Pretreatment of rhesus monkeys with transdermal patches containing physostigmine and procyclidine: implications of the delivery system for the potential application against VX nerve agent intoxication in humans.

Physostigmine (Phs) is a reversible inhibitor of acetylcholinesterase (AChE) that penetrates the blood-brain barrier (BBB) and could be used to protect the central nervous system (CNS) against the effects of nerve agents. For prophylactic effectiveness, long, steady, and adequate inhibition of AChE activity by Phs is needed to broadly protect against the CNS effects of nerve agents. Here, we evaluated the efficacy of transdermal patches containing Phs and procyclidine (PC) as prophylactic agents. Patches (25 cm2) containing 4.4 mg Phs and 17.8 mg PC had a protective ratio of approximately 78.6-fold in rhesus monkeys challenged with VX nerve agent and given an antidote. Physiologically based pharmacokinetic model in conjunction with an indirect pharmacodynamic (PBPK/PD) was developed for Phs and scaled to rhesus monkeys. The model was able to reproduce the concentration profile and inhibitory effect on AChE of Phs in monkeys, as evidenced by correlation coefficients of 0.994 and 0.992 for 25 cm2 and 49 cm2 patches, respectively (i.e., kinetic data), and 0.989 and 0.968 for 25 cm2 and 49 cm2 patches, respectively (i.e., dynamic data). By extending the monkey PBPK/ PD model to humans, the effective human dose was predicted to be five applications of a 25 cm2 patch (i.e., 22 mg Phs), and two applications of a 49 cm2 patch (i.e., 17.4 mg Phs). Therefore, given that patch application of Phs in rhesus monkeys has a prolonged effect (namely, AChE inhibition of 19.6% for the 25 cm2 patch and 23.0% for the 49 cm2 patch) for up to 216 h, patch formulation of Phs may provide similar protection against nerve agent intoxication in humans.

Crystal structures of human NSDHL and development of its novel inhibitor with the potential to suppress EGFR activity.

NAD(P)-dependent steroid dehydrogenase-like (NSDHL), an essential enzyme in human cholesterol synthesis and a regulator of epidermal growth factor receptor (EGFR) trafficking pathways, has attracted interest as a therapeutic target due to its crucial relevance to cholesterol-related diseases and carcinomas. However, the development of pharmacological agents for targeting NSDHL has been hindered by the absence of the atomic details of NSDHL. In this study, we reported two X-ray crystal structures of human NSDHL, which revealed a detailed description of the coenzyme-binding site and the unique conformational change upon the binding of a coenzyme. A structure-based virtual screening and biochemical evaluation were performed and identified a novel inhibitor for NSDHL harboring suppressive activity towards EGFR. In EGFR-driven human cancer cells, treatment with the potent NSDHL inhibitor enhanced the antitumor effect of an EGFR kinase inhibitor. Overall, these findings could serve as good platforms for the development of therapeutic agents against NSDHL-related diseases.

Effect of treatment period with LC478, a disubstituted adamantayl derivative, on P-glycoprotein inhibition: its application to increase docetaxel absorption in rats.

1. Treatment periods of P-glycoprotein (P-gp) inhibitors have revealed different efficacies. We have previously reported dose-dependent inhibition of P-gp in single-treatment with LC478. However, whether repeated treatment with LC478 can inhibit P-gp even at its ineffective single-treatment dose remains unknown. 2. Therefore, the purpose of this study was to assess the effect of repeated treatment (i.e., 7-day treatment) with LC478 on P-gp known to affect docetaxel bioavailability in rats. Effects of LC478 on P-gp mediated efflux and expression in MDCK-MDR1 cells, P-gp ATPase activity, and binding site with P-gp were evaluated.3. The 7-day treatment with LC478 increased docetaxel absorption via intestinal P-gp inhibition in rats. Intestinal concentrations of LC478 were 8.31-10.3 µM in rats after 7-day treatment of LC478. These concentrations were close to 10 µM that reduced P-gp mediated docetaxel efflux and P-gp expression in MDCK-MDR1 cells. Considering that intestinal LC478 concentrations after 1-day treatment were 2.68-4.19 µM, higher LC478 concentrations after 7-day treatment might have driven P-gp inhibition and increased docetaxel absorption. LC478 might competitively inhibit P-gp considering its stimulated ATPase activity and its binding site with nucleotide binding domain of P-gp. 4. Therefore, repeated treatment with LC478 can determine its feasibility for P-gp inhibition and changing docetaxel bioavailability.

Development and Validation of Analytical Method for SH-1242 in the Rat and Mouse Plasma by Liquid Chromatography/Tandem Mass Spectrometry.

SH-1242, a novel inhibitor of heat shock protein 90 (HSP90), is a synthetic analog of deguelin: It was previously reported that the treatment of SH-1242 led to a strong suppression of hypoxia-mediated retinal neovascularization and vascular leakage in diabetic retinas by inhibiting the hypoxia-induced upregulation of expression in hypoxia-inducible factor 1α (HIF-1ɑ) and vascular endothelial growth factor (VEGF). In this study, an analytical method for the quantification of SH-1242 in biological samples from rats and mice was developed/validated for application in pharmacokinetic studies. SH-1242 and deguelin, an internal standard of the assay, in plasma samples from the rodents were extracted with methanol containing 0.1% formic acid and analyzed at m/z transition values of 368.9→151.0 and 395.0→213.0, respectively. The method was validated in terms of accuracy, precision, dilution, matrix effects, recovery, and stability and shown to comply with validation guidelines when it was used in the concentration ranges of 1-1000 ng/mL for rat plasma and of 2-1000 ng/mL for mouse plasma. SH-1242 levels in plasma samples were readily determined using the developed method for up to 480 min after the intravenous administration of 0.1 mg/kg SH-1242 to rats and for up to 120 min to mice. These findings suggested that the current method was practical and reliable for pharmacokinetic studies on SH-1242 in preclinical animal species.

Suppression of Canine ATP Binding Cassette ABCB1 in Madin-Darby Canine Kidney Type II Cells Unmasks Human ABCG2-Mediated Efflux of Olaparib.

Endogenous canine ATP binding cassette B1 (cABCB1) is expressed abundantly in Madin-Darby canine kidney type II (MDCKII) cells, and its presence often complicates phenotyping of the transport process. Errors in estimating the corrected efflux ratio (cER), as a result of the variable expression of cABCB1, were examined for the dual substrates of ABCB1 and ABCG2 in MDCKII cells expressing human ABCG2 (hABCG2). cABCB1 mRNA and protein expression was 60% and 55% lower, respectively, in MDCKII cells expressing hABCG2 compared with the wild type, suggesting that the expression of endogenous cABCB1 became variable after the expression of hABCG2. To minimize the contribution of endogenous efflux, cABCB1 was suppressed kinetically (using verapamil as a selective inhibitor) or biochemically (transfecting short-hairpin RNA against cABCB1). Under these suppression conditions, cER values for irinotecan and topotecan (dual substrates of ABCB1 and ABCG2) were elevated by more than 4-fold and 2-fold, respectively, compared with cER values without the suppression. The cER of olaparib was similarly increased to 3- and 5-fold in MDCKII cells under the kinetic and biochemical suppression of cABCB1, respectively, suggesting that hABCG2-mediated efflux cannot be ruled out for olaparib. Since the substrate selectivity for ABCB1 and ABCG2 overlapped considerably, the possibility of an inaccurate estimation of cER must be considered for dual substrates in the case of the variable expression of cABCB1 in MDCKII cells.

Z-ligustilide and n-Butylidenephthalide Isolated from the Aerial Parts of Angelica tenuissima Inhibit Lipid Accumulation In Vitro and In Vivo.

Abnormal lipid metabolism, such as increased fatty acid uptake and esterification, is associated with nonalcoholic fatty liver disease (NAFLD). The aqueous extract of the aerial part of Angelica tenuissima Nakai (ATX) inhibited high-fat diet-induced hepatic steatosis in mice as well as oleic acid-induced neutral lipid accumulation in HepG2 cells. ATX decreased the mRNA and protein levels of CD36 and diglyceride acyltransferase 2 (DGAT2), the maturation of sterol regulatory element-binding proteins (SREBP), and the expression of the lipogenic target genes fasn and scd1. The ATX components, Z-ligustilide and n-butylidenephthalide, inhibited the expression of FATP5 and DGAT2 and thus oleic acid-induced lipid accumulation in HepG2 cells. These results suggest that ATX and its active components Z-ligustilide and n-butylidenephthalide inhibit fatty acid uptake and esterification in mice and have potential as therapeutics for NAFLD.

Specific Inhibition of the Distribution of Lobeglitazone to the Liver by Atorvastatin in Rats: Evidence for a Rat Organic Anion Transporting Polypeptide 1B2-Mediated Interaction in Hepatic Transport.

Cytochrome P450 enzymes and human organic anion transporting polypeptide (OATP) 1B1 are reported to be involved in the pharmacokinetics of lobeglitazone (LB), a new peroxisome proliferator-activated receptor γ agonist. Atorvastatin (ATV), a substrate for CYP3A and human OATP1B1, is likely to be coadministered with LB in patients with the metabolic syndrome. We report herein on a study of potential interactions between LB and ATV in rats. When LB was administered intravenously with ATV, the systemic clearance and volume of distribution at steady state for LB remained unchanged (2.67 ± 0.63 ml/min per kg and 289 ± 20 ml/kg, respectively), compared with that of LB without ATV (2.34 ± 0.37 ml/min per kg and 271 ± 20 ml/kg, respectively). Although the tissue-to-plasma partition coefficient (Kp) of LB was not affected by ATV in most major tissues, the liver Kp for LB was decreased by ATV coadministration. Steady-state liver Kp values for three levels of LB were significantly decreased as a result of ATV coadministration. LB uptake was inhibited by ATV in rat OATP1B2-overexpressing Madin-Darby canine kidney cells and in isolated rat hepatocytes in vitro. After incorporating the kinetic parameters for the in vitro studies into a physiologically based pharmacokinetics model, the characteristics of LB distribution to the liver were consistent with the findings of the in vivo study. It thus appears that the distribution of LB to the liver is mediated by the hepatic uptake of transporters such as rat OATP1B2, and carrier-mediated transport is involved in the liver-specific drug-drug interaction between LB and ATV in vivo.

Simultaneous quantification of volitinib and gefitinib in rat plasma by HPLC-MS/MS for application to a pharmacokinetic study in rats.

A rapid, simple, and accurate procedure was developed and validated for the simultaneous quantification of two anticancer agents, volitinib and gefitinib in rat plasma by high-performance liquid chromatography with tandem mass spectrometry. The samples were separated by gradient elution from a cyano column within five minutes, using 0.1% formic acid in acetonitrile and 10 mM ammonium formate solution (pH 3.0) as mobile phase. When plasma samples were deproteinated by adding methanol, the analytes in the extract were detected in the positive ionization mode with the tracer ion mass of 346.1 → 145.1 for volitinib and 446.8 → 128.1 for gefitinib. The assay was determined to be valid in the concentration ranges of 2 to 1000 ng/mL for volitinib, and of 1 to 500 ng/mL for gefitinib. Intra- and interday accuracies ranged from 88.0 to 104.7% for volitinib and from 90.3 to 101%, for gefitinib. The precision of the assay ranged from 2.1 to 9.71% for volitinib and 2.31 to 12.1% for gefitinib. This method was successfully applied to a pharmacokinetic study of volitinib and gefitinib after the administration of an intravenous or oral dose, indicating that the developed assay can be used to simultaneously determine the concentrations of volitinib and gefitinib in rat plasma.

High body clearance and low oral bioavailability of alantolactone, isolated from Inula helenium, in rats: extensive hepatic metabolism and low stability in gastrointestinal fluids.

Alantolactone (ALA) is a major bioactive sesquiterpene lactone present in the roots of Inula helenium L. (Asteraceae) which has been used widely in traditional medicine against various diseases such as asthma, cancer and tuberculosis. The pharmacologic activities of alantolactone have been well characterized, yet information on the physicochemical and pharmacokinetic properties of alantolactone and their mechanistic elucidation are still limited. Thus, this study aims to investigate the oral absorption and disposition of alantolactone and their relevant mechanisms. Log P values of alantolactone ranged from 1.52 to 1.84, and alantolactone was unstable in biological samples such as plasma, urine, bile, rat liver microsomes (RLM) and simulated gastrointestinal fluids. The metabolic rate of alantolactone was markedly higher in rat liver homogenates than in the other tissue homogenates. A saturable and concentration-dependent metabolic rate profile of alantolactone was observed in RLM, and rat cytochrome P450 (CYP) 1 A, 2C, 2D and 3 A subfamilies were significantly involved in its hepatic metabolism. Based on the well-stirred model, the hepatic extraction ratio (HER) was estimated to be 0.890-0.933, classifying alantolactone as a drug with high HER. Moreover, high total body clearance (111 ± 41 ml/min/kg) and low oral bioavailability (0.323%) of alantolactone were observed in rats. Taken together, the present study demonstrates that the extensive hepatic metabolism, at least partially mediated by CYP, is primarily responsible for the high total body clearance of alantolactone, and that the low oral bioavailability of alantolactone could be attributed to its low stability in gastrointestinal fluids and a hepatic first-pass effect in rats. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolic interactions of magnolol with cytochrome P450 enzymes: uncompetitive inhibition of CYP1A and competitive inhibition of CYP2C.

Magnolol (MAG; 5,5'-diallyl-2,2'-biphenyldiol) is a major bioactive component of Magnolia officinalis. We investigated the metabolic interactions of MAG with hepatic cytochrome P450 monooxygenase (CYP) through in vitro microsomal metabolism study using human (HLM) and rat liver microsomes (RLM). CYP2C and 3A subfamilies were significantly involved in the metabolism of MAG, while CYP1A subfamily was not in HLM and RLM. The relative contribution of phase I enzymes including CYP to the metabolism of MAG was comparable to that of uridine diphosphate glucuronosyltransferase (UGT) in RLM. Moreover, MAG potently inhibited the metabolic activity of CYP1A (IC50 of 1.62 µM) and 2C (IC50 of 5.56 µM), while weakly CYP3A (IC50 of 35.0 µM) in HLM and RLM. By the construction of Dixon plot, the inhibition type of MAG on CYP activity in RLM was determined as follows: uncompetitive inhibitor for CYP1A (Ki of 1.09-12.0 µM); competitive inhibitor for CYP2C (Ki of 10.0-15.2 µM) and 3A (Ki of 93.7-183 µM). Based on the comparison of the current IC50 and Ki values with a previously reported liver concentration (about 13 µM) of MAG after its seven times oral administration at a dose of 50 mg/kg in rats, it is suggested that MAG could show significant inhibition of CYP1A and 2C, but not CYP3A, in the in vivo rat system. These results could lead to further studies in clinically significant metabolism-mediated MAG-drug interactions.

Gender differences in the hepatic elimination and pharmacokinetics of lobeglitazone in rats.

The pharmacokinetics of lobeglitazone (LB) was studied after intravenous administration at a dose of 1 mg/kg and oral administration at doses of 0.1, 1 and 10 mg/kg in male and female rats. The area under the plasma concentration-time curve from time zero to infinity (AUCinf ) after intravenous administration was approximately 7.1 times higher in female rats than in male rats. In addition, the AUCinf in the case of oral administration was at least 4.4 times higher in female rats and appeared to increase in proportion to the dose in both genders. The in vitro half-lives were 18.8 ± 4.45 min and 60.7 ± 11.2 min, as evidenced by incubating liver microsomes obtained from male and female rats, respectively. As a result, the estimated CLint for LB for male rat liver microsomes (0.0779 ± 0.0233 ml/min/mg protein) was much higher than that for female rat liver microsomes (0.0233 ± 0.0039 ml/min/mg protein, p < 0.05). These observations suggest that there are gender differences in the pharmacokinetics and hepatic metabolism for LB in rats. Copyright © 2015 John Wiley & Sons, Ltd.

In vitro and in vivo evaluation of the effect of puerarin on hepatic cytochrome p450-mediated drug metabolism.

Puerarin (8-ß-D-glucopyranosyl-7-hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a major pharmacological component of Puerariae Radix, the root of Pueraria lobata. We investigated the effect of puerarin on hepatic cytochrome P450-mediated drug metabolism in rats and humans. The in vitro cytochrome P450 inhibitory effect of puerarin in human and rat liver microsomes was evaluated using the following model cytochrome P450 substrates: phenacetin for CYP1A, diclofenac for CYP2C, dextromethorphan for CYP2D, and testosterone for CYP3A. The in vivo pharmacokinetics of intravenous and oral buspirone, a probe substrate for CYP3A, was studied with single simultaneous intravenous coadministration of puerarin in rats. In the in vitro cytochrome P450 inhibition study, the rate of disappearance of testosterone was significantly reduced in the presence of 10 µM PU, while that of other cytochrome P450 substrates was not significantly affected in both human and rat liver microsomes, suggesting that puerarin inhibits the in vitro hepatic CYP3A-mediated metabolism in the human and rat systems (IC50 = 15.5 ± 3.9 µM). After intravenous administration of buspirone with single simultaneous coadministration of intravenous puerarin at a dose of 10 mg/kg in rats, the total area under the plasma concentration-time curve from time zero to time infinity was increased while time-averaged total body clearance decreased. When buspirone was orally administered in rats with the 10 mg/kg intravenous puerarin coadministration, both total area under the plasma concentration-time curve from time zero to time infinity and the extent of absolute oral bioavailability were significantly increased. Therefore, results of the in vitro microsomal and in vivo pharmacokinetic studies suggest the possible inhibition of hepatic CYP3A-mediated drug metabolism by puerarin administration, potentially leading to metabolism-mediated herb-drug interactions with clinical significance.

AAPE proliposomes for topical atopic dermatitis treatment.

CONTEXT: Anti-inflammatory effect of advanced adipose stem cell derived protein extract (AAPE) could be improved by minimising protein degradation. OBJECTIVE: To develop a proliposomal formulation of AAPE for the treatment of topical atopic dermatitis. MATERIALS AND METHODS: Proliposomal powder was manufactured by evaporating a solution of soy phosphatidyl choline, AAPE and Poloxamer 407 in ethanol under vacuum on sorbitol powder. Characterisation of proliposomes (zeta potential, diameter, stability and flowability) as well as in vivo efficacy in a dermatitis mouse model was investigated. RESULTS AND DISCUSSION: Reconstitution of the proliposomal powder formed liposomes of 589 ± 3.6 nm diameter with zeta potential of -51.33 ± 0.36 mV. Protein stability was maintained up to 90 days at 25 °C as proliposomes. In vivo studies on atopic dermatitis mouse model showed a significant reduction in IgE levels after topical AAPE proliposome treatment. CONCLUSION: AAPE proliposomes maintained protein stability and showed promising results for atopic dermatitis treatment.

Kinetic modeling of the plasma pharmacokinetic profiles of ADAMTS13 fragment and its Fc-fusion counterpart in mice.

Introduction: Fusion of the fragment crystallizable (Fc) to protein therapeutics is commonly used to extend the circulation time by enhancing neonatal Fc-receptor (FcRn)-mediated endosomal recycling and slowing renal clearance. This study applied kinetic modeling to gain insights into the cellular processing contributing to the observed pharmacokinetic (PK) differences between the novel recombinant ADAMTS13 fragment (MDTCS) and its Fc-fusion protein (MDTCS-Fc). Methods: For MDTCS and MDTCS-Fc, their plasma PK profiles were obtained at two dose levels following intravenous administration of the respective proteins to mice. The plasma PK profiles of MDTCS were fitted to a kinetic model with three unknown protein-dependent parameters representing the fraction recycled (FR) and the rate constants for endocytosis (kup, for the uptake into the endosomes) and for the transfer from the plasma to the interstitial fluid (kpi). For MDTCS-Fc, the model was modified to include an additional parameter for binding to FcRn. Parameter optimization was done using the Cluster Gauss-Newton Method (CGNM), an algorithm that identifies multiple sets of approximate solutions ("accepted" parameter sets) to nonlinear least-squares problems. Results: As expected, the kinetic modeling results yielded the FR of MDTCS-Fc to be 2.8-fold greater than that of MDTCS (0.8497 and 0.3061, respectively). In addition, MDTCS-Fc was predicted to undergo endocytosis (the uptake into the endosomes) at a slower rate than MDTCS. Sensitivity analyses identified the association rate constant (kon) between MDTCS-Fc and FcRn as a potentially important factor influencing the plasma half-life in vivo. Discussion: Our analyses suggested that Fc fusion to MDTCS leads to changes in not only the FR but also the uptake into the endosomes, impacting the systemic plasma PK profiles. These findings may be used to develop recombinant protein therapeutics with extended circulation time.

The limited intestinal absorption via paracellular pathway is responsible for the low oral bioavailability of doxorubicin.

1. Doxorubicin exhibited dose-independent pharmacokinetics after intravenous (5-20 mg/kg) and oral (20-100 mg/kg) administration to rats. Nearly all (82.1-99.7%) of the orally administered doxorubicin remained unabsorbed, and the hepatic first-pass extraction ratio and oral bioavailability of doxorubicin were approximately 0.5% and 1%, respectively. Based on these results, it is likely that the primary factor responsible for the low oral bioavailability of doxorubicin is the limited intestinal absorption, rather than the CYP3A4-mediated first-pass metabolism. 2. Moreover, the in vitro transport and cellular uptake studies using Caco-2 cell monolayers have revealed that doxorubicin crosses the intestinal epithelium primarily via the paracellular pathway (accounting for 85.6% of the overall absorptive transport) probably due to its physicochemical properties (hydrophilic cation; pKa = 9.67, log P = -0.5). These results suggest that P-glycoprotein (P-gp)-mediated efflux activity does not play a significant role in limiting the intestinal absorption of doxorubicin, attenuating the absorptive transport by only 5.56-13.2%. 3. Taken together, the present study demonstrated that the limited and paracellular intestinal absorption of doxorubicin was a major factor responsible for its low oral bioavailability, restricting the role of CYP3A4-mediated first-pass metabolism and P-gp-mediated efflux.

Transport of gemifloxacin, a 4th generation quinolone antibiotic, in the Caco-2 and engineered MDCKII cells, and potential involvement of efflux transporters in the intestinal absorption of the drug.

The oral (po) bioavailability of gemifloxacin mesylate in rats and its possible association with efflux transporters was investigated. The apparent permeabilities (Papp) of gemifloxacin across the Caco-2 cell monolayer were 1.20 ± 0.09 × 10(-5) cm/s for apical to basal (absorptive) transport, and 2.13 ± 0.6 × 10(-5) cm/s for basal to apical (secretory) transport for a 5-500 µM concentration range, suggesting the involvement of a carrier-mediated efflux in the secretory transport. The secretory transport in Caco-2 cells was significantly decreased by MRP2 (MK571) and BCRP (Ko143) inhibitors. The secretory transport was distinct in MDCKII/P-gp, MDCKII/MRP2 and MDCKII/BCRP cells, and the affinity was highest for MRP2, followed by BCRP and P-gp. The efflux was significantly decreased by verapamil and Ko143, but not significantly by MK571. The comparative po bioavailability in rats was increased by the preadministration of Ko143 (four-fold), MK571 (two-fold) and verapamil (two-fold). Efflux transporters appeared to significantly limit the bioavailability of gemifloxacin in rats, suggesting their possible contribution to the low bioavailability of the drug in the human (70%).

Prediction of the Time to Reach Equilibrium for Improved Estimation of the Unbound Fraction of Compounds in Equilibrium Dialysis Using kinetic Modeling.

Equilibrium dialysis (ED) is widely used in pharmacokinetics to determine the fraction of unbound (fu) compounds in plasma; however, the kinetics of drugs in the ED system with respect to their permeation across semi-permeable membranes has not been systemically studied. Here, the kinetics of the ED system, including the binding of drugs to plasma proteins, non-specific binding, and permeation across the membrane, was described to enable verification of the equilibrium, prediction of the time to reach equilibrium, and estimations of fu with data obtained during pre-equilibrium. Using data obtained during pre-equilibrium, the time to reach 90% equilibrium (t90%) and fu were estimated with reasonable accuracy. Notably, fu could be estimated reasonably well using one-time-point data for the calculation. Furthermore, the current modeling approach allowed concurrent estimations of fu and the decomposition rate of compounds that were metabolically unstable in the plasma. Reasonable metabolic rate constants were determined for cefadroxil and diltiazem, demonstrating the practicality of this method for determining kinetics related to fu characterization. Because the determination of fu of compounds with 'unfavorable' physicochemical properties is known to be experimentally challenging, the current method may be useful in determining the fu of compounds in vitro.

Involvement of CYP3A4 and MDR1 in altered metabolism and transport of indinavir in 1,25(OH)2D3-treated Caco-2 cells.

Altered drug concentrations may induce unexpected toxicity or treatment failure; thus, understanding the factors that alter the pharmacokinetic profiles of drugs is crucial for optimal disease treatment. Vitamin D receptor (VDR), a nuclear receptor, regulates the expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1), which are crucial determinants of drug pharmacokinetics. In this study, we investigated the effects of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], a VDR ligand, on the metabolism, transport, and pharmacokinetics of indinavir, a dual substrate of CYP3A4 and MDR1. 1,25(OH)2D3 treatment for three days upregulated the expression levels of CYP3A4 and MDR1 in Caco-2 cells and consequently led to an increase in the level of a metabolite formed via CYP3A4 (indinavir M6) and the efflux ratio of indinavir in transport study. The increase in the metabolic reaction was also confirmed through a metabolism assay performed using the lysate of 1,25(OH)2D3-treated Caco-2 cells. In the Ussing chamber study conducted with the rat intestine, 1,25(OH)2D3 treatment did not alter the transport of indinavir into the basolateral side but increased indinavir M6 formation. Similarly, plasma levels of the metabolite increased in 1,25(OH)2D3-treated rats; however, systemic exposure to indinavir led to insignificant alterations. Considering the overlapping substrate specificities for CYP3A4 and MDR1 and their significant roles in drug pharmacokinetics, VDR may play an important role in drug interactions of CYP3A4 and MDR1 substrates for accessing more effective and safe disease treatments.

Physiologically Based Pharmacokinetic Modelling to Predict Pharmacokinetics of Enavogliflozin, a Sodium-Dependent Glucose Transporter 2 Inhibitor, in Humans.

Enavogliflozin is a sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor approved for clinical use in South Korea. As SGLT2 inhibitors are a treatment option for patients with diabetes, enavogliflozin is expected to be prescribed in various populations. Physiologically based pharmacokinetic (PBPK) modelling can rationally predict the concentration-time profiles under altered physiological conditions. In previous studies, one of the metabolites (M1) appeared to have a metabolic ratio between 0.20 and 0.25. In this study, PBPK models for enavogliflozin and M1 were developed using published clinical trial data. The PBPK model for enavogliflozin incorporated a non-linear urinary excretion in a mechanistically arranged kidney model and a non-linear formation of M1 in the liver. The PBPK model was evaluated, and the simulated pharmacokinetic characteristics were in a two-fold range from those of the observations. The pharmacokinetic parameters of enavogliflozin were predicted using the PBPK model under pathophysiological conditions. PBPK models for enavogliflozin and M1 were developed and validated, and they seemed useful for logical prediction.

Poly-L-arginine and dextran sulfate-based nanocomplex for epidermal growth factor receptor (EGFR) siRNA delivery: its application for head and neck cancer treatment.

PURPOSE: A poly-L-arginine (PLR) and dextran sulfate (DEX)-based nano-sized polyelectrolyte complex (nanocomplex) was developed for epidermal growth factor receptor (EGFR) siRNA delivery for the treatment of head and neck cancer. METHODS: PLR and DEX-based nanocomplex including EGFR siRNA was prepared and characterized. In vitro cellular uptake efficiency and EGFR gene silencing effect of nanocomplex including EGFR siRNA were evaluated in Hep-2 and FaDu cells. Its in vivo anti-tumor efficacy was also assessed in FaDu tumor xenografted mouse model. RESULTS: The weight ratio of polymer:RNA was 15:1 and a nanocomplex system consisting of <200 nm in mean diameter and a positive surface charge was prepared. According to the results of confocal laser scanning microscopy (CLSM) and flow cytometry analyses, the PLR-DEX complex exhibited the best cellular uptake efficiency of EGFR siRNA in Hep-2 and FaDu cells, which led to the highest EGFR gene silencing efficiency in both cell lines. PLR-DEX/EGFR siRNA complex exhibited efficient tumor growth inhibition and EGFR silencing effect in a tumor xenografted mouse model. CONCLUSION: PLR and DEX-based nanocomplex containing EGFR siRNA was successfully developed. The new formulation was effective in EGFR gene silencing and tumor growth inhibition in head and neck cancer cells.