Kaustubh H. Kulkarni

First-pass metabolism via UDP-glucuronosyltransferase: a barrier to oral bioavailability of phenolics

Glucuronidation mediated by UDP-glucuronosyltransferases (UGTs) is a significant metabolic pathway that facilitates efficient elimination of numerous endobiotics and xenobiotics, including phenolics. UGT genetic deficiency and polymorphisms or inhibition of glucuronidation by concomitant use of drugs are associated with inherited physiological disorders or drug-induced toxicities. Moreover, extensive glucuronidation can be a barrier to oral bioavailability as the first-pass glucuronidation (or premature clearance by UGTs) of orally administered agents usually results in the poor oral bioavailability and lack of efficacies. This review focused on the first-pass glucuronidation of phenolics including natural polyphenols and pharmaceuticals. The complexity of UGT-mediated metabolism of phenolics is highlighted with species-, gender-, organ- and isoform-dependent specificity, as well as functional compensation between UGT1A and 2B subfamily. In addition, recent advances are discussed with respect to the mechanisms of enzymatic actions, including the important properties such as binding pocket size and phosphorylation requirements.

Effect of Hydrogen Sulfide on Sympathetic Neurotransmission and Catecholamine Levels in Isolated Porcine Iris-Ciliary Body

In the present study, we investigated the pharmacological action of hydrogen sulfide (H2S, using sodium hydrosulfide, NaHS, and/or sodium sulfide, Na2S as donors) on sympathetic neurotransmission from isolated, superfused porcine iris-ciliary bodies. We also examined the effect of H2S on norepinephrine (NE), dopamine and epinephrine concentrations in isolated porcine anterior uvea. Release of [3H]NE was triggered by electrical field stimulation and basal catecholamine concentrations was measured by high performance liquid chromatography (HPLC). Both NaHS and Na2S caused a concentration-dependent inhibition of electrically evoked [3H]NE release from porcine iris-ciliary body without affecting basal [3H]NE efflux. The inhibitory action of H2S donors on NE release was attenuated by aminooxyacetic acid (AOA) and propargyglycine (PAG), inhibitors of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), respectively. With the exception of dopamine, NaHS caused a concentration-dependent reduction in endogenous NE and epinephrine concentrations in isolated iris-ciliary bodies. We conclude that H2S can inhibit sympathetic neurotransmission from isolated porcine anterior uvea, an effect that is dependent, at least in part, on intramural biosynthesis of this gas. Furthermore, the observed action of H2S donors on sympathetic transmission may be due to a direct action of this gas on neurotransmitter pools.

Bioavailability and Pharmacokinetics of Genistein: Mechanistic Studies on its ADME

Genistein, one of the most active natural flavonoids, exerts various biological effects including chemoprevention, antioxidation, antiproliferation and anticancer. More than 30 clinical trials of genistein with various disease indications have been conducted to evaluate its clinical efficacy. Based on many animals and human pharmacokinetic studies, it is well known that the most challenge issue for developing genistein as a chemoprevention agent is the low oral bioavailability, which may be the major reason relating to its ambiguous therapeutic effects and large interindividual variations in clinical trials. In order to better correlate pharmacokinetic to pharmacodynamics results in animals and clinical studies, an in-depth understanding of pharmacokinetic behavior of genistein and its ADME properties are needed. Numerous in vitro/in vivo ADME studies had been conducted to reveal the main factors contributing to the low oral bioavailability of genistein. Therefore, this review focuses on summarizing the most recent progress on mechanistic studies of genistein ADME and provides a systemic view of these processes to explain genistein pharmacokinetic behaviors in vivo. The better understanding of genistein ADME property may lead to development of proper strategy to improve genistein oral bioavailability via mechanism-based approaches.

Disposition of naringenin via glucuronidation pathway is affected by compensating efflux transporters of hydrophilic glucuronides.

The purposes of this study were to investigate how efflux transporters and UDP-glucuronosyltransferases (UGT) affect the disposition of naringenin. A rat intestinal perfusion model with bile duct cannulation was used along with rat intestinal and liver microsomes. In the intestinal perfusion model, both absorption and subsequent excretion of naringenin metabolites were rapid and site-dependent (p < 0.05). Naringenin was absorbed the most in colon, and its glucuronides were excreted the most in duodenum. In metabolism studies, the intrinsic clearance value of naringenin glucuronidation was the highest in jejunum microsomes, followed by liver, ileal and colonic microsomes. The rapid metabolism in microsomes did not always translate into more efficient excretion in the rat perfusion model, however, because of presence of rate-limiting efflux transporters. When used separately, MK-571 (an inhibitor of multidrug resistance-related protein 2 or Mrp2) or dipyridamole (an inhibitor of breast cancer resistance protein or Bcrp1) did not affect excretion of naringenin glucuronides, but when used together, they significantly (p < 0.05) decreased intestinal and biliary excretion of naringenin glucuronides. In conclusion, efflux transporters Mrp2 and Bcrp1 are shown to compensate for each other and enable the intestinal excretion of flavonoid (i.e., naringenin) glucuronides.

Butanol fraction containing berberine or related compound from Nexrutine® inhibits NFκB signaling and induces apoptosis in prostate cancer cells

Epidemiological and laboratory studies support the hypothesis that several plant components influence prostate carcinogenesis and holds promise for disease prevention. Previously we reported that Nexrutine® (bark extract from Phellodendron amurense) inhibits proliferation of prostate cancer cells and prostate tumor development in the transgenic adenocarcinoma of mouse prostate (TRAMP) model through modulation of Akt signaling pathway. In the present investigation we conducted studies to further define the mechanism of action of Nexrutine® and to identify the active component associated with its biological activity.

Disposition of flavonoids via enteric recycling: UDP-glucuronosyltransferase (UGT) 1As deficiency in Gunn rats is compensated by increases in UGT2Bs activities.

Flavonoids have poor bioavailabilities largely because of metabolism via UDP-glucuronosyltransferases (UGTs). This study aims to further understand the functions of UGT in metabolizing genistein and apigenin, two compounds metabolized more extensively in the gut than in the liver. Because Gunn rats are deficient in UGT1As, we determined whether this deficiency would result in less flavonoid glucuronidation, using rat intestinal perfusion model and microsomes prepared from rat liver and intestine. In yeast-expressed rat UGT isoforms, rat UGT1A isoforms (especially UGT1A7) were mainly responsible for flavonoid metabolism. In perfusion studies, the two flavonoids were rapidly absorbed at comparable rates, but the intestinal excretions of glucuronides in Gunn rats compared with Wistar rats were not only comparable for genistein but also were higher (p < 0.05) for apigenin, suggesting up-regulation of UGT isoforms in Gunn rats. To determine the possible compensatory UGT isoforms, we first verified that UGT1A activities were significantly lower (p < 0.05) in Gunn rats by using UGT1A-specific probes 7-ethyl-10-hydroxycamptothecin (SN-38) and prunetin. We then demonstrated using UGT2B probes testosterone, ezetimibe, and indomethacin that UGT2B activities were usually significantly higher in Gunn rats. In addition, testosterone was metabolized much faster in liver microsomes than in intestinal microsomes, and in microsomes prepared from Gunn rats compared with Wistar rats. In conclusion, flavonoids are efficiently metabolized by UGT1A-deficient Gunn rats because of compensatory up-regulation of intestinal UGT2Bs and hepatic anion efflux transporters, which increases their disposition and limits their oral bioavailabilities.

Inhibitory action of hydrogen sulfide on muscarinic receptor-induced contraction of isolated porcine irides

We investigated the pharmacological actions of hydrogen sulfide (H(2)S) using sodium hydrosulfide (NaHS) and sodium sulfide (Na(2)S) as donors on isolated porcine irides in the presence of tone induced by muscarinic receptor stimulation. Furthermore, we also investigated the mechanism of action of H(2)S in this smooth muscle. Isolated porcine iris muscle strips were set up in organ baths and prepared for measurement of longitudinal isometric tension. The relaxant action of NaHS or Na(2)S on carbachol-induced tone was studied in the absence and presence of a K(+)-channel inhibitor and inhibitors/activators of enzymes of the biosynthetic pathways for H(2)S, prostanoid and nitric oxide production. In the concentration range, 10 nM to 100 microM, NaHS produced a concentration-dependent relaxation of carbachol-induced tone reaching a maximum of inhibition of 28% at 30 microM. The cyclooxygenase inhibitor, flurbiprofen (1 microM), enhanced relaxations induced by both NaHS and Na(2)S yielding IC(50) values of 7 microM and 70 microM, respectively. With exception of l-NAME (300 muM) inhibitors of cystathionine gamma-lyase, propargylglycine, (PAG) (1 mM) and beta-cyanoalanine, (BCA) (1 mM) and inhibitors of cystathionine beta-synthase, aminooxyacetic acid (AOA) (30 microM) and hydroxylamine (HOA) (30 microM) caused significant (P < 0.001) rightward shifts in the concentration-response curves to NaHS. An activator of cystathionine beta-synthase, SAM (100 microM), enhanced relaxations elicited by low concentrations of NaHS but attenuated responses caused by the higher concentrations of this H(2)S donor. The inhibitor of K(ATP) channel, glibenclamide (100 and 300 microM), blocked relaxations induced by NaHS. We conclude that the observed inhibitory action of NaHS and Na(2)S in isolated porcine irides is dependent on endogenous production of prostanoids and the biosynthesis of H(2)S by cystathionine gamma-lyase and cystathionine beta-synthase. Furthermore, relaxation induced by H(2)S is mediated, at least in part, by K(ATP) channels. Nitric oxide is not involved in the relaxation induced by this gas in the isolated porcine irides.

Biopharmaceutical and pharmacokinetic characterization of matrine as determined by a sensitive and robust UPLC-MS/MS method.

The purpose of this research was to develop a sensitive and reproducible UPLC-MS/MS method to analyze matrine, an anticancer compound, and to use it to investigate its biopharmaceutical and pharmacokinetic behaviors in rats. A sensitive and fast UPLC-MS/MS method was successfully applied to determine matrine in rat plasma, intestinal perfusate, bile, microsomes, and cell incubation media. The absolute oral bioavailability of matrine is 17.1+/-5.4% at a dose of 2mg/kg matrine. Matrine at 10microM was shown to have good permeability (42.5x10(-6)cm/s) across the Caco-2 cell monolayer, and the ratio of P(A-B) to P(B-A) was approximately equal to 1 at two different concentrations (1 and 10microM). Perfusion study showed that matrine displayed significant differences (P<0.05) in permeability at different intestinal regions. The rank order of permeability was ileum (highest, P(w)=6.18), followed by colon (P(w)=2.07), duodenum (P(w)=0.61) and jejunum (P(w)=0.52). Rat liver microsome studies showed that CYP and UGTs were not involved in matrine metabolism. In conclusion, a sensitive and reliable method capable of measuring matrine in a variety of matrixes was developed and successfully used to determine absolute oral bioavailability of matrine in rats, transport across Caco-2 cell monolayers, absorption in rat intestine, and metabolism in rat liver microsomes.

Inhibition of potassium- and ischemia-evoked [3H] D-aspartate release from isolated bovine retina by cannabinoids

We investigated the effect of cannabinoids on potassium chloride (K+)- and ischemia-induced [3H]D-aspartate release from isolated bovine retinae. The superfusion method was employed for studies of [3H]-neurotransmitter release. Cannabinoid receptor CB1 agonists, but not the CB2 agonist JWH 015, inhibited K+-induced [3H]D-aspartate release from bovine retinae with the following rank order of activity: anandamide > ACEA > methanandamide > WIN 55,212-2. In the ischemic model, the rank order of activity was as follows: methanandamide > ACEA > WIN 55,212-2. The CB1 receptor antagonist AM 251 blocked inhibitory responses produced by cannabinoids in both experimental conditions. In conclusion, cannabinoids inhibit evoked [3H]D-aspartate release from isolated bovine retinae via an effect on CB1 receptors.

Arachidonic acid metabolites and peroxide-induced inhibition of [ 3H]D-aspartate release from bovine isolated retinae

We have previously shown that hydrogen peroxide (H 2 O 2) can inhibit K + -depolarization-evoked [ 3 H]D-aspartate release from bovine isolated retinae. In the present study, we investigated the role of arachidonic acid metabolites in the inhibitory response elicited by H 2 O 2 in the bovine retinae. Furthermore, we examined the direct effect of H 2 O 2 on the production of prostaglandins and isoprostanes in this tissue. Isolated bovine retinae were prepared for studies of [ 3 H]D-aspartate release using the Superfusion Method. Release of [ 3 H]D-aspartate was elicited by Krebs solution containing an iso-osmotic concentration of KCl (50mM). A direct action of H 2 O 2 on prostaglandin E 2 (PGE2) and 8-isoprostane F 2a (8-iso-PGF 2a) was also measured by enzyme-linked immunosorbant assay (ELISA). The cyclooxygenase inhibitor, flurbiprofen (3µM), or the thromboxane-receptor antagonist, SQ 29548 (10µM) had no significant (p > 0.05) effect on K + -evoked [ 3 H]D-aspartate release. On the other hand, both flurbiprofen (3µM) and SQ 29548 (10µM) blocked the inhibition of K + -evoked [ 3 H]D-aspartate induced by H 2 O 2 (30µM). In concentrations up to 100µM, H 2 O 2 caused an increase in PGE 2 and 8-iso-PGF 2a over basal levels. For instance, H 2 O 2 (100µM) increased PGE 2 and 8-iso-PGF 2a over basal levels by 348 ± 41% and 185 ± 26 (n = 4), respectively. We conclude that the peroxide-mediated inhibition of [ 3 H]D-aspartate may involve the production of prostaglandins and isoprostanes in the bovine isolated retinae.