Gene Polymorphisms and Drug-Drug Interactions Determine the Metabolic Profile of Blonanserin.

This study aimed to evaluate the effects of cytochrome P450 3A4 (CYP3A4) gene polymorphism and drug interaction on the metabolism of blonanserin. Human recombinant CYP3A4 was prepared using the Bac-to-Bac baculovirus expression system. A microsomal enzyme reaction system was established, and drug-drug interactions were evaluated using Sprague-Dawley rats. Ultra-performance liquid chromatography-tandem mass spectrometry was used to detect the concentrations of blonanserin and its metabolite. Compared with wild type CYP34A, the relative clearance of blonanserin by CYP3A4.29 significantly increased to 251.3%, while it decreased notably with CYP3A4.4, 5, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 23, 24, 28, 31, 33, and 34, ranging from 6.09% to 63.34%. Among 153 tested drugs, nimodipine, felodipine, and amlodipine were found to potently inhibit the metabolism of blonanserin. Moreover, the inhibitory potency of nimodipine, felodipine, and amlodipine varied with different CYP3A4 variants. The half-maximal inhibitory concentration and enzymatic kinetics assay demonstrated that the metabolism of blonanserin was noncompetitively inhibited by nimodipine in rat liver microsomes and was inhibited in a mixed manner by felodipine and amlodipine in both rat liver microsomes and human liver microsomes. When nimodipine and felodipine were coadministered with blonanserin, the area under the blood concentration-time curve (AUC)(0-t), AUC(0-∞), and C max of blonanserin increased. When amlodipine and blonanserin were combined, the C max of blonanserin C increased remarkably. The vast majority of CYP3A4 variants have a low ability to catalyze blonanserin. With combined administration of nimodipine, felodipine, and amlodipine, the elimination of blonanserin was inhibited. This study provides the basis for individualized clinical use of blonanserin. SIGNIFICANCE STATEMENT: The enzyme kinetics of novel CYP3A4 enzymes for metabolizing blonanserin were investigated. Clearance of blonanserin by CYP3A4.4, 5, 7-10, 12-14, 16-18, 23-24, 28, 31, 33, and 34 decreased notably, but increased with CYP3A4.29. Additionally, we established a drug interaction spectrum for blonanserin, in which nimodipine, felodipine, and amlodipine kinetics exhibited mixed inhibition. Moreover, their inhibitory potencies decreased with CYP3A4.4 and 5 compared to CYP3A4.1. This study provides essential data for personalized clinical use of blonanserin.

Identification of novel pregnane X receptor (PXR) agonists by In silico and biological activity analyses and reversal of cigarette smoke-induced PXR downregulation.

Cigarette smoke (CS) contains many toxins that collectively harm nearly every organ in the body, and smoking is a key risk factor for many chronic diseases. Aside from its toxic actions, CS may alter expression of the drug- and steroid-binding pregnane X receptor (PXR), which when activated upregulates expression of cytochrome P450 (CYP) enzymes, glutathione transferases (GSTs), and multidrug resistance protein 1 (MDR1), an adaptive metabolic array that mediates clearance of CS component toxins. We sought to identify new PXR agonists that may be useful for restoring PXR activity in conditions wherein it is suppressed, and their mechanisms of PXR binding and activation. PXR has a uniquely larger, hydrophobic, and highly flexible ligand-binding domain (LBD) vs. other nuclear receptors, enabling it to interact with structurally diverse molecules. We tested certain calcium channel blockers (CCBs) as a pharmacological subset of potential PXR ligands, analyzing by molecular docking methods, and identified a putative active site in the PXR LBD, along with the relevant bonds and bonding energies. We analyzed felodipine binding and agonist activity in detail, as it showed the lowest binding energy among CCBs tested. We found felodipine was a potent PXR agonist as measured by luciferase reporter assay, whereas CCBs with higher binding energies were less potent (amlodipine) or nearly inactive (manidipine), and it induced CYP3A4 expression in HepG2 cells, a known target of PXR agonism. Felodipine also both induced PXR mRNA in HepG2 hepatocytes and reduced CS extract-induced diminution of PXR levels, indicating it modulates PXR expression. The results illuminate mechanisms of ligand-induced PXR activation and identify felodipine as a novel PXR agonist.

Application of biorelevant saliva-based dissolution for optimisation of orally disintegrating formulations of felodipine.

The oral cavity is of great importance to the performance of orally retained formulations, including: orally disintegrating tablets, taste-masked formulations, and buccal/sublingual delivery systems. With regards to in vitro dissolution assessment of these dosage forms, human saliva should be represented by the dissolution media. Currently there is no general consensus regarding oral cavity dissolution. In this study pooled human saliva was characterised and utilised as dissolution media for biorelevant oral cavity dissolution studies and to assess drug release. Lipophilic drug felodipine with challenging biopharmaceutical properties was selected for assessment in oral cavity dissolution studies. These saliva dissolution studies investigated for the first time how biorelevant dissolution can be implemented as a screening tool to guide the formulation development process and to predict dosage form performance within the mouth. In this study a combination of three dissolution enhancement strategies (cryomilling, solid dispersion, and inclusion complexation) were employed to eventually increase the concentration of felodipine in saliva 150-fold. Using this successful formulation strategy orally disintegrating tablets of felodipine were produced. Interestingly, the percentage release of felodipine in compendial dissolution apparatus was shown to be over 80% after 10 min. On the other hand, saliva-based dissolution showed that percentage release of felodipine was only 0.2% after 10 min using the same formulation. This discrepancy in drug release between dissolution media highlights the need for biorelevant dissolution apparatus for the oral cavity to reliably assess performance of relevant dosage forms in vitro.

Dry Gel Containing Optimized Felodipine-Loaded Transferosomes: a Promising Transdermal Delivery System to Enhance Drug Bioavailability.

Felodipine has a very low bioavailability due to first-pass metabolism. The aim of this study was to enhance its bioavailability by transdermal application. Felodipine-loaded transferosomes were prepared by thin-film hydration using different formulation variables. An optimized formula was designed using statistical experimental design. The independent variables were the used edge activator, its molar ratio to phosphatidylcholine, and presence or absence of cholesterol. The responses were entrapment efficiency of transferosomes, their size, polydispersity index, zeta potential, and percent drug released after 8 h. The optimized formula was subjected to differential scanning calorimetry studies and its stability on storage at 4°C for 6 months was estimated. This formula was improved by incorporation of different permeation enhancers where ex vivo drug flux through mice skin was estimated and the best improved formula was formulated in a gel and lyophilized. The prepared gel was subjected to in vivo study using Plendil® tablets as a reference. According to the calculated desirability, the optimized transferosome formula was that containing sodium deoxycholate as edge activator at 5:1 M ratio to phosphatidylcholine and no cholesterol. The thermograms of this formula indicated the incorporation of felodipine inside the prepared vesicles. None of the tested parameters differed significantly on storage. The lyophilized gel of labrasol-containing formula was chosen for in vivo study. The relative bioavailability of felodipine from the designed gel was 1.7. In conclusion, topically applied lyophilized gel containing felodipine-loaded transferosomes is a promising transdermal delivery system to enhance its bioavailability.

Interactions of dimethylaminoethyl methacrylate copolymer with non-acidic drugs demonstrated high solubilization in vitro and pronounced sustained release in vivo.

Recent work demonstrated remarkable solubilization effects of methacrylate-copolymer Eudragit EPO (EPO) not only with acidic drugs but interestingly also with poorly soluble basic compounds. The current work studied EPO-mediated solubilization effects first in vitro using felodipine (FLP) and tamoxifen (TMX) as model compounds. EPO-containing solutions were subsequently compared in a rat pharmacokinetic study against reference solutions and suspensions. Surprisingly, solution formulations with EPO did not result in an increased relative oral bioavailability. Exposure was reduced for both drugs and plasma-profiles of the EPO solutions showed a delayed and lower maximum plasma concentration compared to the reference formulations. This sustained in vivo release was likely due to combined effects of strong drug-polymer interactions and pH-dependent precipitation of the polymer in the rat intestine. Remarkable was that in vitro drug-polymer coprecipitates did not reveal crystalline drug by polarized light microscopy. Thus, such a formulation approach provides a rather simple opportunity to modify drug release in vivo. However, this may be rather an approach for preclinical formulations, if high peak-to-trough ratios of plasma levels are problematic regarding adverse effects related to Cmax or if plasma concentrations drop too fast below required pharmacological concentrations.

Biomimetic Dissolution: A Tool to Predict Amorphous Solid Dispersion Performance.

The presented study describes the development of a membrane permeation non-sink dissolution method that can provide analysis of complete drug speciation and emulate the in vivo performance of poorly water-soluble Biopharmaceutical Classification System class II compounds. The designed membrane permeation methodology permits evaluation of free/dissolved/unbound drug from amorphous solid dispersion formulations with the use of a two-cell apparatus, biorelevant dissolution media, and a biomimetic polymer membrane. It offers insight into oral drug dissolution, permeation, and absorption. Amorphous solid dispersions of felodipine were prepared by hot melt extrusion and spray drying techniques and evaluated for in vitro performance. Prior to ranking performance of extruded and spray-dried felodipine solid dispersions, optimization of the dissolution methodology was performed for parameters such as agitation rate, membrane type, and membrane pore size. The particle size and zeta potential were analyzed during dissolution experiments to understand drug/polymer speciation and supersaturation sustainment of felodipine solid dispersions. Bland-Altman analysis was performed to measure the agreement or equivalence between dissolution profiles acquired using polymer membranes and porcine intestines and to establish the biomimetic nature of the treated polymer membranes. The utility of the membrane permeation dissolution methodology is seen during the evaluation of felodipine solid dispersions produced by spray drying and hot melt extrusion. The membrane permeation dissolution methodology can suggest formulation performance and be employed as a screening tool for selection of candidates to move forward to pharmacokinetic studies. Furthermore, the presented model is a cost-effective technique.

Multicomponent solid forms of felodipine: preparation, characterisation, physicochemical and in-vivo studies.

OBJECTIVES: This study aimed to improve biopharmaceutical parameters of the poorly soluble antihypertensive drug, felodipine, by preparing multicomponent solid forms using three coformers, viz. imidazole, nicotinamide and malonic acid. METHODS: The multicomponent solid forms were prepared by mechanochemical synthesis and characterised by various analytical techniques. These solid forms were further assessed for their physicochemical parameters. Pharmacokinetic and in-vivo antihypertensive activity was performed in rats. KEY FINDINGS: Felodipine (FEL) was found to be cocrystallised with imidazole (FEL-IM) while it formed eutectic with nicotinamide (FEL-NCT) and malonic acid (FEL-MA). Cocrystal was sustained by NH…N and NH….O hydrogen-bonded network. Solubility and intrinsic dissolution studies in 0.1 N HCl (pH 1.2) revealed that eutectics exhibited higher solubility and release rate than cocrystal vis-a-vis pure drug and were found to be stable under accelerated storage condition. Significant enhancement of bioavailability was observed in eutectics (3.5- to twofold) and cocrystal (1.3-fold) compared with the pure drug. Antihypertensive activity of new solid forms in an animal model showed a marked decrease in systolic blood pressure. CONCLUSIONS: Mechanochemical approach was successful to prepare multicomponent solid forms that have the potential to improve biopharmaceutical parameters of the poorly soluble drug, FEL.

Enhancement of the Oral Bioavailability of Felodipine Employing 8-Arm-Poly(Ethylene Glycol): In Vivo, In Vitro and In Silico Evaluation.

Poor oral bioavailability is the single most important challenge in drug delivery. Prominent among the factors responsible for this is metabolic activity of the intestinal and hepatic cytochrome P450 (CYP450) enzymes. In preliminary studies, it was demonstrated that 8-arm-PEG was able to inhibit the felodipine metabolism. Therefore, this report investigated the oral bioavailability-enhancing property of 8-arm-PEG employing detailed in vitro, in vivo, and in silico evaluations. The in vitro metabolism of felodipine by cytochrome P450 3A4-expressed human liver microsomes (HLM) was optimized yielding a typical Michaelis-Menten plot through the application of Enzyme Kinetic Module software from where the enzyme kinetic parameters were determined. In vitro investigation of 8-arm-poly(ethylene glycol) against CYP3A4-catalyzed felodipine metabolism employing human liver microsomes compared closely with naringenin, a typical grapefruit flavonoid, yielding IC50 values of 7.22 and 121.97 µM, respectively. The investigated potential of 8-arm-poly(ethylene glycol) in oral drug delivery yielded satisfactory in vitro drug release results. The in vivo studies of the effects of 8-arm-poly(ethylene glycol) on the oral bioavailability of felodipine as performed in the Large White pig model showed a >100% increase in plasma felodipine levels compared to controls, with no apparent effect on systemic felodipine clearance. The outcome of this research presents a novel CYP3A4 inhibitor, 8-arm-poly(ethylene glycol) for oral bioavailability enhancement.

Molecular Dynamics Simulation of Amorphous Hydroxypropylmethylcellulose and Its Mixtures With Felodipine and Water.

Understanding drug-polymer molecular interactions, their miscibility, supersaturation potential, and the effects of water uptake may be invaluable for selecting amorphous polymer dispersions that can maximize the oral bioavailability of poorly water-soluble drugs. Molecular dynamics simulations were performed using a model for hydroxypropylmethylcellulose (HPMC) resembling the substitution patterns found experimentally. HPMC at low and high water contents (0.9%-23.0% wt/wt) and mixtures with a hydrophobic drug, felodipine (FEL), were constructed. Tg values and densities after ∼30 ns aging at 298 K were close to published results. Except for hydrogen bonds (HBs) between the 5-O- and a 3-OH group in a neighboring repeat unit, HPMC oxygen atoms have a low HB probability (p < 0.1) perhaps due to shielding by surrounding substituents. Water molecules tend to be isolated at low water content while clusters were prevalent at ≥10.7% water. The Flory-Huggins FEL-HPMC interaction parameter (-0.20 ± 0.07) predicts complete miscibility at all HPMC compositions, in agreement with experiments. However, HBs between the FEL-N-H and HPMC favoring miscibility are disrupted with increasing water. Apparent diffusion coefficients versus water content were generated for water and FEL and a theory for the non-Einsteinian nature of water diffusion is proposed.

Probing adsorption of DSPE-PEG2000 and DSPE-PEG5000 to the surface of felodipine and griseofulvin nanocrystals.

Nanosized formulations of poorly water-soluble drugs show great potential due to improved bioavailability. In order to retain colloidal stability, the nanocrystals need to be stabilized. Here we explore the use of the poly(ethylene glycol) (PEG) conjugated phospholipids DSPE-PEG2000 and DSPE-PEG5000 as stabilizers of felodipine and griseofulvin nanocrystals. Nanocrystal stability and physicochemical properties were examined and the interaction between the PEGylated lipids and the nanocrystal surface as well as a macroscopic model surface was investigated. Using quartz crystal microbalance with dissipation monitoring both mass adsorption and the thickness of the adsorbed layer were estimated. The results indicate that the PEGylated lipids are adsorbed as flat layers of around 1-3nm, and that DSPE-PEG5000 forms a thicker layer compared with DSPE-PEG2000. In addition, the mass adsorption to the drug crystals and the model surface are seemingly comparable. Furthermore, both DSPE-PEG2000 and DSPE-PEG5000 rendered stable drug nanocrystals, with a somewhat higher surface binding and stability seen for DSPE-PEG2000. These results suggest DSPE-PEG2000 and DSPE-PEG5000 as efficient nanocrystal stabilizers, with DSPE-PEG2000 giving a somewhat higher surface coverage and superior colloidal stability, whereas DSPE-PEG5000 shows a more extended structure that may have advantages for prolongation of circulation time in vivo and facilitation for targeting modifications.

The effect of processing on the surface physical stability of amorphous solid dispersions.

The focus of this study was to investigate the effect of processing on the surface crystallization of amorphous molecular dispersions and gain insight into the mechanisms underpinning this effect. The model systems, amorphous molecular dispersions of felodipine-EUDRAGIT® E PO, were processed both using spin coating (an ultra-fast solvent evaporation based method) and hot melt extrusion (HME) (a melting based method). Amorphous solid dispersions with drug loadings of 10-90% (w/w) were obtained by both processing methods. Samples were stored under 75% RH/room temperatures for up to 10months. Surface crystallization was observed shortly after preparation for the HME samples with high drug loadings (50-90%). Surface crystallization was characterized by powder X-ray diffraction (PXRD), ATR-FTIR spectroscopy and imaging techniques (SEM, AFM and localized thermal analysis). Spin coated molecular dispersions showed significantly higher surface physical stability than hot melt extruded samples. For both systems, the progress of the surface crystal growth followed zero order kinetics on aging. Drug enrichment at the surfaces of HME samples on aging was observed, which may contribute to surface crystallization of amorphous molecular dispersions. In conclusion it was found the amorphous molecular dispersions prepared by spin coating had a significantly higher surface physical stability than the corresponding HME samples, which may be attributed to the increased process-related apparent drug-polymer solubility and reduced molecular mobility due to the quenching effect caused by the rapid solvent evaporation in spin coating.

A useful model capable of predicting the clearance of cytochrome 3A4 (CYP3A4) substrates in humans: validity of CYP3A4 transgenic mice lacking their own Cyp3a enzymes.

The accurate prediction for the body clearance of a novel drug candidate by humans during the preclinical stage contributes to its successful development. To improve the predictability of human hepatic clearance, we focused on CYP3A4, which is involved in the metabolism of more than 50% of all currently marketed drugs. In this study, we investigated the validity of the in vivo model using transgenic mice carrying the human CYP3A4 gene and lacking their own Cyp3a genes (CYP3A4-Tg mice). The CYP3A4 activity toward its substrates in liver microsomes was similar in CYP3A4-Tg mice and humans. As for the clearance, six CYP3A4 substrates (alprazolam, felodipine, midazolam, nifedipine, nitrendipine, and quinidine) were given intravenously to CYP3A4-Tg mice, and their hepatic intrinsic clearance (CLint,h) was evaluated. A regression analysis of the data obtained indicated that the CLint,h values of six substrates in CYP3A4-Tg mice were highly correlated with those in humans (R(2) = 0.95). This correlation could be improved by correcting the CLint,h values by the relative contribution of artificially expressed CYP3A4 to the overall metabolism in the mice. From these findings, it is reasonable to expect that the CLint,h of a particular drug in humans is predictable by applying the CLint,h obtained in CYP3A4-Tg mice to a regression line prepared in advance. The variance of the CLint,h prediction by this method was evaluated and found to be within a range of 2-fold of the regression value. These results suggest that the CYP3A4-Tg mouse model has the potential to accurately predict the human hepatic clearance of CYP3A4 substrates.

QSAR model for human pregnane X receptor (PXR) binding: screening of environmental chemicals and correlations with genotoxicity, endocrine disruption and teratogenicity.

The pregnane X receptor (PXR) has a key role in regulating the metabolism and transport of structurally diverse endogenous and exogenous compounds. Activation of PXR has the potential to initiate adverse effects, causing drug-drug interactions, and perturbing normal physiological functions. Therefore, identification of PXR ligands would be valuable information for pharmaceutical and toxicological research. In the present study, we developed a quantitative structure-activity relationship (QSAR) model for the identification of PXR ligands using data based on a human PXR binding assay. A total of 631 molecules, representing a variety of chemical structures, constituted the training set of the model. Cross-validation of the model showed a sensitivity of 82%, a specificity of 85%, and a concordance of 84%. The developed model provided knowledge about molecular descriptors that may influence the binding of molecules to PXR. The model was used to screen a large inventory of environmental chemicals, of which 47% was found to be within domain of the model. Approximately 35% of the chemicals within domain were predicted to be PXR ligands. The predicted PXR ligands were found to be overrepresented among chemicals predicted to cause adverse effects, such as genotoxicity, teratogenicity, estrogen receptor activation and androgen receptor antagonism compared to chemicals not causing these effects. The developed model may be useful as a tool for predicting potential PXR ligands and for providing mechanistic information of toxic effects of chemicals.

Selection of alternative CYP3A4 probe substrates for clinical drug interaction studies using in vitro data and in vivo simulation.

Understanding the potential for cytochrome P450-mediated drug-drug interactions (DDIs) is a critical step in the drug discovery process. DDIs of CYP3A4 are of particular importance because of the number of marketed drugs that are cleared by this enzyme. In response to studies that suggested the presence of several binding regions within the CYP3A4 active site, multiple probe substrates are often used for in vitro CYP3A4 DDI studies, including midazolam (the clinical standard), felodipine/nifedipine, and testosterone. However, the design of clinical CYP3A4 DDI studies may be confounded for cases such as 1-(2-hydroxy-2-methylpropyl)-N-[5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl]-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (AMG 458), with which testosterone is predicted to exhibit a clinically relevant DDI whereas midazolam and felodipine/nifedipine are not. To develop an appropriate path forward for such clinical DDI studies, the inhibition potency of 20 known inhibitors of CYP3A4 were measured in vitro using 8 clinically relevant CYP3A4 probe substrates and testosterone. Hierarchical clustering suggested four probe substrate clusters: testosterone; felodipine; midazolam, buspirone, quinidine, and sildenafil; and simvastatin, budesonide, and fluticasone. The in vivo sensitivities of six clinically relevant CYP3A4 probe substrates (buspirone, cyclosporine, nifedipine, quinidine, sildenafil, and simvastatin) were determined in relation to midazolam from literature DDI data. Buspirone, sildenafil, and simvastatin exhibited similar or greater sensitivity than midazolam to CYP3A4 inhibition in vivo. Finally, Simcyp was used to predict the in vivo magnitude of CYP3A4 DDIs caused by AMG 458 using midazolam, sildenafil, simvastatin, and testosterone as probe substrates.

Drug metabolism enzyme expression and activity in primary cultures of human proximal tubular cells.

We previously catalogued expression and activity of organic anion and cation, amino acid, and peptide transporters in primary cultures of human proximal tubular (hPT) cells to establish them as a cellular model to study drug transport in the human kidney [Lash, L.H., Putt, D.A., Cai, H., 2006. Membrane transport function in primary cultures of human proximal tubular cells. Toxicology 228, 200-218]. Here, we extend our analysis to drug metabolism enzymes. Expression of 11 cytochrome P450 (CYP) enzymes was determined with specific antibodies. CYP1B1, CYP3A4, and CYP4A11 were the only CYP enzymes readily detected in total cell extracts. These same CYP enzymes, as well as CYP3A5 and possibly CYP2D6, were detected in microsomes from confluent hPT cells, although expression levels varied among kidney samples. In agreement with Western blot data, only activity of CYP3A4/5 was detected among the enzyme activities measured. Expression of all three glutathione S-transferases (GSTs) known to be found in hPT cells, GSTA, GSTP, and GSTT, was readily detected. Variable expression of three sulfotransferases (SULTs), SULT1A3, SULT1E, and SULT2A1, and three UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A6, and UGT2B7, was also detected. When examined over the course of cell growth to confluence, expression of all enzymes was generally maintained at readily measurable levels, although they were often lower than in fresh tissue. These results indicate that primary cultures of hPT cells possess significant capacity to metabolize many classes of drugs, and can be used as an effective model to study drug metabolism.

Urinary excretion of aquaporin-2 after furosemide and felodipine in healthy humans.

OBJECTIVE: Furosemide inhibits renal sodium and chloride reabsorption in the loop of Henle. A compensatory increased reabsorption of sodium and water takes place in the collecting duct. It is not known whether aquaporin-2 (AQP2) renal water channels are involved in this compensatory reabsorption. In animals, dihydropyridine derivatives of calcium channel blockers down-regulate AQP2 in the collecting duct, but the effect has not been studied in humans. We sought to test the hypotheses that urinary excretion of aquaporin-2 (U-AQP2) increases after a single intravenous dose of furosemide, and that U-AQP2 decreases after a single oral dose of felodipine. MATERIAL AND METHODS: In two randomized, single-blind, placebo-controlled, cross-over studies, we measured the effect of furosemide and felodipine on U-AQP2, urine volume, free water clearance (CH2O), and fractional excretion of sodium (FENa) in 13 healthy subjects in each study. Plasma concentrations of vasopressin (AVP), renin (PRC), angiotensin II (ang II), aldosterone (aldo), atrial (ANP), and brain natriuretic peptides (BNP) were measured during the study. Glomerular filtration rate (GFR) was measured by constant infusion technique. U-AQP2 and hormones were determined by radioimmunoassay. RESULTS: Furosemide treatment increased U-AQP2 (202%), urine volume (214%), and FENa by a factor of 11, (p < 0.001 for all), whereas CH2O and GFR were unchanged. After treatment with placebo, no differences were seen. Furosemide treatment increased AVP (18%), PRC (60%), ang II (100%), and aldo (98%) (p < 0.032); ANP was decreased by 29% (p < 0.001), whereas there was no change in BNP. The hormones were unchanged after placebo except for a minor decrease in ANP after placebo. Felodipine tended to increase U-AQP2, to decrease CH2O and urine volume and GFR, and to increase FENa, but the effect was not significantly different from placebo. Felodipine increased PRC (82%) (p < 0.003) and ang II, but decreased aldo, and increased AVP. After placebo, PRC was unchanged, whereas ang II, aldo and AVP were changed as after felodipine. CONCLUSIONS: Furosemide treatment increased U-AQP2, AVP, and the activity of the renin-angiotensin-aldosterone system. These changes are most likely compensatory phenomena, which prevent an excess loss of sodium and water. Felodipine tended to increase U-AQP2.

Multisite kinetic analysis of interactions between prototypical CYP3A4 subgroup substrates: midazolam, testosterone, and nifedipine.

The potential of substrates and modifiers of CYP3A4 to show differential effects, attributed to the existence of multiple binding sites, confounds the straightforward prediction of in vivo drug-drug interactions from in vitro data. A set of in vitro interaction studies was performed in human lymphoblast-expressed CYP3A4 involving representatives of two CYP3A4 subclasses, midazolam (MDZ) and testosterone (TST); a distinct subgroup, nifedipine (NIF); and its structural analog, felodipine (FEL). Mechanistic insight into the interaction of each pair of substrates was provided by employing a range of multisite kinetic models; most were subtypes of a generic two-site model, but a three-site model was required for TST interactions. The complexity of the inhibition profiles and the selection of the kinetic model with appropriate interaction factors were dependent upon the kinetics of substrates involved (hyperbolic, substrate inhibition, or sigmoidal for MDZ/FEL, NIF, and TST, respectively). In no case was a simple reciprocity seen between pairs of substrates. The interaction profiles observed between TST, MDZ, NIF, and FEL involved several atypical inhibition features (partial, cooperative, concentration-dependent loss of characteristic homotropic behavior) and pathway-differential effects reflecting an 80-fold difference in Ki values and a delta factor (defining the alteration in the binding affinity in the presence of a modifier) ranging from 0.04 to 2.3. The conclusions from the multisite kinetic analysis performed support the hypothesis of distinct binding domains for each substrate subgroup. Furthermore, the analysis of intersubstrate interactions strongly indicates the existence of a mutual binding domain common to each of the three CYP3A4 substrate subclasses.

Unmasking the dynamic interplay between intestinal P-glycoprotein and CYP3A4.

Drug efflux by intestinal P-glycoprotein (P-gp) is known to decrease the oral bioavailability of many CYP3A4 substrates. We hypothesized that the interplay occurring between P-gp and CYP3A4 at the apical membrane would increase the opportunity for drug metabolism. To define the roles of P-glycoprotein (P-gp) and CYP3A4 in controlling the extent of intestinal absorption and metabolism, two substrates were tested. The transport, metabolism, and intracellular levels of N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K77, a cysteine protease inhibitor; P-gp and CYP3A4 substrate) and felodipine (CYP3A4 substrate only) were measured across CYP3A4-transfected Caco-2 cells in the presence of an inhibitor of CYP3A4 and P-gp, cyclosporine (CsA), or an inhibitor of P-gp and not CYP3A4, GG918 (N-[4-[2-(1,2,3,4-tetrahydro-6,7- dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine). The extent of metabolism was measured by calculating the extraction ratio (ER) across the cells, while accounting for intracellular changes occurring with P-gp inhibition. The (A)pical to (B)asolateral and B-->A ERs for K77 were 0.33 and 0.06, respectively. These changed with GG918 to 0.14 and 0.12 and with CsA to 0.06 and 0.04. Felodipine ERs were similar in both directions, 0.26 and 0.24 (A-->B and B-->A), and were unchanged in the presence of GG918 but decreased with CsA (0.14 and 0.11). The K77 absorption rate was increased 5 and 4.2-fold in the presence of CsA and GG918, respectively, whereas no change was observed for felodipine absorption. The decreased A-->B ER and increased absorption of K77 with GG918 suggest that P-gp influences the extent of drug metabolism in the intestine via prolonging the access of drugs to CYP3A4 near the apical membrane and decreasing transport across the cells.

In vitro metabolism and interaction of cilostazol with human hepatic cytochrome P450 isoforms.

1. Cilostazol (OPC-13013) undergoes extensive hepatic metabolism. The hydroxylation of the quinone moiety of cilostazol to OPC-13326 was the predominant route in all the liver preparations studies. The hydroxylation of the hexane moiety to OPC-13217 was the second most predominant route in vitro. 2. Ketoconazole (1 microM) was the most potent inhibitor of both quinone and hexane hydroxylation. Both the CYP2D6 inhibitor quinidine (0.1 microM) and the CYP2C19 inhibitor omeprazole (10 microM) failed to consistently inhibit metabolism of cilostazol via either of these two predominant routes. 3. Data obtained from a bank of pre-characterized human liver microsomes demonstrated a stronger correlation (r2=0.68, P < 0.01) between metabolism of cilostazol to OPC-13326 and metabolism of felodipine, a CYP3A probe, that with probes for any other isoform. Cimetidine demonstrated concentration-dependent competitive inhibition of the metabolism of cilostazol by both routes. 4. Kinetic data demonstrated a Km value of 101 microM for cilostazol, suggesting a relatively low affinity of cilostazol for CYP3A. While recombinant CYP1A2, CYP2D6 and CYP2C19 were also able to catalyze formation of specific cilostazol metabolites, they did not appear to contribute significantly to cilostazol metabolism in whole human liver microsomes.