Potential application of D-optimal designs in the efficient investigation of cytochrome P450 inhibition kinetic models.

Correctly chosen d-optimal designs provide efficient experimental schemes when the aim of the investigation is to obtain precise estimates of parameters. In the current work, estimates of parameters refer to the enzyme kinetic parameters V(max) and K(m), but they also refer to the inhibition constant K(i). In general, this experimental approach is performed on a grid of values of the design variables. However, this approach may not be very efficient, in the sense that the parameter estimates (V(max), K(m), and K(i)) have unnecessarily high variances. For good estimates of parameters, the most efficient designs consist of clusters of replicates of a few sets of experimental conditions. The current study compares the application of such d-optimal designs with that of a conventional approach in assessing the competitive inhibitory potency of fluconazole and sertraline toward CYP2C9 and 2D6, respectively. In each instance, the parameter estimates, namely V(max), K(m), and K(i), were predicted well using the d-optimal design compared with those measured using the rich data sets, for both inhibitors. We show that d optimality can provide more efficient designs for estimating the model parameters, including K(i). We also show that real cost savings can be made by carefully planning studies that use the theory of optimal experimental design.

A review of LC-MS techniques and high-throughput approaches used to investigate drug metabolism by cytochrome P450s.

One of the major challenges facing the pharmaceutical industry today is finding new ways to increase productivity, decrease costs whilst still ultimately developing new therapies that enhance human health. To help address these challenges the utilisation of analytical technologies and high-throughput automated platforms has been employed; in order to perform more experiments in a shorter time frame with increased data quality. One of the main in vitro techniques to assess new chemical entities in a discovery setting has been the use of recombinant liver enzymes, microsomes and hepatocytes. These techniques can help predict in vivo metabolism, clearance and potential drug-drug interactions of these new compounds by cytochrome P450s (the major drug metabolising enzymes). This in vitro methodology has been totally transformed in recent times by the use of automated liquid handling and HPLC tandem mass spectrometry detection techniques (LC-MS/MS). This review aims looking at recent advances in the methodology used to investigate drug metabolism by cytochrome P450s; including an up to date summary of high-throughput platforms including the use of automation and LC-MS/MS to facilitate greater throughput, chromatographic resolution and data quality.

An automated, high-throughput, 384 well Cytochrome P450 cocktail IC50 assay using a rapid resolution LC-MS/MS end-point.

The current study focused on the development of an automated IC50 cocktail assay in a miniaturized 384 well assay format. This was developed in combination with a significantly shorter high pressure liquid chromatography (HPLC) separation and liquid chromatography-mass spectrometry (LC-MS/MS) run-time; than those currently reported in the literature. The 384-well assay used human liver microsomes in conjunction with a cocktail of probe substrates metabolized by the five major CYPs (tacrine for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4). To validate the usefulness of the automated and analytical methodologies, IC50 determinations were performed for a series of test compounds known to exhibit inhibition across these five major P450s. Eight compounds (sertraline, disulfuram, ticlopidine fluconazole, fluvoxamine, ketoconazole, miconazole, paroxetine, flunitrazepam) were studied as part of a cocktail assay, and against each CYPs individually. The data showed that the IC50s generated with cocktail incubations did not differ to a great extent from those obtained in the single probe experiments and hence unlikely to significantly influence the predicted clinical DDI risk. In addition the present method offered a significant advantage over some of the existing cocktail analytical methodology in that separation can be achieved with run times as short as 1 min without compromising data integrity. Although numerous studies have been reported to measure CYP inhibition in a cocktail format the need to support growing discovery libraries not only relies on higher throughput assays but quicker analytical run times. The current study reports a miniaturized high-throughput cocktail IC50 assay, in conjunction with a robust, rapid resolution LC-MS/MS end-point offered increased sample throughput without compromising analytical sensitivity or analyte resolution.

Application of CYP3A4 in vitro data to predict clinical drug-drug interactions; predictions of compounds as objects of interaction.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Numerous retrospective analyses have shown the utility of in vitro systems for predicting potential drug-drug interactions (DDIs). Prediction of DDIs from in vitro data is commonly obtained using estimates of enzyme K(i), inhibitor and substrate concentrations and absorption rate for substrate and inhibitor. WHAT THIS STUDY ADDS: Using a generic approach for all test compounds, the findings from the current study showed the use of recombinant P450s provide a more robust in vitro measure of P450 contribution (fraction metabolized, f(m)) than that achieved when using chemical inhibitors in combination with human liver microsomes, for the prediction of potential CYP3A4 drug-drug interactions prior to clinical investigation. The current study supported the use of SIMCYP(R), a modelling and simulation software in utilizing the in vitro measures in the prediction of potential drug-drug interactions. AIMS: The aim of this study was to explore and optimize the in vitro and in silico approaches used for predicting clinical DDIs. A data set containing clinical information on the interaction of 20 Pfizer compounds with ketoconazole was used to assess the success of the techniques. METHODS: The study calculated the fraction and the rate of metabolism of 20 Pfizer compounds via each cytochrome P450. Two approaches were used to determine fraction metabolized (f(m)); 1) by measuring substrate loss in human liver microsomes (HLM) in the presence and absence of specific chemical inhibitors and 2) by measuring substrate loss in individual cDNA expressed P450s (also referred to as recombinant P450s (rhCYP)) The fractions metabolized via each CYP were used to predict the drug-drug interaction due to CYP3A4 inhibition by ketoconazole using the modelling and simulation software SIMCYP. RESULTS: When in vitro data were generated using Gentest supersomes, 85% of predictions were within two-fold of the observed clinical interaction. Using PanVera baculosomes, 70% of predictions were predicted within two-fold. In contrast using chemical inhibitors the accuracy was lower, predicting only 37% of compounds within two-fold of the clinical value. Poorly predicted compounds were found to either be metabolically stable and/or have high microsomal protein binding. The use of equilibrium dialysis to generate accurate protein binding measurements was especially important for highly bound drugs. CONCLUSIONS: The current study demonstrated that the use of rhCYPs with SIMCYP provides a robust in vitro system for predicting the likelihood and magnitude of changes in clinical exposure of compounds as a consequence of CYP3A4 inhibition by a concomitantly administered drug.

Induction of cytochrome P450: assessment in an immortalized human hepatocyte cell line (Fa2N4) using a novel higher throughput cocktail assay.

Over recent years the application of cocktail studies to measure biological markers has become increasingly popular. The current study investigated a novel approach in assessing cytochrome P450 (P450) enzyme induction in an immortalized cell line using a cocktail of five P450 substrate probes compared with the traditional single-probe approach. The findings reported herein support use of a cocktail approach to assess the induction of the major P450s, namely, CYP3A4, CYP1A2, and CYP2C9. CYP2C19 and CYP2D6 could also be followed as part of the cocktail approach reported. Response to prototypical inducers did not differ to those observed in the presence of the specific probes alone. Consequently, this approach requires significantly fewer sample numbers if screening the induction potential of more than one P450. Moreover, these studies highlight the utility of the immortalized cell line Fa2N4 as a robust model system for induction studies. In conclusion, the current experimental setup is an improvement on current approaches used to assess P450 induction, significantly increasing sample throughput.

The reaction of flavanols with nitrous acid protects against N-nitrosamine formation and leads to the formation of nitroso derivatives which inhibit cancer cell growth.

Studies have suggested that diets rich in polyphenols such as flavonoids may lead to a reduced risk of gastrointestinal cancers. We demonstrate the ability of monomeric and dimeric flavanols to scavenge reactive nitrogen species derived from nitrous acid. Both epicatechin and dimer B2 (epicatechin dimer) inhibited nitrous acid-induced formation of 3-nitrotyrosine and the formation of the carcinogenic N-nitrosamine, N-nitrosodimethylamine. The reaction of monomeric and dimeric epicatechin with nitrous acid led to the formation of mono- and di-nitroso flavanols, whereas the reaction with hesperetin resulted primarily in the formation of nitrated products. Although, epicatechin was transferred across the jejunum of the small intestine yielding metabolites, its nitroso form was not absorbed. Dimer B2 but not epicatechin monomer inhibited the proliferation of, and triggered apoptosis in, Caco-2 cells. The latter was accompanied by caspase-3 activation and reductions in Akt phosphorylation, suggesting activation of apoptosis via inhibition of prosurvival signaling. Furthermore, the dinitroso derivative of dimer B2, and to a lesser extent the dinitroso-epicatechin, also induced significant toxic effects in Caco-2 cells. The inhibitory effects on cellular proliferation were paralleled by early inhibition of ERK 1/2 phosphorylation and later reductions in cyclin D1 levels, indicating modulation of cell cycle regulation in Caco-2 cells. These effects highlight multiple routes in which dietary derived flavanols may exert beneficial effects in the gastrointestinal tract.

Flavonoids and the brain: interactions at the blood-brain barrier and their physiological effects on the central nervous system.

Over the past few years there has been an exponential growth in the number of reports describing the effects of nutritional modulation on aging and age-related diseases. Specific attention has been directed toward the beneficial effects afforded by dietary antioxidants, in particular those from fruit and vegetables, in ameliorating age-related deficits in brain performance. The rationale for studying the effects of dietary intervention stems from evidence implicating free radicals in aspects related to the aging process. Age-dependent neuropathology is a cumulative response to alterations induced by reactive oxygen species. Therefore cognitive aging, according to this hypothesis, should be slowed, and possibly even reversed, by appropriately increasing levels of antioxidants or decreasing overproduction of free radicals in the body.

The flavone hispidulin, a benzodiazepine receptor ligand with positive allosteric properties, traverses the blood-brain barrier and exhibits anticonvulsive effects.

The functional characterization of hispidulin (4',5,7-trihydroxy-6-methoxyflavone), a potent benzodiazepine (BZD) receptor ligand, was initiated to determine its potential as a modulator of central nervous system activity. After chemical synthesis, hispidulin was investigated at recombinant GABA(A)/BZD receptors expressed by Xenopus laevis oocytes. Concentrations of 50 nm and higher stimulated the GABA-induced chloride currents at tested receptor subtypes (alpha(1-3,5,6)beta(2)gamma(2)S) indicating positive allosteric properties. Maximal stimulation at alpha(1)beta(2)gamma(2)S was observed with 10 microm hispidulin. In contrast to diazepam, hispidulin modulated the alpha(6)beta(2)gamma(2)S-GABA(A) receptor subtype. When fed to seizure-prone Mongolian gerbils (Meriones unguiculatus) in a model of epilepsy, hispidulin (10 mg kg(-1) body weight (BW) per day) and diazepam (2 mg kg(-1) BW per day) markedly reduced the number of animals suffering from seizures after 7 days of treatment (30 and 25% of animals in the respective treatment groups, vs 80% in the vehicle group). Permeability across the blood-brain barrier for the chemically synthesized, (14)C-labelled hispidulin was confirmed by a rat in situ perfusion model. With an uptake rate (K(in)) of 1.14 ml min(-1) g(-1), measurements approached the values obtained with highly penetrating compounds such as diazepam. Experiments with Caco-2 cells predict that orally administered hispidulin enters circulation in its intact form. At a concentration of 30 microm, the flavone crossed the monolayer without degradation as verified by the absence of glucuronidated metabolites.

Flavonoid permeability across an in situ model of the blood-brain barrier.

Understanding mechanisms associated with flavonoid neuroprotection is complicated by the lack of information on their ability to enter the CNS. This study examined naringenin and quercetin permeability across the blood-brain barrier (BBB), using in vitro (ECV304/C6 coculture) and in situ (rat) models. We report measurable permeabilities (P(app)) for both flavonoids across the in vitro BBB model, consistent with their lipophilicity. Both flavonoids showed measurable in situ BBB permeability. The rates of uptake (K(in)) into the right cerebral hemisphere were 0.145 and 0.019 ml min(-1) g(-1) for naringenin and quercetin, respectively. Quercetin K(in) was comparable to that of colchicine (0.006 ml min(-1) g(-1)), a substrate for P-glycoprotein (P-gp). Preadministration of the P-gp inhibitor PSC833 or GF120918 (10 mg/kg body wt) significantly increased colchicine K(in), but only GF120918 (able to inhibit breast cancer resistance protein, BCRP) affected K(in) for quercetin. Naringenin K(in) was not affected. The influence of efflux transporters on flavonoid permeability at the BBB was further studied using MDCK-MDR1 and immortalized rat brain endothelial cells (RBE4). Colchicine, quercetin, and naringenin all showed measurable accumulation (distribution volume, V(d) (microl/mg protein)) in both cell types. The V(d) for colchicine increased significantly in both cell lines following coincubation with either PSC833 (25 microM) or GF120918 (25 microM). Both inhibitors also caused an increase in naringenin V(d); by contrast only GF120918 coincubation significantly increased quercetin V(d). In conclusion, the results demonstrate that flavonoids are able to traverse the BBB in vivo. However, the permeability of certain flavonoids in vivo is influenced by their lipophilicity and interactions with efflux transporters.

In vitro trans-monolayer permeability calculations: often forgotten assumptions.

In designing effective therapeutic strategies, novel drugs must exhibit favorable pharmacokinetic properties. The physicochemical characteristics of a drug, such as pK(a), molecular weight, solubility and lipophilicity, will influence the way the drug partitions from the aqueous phase into membranes, and thus, will influence its ability to cross cellular barriers, such as the lining of the gastrointestinal tract and the blood-brain barrier. Physicochemical characteristics also influence the degree to which a drug is able to cross a barrier layer, and the route by which it does this; whether transcellular (across the cells)-by diffusion, carrier-mediated transport or transcytosis-or paracellular-by diffusing through the tight junctions between the cells. The in vitro model systems that are currently employed to screen the permeation characteristics of a drug often represent a compromise between high throughput with low predictive potential and low throughput with high predictive potential. Here, we will examine the way in which in vitro cellular permeability assays are often performed and the assumptions that are implied but sometimes forgotten, and we will make simple suggestions for improving the methodological techniques and mathematical equations used to determine drug permeability.

Interaction between flavonoids and the blood-brain barrier: in vitro studies.

There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood-brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (Papp) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity.

Dietary flavonoids as potential neuroprotectants.

There is an increasing awareness of the role of certain nutritional components, including dietary flavonoids found in fruit, vegetables and beverages, in the maintenance of health and prevention of chronic diseases. In this regard, recent studies highlight an exciting role with respect to their potential neuroprotective actions, in particular towards deficits commonly observed with aging, such as reduced performance of cognitive, memory and learning tasks. These neurological functions, and possible mechanisms involved in controlling them, can be influenced by supplementation of single dietary flavonoids, or as part of a flavonoid-rich preparation. With this, a renewed emphasis is aimed at further understanding their modes and sites of action. Moreover a common theme among many in vitro studies examining mechanisms of neuroprotection is the failure to include biologically relevant metabolites of the flavonoids known to enter the circulation, and thus most likely to be bioavailable to cells and tissues. This oversight will ultimately influence the mechanisms of action proposed to explain the neuroprotection observed in animals and human studies. As such, emerging findings suggest a variety of potential mechanisms of action of flavonoids and their bioavailable metabolites in cytoprotection against oxidative stress, which may be independent of conventional antioxidant reducing activities. Such mechanisms might involve their interaction with cell signalling cascades, their influence on gene expression and the down regulation of pathways leading to cell death.

Potential role of dietary flavonoids in reducing microvascular endothelium vulnerability to oxidative and inflammatory insults ( small star, filled).

Although antioxidant systems help control the level of reactive oxygen species they may be overwhelmed during periods of oxidative stress. Evidence suggests that oxidative stress components as well as inflammatory mediators may be involved in the pathogenesis of vascular disorders, where localized markers of oxidative damage have been found. In this regard we investigated the putative antioxidant and anti-inflammatory effects of blueberry and cranberry anthocyanins and hydroxycinnamic acids against H(2)O(2) and TNFalpha induced damage to human microvascular endothelial cells. Polyphenols from both berries were able to localize into endothelial cells subsequently reducing endothelial cells vulnerability to increased oxidative stress at both the membrane and cytosol level. Furthermore, berry polyphenols also reduced TNFalpha induced up-regulation of various inflammatory mediators (IL-8, MCP-1 and ICAM-1) involved in the recruitment of leukocytes to sites of damage or inflammation along the endothelium. In conclusion, polyphenols isolated from both blueberry and cranberry were able to afford protection to endothelial cells against stressor induced up-regulation of oxidative and inflammatory insults. This may have beneficial actions against the initiation and development of vascular diseases and be a contributing factor in the reduction of age-related deficits in neurological impairments previously reported by us.

Fruit polyphenolics and brain aging: nutritional interventions targeting age-related neuronal and behavioral deficits.

Nutritional interventions, in this case, increasing dietary intake of fruits and vegetables, can retard and even reverse age-related declines in brain function and in cognitive and motor performance in rats. Our lab has shown that as Fischer 344 rats age their brains are increasingly vulnerable to oxidative stress. Dietary supplementation with fruit or vegetable extracts high in antioxidants (e.g., blueberry, BB, spinach, respectively) can decrease this vulnerability to oxidative stress as assessed in vivo by examining reductions in neuronal signaling and behavioral deficits and in vitro via H2O2-induced decrements in striatal synaptosomal calcium buffering. Examinations have also revealed that BB supplementations are effective in antagonizing other age-related changes in brain and behavior, as well as decreasing indices of inflammation and oxidative stress in gastrocnemius and quadriceps muscles. In ongoing studies we are attempting to determine the most effective BB polyphenolic components. To date, the anthocyanins show the most efficacy in penetrating the cell membrane and in providing antioxidant protection. In sum, our results indicate that increasing dietary intake of fruits and vegetables high in antioxidant activity may be an important component of a healthy living strategy designed to maximize neuronal and cognitive functioning into old age.