PEGylated chitosan-based polymer micelle as an intracellular delivery carrier for anti-tumor targeting therapy

Stearic acid-grafted chitosan oligosaccharide (CSO-SA) micelles presented a potential candidate for intracellular drug delivery carrier due to its special spatial structure. In this article, CSO-SA was further modified by polyethylene glycol (PEG). The physicochemical properties of PEGylated CSO-SA (PEG-CSO-SA) micelles were characterized. After PEGylation, the critical micelle concentration (CMC) of PEG-CSO-SA had no significant change; the micelle size increased; and the zeta potential decreased. The cellular uptake of CSO-SA micelles before and after PEGylation in macrophage RAW264.7, immortalized rat liver cells BRL-3A and human liver tumor cells HepG2 was studied. About 58.4 ± 0.63% of CSO-SA micelles were uptaked by RAW264.7 in 24 h, however, only 17.7 ± 0.94% of PEG-CSO-SA micelles were internalized into RAW264.7 after the CSO-SA was modified with PEG in five molar times. Meanwhile, there were no changes in the uptake after PEGylation of CSO-SA in BRL-3A and HepG2. Using mitomycin C as a model drug, the in vitro anti-tumor activities of the drug loaded in the micelles were investigated. The 50% cellular growth inhibition (IC₅₀) of the drug decreased from 1.97 ± 0.2 to 0.13 ± 0.02 μg/mL after mitomycin C was loaded into CSO-SA micelles, and the IC₅₀ value of the drug had no obvious change when the CSO-SA was modified by PEG.     

Enhanced Oral Absorption of Paclitaxel in N-Deoxycholic Acid-N,O-Hydroxyethyl Chitosan Micellar System

The overall goal of this study was to develop a micellar system of paclitaxel (PTX) to enhance its oral absorption. An amphiphilic chitosan derivative, N-deoxycholic acid-N, O-hydroxyethyl chitosan (DHC), was synthesized and characterized by FTIR, 1H NMR, elemental analysis, and X-ray diffraction (XRD) techniques. The degree of substitution (DS) of hydroxyethyl group and deoxycholic acid group ranged from 89.5–114.5% and 1.11-8.17%, respectively. The critical micelle concentration (CMC) values of DHC decreased from 0.26 to 0.16 mg/mL as the DS of deoxycholic acid group increased. PTX was successfully loaded in DHC micelles with a high drug loading (31.68 ± 0.14%) and entrapment efficiency (77.57 ± 0.51%). The particle size of PTX-loaded DHC micelles ranged from 203.35 ± 2.19 to 236.70 ± 3.40 nm as the DS of deoxycholic acid group increased. After orally administration of PTX-loaded DHC micelles, the bioavailability was threefold compared with that of an orally dosed Taxol®. The single-pass intestinal perfusion studies (SPIP) showed that the intestinal absorption of micelles was via endocytosis involving a saturable process and a p-glycoprotein (P-gp)-inde-pendent way. All these indicated that the DHC micelles might be a promising tool for oral delivery of poorly water-soluble drugs. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:4543–4553, 2010     

Differential Involvement of P-Glycoprotein (ABCB1) in Permeability,Tissue Distribution,and Antinociceptive Activity of Methadone,Buprenorphine, and Diprenorphine: In Vitro and In Vivo Evaluation

Conclusions based on either in vitro or in vivo approach to evaluate the P-gp affinity status of opioids may be misleading. For example, in vitro studies indicated that fentanyl is a P-gp inhibitor while in vivo studies indicated that it is a P-gp substrate. Quite the opposite was evident for meperidine. The objective of this study was to evaluate the P-gp affinity status of methadone, buprenorphine and diprenorphine to predict P-gp-mediated drug-drug interactions and to determine a better candidate for management of opioid dependence. Two in vitro (P-gp ATPase and monolayer efflux) assays and two in vivo (tissue distribution and antinociceptive evaluation in mdr1a/b (?/?) mice) assays were used. Methadone stimulated the P-gp ATPase activity only at higher concentrations, while verapamil and GF120918 inhibited its efflux (p < 0.05). The brain distribution and antinociceptive activity of methadone were enhanced (p < 0.05) in P-gp knockout mice. Conversely, buprenorphine and diprenorphine were negative in all assays. P-gp can affect the PK/PD of methadone, but not buprenorphine or diprenorphine. Our report is in favor of buprenorphine over methadone for management of opioid dependence. Buprenorphine most likely is not a P-gp substrate and concerns regarding P-gp-mediated drug-drug interaction are not expected. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4928–4940, 2009     

Supersaturated polymeric micelles for oral cyclosporine A delivery

Polymeric micelles provide a promising platform for improving oral absorption of poorly soluble drugs. However, improved understanding of how drug retention within the hydrophobic micelle core can reduce drug absorption is required. We designed supersaturated polymeric micelles (Super-PMs) to increase molecularly dissolved drug concentration and gain an insight into the effect of the degree of supersaturation on oral absorption of cyclosporine A (CsA) in rats. The drug release from Super-PMs increased with an increase in initial supersaturation degrees in micelles. The cellular uptake of coumarin-6 was reduced by the retention of drug in polymer micelles. The transport flux of CsA across Caco-2 monolayer was increased with initial supersaturation degrees of 0.81–3.53 (p < 0.05). However, increase in supersaturation to 5.64 actually resulted in decreased CsA transport. The same trend was observed in a rat in vivo absorption study, in which the highest bioavailability of 134.6 ± 24.7% (relative to a commercial product, Sandimmun Neoral®, p < 0.01) was achieved when the supersaturation degree was 3.53. These results demonstrated that Super-PMs were a promising drug delivery system for compounds with low aqueous solubility. This study also provided an experimental proof for the hypothesis that moderately supersaturated formulations are valuable alternative to high supersaturation formulations, resulting in optimal in vivo performance, and the degree of supersaturation should be carefully controlled to optimize drug absorption.     

Distribution of Doxorubicin in Rats Undergoing Ultrasonic Drug Delivery

Ultrasound (US) increases efficacy of drugs delivered from micelles, but the pharmacokinetics have not been studied previously. In this study, US was used to deliver doxorubicin (Dox) sequestered in micelles in an in vivo rat model with bilateral leg tumors. One of two frequencies with identical mechanical index and intensity was delivered for 15 min to one tumor immediately after systemic injection of micellar Dox. Pharmacokinetics in myocardium, liver, skeletal muscle, and tumors were measured for 1 week. When applied in combination with micellar Dox, the ultrasoincated tumor had higher Dox concentrations at 30 min, compared to bilateral noninsonated controls. Initially, concentrations were highest in heart and liver, but within 24 h they decreased significantly. From 24 h to 7 days, concentrations remained highest in tumors, regardless of whether they received US or not. Comparison of insonated and noninsonated tumors showed 50% more Dox in the insonated tumor at 30 min posttreatment. Four weekly treatment produced additional Dox accumulation in the myocardium but not in liver, skeletal leg muscle, or tumors compared to single treatment. Controls showed that neither US nor the empty carrier impacted tumor growth. This study shows that US causes more release of drug at the targeted tumor. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3122–3131, 2010     

A novel mifepristone-loaded implant for long-term treatment of endometriosis: In vitro and in vivo studies

The objective of this study was to prepare a novel mifepristone-loaded PCL/Pluronic F68 implant to achieve long-term treatment of endometriosis. PCL/Pluronic F68 compound (90/10, w/w) with viscosity average molecular weight of 65,000 was successfully synthesized. The end-capped Pluronic F68 was incorporated in PCL matrixes as molecular dispersion without forming a copolymer. The mifepristone-loaded implant made of PCL/Pluronic F68 compound was a cylindrical capsule with an outer diameter of 2.5 mm and an inner diameter of 2.2 mm. The surface of PCL/Pluronic F68 compound appears porous because Pluronic F68 which is water soluble could leach out due to the water phase. Drug loading of 0.75-, 1.5- and 3.0-cm length implants was 3.05 ± 0.18, 6.06 ± 0.41 and 11.87 ± 0.39 mg, respectively. A sustained mifepristone release rate without obvious initial burst and later decline over a period of 180 d was observed. The cumulative drug release showed a linear relationship with time, indicating that mifepristone release from the implants followed zero-order kinetics (R² > 0.99). The data showed that the C_max and AUC_0–inf were proportional to imlant length and dose, and all groups reached plasma C_max at about the same time (approximately 7 d) and had similar T_1/2 (approximately 150 d) and MRT (approximately 220 d). There were obvious inhibitory effects on the growth of endometrial explants in Wister rats in a dose-dependent manner after administration of mifepristone-loaded implants with implant length from 1.5 to 9.0 cm for 1–3 months. However, mifepristone-loaded implants with implant length of 12.0 cm had no better inhibitory effects on the growth of endometrium when compared with the implants with implant length of 9.0 cm (P > 0.05). In conclusion, subcutaneous implantation of mifepristone-loaded PCL/Pluronic F68 capsules was proven an effective means for long-term treatment of chronic endometriosis.     

Pesticides and their binary combinations as P-glycoprotein inhibitors in NIH 3T3/MDR1 cells

The purpose of the present study was to assess do selected pesticides as well as their binary combinations act as inhibitors of P-glycoprotein (P-gp) activity of NIH 3T3 mouse fibroblasts stably transfected with human MDR1 gene (NIH 3T3/MDR1). As a result of P-gp inhibition, the increase of intracellular accumulation of a model P-gp substrate fluorescent calcein acetoxymethyl ester was measured. Pesticide and verapamil individual dose–response data were scaled and expressed as percent of maximum effect. Results showed that out of 14 pure pesticides tested, endosulfan, phosalone and propiconazole were nearly as potent as model inhibitor verapamil (EC₅₀ = 1.5 μM), while diazinon showed a lower potency of inhibiting P-gp transport activity (EC₅₀ = 58.4 μM). Concentrations of pesticides that produced the same inhibiting effect (isoboles) were combined binary. Results calculated using the isobole method revealed that diazinon caused synergistic effect in inhibiting P-gp transport activity in all combinations.     

P-glycoprotein function in peripheral T lymphocyte subsets of myasthenia gravis patients: Clinical implications and influence of glucocorticoid administration

Myasthenia gravis (MG) is an autoimmune neuromuscular disorder with a chronic clinical course that requires long-term glucocorticoid (GC) therapy. A drug efflux pump, P-glycoprotein (P-gp), actively transports GC out of target cells, thereby reducing its efficacy. We evaluated the P-gp function of peripheral-blood mononuclear cells in 59 MG patients. P-gp function was estimated from a decrease in fluorescent P-gp substrate Rhodamine 123 and its inhibition by the conformation-sensitive UIC2 monoclonal antibody. P-gp function on CD8⁺ T cells in 21 MG patients having experienced GC therapy was higher than that in 19 MG patients having no history of GC therapy (p = 0.026). There was a significant correlation between P-gp function in CD3⁺ (r = 0.55, p = 0.014) or CD4⁺ (r = 0.48, p = 0.034) T cells and the total dose of prednisolone for treatment. P-gp function on CD4⁺ T cells in MG patients who showed low responses to prednisolone therapy (n = 8) was higher than that in patients who showed relatively high responses to prednisolone therapy (n = 10) (p = 0.045). These results suggest that higher P-glycoprotein activity on CD3⁺ or CD4⁺ cells necessitated treatment with higher steroid doses in order to achieve a clinical response. The measurement of P-gp function on CD4⁺ T cells is useful in the assessment of clinical response to GC therapy.     

Self-Assembled Methoxy Poly(Ethylene Glycol)-Cholesterol Micelles for Hydrophobic Drug Delivery

To promote the application of methoxy poly(ethylene glycol)-cholesterol (mPEG—Chol), mPEG–Chol was used to prepare core-shell micelles encapsulating poorly water-soluble docetaxel (DTX-PM) by modified cosolvent evaporation method. Approaches to enhance DTX entrapment efficiency (EE) and minimize particle size were investigated in detail, including organic and aqueous phase composition, organic/aqueous phase ratio, and polymer concentration. In optimal formulation, micelles had higher EE (97.6%) and drug loading (4.76%) with the diameter of 13.76 ± 0.68 nm and polydispersity index of 0.213 ± 0.006. Transmission electron microscopy (TEM) showed that the micelles were spherical, and differential scanning calorimetry (DSC) analysis proved that DTX was successfully entrapped into mPEG–Chol micelles. The in vitro cytotoxicity experiments displayed that blank micelles had no effect on the growth of SKOV-3, BXPC-3, A549, and HepG-2 cells, demonstrating that mPEG–Chol was one of the biocompatible biomaterials. The half inhibition concentration of DTX-PM on SKOV-3, BXPC-3, A549, and HepG-2 cells were 10.08, 7.6, 28.37, and 125.75 ng/mL, respectively. DTX-PM had the similar antitumor activity to free DTX, indicating that mPEG–Chol was a promising micellar vector for hydrophobic drug delivery. In addition, this work provided a new and facile approach to prepare drug-loaded micelles with controllable performances. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1054–1062, 2013     

Effects of statins on nitric oxide/cGMP signaling in human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVECs) were established as in vitro models for the modulation of endothelial function and cell viability by statins. Emphasis was placed on the biphasic effects of the drugs on nitric oxide (NO) bioavailability and cytotoxicity, as well as drug interference with the interaction of endothelial NO synthase (eNOS) with caveolin-1 (Cav-1). Incubation of HUVECs with fluvastatin, lovastatin or cerivastatin for 24 h caused an approximately 3-fold upregulation of eNOS expression that was associated with increased eNOS activity and accumulation of cGMP. Cerivastatin exhibited the highest potency with an EC50 of 13.8 ± 2nM after 24 h, while having no effect after only 30 min. The effects of statins on eNOS expression were similar in control and Cav-1 knockdown cells, but the increase in eNOS activity was less pronounced in Cav-1-deficient cells. Statin-triggered cyto-toxicity occurred at ~10-fold higher drug concentrations (maximal toxicity at 1–10 µM), was sensitive to mevalonate, and was significantly enhanced in the presence of N^G-nitro-L-arginine. The overexpression of eNOS induced by clinically relevant concentrations of statins may contribute to the beneficial vascular effects of the drugs in patients. Stimulation of NO synthesis and cytotoxicity appear to share a common initial mechanism but involve distinct downstream signaling cascades that exhibit differential sensitivity to HMG-CoA reductase inhibition.     

The role of MAPK in the biphasic dose-response phenomenon induced by cadmium and mercury in HEK293 cells

Some studies show that Cd²⁺ and Hg²⁺ may induce cell proliferation and apoptosis via biphasic dose-response relationship in human cells. However, mechanisms underlying this phenomenon are still in puzzle. In this study, we aim at detecting the biphasic effects of Cd²⁺ and Hg²⁺ on proliferation and apoptosis of human embryonic kidney 293 (HEK293) cells, analyzing the change of the mitogen-activated protein kinase (MAPK) pathways, and discussing the relationship between them. The results demonstrate that Cd²⁺ and Hg²⁺ can stimulate cell proliferation at lower concentrations (0.05 and 0.5 μM) but inhibit it at higher concentrations (50 and 500 μM). Apoptosis increases at higher concentrations (50 and 500 μM) of Cd²⁺ and Hg²⁺. While 0.5 μM Cd²⁺ and Hg²⁺ decrease the JNK phosphorylation, 50 μM Cd²⁺ and Hg²⁺ increase the JNK and P38 phosphorylation. When HEK293 cells are treated with 20 μM JNK inhibitor or 100 μM ERK1/2 inhibitor, the cell proliferation do not increase significantly at low concentrations (0.05 and 0.5 μM), but still decrease at high concentrations (50 and 500 μM). When HEK293 cells are treated with 20 μM P38 inhibitor, the tendency of cell proliferation is not affected. Data in our study suggests that activation of MAPK pathway may be involved in the biphasic effect induced by Cd²⁺ and Hg²⁺.     

Comparative evaluation of two dye probes in the rat everted gut sac model for unambiguous classification of P-gp substrate and inhibitor

IntroductionP-glycoprotein (P-gp) plays a crucial role in beta-amyloid efflux from the blood–brain barrier thus becoming a promising pharmacological target in the treatment of Alzheimer's disease (AD). The increase of P-glycoprotein expression and activity by a P-gp inducer could be an effective pharmacological strategy in slowing or halting the progression of AD. Commonly used in vitro methods to classify a P-gp interacting molecule as substrate, inhibitor, modulator or inducer are not always confirmed by in vivo experiments. Here we validate the new dye-probe beta-amyloid (1–40) HiLyte Fluor? TR-labeled (Ab-HiLyte) (Anaspec) P-gp mediated transport in the ex vivo rat everted gut sac assay by using MC18 or MC266, a fully characterized P-gp inhibitor and substrate, respectively, and compare it with the commonly used dye rhodamine.MethodsMale Wistar rats' everted intestines were divided into sacs, each sac was filled with 10 μM Ab-HiLyte with or without 50 μM of MC18 or MC266. Ab-HiLyte concentrations in mucosal fluid were measured spectrophotometrically at 594 nm at each appropriate time.ResultsThe Ab-HiLyte P-gp mediated efflux had a K = 1.00 × 10^? 2 min^? 1 and t_1/2 = 68.74 min, while in the presence of MC18, the Ab-HiLyte efflux turned out to be reduced by an order of magnitude (K = 1.65 × 10^? 3 min^? 1) and the half life is extremely increased (t_1/2 = 419 min). A P-gp substrate, like MC266, determines no change in the efflux of Ab: the kinetic constant and the half life turned out to be unmodified (K = 1.81 × 10^? 2 min^? 1 and t_1/2 = 38.28 min).DiscussionThe results demonstrate that the new dye probe, Ab-HiLyte, could be a probe of choice to unequivocally distinguish between a P-gp substrate and an inhibitor. This is particularly important as different groups obtain a controversial classification of the same compound.     

Effects of Pluronic Block Copolymers on Drug Absorption in Caco-2 Cell Monolayers

Purpose. The present work characterizes the effects of Pluronic copolymers on the transport of a P-gp-dependent probe, rhodamine 123 (R123) in Caco-2 cell monolayers. Methods. The accumulation and efflux studies were performed on the confluent Caco-2 monolayers using fluorescent probes with and without Pluronic copolymers. Results. At concentrations below the critical micelle concentration single chains ("unimers) of Pluronic P85 enhanced the accumulation and inhibited the efflux of R123 in Caco-2 monolayers. The transport of the P-gp-independent probe, rhodamine 110 was not altered under these conditions. In contrast the micelles increased R123 accumulation to a much lower extent when compared to the unimers and enhanced R123 efflux in Caco-2 monolayers. Conclusions. Pluronic P85 unimers increase accumulation of a P-gp-dependent drug in Caco-2 monolayers through inhibition of the P-gp efflux system. The mechanism of the micelle effect is not known, however, it is very similar to the micelle effects in BBMEC. This has been previously shown to involve vesicular transport of the micelle-incorporated drug. The study suggests that Pluronic copolymers can be useful in increasing oral absorption of select drugs.     

Comparison of bidirectional lamivudine and zidovudine transport using MDCK,MDCK–MDR1, and Caco-2 cell monolayers

Bidirectional transport studies were conducted using Caco-2, MDCK, and MDCK–MDR1 to determine P-gp influences in lamivudine and zidovudine permeability and evaluate if zidovudine permeability changes with the increase of zidovudine concentration and/or by association of lamivudine. Transport of lamivudine and zidovudine separated and coadministrated across monolayers based on these cells were quantified using LC–MS–MS. Drug efflux by P-gp was inhibited using GG918. Bidirectional transport of lamivudine and zidovudine was performed across MDCK–MDR1 and Caco-2 cells. Statistically significant transport decrease in B → A direction was observed using MDCK–MDR1 for zidovudine and MDCK–MDR1 and Caco-2 for lamivudine. Results show increased transport in B → A and A → B directions as concentration increases but data from P_app increase in both directions for both drugs in Caco-2, decrease in MDCK, and does not change significantly in MDCK–MDR1. Zidovudine transport in A → B direction increases when coadministrated with increasing lamivudine concentration but does not change significantly in B → A direction. Zidovudine and lamivudine are P-gp substrates, but results assume that P-gp does not affect significantly lamivudine and zidovudine. Their transport in monolayers based on Caco-2 cells increase proportionally to concentration (in both directions) and zidovudine transport in Caco-2 cell monolayer does not show significant changes with lamivudine increasing concentrations. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4413–4419, 2009     

Pharmacokinetic drivers of toxicity for basic molecules: Strategy to lower pKa results in decreased tissue exposure and toxicity for a small molecule Met inhibitor

Several toxicities are clearly driven by free drug concentrations in plasma, such as toxicities related to on-target exaggerated pharmacology or off-target pharmacological activity associated with receptors, enzymes or ion channels. However, there are examples in which organ toxicities appear to correlate better with total drug concentrations in the target tissues, rather than with free drug concentrations in plasma. Here we present a case study in which a small molecule Met inhibitor, GEN-203, with significant liver and bone marrow toxicity in preclinical species was modified with the intention of increasing the safety margin. GEN-203 is a lipophilic weak base as demonstrated by its physicochemical and structural properties: high LogD (distribution coefficient) (4.3) and high measured pKa (7.45) due to the basic amine (N-ethyl-3-fluoro-4-aminopiperidine). The physicochemical properties of GEN-203 were hypothesized to drive the high distribution of this compound to tissues as evidenced by a moderately-high volume of distribution (Vd > 3 l/kg) in mouse and subsequent toxicities of the compound. Specifically, the basicity of GEN-203 was decreased through addition of a second fluorine in the 3-position of the aminopiperidine to yield GEN-890 (N-ethyl-3,3-difluoro-4-aminopiperidine), which decreased the volume of distribution of the compound in mouse (Vd = 1.0 l/kg), decreased its tissue drug concentrations and led to decreased toxicity in mice. This strategy suggests that when toxicity is driven by tissue drug concentrations, optimization of the physicochemical parameters that drive tissue distribution can result in decreased drug concentrations in tissues, resulting in lower toxicity and improved safety margins.     

Pre-clinical pharmacokinetics evaluation of an anticonvulsant candidate benzaldehyde semicarbazone free and included in β-cyclodextrin

The study aimed to investigate the pharmacokinetics and tissue distribution of the benzaldehyde semicarbazone (BS) a potential antiepileptic drug, administered as a free drug or complexed β-cyclodextrin (BS/β-CD). Free BS and BS/β-CD were administered to male Wistar rats as a 10 mg/kg intravenous bolus dose. For the oral route, 50 mg/kg and 100 mg/kg doses of the free drug and 50 mg/kg of the complex were administrated and plasma concentrations were determinated by a validated HPLC-UV method. Individual profiles were evaluated by non-compartmental and compartmental analysis using Excel^® and Scientist^®, respectively. Free BS plasma protein binding was 34 ± 5%. A one-compartmental model adequately described all the plasma profiles for both formulations. After intravenous (10 mg/kg) and oral (50 mg/kg) administration, the V_d (1.6 ± 0.5 and 2.2 ± 0.8 L/kg, respectively) and the Cl_tot (1.4 ± 0.5 and 1.8 ± 0.5 L/h kg, respectively) determinated for the BS/β-CD complex were higher than those obtained for the free drug, but the t_1/2 (0.8 ± 0.1 h) was similar (p < 0.05). The oral bioavailability of the BS/β-CD complex (～37%) was approximately 2-fold of the free BS (～20%). The higher drug brain penetration (2.8) after BS/β-CD dosing and the longer mean residence time in this organ, regardless of the administration route, reveals that the complex may be a potential drug carrier for the central nervous system delivery of BS.     

Evaluation of the maternal–fetal transfer of granisetron in an ex vivo placenta perfusion model

The objective of this study was to estimate maternal–fetal transplacental passage of granisetron in an ex vivo placental perfusion model. Term human placentas (N = 8) were collected immediately after delivery. A single cotyledon from each placenta was perfused granisetron concentration to mimic systemic maternal peak plasma concentrations following either IV (50 ng/mL) or transdermal administration (5 ng/mL). To assess drug transfer and accumulation, samples were collected from maternal and fetal compartments.In the 50 ng/mL open model, the mean transport fraction was 0.21 ± 0.08 with clearance index of 0.53 ± 0.66. Fetal peak concentrations achieved was 5.6 ± 6.6 ng/mL with mean accumulation of 5.35 ± 6.4 ng/mL. No drug was detected in the fetal compartment with the 5 ng/mL models.Transplacental passage of granisetron was inconsistent at the 50 ng/mL concentration that achieved with IV dosing. However, there consistently was no detectable passage in all the placentas evaluated of the granisetron at 5 ng/mL concentration that would be achieved after transdermal patch administration.     

Transport of 5,5-diphenylbarbituric acid and its precursors and their effect on P-gp,MRP2 and CYP3A4 in Caco-2 and LS180 cells

AimTo examine the transport of 5,5-diphenylbarbituric acid sodium (T2007) and its mono- (MMMDPB) and di- (T2000) methoxymethylated precursors and their inducibility potential in Caco-2 and LS180 cells.MethodsTransport studies of T2000, MMMDPB and T2007 in Caco-2 cells were performed in Transwells. P-gp and CYP3A4 activities were assayed by [³H]digoxin and rhodamine 123 cellular retention and testosterone 6β-hydroxylation, respectively. Expressions of PXR, VDR and CAR mRNA and CYP3A4, MDR1/P-gp and MRP2 mRNA and protein were determined by qPCR and Western blotting, respectively. PXR siRNA was used to assess the involvement of PXR.ResultsThe P_app(A→B)s and P_app(B→A)s of T2000, MMMDPB and T2007 were similar (30–35 × 10^?6 cm/s) in Caco-2 cells. Treatment for 3 days with T2000 (15 μM), MMMDPB (70 μM) and T2007 (300 μM) generally furnished a greater induction in LS180 cells over the Caco-2 cells due to the higher, natural abundance of PXR. Changes in expression were confined mostly to MDR1 and CYP3A4: in LS180 cells, treatment for 3 days increased MDR1 and CYP3A4 but not MRP2 mRNA, and elevated P-gp and CYP3A4 protein expression that led to decreased cellular accumulation of [³H]digoxin and rhodamine 123, and enhanced testosterone 6β-hydroxylase activity towards T2007, respectively. The silencing of PXR by PXR siRNA in LS180 cells significantly attenuated the induction of MDR1 and CYP3A4.ConclusionsT2000, MMMDPB, and T2007 exhibited high permeabilities but are not P-gp substrates. T2007 and its analogs upregulated CYP3A4 and MDR1 modestly via the PXR.     

Xenopus laevis oocytes expressing human P-glycoprotein: Probing trans- and cis-inhibitory effects on [3H]vinblastine and [3H]digoxin efflux

P-glycoprotein (P-gp; MDR1) recognizes and actively transports many structurally diverse compounds (hydrophobic neutral and cationic). We studied MDR1-mediated drug transport using a high-throughput (96-well) oocyte expression system. MDR1-expressing oocytes contained sufficient ATP levels to conduct fundamental efflux studies; the optimal experimental temperature was 25 °C. [³H]Vinblastine efflux by MDR1-expressing oocytes was detectable and afforded a K_m of 145.5 ± 25.4 μM. [³H]Vinblastine (5.6 ± 0.3 μM) and [³H]digoxin (1.0 ± 0.1 μM) were individually injected into MDR1-expressing oocytes and their efflux monitored. Quinidine and verapamil, known MDR1 substrates/inhibitors, showed trans-inhibition on MDR1-mediated [³H]vinblastine and [³H]digoxin efflux. Conversely, doxorubicin demonstrated cis-inhibition without trans-inhibition on MDR1-mediated [³H]vinblastine efflux. The MDR1-expressing oocyte system offers researchers with an alternative in vitro method to screen compounds and may allow one to probe P-gp drug–drug and/or drug–inhibitor interactions.     

Spray-dried casein-based micelles as a vehicle for solubilization and controlled delivery of flutamide: Formulation,characterization, and in vivo pharmacokinetics

Novel casein (CAS)-based micelles loaded with the poorly soluble anti-cancer drug, flutamide (FLT), were successfully developed in a powdered form via spray-drying technique. Genipin (GNP) was used to crosslink CAS micelles as demonstrated by color variation of the micelles. Drug solubilization was enhanced by incorporation within the hydrophobic micellar core which was confirmed by solubility study and UV spectra. Spherical core–shell micelles were obtained with a particle size below 100 nm and zeta potential around −30 mV. At low drug loading, FLT was totally incorporated within micellar core as revealed by thermal analysis. However, at higher loading, excess non-incorporated drug at micelle surface caused a significant reduction in the surface charge density. Turbidity measurements demonstrated the high physical stability of micelles for 2 weeks dependent on GNP-crosslinking degree. In a dry powdered form, the micelles were stable for 6 months with no significant changes in drug content or particle size. A sustained drug release from CAS micelles up to 5 days was observed. After i.v. administration into rats, CAS micelles exhibited a prolonged plasma circulation of FLT compared to drug solution. Furthermore, a more prolonged drug systemic circulation was observed for GNP-crosslinked micelles. Overall, this study reports the application of spray-dried natural protein-based micelles for i.v. delivery of hydrophobic anti-cancer drugs such as FLT.