The purpose of this study was to evaluate in vitro interactions of commercially obtained pure herbal constituents with p-glycoprotein P-gp and cytochrome P-450 3A4 (CYP3A4) activities, which can further modulate the transcellular transport and metabolism kinetics of orally administered drugs. Caco-2 cells grown in the presence of 0.25 micromol/L 1alpha,25-dihydroxy vitamin D3 and multidrug-resistant 1 (MDR1) transfected MDCK cells were used as models to evaluate the effect of purified herbal constituents (quercetin, hypericin, hyperforin from St. John's wort, kaempferol from ginseng, silibinin from milk thistle, and allicin from garlic) on P-gp-mediated efflux of the human immunodeficiency virus (HIV) protease inhibitor ritonavir. In addition, the inhibitory effect of these constituents on CYP3A4-mediated metabolism was determined by using cortisol as a model compound. Silibinin and hyperforin did not significantly alter cellular uptake of H-ritonavir in Caco-2 cells. A similar result was also observed for silibinin when tested in MDR1-MDCK cells. Quercetin, hypericin, and kaempferol exhibited a remarkable inhibition of P-gp-mediated efflux of ritonavir by increasing its cellular uptake in these models. These values were also comparable with the inhibitory effect of quinidine in Caco-2 cells, a well-known inhibitor of P-gp, on ritonavir efflux from Caco-2 cells. Allicin exhibited a concentration-dependent inhibition of ritonavir efflux when tested on MDR1-MDCK cells. There was a significant decrease in the Apical to Basal/Basal to Apical (AP-BL/BL-AP) transport ratio of ritonavir in presence of hypericin, kaempferol, and quercetin. These herbal constituents inhibited the CYP3A4 activity when tested with the Vivid CYP3A4 assay kit, whereas silibinin did not alter cortisol metabolism. Hypericin showed a significant inhibition in reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent metabolism of cortisol with 64.6% of intact drug at the end of a 1-hour study. Similarly, kaempferol and quercetin also caused substantial inhibition of cortisol metabolism with 89.7% and 90.1% of intact cortisol, respectively, compared with 45.9% in the control. Prolonged exposure of quercetin resulted in significant increase of mRNA expression of both MDR1 and CYP3A4 levels in Caco-2 cells. However, hyperforin caused upregulation of CYP3A4 and downregulation of MDR1, whereas the effect of silibinin and kaempferol remained inconclusive on these gene expressions. Hypericin, kaempferol, quercetin, and allicin inhibit the efflux and CYP3A4-mediated metabolism of xenobiotics in vitro. Hence, this study warns against the use of herbal constituents along with prescribed HIV protease inhibitors that are substrates for P-gp and/or CYP3A4.

Functional and molecular characterization showed the existence of P-gp in human cornea, rabbit cornea, and a rabbit corneal cell line. This knowledge of the existence of P-gp will help in development of better ocular drug delivery strategies.

The total syntheses of the PKC inhibitors (+)-calphostin D, (+)-phleichrome, cercosporin, and 10 novel perylenequinones are detailed. The highly convergent and flexible strategy developed employed an enantioselective oxidative biaryl coupling and a double cuprate epoxide opening, allowing the selective syntheses of all the possible stereoisomers in pure form. In addition, this strategy permitted rapid access to a broad range of analogs, including those not accessible from the natural products. These compounds provided a powerful means for evaluation of the perylenequinones structural features necessary to PKC activity. Simpler analogs were discovered with superior PKC inhibitory properties and superior photopotentiation in cancer cell lines relative to the more complex natural products.

No abstract

Efflux pump like P-glycoprotein (P-gp) is known to be a major barrier to drug delivery. Functional P-glycoprotein has been recently identified in cornea and corneal cell lines. Thus, it is probable that P-glycoprotein may restrict in vivo ocular drug absorption, resulting in low ocular bioavailability. Experiments were designed using New Zealand albino (New Zealand White) rabbits to assess inhibitors of P-gp efflux to increase drug absorption. Anesthetized rabbits were given constant topical infusions of [14 C]erythromycin in the presence and absence of inhibitors. Testosterone, verapamil, quinidine, and cyclosporine A were selected as P-gp inhibitors. Transport experiments were conducted in Madin-Darby canine kidney cells transfected with the human mdr1 gene (MDCK-MDR1). Erythromycin exhibited significant efflux out of MDCK-MDR1 cells, suggesting that erythromycin is a good substrate for P-gp. Ocular pharmacokinetic studies were conducted using a topical single-dose infusion method. Maximum inhibition of P-gp mediated efflux was observed with 500 M testosterone. Area under the curve (AUC) 0-ϱ of erythromycin with 500 M testosterone was almost 4 times higher than AUC 0-ϱ without any inhibitor. Rate of elimination (k 10 ) for erythromycin and those with inhibitors was found to be similar (141 Ϯ 23 min), suggesting that elimination pathways were not altered. All the inhibitors were found to be nontoxic. Verapamil also inhibited the efflux pump with moderate change in AUC 0-ϱ and C max compared with control. Thus, P-gp is found to be active in vivo, and it restricts topical erythromycin absorption across the cornea, which can be inhibited by known P-gp inhibitors. Therefore, ocular bioavailability of P-gp substrates can be significantly enhanced by proper selection of P-gp inhibitors.

The first examples of asymmetric oxidative coupling of simple phenols and 2-hydroxycarbazoles are outlined. Generation of a more vanadium catalyst by ligand design and by addition of an exogenous Brønsted or Lewis acid was found to be key to coupling the more oxidatively resistant phenols. The resultant vanadium complex is both more Lewis acidic and more strongly oxidizing. Good to excellent levels of enantioselectivity could be obtained, and simple trituration readily provided the products with ≥95% ee.

No abstract

Described is a new method for the screening of an array of catalysts, in situ, to estimate enantioselectivity and relative rates. We term this approach "double-cuvette ISES (in situ enzymatic screening)". The Co(III)-salen mediated hydrolytic kinetic resolution (HKR) of (+/-)-propylene oxide is used as a model reaction to demonstrate proof of principle. In two parallel cuvettes, a lower CHCl3-based organic layer is loaded with the epoxide and the chiral salen catalyst. Aqueous reporting layers, containing distinct "reporting enzymes" and their nicotinamide cofactors, are layered above the organic layers. The 1,2-propanediol enantiomers formed by the chiral catalyst diffuse into the aqueous layer and are oxidized there by the reporting enzymes at rates dependent upon the diol concentration, the R:S ratio of the diol, and the enantioselectivity of the reporting enzymes. A focused chiral salen library was constructed from seven chiral 1,2-diamines, derived from amino acid, terpenoid, and carbohydrates skeletons, and seven salicylaldehyde derivatives. Double-cuvette ISES identified a couple of interesting combinatorial hits in this salen array, wherein either the sense or magnitude of enantioselection for a given chiral diamine depends significantly upon the choice of "salicylaldehyde" partner. A comparison of predicted ee's and relative rates using this new screening tool with those independently measured is provided.