BINDING CHARACTERISTICS OF KNI-272 TO PLASMA PROTEINS,A NEW POTENT TRIPEPTIDE HIV PROTEASE INHIBITOR

The binding characteristics of KNI-272, a potent and selective human immuno-deficiency virus (HIV) protease inhibitor, were evaluated in rat and human plasma, and in solutions of human α₁-acid glycoprotein (AAG) and human serum albumin (HSA). The unbound fractions (F_u ) of KNI-272 were 12·13 and 2·24% in rat and human plasma, respectively, at the drug concentration of 1·0 μg mL⁻¹. Although KNI-272 binds to both AAG and HSA, the F_u of KNI-272 in AAG solution was 1·83%, and only one-quarter of that in HSA solution (F_u6·78%). Binding displacing agents, such as disopyramide, warfarin, diazepam, and digitoxin, were used to determine the binding site of KNI-272 on these plasma proteins. The F_u of KNI-272 in AAG solution increased 14-fold when disopyramide was added to the AAG solution. In addition, warfarin, diazepam, and digitoxin were added to HSA solution as representative drugs bound to distinct binding sites on HSA, namely sites I, II, and III, respectively. The F_u values of KNI-272 in HSA solution significantly increased when warfarin and diazepam were added. In particular, with the addition of warfarin to HSA solution, the F_u of KNI-272 increased to 16%. The modified Scatchard plots of KNI-272 binding to AAG and HSA both showed biphasic curves, and the KNI-272 binding sites at low concentration range on AAG and HSA disappeared with the addition of disopyramide and warfarin, respectively. Therefore, it is considered that KNI-272 binds to the identical site as disopyramide on AAG and site I on HSA in the low KNI-272 concentration range. By comparing the KNI-272 binding parameters obtained in human plasma and these protein solutions, we can assume that KNI-272 binding at low concentration in human plasma is mainly concerned with the binding on AAG. As KNI-272 concentration in plasma increases, HSA becomes concerned with KNI-272 binding.     

In silico prediction of human serum albumin binding for drug leads

Introduction: Binding of drugs to human serum albumin (HSA) strongly influences their pharmacokinetic behavior and is associated with drug safety issues, low clearance, low brain penetration, as well as drug-drug interactions. Thus, in silico prediction of HSA binding contributes significantly to the discovery of new drug candidates.

Areas covered: The authors provide a short overview on the principles of HSA binding and the crystal structure of HSA, as well as discussing and analyzing the recent structure- and ligand-based HSA binding models. The authors also present the advantages and limitations of each methodology to construct efficient local or global models and outline the critical structural features contributing to HSA.

Expert opinion: The in silico estimation of drug binding to HSA in early drug discovery contributes to the lead optimization process. Local models are useful for the design of new compounds with reduced HSA binding for a particular target receptor, while real-time quantitative structure-activity relationships or global models combining structure- and ligand-based approaches serve for compound libraries screening. However, research efforts on other important plasma proteins should be strengthened in the perspective to enable predictions of total plasma protein binding for clinical candidates.     

Antifungal efficiency of miconazole and econazole and the interaction with transport protein: A comparative study

Abstract

Context: Miconazole (MIZ) and econazole (ECZ) are clinically used as antifungal drugs.

Objective: The drug effect and binding property with transport protein human serum albumin of MIZ and ECZ were studied.

Materials and methods: The antifungal efficiency was investigated by microdiluting drug solutions from 0 to 48?μmol?L^?1 through microcalorimetry and voltammetry studies. Transmission electron microscopy was used for morphological observations of C. albicans. The interaction with HSA was studied by electrochemical methods, fluorescence spectrometry, electron microscopy, and molecular simulation.

Results: IC₅₀ of MIZ and ECZ for C. albicans were obtained as 19.72 and 29.90?μmol?L^?1. Binding constants of MIZ and ECZ with HSA of 2.36?×?10⁴ L?mol^?1 and 3.73?×?10⁴ L?mol^?1 were obtained. After adding MIZ solution of 12 and 40?μmol?L^?1, the peak currents increased to 4.887 and 6.024?μA. The peak currents of C. albicans in the presence of 20 and 48?μmol?L^?1 ECZ were 4.701 and 5.544?μA. The docking scores for MIZ and ECZ of the best binding conformation in site I and site II were 5.60, 4.79, 5.63, and 5.85.

Discussion and conclusion: Strong inhibition to the metabolism of C. albicans and destructive effect was proved for both drugs. The lower IC₅₀, growth rate constant of C. albicans, and higher peak current, reveal stronger antifungal activity of MIZ. Both drugs show an efficient quenching effect to intrinsic fluorescence residues of protein. MIZ mainly binds on site I while ECZ on site II. Molecular modeling experiments give further insight of the binding mechanism.     

Mechanistic investigations into the species differences in pinometostat clearance: impact of binding to alpha-1-acid glycoprotein and permeability-limited hepatic uptake

1.?The plasma clearance of the first-in-class DOT1L inhibitor, EPZ-5676 (pinometostat), was shown to be markedly lower in human compared to the preclinical species, mouse, rat and dog.

2.?This led to vertical allometry where various interspecies scaling methods were applied to the data, with fold-errors between 4 and 13. We had previously reported the elimination and metabolic pathways of EPZ-5676 were similar across species. Therefore, the aim of this work was to explore the mechanistic basis for the species difference in clearance for EPZ-5676, focusing on other aspects of disposition.

3.?The protein binding of EPZ-5676 in human plasma demonstrated a non-linear relationship suggesting saturable binding at physiologically relevant concentrations. Saturation of protein binding was not observed in plasma from preclinical species. Kinetic determinations using purified serum albumin and alpha-1-acid glycoprotein (AAG) confirmed that EPZ-5676 is a high affinity ligand for AAG with a dissociation constant (K_d) of 0.24?μM.

4.?Permeability limited uptake was also considered since hepatocyte CL_int was much lower in human relative to preclinical species. Passive unbound CL_int for EPZ-5676 was estimated using a correlation analysis of logD and data previously reported on seven drugs in sandwich cultured human hepatocytes.

5.?Incorporation of AAG binding and permeability limited hepatic uptake into the well-stirred liver model gave rise to a predicted clearance for EPZ-5676 within 2-fold of the observed value of 1.4?mL min^?1?kg^?1. This analysis suggests that the marked species difference in EPZ-5676 clearance is driven by high affinity binding to human AAG as well as species-specific hepatic uptake invoking the role of transporters.     

A unified strategy in selection of the best allometric scaling methods to predict human clearance based on drug disposition pathway

1.?It is critical to develop a unified strategy to select the best allometric scaling (AS) method for a given group of drugs.

2.?A total of 446 drugs with known human CL_iv, clear disposition pathway and animal (rat, dog, monkey) CL_iv were analyzed. All drugs were stratified based on their disposition pathway, liver extraction ratio (ER_H) and ratios of unbound clearance to renal glomerular filtration rate (R_GFR). Up to 22 AS methods were applied and compared in prediction of human CL_iv to each group of drugs.

3.?AS methods that give the best prediction of human CL_iv, were identified for drugs primarily eliminated through liver with a fraction of renal elimination (f_renal) within 0.3–0.5 or ER_H?>?0.3, where human CL_iv of more than 80% or 90% drugs could be accurately (within 2- or 3-fold error) predicted. For drugs with ER_H?<?0.3, acceptable accuracy could be achieved by a two species method TS_R,D resulting more than 60% or 75% drugs were predicted within 2- or 3-fold error.

4.?By stratified analysis of drugs, according to their disposition pathway and organ extraction ratio, a unified strategy was developed to select the best AS method in prediction of human CL_iv.     

Effect of Plasma Protein Binding on Kinetics of Capillary Uptake and Efflux

We examined the effect of plasma protein binding on the kinetics of organ accumulation and washout of drugs using the single-pass Kety–Renkin–Crone capillary model. An equation relating the accumulation and washout rate constant (k) with the plasma unbound fraction (f _u) was derived. Simulations showed that k was highly dependent on f _u if capillary permeability was high but was independent of f _u if permeability was low. The effect of plasma protein binding was to increase the rate of tissue accumulation and washout of drug but to decrease the equilibrium amount of drug taken up by the tissue, both effects mediated via a decrease in the volume of distribution. This model was used to analyze published data on the effect of plasma protein binding on the kinetics of accumulation and washout of isradipine and propafenone in the isolated perfused heart preparation. The relationship between k and f _u and the directly measured volume of distribution were in accordance with the model. Although more complex models relating k and f _u could be proposed, taking into account unequal flows in capillaries, slow dissociation of ligand from protein, and unstirred layer constraints, this simple model appears adequate for describing the effect of f _u on myocardial accumulation and washout of isradipine and propafenone.     

Quantitative projection of human brain penetration of the H3 antagonist PF-03654746 by integrating rat-derived brain partitioning and PET receptor occupancy

1.?Unbound brain drug concentration (C_b,u), a valid surrogate of interstitial fluid drug concentration (C_ISF), cannot be directly determined in humans, which limits accurately defining the human C_b,u:C_p,u of investigational molecules.

2.?For the H₃R antagonist (1R,3R)-N-ethyl-3-fluoro-3-[3-fluoro-4-(pyrrolidin-1-lmethyl)phenyl]cyclobutane-1-carboxamide (PF-03654746), we interrogated C_b,u:C_p,u in humans and nonhuman primate (NHP).

3.?In rat, PF-03654746 achieved net blood–brain barrier (BBB) equilibrium (C_b,u:C_p,u of 2.11).

4.?In NHP and humans, the PET receptor occupancy-based C_p,u IC₅₀ of PF-03654746 was 0.99?nM and 0.31?nM, respectively, which were 2.1- and 7.4-fold lower than its in vitro human H₃ K_i (2.3?nM).

5.?In an attempt to understand this higher-than-expected potency in humans and NHP, rat-derived C_b,u:C_p,u of PF-03654746 was integrated with C_p,u IC₅₀ to identify unbound (neuro) potency of PF-03654746, nIC₅₀.

6.?The nIC₅₀ of PF-03654746 was 2.1?nM in NHP and 0.66?nM in human which better correlated (1.1- and 3.49-fold lower) with in vitro human H₃ K_i (2.3?nM).

7.?This correlation of the nIC₅₀ and in vitro hH₃ K_i suggested the translation of net BBB equilibrium of PF-03654746 from rat to NHP and humans, and confirmed the use of C_p,u as a reliable surrogate of C_b,u.

8.?Thus, nIC₅₀ quantitatively informed the human C_b,u:C_p,u of PF-03654746.     

Binding of Sulfinpyrazone and its Metabolites in Human Serum and in Solutions of Human Serum Albumin

Abstract: The binding of sulfinpyrazone, its sulfone metabolite and its sulfide metabolite to serum protein was studied by equilibrium dialysis. At 20 μg/ml 99.1% of the parent compound was bound in serum, whereas 99.8% of the sulfide and 98.3% of the sulfone were bound at this concentration. The binding of the three compounds were studied in diluted serum and in solutions of human serum albumin (HSA). There was no evidence of binding to proteins other than albumin. The association constants to primary and secondary binding sites and the number of binding sites were calculated. For the sulfide a lower K₁-value in serum (0.76·10⁶ M^?1) than in the HSA solution (1.8·10⁶ M^?1) indicated the possible presence of a competitively bound substance in serum. In undiluted serum no displacing effect of the sulfide on sulfinpyrazone binding was found when both compounds were present in a concentration of 20 μg/ml, but in a HSA solution a pronounced sulfide induced displacement of the sulfinpyrazone from its primary binding site was shown. Acetylation of HSA depressed the binding of sulfinpyrazone but in undiluted serum there was no other effect on sulfinpyrazone binding by the addition of acetylsalicylic acid than could be explained by the displacing effect of salicylic acid. At concentrations at 20 μg/ml of sulfinpyrazone and above 50 μg/ml of the displacing agent significant displacement was demonstrated with phenylbutazone, tolbutamide and salicylic acid.     

Predicting Drug Binding to Human Serum Albumin and Alpha One Acid Glycoprotein in Diseased and Age Patient Populations

Plasma protein binding, namely the fraction unbound (f_u), can be an important determinant of the disposition and response of drugs. The primary objective of this study was to predict f_u values of 183 drugs utilizing either a single binding protein model, where the predominant binding protein had been established, or a multiple binding protein model (MBPM), where the relative binding contribution of human serum albumin (HSA) or alpha 1 acid glycoprotein (AAG) is known. Mean protein concentrations, dependent on disease or age, were used to account for changes in f_u. A simple scaling approach for binding protein concentration was employed to account for quantitative changes in molar concentrations of either HSA or AAG in their respective conditions. The MBPM predictive model works best if the relative binding contribution of HSA and AAG is known, and a scaler for the change in protein concentration can be adjusted accordingly. The value of MBPM was most evident when considering reported changes in lidocaine binding because of increasing AAG concentration in response to trauma. The present approach enhances the ability to predict f_u in diseased and age populations because of quantitative changes in major binding proteins.     

Interactions of Aldosterone Antagonist Diuretics with Human Serum Proteins

Purpose. The purpose of this study was to investigate the binding mechanism of aldosterone antagonist diuretics with human serum proteins, human serum albumin (HSA) and ₁-acid glycoprotein (AGP), as well as to identify the binding sites of the drugs on these proteins. Methods. Binding activities of spironolactone (SP) and its pharmacologically active metabolite canrenone (CR) to serum and serum protein were examined by ultrafiltration and spectroscopic techniques. The data for the binding of these drugs to HSA were analyzed on the basis of a theoretical model of simultaneous binding of the ligands. Results. The binding percentages of antagonist diuretics SP and CR to human serum proteins were 88.0% and 99.2%, respectively, at therapeutic concentrations. SP bound strongly and almost equally to both HSA and AGP, but CR bound strongly only to HSA. In addition, the displacement results found using fluorescent probes and ultrafiltration methods demonstrated that SP bound to site I, particularly to the warfarin region on HSA, and to the basic binding site on AGP, while CR bound to the warfarin region on HSA. Conclusions. The limited results presented here stress the need for caution on coadministration of acidic drugs which bind to the warfarin region on HSA and basic drugs which bind to AGP with SP and its metabolite CR.     

Investigation of the prerequisites for the optimization of specific plasma protein binding as a strategy for the reduction of first-pass hepatic metabolism

Abstract

1. It is hypothesized that the deliberate structural tailoring of compounds designed for drug use to increase the specific plasma protein binding can be used to reduce first-pass hepatic metabolism. To test the feasibility of this hypothesis, a dataset of drugs with plasma protein binding of 90% or above divided into three classes including 50 acids, 44 bases and 69 neutrals was analyzed.

2. Among the drugs with ≥99% plasma protein binding, the fraction of the total dose existing in free form in vivo (free dose fraction) decreased in the following order: acids (0.55%)?>?neutrals (0.16%)?>?bases (0.08%). The order was different for the fraction of the total dose that existed in plasma protein bound form (plasma protein bound dose fraction): acids (58%)?>?neutrals (17%)?=?bases (18%).

3. The free fraction was poorly correlated with the partition coefficient (Log P). The lower aqueous solubility associated with high plasma protein binding was explained by differences in Log P and not by the plasma protein binding per se. The logarithm of the extrarenal clearance was correlated with Log P. For acids and bases, extrarenal clearance was also correlated with f_u. For neutrals, plasma protein binding had no protective effect.     

Generalized pharmacokinetic modeling for drugs with nonlinear binding: I. Theoretical framework

The following integrodifferential equation is proposed as the basis for a generalized treatment of pharmacokinetic systems in which nonlinear binding occurs $$\phi '(c_u )c'_u = - q(c_u ) + g*c_u + f$$ where c_u≡unbound plasma drug concentration, f≡drug input rate,'indicates the derivative of a function, and * indicates the convolution operation: (g* c_u)(t)=∫ ₀ ^t g(t?u)c_udu.Possible physical interpretations of the functions q, g and f are: q (c_u)≡ rate at which drug leaves the sampling compartment, g * c_u ≡ rate at which drug returns to the sampling compartment from the peripheral system (tissues that are kinetically distinct from the sampling compartment), and φ(c_u) ≡ amount of drug in the sampling compartment. The approach assumes that drug binding is sufficiently rapid that it may be treated as an equilibrium process. It may be applied to systems in which nonlinear binding occurs within the sampling compartment, i.e., in the systemic circulation or in tissues to which drug is rapidly distributed. The proposed relationship is a generalization of most existing models for drugs with nonlinear binding. It can serve as a general theoretical framework for such models or as the basis for “model-independent” methods for analyzing the pharmacokinetics of drugs with nonlinear binding. Computer programs for the numerical solution of the integrodifferential equation are presented. Methods for pharmacokinetic system characterization, prediction and bioavailability are presented and demonstrated.     

Validation of a total IC50 method which enables in vitro assessment of transporter inhibition under semi-physiological conditions

1.?Accurate predictions of clinical transporter-mediated drug–drug interactions (DDI) from in vitro data can be challenging when compounds have poor solubility and/or high nonspecific binding. Additionally, current DDI predictions for compounds with high plasma–protein binding assume that the unbound fraction in plasma is 0.01, if the experimental value is less than 0.01 or cannot be determined. This approach may result in an overestimation of DDI risk. To overcome these challenges, it may be beneficial to conduct inhibition studies under physiologically relevant conditions.

2.?Here, IC₅₀ values, determined in the presence of 4% bovine serum albumin approximating human plasma albumin concentrations, were successfully used to predict DDI for uptake transporters, OATP1B1/1B3, OCT1/2, OAT1/3 and MATE1/2K.

3.?The IC₅₀ values of reference inhibitors with 4% bovine serum albumin, considered total IC₅₀, were comparable to the predicted values based on nominal IC₅₀ values determined under protein-free conditions and unbound fraction in plasma. Calculation of R-total and C_max/IC_50,total values using total plasma exposure and total IC₅₀ values explained the clinical DDI or absence of it for these inhibitors.

4.?These results suggest that IC₅₀ determinations in the presence of 4% albumin can be used, in the context of clinical total exposure, to predict DDI involving uptake transporters.     

Irreversible binding of an anticancer compound (BI-94) to plasma proteins

Abstract

1.?We investigated the mechanisms responsible for the in vivo instability of a benzofurazan compound BI-94 (NSC228148) with potent anti-cancer activity.

2.?BI-94 was stable in MeOH, water, and in various buffers at pHs 2.5–5, regardless of the buffer composition. In contrast, BI-94 was unstable in NaOH and at pHs 7–9, regardless of the buffer composition. BI-94 disappeared immediately after spiking into mice, rat, monkey, and human plasma. BI-94 stability in plasma can be only partially restored by acidifying it, which indicated other mechanisms in addition to pH for BI-94 instability in plasma.

3.?BI-94 formed adducts with the trapping agents, glutathione (GSH) and N-acetylcysteine (NAC), in vivo and in vitro via nucleophilic aromatic substitution reaction. The kinetics of adduct formation showed that neutral or physiological pHs enhanced and accelerated GSH and NAC adduct formation with BI-94, whereas acidic pHs prevented it. Therefore, physiological pHs not only altered BI-94 chemical stability but also enhanced adduct formation with endogenous nucleophiles. In addition, adduct formation with human serum albumin-peptide 3 (HSA-T3) at the Cys34 position was demonstrated.

4.?In conclusion, BI-94 was unstable at physiological conditions due to chemical instability and irreversible binding to plasma proteins.     

Leishmanicidal candidate LASSBio-1736, a cysteine protease inhibitor with favorable pharmacokinetics: low clearance and good distribution

Abstract

1.?LASSBio-1736 ((E)-1-4(trifluoromethyl) benzylidene)-5-(2-4-dichlorozoyl) carbonylhydrazine) is proposed to be an oral cysteine protease leishmanicidal inhibitor.

2.?This work aimed to investigate plasma pharmacokinetics, protein binding and tissue distribution of LASSBio-1736 in male Wistar rats.

3.?LASSBio-1736 was administered to male Wistar rats at doses of 3.2?mg/kg intravenously and 12.6?mg/kg oral and intraperitoneal. The individual plasma-concentration profiles were determined by HPLC-UV and evaluated by non-compartmental and population pharmacokinetic analysis (Monolix 2016R1, Lixoft). Tissue distribution was evaluated after iv injection of 3.2?mg/kg drug by non-compartmental approach.

4.?After intravenous administration, Vd_ss (1.79?L/kg), t _½ (23.1?h) and CL_tot (56.1?mL/h/kg) were determined, and they were statistically similar (α?=0.05) to oral and intraperitoneal pharmacokinetic parameters. The plasma profiles obtained after intravenous, oral and intraperitoneal administration of the compound were best fitted to a three-compartment and one-compartment open model with first-order absorption.

5.?The intraperitoneal and oral bioavailability were around 40 and 15%, respectively.

6.?Liver, spleen and skin tissues showed penetration of 340, 130 and 40%, respectively, with t _½ like plasma values.

7.?LASSBio-1736 protein binding was 95?±?2%.

8.?The t _½, CL_tot and tissue distribution of the compound agreed with the desired drug characteristics for leishmanicidal activity.     

Prediction of in vivo drug interactions with eplerenone in man from in vitro metabolic inhibition data

1.?The inhibition kinetics of eplerenone (EP) 6β-hydroxylation by 10 drugs were determined in vitro using human liver microsomes. Inhibition factors were calculated from in vitro inhibition constant (K_i) and three different inhibitor C_max values (liver C_max of total and unbound inhibitor, and maximum influx concentration of inhibitor into the liver). Subsequently, the inhibition factors were compared with available pharmacokinetic data derived from clinical interaction trials conducted by Pfizer involving EP and these drugs. EP was also evaluated for its effect on the metabolism of 10 drugs in vitro, and again the in vitro data were compared with results from the clinical trials.

2.?The K_i values for the inhibition of EP 6β-hydroxylation by cisapride, cyclosporine, digoxin, erythromycin, fluconazole, ketoconazole, midazolam, saquinavir, simvastatin and verapamil were 2.90, 1.24,>75.0, 9.50, 59.0, 0.160, 8.10, 0.546, 6.23 and 13.3?μM, respectively. Among the three methods, inhibition factors (R_b) calculated using the K_i and estimated liver C_max values of the unbound drug were best correlated with the in vivo area under the curve-fold increases of EP in humans. The R_b values for the drugs listed above were 1.04, 1.69, 1.00, 2.17, 2.24, 4.90, 1.00, 1.82, 1.01 and 1.04, respectively, and the in vivo area under the curve-fold increases of EP by these drugs were 1.04, 1.16, 0.930, 2.87, 2.24, 5.39, 1.00, 2.07, 1.03 and 1.98, respectively.

3.?EP did not have any significant effects on the drugs tested in vitro or in the clinic.

4.?Using in vitro metabolic interaction data, human in vivo pharmacokinetic interactions involving EP could be predicted nearly quantitatively. The lack of effects of EP on the pharmacokinetics of other drugs in man was also suggested in the in vitro data.     

Spectroscopic and molecular docking studies for characterizing binding mechanism and conformational changes of human serum albumin upon interaction with Telmisartan

Human serum albumin (HSA), one of the most copious plasma proteins is responsible for binding and transportation of many exogenous and endogenous ligands including drugs. In this study, we intended to explore the extent and types of binding interaction present between HSA and the antihypertensive drug, telmisartan (TLM). The conformational changes in HSA due to this binding were also studied using different spectroscopic and molecular docking techniques. The spectral shifting and intensity variations upon interaction with TLM were studied using FT-IR spectroscopy. Binding constant and the change in absorption of HSA at its λ_max was analyzed using absorption spectroscopy. Eventually, the types and extent of binding interactions were confirmed using molecular docking technique. Results have shown that TLM significantly interacts with the binding site-1 of HSA utilizing strong hydrogen bonding with Glu292, and Lys195 residues. The UV-absorption intensities were found to be decreased serially as the drug concentration increased with a binding constant of 1.01 × 10³ M⁻¹. The secondary structure analysis using FT-IR spectroscopy also revealed a marked reduction in the α-helix (56%) component of HSA on interaction. This study gives critical insights into the interaction of TLM with HSA protein which eventually affects the concentration of TLM reaching the site of action and ultimately its therapeutic profile.     

Predictions of bisphenol A hepatic clearance in the isolated perfused rat liver (IPRL): impact of albumin binding and of co-administration with naproxen

1.?This study aimed (i) to characterise hepatic clearance (CL) of bisphenol A (BPA) and naproxen (NAP) administered alone or in binary mixtures to highlight the influence of a binding to albumin (ALB) using an isolated perfused rat liver (IPRL) system; and (ii) to compare results of prediction algorithms with measured clearance rates.

2.?The IPRL system and liver microsomes were used to determine the metabolic constants of BPA and NAP either in the presence or absence of ALB. In this study, the IPRL was used as proxy for the in vivo situation. Accordingly, diverse in vitro-to-in vivo and in vivo-to-in vivo extrapolations (IVIVEs) were made to predict CL of BPA determined in situ/in vivo with ALB from metabolic data determined without ALB by using different binding correction methods (i.e., direct and conventional scaling as well as a novel scaling considering an ALB-facilitated uptake mechanism).

3.?The addition of ALB significantly influenced the liver kinetics of BPA and NAP either administered alone or in binary mixtures, which was reflected in the Michaelis-Menten constants. Analysis of concomitant exposures of BPA and NAP gave a fully competitive inhibition. Furthermore, the IVIVE method based on the ALB-facilitated uptake mechanism provided the most accurate predictions of CL_{in vivo} as compared with the other IVIVE methods when the impact of ALB is considered.

4.?Our findings support the notion that high binding to ALB reduces the biotransformation of BPA and NAP when administered alone or in mixtures in the IPRL system. However, the free drug concentration in liver in vivo is probably higher than expected since the IVIVE method based on a potential ALB-facilitated uptake mechanism is the most robust prediction method. Overall, this study should improve the physiologically-based pharmacokinetic (PBPK) modelling of chemical–drug interactions.     

Bumetanide binding to normal albumins and to albumin in renal failure

Binding of bumetanide, a loop diuretic, to partially purified albumins from renal failure patients (RF-HA), and healthy subjects (N-HA), human serum albumin (HSA) and defatted-HSA (D-HSA), was studied with equilibrium dialysis at a constant albumin concentration and various ligand concentrations. Binding parameters (n and K) were estimated from Scatchard plots and with a nonlinear two-binding site model computer program, assuming two classes of independent sites. The binding capacities (n₁K₁) decreased in the order N-HA>RF-HA>D-HSA>HSA. Computer estimates of K₁ for the partially purified albumin preparations were not markedly different. However, the graphical estimate of K₁ for N-HA was greater than that for RF-HA. When the degree of binding (r) was plotted as a function of the logarithm of the free bumetanide concentration, an asymptotic plateau was not observed, indicating that the protein binding sites were not saturated. Consequently, the calculated binding estimates may not adequately describe the binding of bumetanide.     

Study on influence of piperine treatment on the pharmacokinetics of diclofenac in healthy volunteers

1.?Diclofenac sodium (DIC) is a widely used anti-inflammatory drug and its administration in humans receiving long-term therapy with herbal drugs containing piperine (PIP) may occur, which leads to drug–phytochemical interactions. The purpose of the present study was to investigate the influence of PIP treatment on the pharmacokinetics of DIC in healthy volunteers.

2.?The open-label, two period, sequential study was conducted in 12 healthy volunteers. PIP 20?mg was administered once daily for 10 days during treatment phase. A single dose of DIC 100?mg was administered during control and after treatment phases under fasting conditions. The blood samples were collected after DIC dosing at predetermined time intervals and analyzed by HPLC.

3.?Treatment with PIP significantly enhanced maximum plasma concentration (C_max) (2.24–3.68?μg/mL, p?<?0.05), area under the curve (AUC) (7.09–11.81?μg h/mL, p?<?0.05), half-life (T_1/2) (1.23–1.65?h, p?<?0.05) and significantly decreased elimination rate constant (K_el) (0.62–0.41?h^?1, p?<?0.05), apparent oral clearance (CL/F) (7.57–4.52?L/h, p?<?0.05) of DIC as compared to that of control phase.

4.?The results suggest that the altered pharmacokinetics of DIC might be attributed to PIP mediated inhibition of CYP2C9 enzyme, which indicates the clinically significant interaction present between DIC and PIP. Therefore, the combination therapy of DIC along with PIP may represent a novel approach to reduce dosage and result in reduced incidence of gastrointestinal side effects seen with DIC alone at higher doses.