigated. Pharmacokinetics of AC after oral administration of AC at 1.5 mg/kg either with pre-treatment of liquorice water extracts at 0.433 or 1.299 g/kg (crude drug), respectively, for one week or not were studied. Additionally, LS-180 cells and human primary hepatocytes were utilized to explore the potential effects of bioactive ingredients of liquorice on P-glycoprotein (P-gp) and Cytochromes P450 (CYPs), respectively. The results revealed that exposure of AC after pre-treatment with liquorice was altered remarkably. Area under the concentration-time curve (AUC) decreased from 161 ± 37.8 to 58.8 ± 8.97 and 44.7 ± 8.20 ng/mL*h, respectively. Similarly, Cmax decreased from 26.2 ± 5.19 to 11.8 ± 1.15 and 6.86 ± 0.600 ng/mL, respectively. In addition, expressions of CYPs of human primary hepatocytes were enhanced to various contents after induction. Moreover, accumulation of AC and hypaconitine (HA), not mesaconitine (MA) inside of LS-180 cells were reduced after pre-treatment by comparison with control. In conclusion, the exposure of AC in vivo declined after pre-treatment with liquorice extract, which may be highly associated with upregulated expression and/or function of CYPs and P-gp.

Potential detoxification effect of active ingredients in liquorice by upregulating efflux transporter.

BACKGROUND: As one of most widely used herbal medicine, liquorice exhibits diverse pharmacological activities, for instance, analgesic, antitussive, antiarrhythmic, anti-inflammatory, and immune regulation. Additionally, detoxification effects were observed in combination of liquorice with other herbal drugs. The mechanism of detoxification of liquorice has been extensively investigated through material basis and interference with CYPs though, investigations of its effect on transporters were very limited, according to the literature. PURPOSE: The objective of this study was attempt to investigate the effect of active ingredients existing in liquorice on the efflux transporters as to clarify the potential mechanism of detoxification of liquorice. METHODS: Multiple analytical approaches have been explored, including flow cytometry, fluorescent detection, RT-PCR, Western blot to measure the function, activity as well as mRNA/protein expression of efflux transporters on LS-180 cell model after treatments with active compounds of liquorice. Additionally, Caco-2 cell model was utilized to further investigate the potential impact of those ingredients on efflux transporter. RESULTS: The resulting data indicated that those active ingredients, including flavonoids (liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin and licochalcone A) and pentacyclic triterpene saponin (glycyrrhetinic acid) were able to upregulate the expression of efflux transporters, for example P-gp, BCRP and MRP2. The gene expressions were approximately over 2.5 folds by comparison with that of control, and up to 13 folds and 16 folds for BCRP by isoliquiritin and isoliquiritigenin, and further confirmed by Western blot. The functional assay also supported up-regulation of efflux transporter by those ingredients. Flow cytometry study showed that the level of rhodamine123 as probe substrate in LS-180 cells decreased to approximately 50% after treatment with active ingredients of liquorice, compared with that of control. The fluorescent assay confirmed that change of rhodamine 123 was correlated with the concentrations of active ingredients given. The efflux transport of rhodamine 123 was enhanced in Caco-2 cell models as well. CONCLUSION: The study clarified potential detoxification mechanism of liquorice by up-regulating efflux transporter as to reduce absorption of xenobiotics across small intestinal membrane, which provided a new insight into pharmacological function of liquorice.

The effect of polymorphism of uric acid transporters on uric acid transport.

The abnormal metabolism of uric acid results in many disease such as chronic kidney disease, hyperuricemia, nephrolithiasis, gout, hypertension, vascular disease and so on. Serum uric acid levels are maintained by the balance between production and elimination. There are many factors that maintain the balance of serum uric acid. One of them is transporters which are responsible for the debouchment of uric acid within blood. The transport and excretion of uric acid is a complicated procedure which is related with various transporters such as OAT1, OAT3, OAT4, URAT1, GLUT9, BCRP, MRP4, NPT1, NTP4, and so on. In recent years, a large number of genome-wide association studies have shown that the single nucleotide polymorphisms of uric acid transporters were closely related to serum uric acid level. What's more, some mutations on these gene locus may also break the balance of serum uric acid. Here, the polymorphisms of uric acid transporters closely related with the serum uric acid balance were reviewed and discussed because of their important significance in clinical therapy for a precision medicine. The mechanism of metabolic diseases with gene variation may provide new strategy for the design and development of innovative drug to treat diseases with uric acid metabolic disturbance.

[The in vitro transport mechanism of bentysrepinine: a novel anti-hepatitis B virus drug candidate].

Bentysrepinine (Y101), a derivative of phenylalanine dipeptide, is a novel drug candidate for the treatment of hepatitis B virus (HBV) infection. Our previous preclinical pharmacokinetic study showed that its in vivo absorption and distribution characteristics were probably related to transmembrane transport after Y101 was administered intragastically in rats. In this study, Caco-2 and MDCK-MDR1 cell models were used to investigate interactions between Y101 and P-gp through the apparent permeation coefficient (P(app)) and efflux ratio (RE); the results showed that Y101 was a substrate of P-gp. In addition, gene-transfected cell models, HEK293-h OATP1B1, HEK293-h OATP2B1 and CHO-PEPT1 were used to evaluate the affinity to OATP1B1, OATP2B1 and PEPT1. The results suggest that Y101 has a weak inhibitory effect on OATP1B1 and OATP2B1, and Y101 may not be substrates of OATP1B1, OATP2B1 or PEPT1. The above results can be used to explain the in vivo absorption and distribution characteristics, and to provide a scientific basis for the further development of Y101.

[The in vitro inhibition and induction of cytochrome P450 activities by bentysrepinine: a novel candidate of anti-hepatitis B virus drug].

Bentysrepinine (Y101), a derivative of phenyalanine dipeptide, has a novel mechanism in the treatment of hepatitis B virus (HBV) infection with a good anti-HBV effect. In the present study, a fluorometric-based high throughput method using cytochrome P450 (CYP) screening kit was adopted to evaluate in vitro inhibition potential of Y101 on CYP isoenzymes by calculating remaining enzyme activities and inhibitory potential (IC(50) values) using the determined values of fluorescence intensity. The result showed that Y101 exhibited little activity in the inhibition of CYP1A2, CYP3A4, CYP2C9, CYP2C19 and CYP2D6 (IC(50) > 100 µmol·L(-1)). Y101 was used to treat human primary hepotocytes for 72 h, and the enzyme activities of CYP1A2, CYP2B6 and CYP3A4 were determined with a cocktail of probe substrates for the three CYP isoforms. The metabolites were simultaneously determined using a LC-MS/MS method. Y101 had no activity in the induction of CYP1A2, CYP2B6 and CYP3A4 on the basis of the following results: 1 The ratio of enzyme activities between test and control groups were all below than 1 (varied from 0.662 to 0.928); 2 The induction potential of Y101 were lower than forty percent compared with that of positive groups. The above results suggest that Y101 has little activity in the regulation of metabolic drug-drug interactions based on the CYP isoform changes following co-administration of drugs.

The Targeted-liposome Delivery System of Antitumor Drugs.

The liposome delivery system has been intensively explored as novel drug delivery system (DDS) for antitumor drugs, due to its safety, selective cytotoxicity, long circulation and slow elimination in blood, which is favorable for cancer therapy. The liposome-based chemotherapeutics are used to treat a variety of cancers to enhance the therapeutic index of antitumor drugs. Here, the author reviewed the important targets for cancer therapy and the pharmacokinetic behavior of liposomal drugs in vivo, as well as the application of the targeting liposomal system in cancer therapy. Considering further application for clinical use, the great challenges of the liposome-based delivery system were also proposed as follows: 1) prepare stealth liposome with steric stabilization and further enhance the therapeutic effects and safety; 2) explore more safe clinical targets and complementary or different types of targeting liposome; 3) thirdly, more investment is needed on the research of pharmacokinetics of the elements such as the ligands (antibody), PEG and lipids of liposome delivery system as well as safety evaluation. Considering the complex process of the liposomal encapsulation drugs in vivo, the author inferred that there are maybe different forms of the encapsulation drug to be internalized by the tumor tissues at the same time and space, although there are little reports on it.

Aspartate-modified doxorubicin on its N-terminal increases drug accumulation in LAT1-overexpressing tumors.

L-type amino acid transporter 1 (LAT1), overexpressed on the membrane of various tumor cells, is a potential target for tumor-targeting therapy. This study aimed to develop a LAT1-mediated chemotherapeutic agent. We screened doxorubicin modified by seven different large neutral amino acids. The aspartate-modified doxorubicin (Asp-DOX) showed the highest affinity (Km = 41.423 µmol/L) to LAT1. Aspartate was attached to the N-terminal of DOX by the amide bond with a free carboxyl and a free amino group on the α-carbon atom of the Asp residue. The product Asp-DOX was characterized by HPLC/MS. In vitro, Asp-DOX exerted stronger inhibition on the cancer cells overexpressing LAT1 and the uptake of Asp-DOX was approximately 3.5-fold higher than that of DOX in HepG2 cells. Pharmacokinetic data also showed that Asp-DOX was expressed over a longer circulation time (t1/2 = 49.14 min) in the blood compared to DOX alone (t1/2 = 15.12 min). In HepG2 and HCT116 tumor-bearing mice, Asp-DOX achieved 3.1-fold and 6.4-fold accumulation of drugs in tumor tissue, respectively, than those of the unmodified DOX. More importantly, treatment of tumor-bearing mice with Asp-DOX showed a significantly stronger inhibition of tumor growth than mice treated with free DOX in HepG2 tumor models. Furthermore, after Asp modification, Asp-DOX avoided MDR mediated by P-glycoprotein. These results suggested that the Asp-DOX modified drug may provide a new treatment strategy for tumors that overexpress LAT1 and MDR1.

[Induction, purification and antifungal activity of beta-1, 3-glucanase from wheat leaves].

Treatment with mercuric chloride (0.01%), salicylic acid (10.0 mg/mL) or riboflavin (1 mmol/L) induced the beta-1, 3-glucanase activity in all the three wheat varieties i.e. 331, Kangdao 680 and Lumai 23 tested, with the strongest inductive effect on variety 331 by treatment with mercuric chloride (0.01%) for 24 h. From leaves of variety 331 treated with mercuric chloride (0.01%) for 24 h, a kind of beta-1, 3-glucanase was purified by fractional precipitation with ammonium sulphate, Phenyl-Sepharose chromatography (Phenyl-Sepharose Fast Flow), ion-exchange chromatography (DEAE-Sepharose Fast Flow) and gel-filtration chromatography (Sephacryl S-100). Through SDS-PAGE and gel filtration, the molecular weight of the purified beta-1, 3-glucanase was determined to be about 52.0-53.6 kD. The purified beta-1, 3-glucanase showed antifungal activity against both Alternaria longipes and Rhizoctonia cerealis on tested plates, and inhibited the germ tube elongation and spore germination of Verticillium dahliae and Fusarium omysporum f.sp cucumerinum.

catena-Poly[[di-mu-chloro-bis[(triphenylphosphine)silver(I)]]-mu-2-aminopyrimidine-kappa 2N1:N3].

In the title supramolecular complex, [Ag(2)Cl(2)(C(4)H(5)N(3))(C(18)H(15)P)(2)](n), a one-dimensional chain is formed by dimeric [Ag(2)Cl(2)(PPh(3))(2)] units bridged by 2-aminopyrimidine moieties. The Ag atoms are four-coordinate, with an AgCl(2)NP core. A crystallographic inversion centre is located in the centre of the Ag(2)Cl(2) chelate ring, while the crystallographic twofold axis bisects the 2-aminopyrimidine ligand.