Towards Prediction of In Vivo Intestinal Absorption Using a 96-Well Caco-2 Assay

We systematically validated a robust 96-well Caco-2 assay via an extended set of 93 marketed drugs with diverse transport mechanisms and quantified by LC/MS/MS, to investigate its predictive utility while dealing with challenging discovery compounds. Utilizing nonlinear fit, the validation led to a good correlation (R² = 0.76) between absorptive permeability, log-P_app(A–B), from in vitro Caco-2 assay and reported human fraction of dose absorbed. We observed that paracellular compounds could be flagged by log P_app(A–B) (<?5.5 cm/s) and physicochemical property space (c log P < 1). Of 8000 Novartis discovery compounds examined 13% were subject to low recovery (< 30%). Compound loss was investigated by comparing cell monolayer and artificial membrane, while 0.5% bovine serum albumin (in both donor and acceptor compartments) was utilized to improve recovery. The second focus of this study was to investigate the advantages and limitations of the current Caco-2 assay for predicting in vivo intestinal absorption. Caco-2 measurements for compounds with high aqueous solubility and low in vitro metabolic clearance were compared to 88 in vivo rat bioavailability studies. Despite the challenges posed by discovery compounds with suboptimal physicochemical properties, Caco-2 data successfully projected low intestinal absorption. This platform sets the stage for mechanistically evaluating compounds towards improving in vitro–in vivo correlations.     

Binding of new aminopropan-2-ol compounds to bovine serum albumin, α1 acid glycoprotein and rat serum using equilibrium dialysis and LC/MS/MS

BackgroundThe binding of three new aminopropan-2-ol compounds briefly called 2F109, ANBL and TWo8 with potential cardiovascular activity to bovine serum albumin (BSA), α₁-acid glycoprotein (AGP) and to rat serum was studied. The chemical structures of these compounds are related to carvedilol. They possess an antiarrhythmic and hypotensive activity, and β- and α-adrenolytic mechanism of action. All analogues are weak bases with pKa values 8.65,8.85 and 8.26 for 2F109, ANBL and TWo8, respectively, and they possess lipophilic character (log P > 1.9584).MethodsThe extent of protein binding was determined using equilibrium dialysis in the range 2.5 – 900 μM, and 2.5 – 300 μM for binding of investigated compounds to BSA and AGP, respectively, and the quantitative measurement was done by LC/ESI-MS/MS assay.ResultsThe studied compounds bound to a single class of binding sites on BSA which was characterized by low affinity (K_d for 2F109 = 8.49 × 10^–5 M, for ANBL = 1.92 × 10^–5 M, and for TWo8 = 1.71 × 10^–5 M) and low capacity(n = 0.53 for 2F109,0.132 for ANBL and 0.13 for TWo8). The binding of 2F109, ANBL and TWo8 to AGP revealed one class of binding sites, with moderate affinity (K_d for 2F109 = 4.67 × 10^–6 M, for ANBL = 3.48 × 10^–5 M, and for TWo8 = 1.13 × 10^–5 M) and higher capacity (n = 2.21 for 2F109, 2.76 for ANBL and 2.28 for TWo8).ConclusionThe obtained data indicate that 2F109, ANBL and TWo8 moderately bind to BSA (34.2 – 71.2%) with low capacity (K_a = 6.21 × 10³–7.61 × 10³ M^–1)and strongly bind to AGP(71.5–85.5%)with moderate affinity (K_a = 7.94 × 104⁴.73 ×10⁵ M^–1).     

In vivo and in vitro evaluation of the estrogenic properties of the 17β-(butylamino)-1,3,5(10)-estratrien-3-ol (buame) related to 17β-estradiol

BackgroundBuame [17β-(butylamino)-1,3,5(10)-estratrien-3-ol] possesses anticoagulant and antiplatelet activities that are potentially antithrombotic. Since its estrogenicity is unknown, it was evaluated by established methods.MethodsBuame (10, 100, 500, and 1,000 μg/kg), 17β-estradiol (E₂) (100 μg/kg), or propylene glycol (10 ml/kg) were subcutaneously (sc) administered for three days to immature Wistar female rats (21 days old). The relative uterotrophic effect to E₂ was 78 (E₂ = 100) with a relative uterotrophic potency of 1.48 (E₂ = 100). Adult ovariectomized Wistar rats received an sc injection at 8:00 h (reversed cycle) of: 7.5 μg of E₂ (≈ 30 μg/kg), buame (≈ 750, 1,500, 3,000 μg/kg), or corn oil (≈ 1.2 ml/kg). After 24 h, progesterone (4–5 mg/kg) was administered. Sexual receptivity was assessed 5 to 7 h later, and the lordosis quotient (LQ; number lordosis/number mounts × 100) was evaluated.ResultsBuame induced lordosis (LQmax 85 ± 9; ED50 952 ± 19 μg/kg) and E₂ LQmax 56 ± 8; ED50 10 ± 2 μg/kg; the relative LQpotency was 0.51 (E₂ = 100). Buame competed with [³H]E₂ for the estrogen receptor (Buame RBA = 0.15 and Ki = 5.9 × 10^?7 M; E₂ RBA = 100; Ki = 6.6 × 10^?9 M). Buame increased MCF-7 cells proliferation, from 10^?11 to 10^?9 M, its proliferative effect was 1.73–1.79 (E₂ = 3.0–3.9); its relative proliferative effect to E₂ was 33–40% (E₂ = 100%) and relative potency 10.4–10.7 (E₂ = 100). Tamoxifen and fulvestrant (ICI 182,780) inhibited buame's proliferation indicating mediation through estrogen receptors in this response.ConclusionBuame is therefore an estrogen partial agonist with a weak estrogenic activity.     

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.     

On the Mechanism and Dynamics of Uptake and Permeation of Polyether-Copolyester Dendrimers Across an In Vitro Blood–Brain Barrier Model

Dendrimers have emerged as a promising drug delivery system due to their well defined size, tailorability, and multifunctional nature. However, their application in brain delivery is relatively a new area of research. The present study was aimed at evaluating the uptake and permeation of polyether-copolyester (PEPE) dendrimers across the blood–brain barrier model and exploring the underlying mechanisms. Saturation was observed in the uptake of rhodamine B labeled PEPE dendrimers by brain vascular endothelial (bEnd.3) cells at high concentrations. Clathrin and caveolin inhibitors produced partial inhibition of the dendrimer uptake, signifying contribution of both pathways in the uptake process. PEPE dendrimers were able to cross in vitro BBB model in high amounts with P_app of 19.7 ± 1.9 × 10^?6 cm/s and 38.6 ± 4.1 × 10^?6 cm/s for den-1-(G2)-400 and den-2-(G2)-400, respectively; and only 11–14% reduction in transendothelial electrical resistance during initial 4 h. The results of this study suggest that architecture of dendrimers plays a major role not only in influencing the extent and mechanism of uptake by bEnd.3 cells but also permeation across the BBB model. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3748–3760, 2009     

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.     

Covalent inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by the carbamates JZL184 and URB597

Carboxylesterase type 1 (CES1) and CES2 are serine hydrolases located in the liver and small intestine. CES1 and CES2 actively participate in the metabolism of several pharmaceuticals. Recently, carbamate compounds were developed to inhibit members of the serine hydrolase family via covalent modification of the active site serine. URB597 and JZL184 inhibit fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively; however, carboxylesterases in liver have been identified as a major off-target. We report the kinetic rate constants for inhibition of human recombinant CES1 and CES2 by URB597 and JZL184. Bimolecular rate constants (k_inact/K_i) for inhibition of CES1 by JZL184 and URB597 were similar [3.9 (±0.2) × 10³ M^?1 s^?1 and 4.5 (±1.3) × 10³ M^?1 s^?1, respectively]. However, k_inact/K_i for inhibition of CES2 by JZL184 and URB597 were significantly different [2.3 (±1.3) × 10² M^?1 s^?1 and 3.9 (±1.0) × 10³ M^?1 s^?1, respectively]. Rates of inhibition of CES1 and CES2 by URB597 were similar; however, CES1 and MAGL were more potently inhibited by JZL184 than CES2. We also determined kinetic constants for spontaneous reactivation of CES1 carbamoylated by either JZL184 or URB597 and CES1 diethylphosphorylated by paraoxon. The reactivation rate was significantly slower (4.5×) for CES1 inhibited by JZL184 than CES1 inhibited by URB597. Half-life of reactivation for CES1 carbamoylated by JZL184 was 49 ± 15 h, which is faster than carboxylesterase turnover in HepG2 cells. Together, the results define the kinetics of inhibition for a class of drugs that target hydrolytic enzymes involved in drug and lipid metabolism.     

The effects of sulfur mustard exposure and freezing on transdermal penetration of tritiated water through ex vivo pig skin

The percutaneous absorption of tritiated water (³H₂O) through sulfur mustard (SM) exposed abdominal pig skin was measured using in vitro Franz-type static diffusion cells. The barrier function to water permeation following exposure to liquid SM for 8 min and excision 3 h later did not change significantly. A small, but statistically significant difference (P < 0.05) in steady state penetration (Jss), permeability coefficient (Kp) and lag time (t_L) of ³H₂O was observed between fresh skin and skin stored frozen (?20 °C) for up to two weeks. Steady-state penetration and Kp values were significantly higher (P < 0.05) in skin stored frozen compared with fresh skin. Fresh naïve skin had an average Kp of 1.65 × 10^?3 cm h^?1, whereas frozen naïve skin was 2.04 × 10^?3 cm h^?1. Fresh SM exposed skin had a mean Kp of 1.72 × 10^?3 cm h^?1, whereas frozen SM exposed skin was 2.31 × 10^?3 cm h^?1. Lag times were also shorter (P < 0.05) in skin that had been stored frozen. Frozen, SM-exposed porcine abdominal skin may be used for in vitro penetration studies, but effects of treatment and storage on the barrier layer should be taken into account.     

High Shear Rheology and Anisotropy in Concentrated Solutions of Monoclonal Antibodies

The high shear rheology of three concentrated solutions of immunoglobulin G1 monoclonal antibodies (mAbl, mAb2, and mAb3), differing only in their complementarity determining regions, was characterized using rotary and capillary rheometry. The more viscous solutions (mAb1 and mAb3) showed non-Newtonian behavior at high shear rates exhibiting both shear thinning and appreciable normal stress differences (NSDs) in the shear rate range γ = 10 to 10⁴ s^? 1. The rheograms were retraced after γ is increased and decreased, suggesting reversible self-associations under shear. In contrast, mAb2 solutions showed Newtonian behavior up to γ = 6 × 10⁴ s^? 1. The critical shear stress τ_c, corresponding to the onset of the reduction in the viscosity η, is a measure of mAb equilibrium cluster strength and increased rapidly with concentration for the high viscosity mAb solutions above 100 mg/mL. In addition, decreasing the temperature from 20 °C to 5 °C increased η at low γ, but shear-thinning was enhanced and its onset occurred at a lower γ_c. Using an Arrhenius model η = A exp(E_a/kT), the activation energy for viscous flow E_a was found to decrease for mAb1 solutions as γ was increased from 10 to 10⁴ s^? 1, suggesting mAb cluster disruption or rearrangement under shear. In contrast, for mAb2, this E_a remained constant in the γ range. Finally, mAb1 and mAb3 solutions showed appreciable NSDs, with their N₁ > 0 scaling linearly with γ in the range 10³ to 10⁴ s^? 1, whereas their |N₂/N₁| was less than 0.25 in this region. These suggest anisotropy and deformation of their solution microstructure toward the extensional quadrant of the flow at high γ. In contrast, the NSDs for mAb2 were close to zero indicating that the solution microstructure under shear is practically isotropic. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2538–2549, 2013     

Contractile effects of endothelins on isolated ampullar segment of human oviduct in luteal phase of menstrual cycle

Endothelin-1 induces contractions of human oviduct ampullar segment in follicular phase of menstrual cycle, acting on ET_A receptors. The aim of our study was to investigate effects of endothelin-1, endothelin-2 and endothelin-3 on isolated ampullar segment of human oviducts, taken from the patients in luteal phase of menstrual cycle. Fallopian tubes were taken from 20 female patients (one tube from each patient) during abdominal hysterectomy with adnexectomy, due to extensive uterine fibroids. The oviduct ampulla was mounted in an organ bath longitudinally, and the tension of the isolated preparation was recorded with the isometric transducer. Endothelin-1 produced concentration-dependent tonic contraction of the isolated ampullar segment (EC₅₀ = 6.80 ± 1.2 × 10^?10 M), and concentration-dependent inhibition of its rhythmic contractions (EC₅₀ = 7.86 ± 2.3 × 10^?10 M). Endothelin-2 produced concentration-dependent tonic contraction of the isolated ampullar segment (EC₅₀ = 4.56 ± 0.3 × 10^?10 M), without affecting its rhythmic contractions. Endothelin-3 did not affect either tone or rhythmic contractions of the isolated preparations. Selective antagonist for ET_A receptor subtype, BQ 123, produced inhibition of endothelin-1 effects on both tone (pA₂ = 9.50) and spontaneous rhythmic contractions (pA₂ = 10.73), while selective antagonist for ET_B receptor subtype, BQ 788, produced only inhibition of endothelin-1 effects on tone (pA₂ = 9.61), while the effect of endothelin-1 on spontaneous rhythmic contractions remained unaffected. The results of our study suggest that in the luteal phase both ET_A and ET_B receptors regulate tone, and only ET_A receptors regulate rhythmic activity of human oviduct's ampullar segment.     

Extractive spectrophotometric assay of cyclizine in a pharmaceutical formulation and biological fluids

Two highly sensitive and simple spectrophotometric methods were developed to quantitate the drug cyclizine (CYC) in its pure form and in a pharmaceutical formulation. The two methods involved ion-associate formation reactions (method A) with mono-acid azo dyes, i.e., sudan (I) and sudan (II), as well as ion-pair reactions (method B) with bi-azo dyes, i.e., sudan (III), sudan (IV) and sudan red 7B (V). The reactions were extracted with chloroform, and the extraction products were quantitatively measured at 480, 550, 500, 530 and 570 nm using reagents I–V, respectively. The reaction conditions were monitored and optimised. The Beer plots for reagents I–V showed linear relationships for the concentrations of 4.2–52.0, 5.4–96.0, 3.5–43.0, 4.4–80.0 and 0.6–18.0 μg mL^?1, respectively, with molar absorptivities of 2.2 × 10⁴, 4.1 × 10⁴, 3.6 × 10⁴, 2.5 × 10⁴ and 1.3 × 10⁴ L mol^?1 cm^?1, respectively. Sandell sensitivities and detection limits were calculated and analysed. The implementation of the two methods to the analysis of a commercial tablet (Valoid) succeeded, and the recovery study suggested that there was no interference from common excipients in the tablet. Regarding the accuracy and precision of the methods, a statistical comparison of the results was performed using Student’s t-test and the F-test at the 95% confidence level. The accuracy and precision of the proposed methods were not significantly different.     

Dry Powder Aerosols Generated by Standardized Entrainment Tubes From Drug Blends With Lactose Monohydrate: 1. Albuterol Sulfate and Disodium Cromoglycate

The major objective of this study was: discriminatory assessment of dry powder aerosol performance using standardized entrainment tubes (SETs) and lactose-based formulations with two model drugs. Drug/lactose interactive physical mixtures (2%w/w) were prepared. Their properties were measured: solid-state characterization of phase behavior and molecular interactions by differential scanning calorimetry and X-ray powder diffraction; particle morphology and size by scanning electron microscopy and laser diffraction; aerosol generation by SETs and characterization by twin-stage liquid impinger and Andersen cascade impactor operated at 60 L/min. The fine particle fraction (FPF) was correlated with SET shear stress (τ_s), using a novel powder aerosol deaggregation equation (PADE). Drug particles were < 5 μm in volume diameter with narrow unimodal distribution (Span < 1). The lowest shear SET (τ_s = 0.624 N/m²) gave a higher emitted dose (ED ~ 84–93%) and lower FPF (FPF_6.4 ~ 7–25%). In contrast, the highest shear SET (τ_s = 13.143 N/m²) gave a lower ED (ED ~ 75–89%) and higher FPF (FPF_6.4 ~ 15–46%). The performance of disodium cromoglycate was superior to albuterol sulfate at given τ_s, as was milled with respect to sieved lactose monohydrate. Excellent correlation was observed (R² ~ 0.9804–0.9998) when pulmonary drug particle release from the surface of lactose carriers was interpreted by PADE linear regression for dry powder formulation evaluation and performance prediction.     

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     

Gabapentin-induced changes of plasma cortisol level and immune status in hysterectomized women

AimWe have examined the effects of gabapentin (GBP) on stress-related changes of cortisol and catecholamines in patients who underwent hysterectomy because of uterine fibrinoids. Additionally, we have observed the effect of GBP on the immune status in the acute stress response to surgery.MethodsSixty patients scheduled for an abdominal hysterectomy were randomly assigned to the GBP administration 1 h before surgery (n = 30 pts), or to the placebo group (n = 30 pts). Blood samples were collected before and 24 h after the surgery. The intensity of pain was assessed by a visual analogue scale (VAS) every 8 h at rest. Immunomodulatory effects of GBP were determined by flow cytometry. We followed the total proportion of CD3⁺ lymphocytes, CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺ B lymphocytes, CD16⁺CD56⁺CD3⁻NK cells and CD16⁺CD56⁺CD3⁺ NKT cells before and 24 h after hysterectomy. The plasma cortisol and catecholamines concentration was used to estimate the level of the stress response.ResultsVAS pain score at rest was significantly lower in the GBP group than in the placebo group (P = 0.003). Application of GBP significantly decreased the plasma cortisol level 24 h after the operation in comparison to the placebo group (P < 0,001). We found significant positive correlation between the VAS pain score and concentration of cortisol in all patients (P = 0.025). GBP reduced the concentration of catecholamines (p < 0.05). The proportion of CD3⁺ (P = 0.027) and CD3⁺CD4⁺cells (P = 0.006) was significantly lower in the GBP group 24 h after operation, while the contribution of CD19⁺ (P = 0.033) was significantly higher.ConclusionPreoperative administration of GBP reduced the pain scores at rest in patients at 0, 16 and 24 h after abdominal hysterectomy. Additionally, GBP reduced the stress response and changed immune parameters in the reaction to surgery.     

Case–control association analysis of polymorphisms in the delta-opioid receptor,OPRD1, with cocaine and opioid addicted populations

BackgroundAddiction susceptibility and treatment responsiveness are greatly influenced by genetic factors. Sequence variation in genes involved in the mechanisms of drug action have the potential to influence addiction risk and treatment outcome. The opioid receptor system is involved in mediating the rewarding effects of cocaine and opioids. The μ-opioid receptor (MOR) has traditionally been considered the primary target for opioid addiction. The MOR, however, interacts with and is regulated by many known MOR interacting proteins (MORIPs), including the δ-opioid receptor (DOR).MethodsThe present study evaluated the contribution of OPRD1, the gene encoding the DOR, to the risk of addiction to opioids and cocaine. The association of OPRD1 polymorphisms with both opioid addiction (OA) and cocaine addiction (CA) was analyzed in African American (OA n = 336, CA n = 503) and European American (OA n = 1007, CA n = 336) populations.ResultsThe primary finding of this study is an association of rs678849 with cocaine addiction in African Americans (allelic p = 0.0086). For replication purposes, this SNP was analyzed in a larger independent population of cocaine addicted African Americans and controls and the association was confirmed (allelic p = 4.53 × 10^?5; n = 993). By performing a meta-analysis on the expanded populations, the statistical evidence for an association was substantially increased (allelic p = 8.5 × 10^?7) (p-values non-FDR corrected).ConclusionThe present study suggests that polymorphisms in OPRD1 are relevant for cocaine addiction in the African American population and provides additional support for a broad role for OPRD1 variants in drug dependence.     

Preparation and characterisation of novel chlorothiazide potassium solid-state salt forms: Intermolecular self assembly suprastructures

Chlorothiazide (CTZ) is a poorly soluble diuretic agent. The aim of the present work was to produce and characterise a potassium salt form of chlorothiazide which has the potential advantages of improved aqueous solubility and potassium supplementation. A number of novel potassium salt forms of CTZ (CTZK) were prepared: CTZK monohydrate (form I), CTZK dihydrate (form II), anhydrous CTZK (form III), CTZK monohydrate hemiethanolate (form IV) and a desolvate of CTZK monohydrate hemiethanolate (form V). These salt forms were characterised by thermal analysis, PXRD, NMR, elemental analysis, FTIR, Karl Fisher titrimetry, ICP-MS and GC–MS. The ethanol-free CTZK forms were also characterised by dynamic vapour sorption analysis (DVS). CTZK form I was stable (in the DVS) over the range 0–60% RH. The dihydrate form of the salt was stable (in the DVS) over a broader range of relative humidities, 10–90% RH at 25 °C. CTZK form II was less hygroscopic at high humidities (70–90% RH) than the previously reported CTZNa dihydrate. Single crystal X-ray analysis indicated that chlorothiazide potassium, crystallised from water or water/acetone mixture, formed a dihydrated polymeric-like intermolecular self-assembly (ISA) suprastructure. The ISA coordination was determined to be: (CTZ)₃·K·(H₂O)₂(CTZ)₂·(H₂O)₂·K·(CTZ)₃ (monoclinic, space group: C2/c, single crystal cell parameters: a = 18.328(4) Å, b = 7.3662(16) Å, c = 19.993(5) Å, α = 90°, β = 99.729(3)°, γ = 90°). When CTZK was crystallised from ethanol, a monohydrate hemiethanolate ISA was formed, described as (CTZ)₃·K·CTZ·(H₂O)₂·CTZ·K·(CTZ)₂ (triclinic, space group: P-1, single crystal cell parameters: a = 7.078(3) Å, b = 9.842(5) Å, c = 21.994(11) Å, α = 87.522(13)°, β = 84.064(14)°, γ = 78.822(12)°). The aqueous solubility of CTZK dihydrate, was determined to be 78.71 ± 1.82 mg/ml, approximately 400-fold higher than chlorothiazide, indicating a biopharmaceutical advantage associated with the potassium salt form.     

Decreased analgesic effect of morphine,but not buprenorphine,in patients with advanced P-glycoprotein+ cancers

BackgroundP-glycoprotein (P-gp) is expressed on the blood-brain barrier (BBB) and acts as a transporter regulating the analgesic effect of morphine. The P-gp is also expressed by different types of tumors. The aim of this study was to determine the potential association of the P-gp expression in malignant tumors with analgesic effects in patients.MethodsThe P-gp expression in 120 malignant tumors was examined by immunohistochemistry. The analgesic responses of individual patients to morphine and buprenorphine (BNP) were evaluated by visual analog scale (VAS). The levels of plasma morphine and BNP were determined by HPLC.ResultsWe found that there was no significant difference in the values of VAS between patients with P-gp⁺ and P-gp^?malignant tumors in responses to 0.000025 g × kg^?2 of BNP administered by patient-controlled intravenous analgesia (PCIA), accompanied by similar levels of plasma BNP in those patients. In contrast, the values of VAS in response to 0.00075 g × kg^?2 of morphine in patients with P-gp⁺ tumors were significantly greater than those in the patients with P-gp^? tumors, although similar levels of plasma morphine were detected in both groups of patients. Furthermore, treatment with a higher dose (0.0011 g × kg^?2) of morphine effectively controlled pain in those with P-gp⁺ tumors.ConclusionOur data indicated that patients with P-gp⁺ tumors required a higher dose of morphine to achieve an analgesic effect and that the P-gp expression in tumors may be valuable for predicting the analgesic responses of patients with severe pain to morphine.     

The efficacy and safety comparison between tenofovir and entecavir in treatment of chronic hepatitis B and HBV related cirrhosis: A systematic review and Meta-analysis

BackgroundThe purpose of this study was to assess the efficacy and safety between tenofovir and entecavir in the treatment of CHB and HBV related cirrhosis through Meta-analysis. MethodsThe electronic databases of PubMed, the Cochrane Library, Nature, CNKI and WanFang data were searched. The key words were: (“tenofovir”, “entecavir”) and (“Chronic Hepatitis B” or “CHB”) and “Liver cirrhosis”. Heterogeneity and report bias were analyzed.ResultsThere was significant difference of ALT norm level in the short-term period of 3 months (RR = 1.43, 95%CI: 1.06–1.94, P < 0.017) and 6 months (RR = 0.89, 95%CI: 0.81–0.97, P < 0.017), and significant difference of undetectable HBV-DNA only in 3 months follow-up period (RR = 1.59, 95%CI: 1.04–2.42, P < 0.017) between TDF and ETV, but no significant difference in the long-term period. There is significant difference between TDF and ETV in eGFR level (RR = 1.601, 95%CI: 1.035–2.478, P = 0.0034) and hypophosphatemia incidence (RR = 4.008, 95%CI: 1.485–10.820, P = 0.006).ConclusionTDF has a better efficacy than ETV in 3 months treatment duration, but intriguingly, TDF might not better than ETV during the 6 months treatment period in the viral suppression and liver function improvement. There's no significant difference between TDF and ETV in the long-term treatment duration and in the treatment of HBV related liver cirrhosis. Both TDF and ETV could influence renal function but patients under TDF therapy may have more risk to suffer from renal damage and hypophosphatemia.     

Absorption mechanism of oxymatrine in cultured Madin–Darby canine kidney cell monolayers

Context Oxymatrine (OMT) is beneficial to human health by exerting various biological effects. Objective To investigate the absorption mechanism of OMT and discover absorption enhancers using Madin–Darby canine kidney (MDCK) cell monolayers.

Materials and methods Concentration effects on the transport of OMT were measured in the range of 1.0?×?10^?5–1.0?×?10^?3 M in 2?h. Then, the effect of time, direction, temperature and pH on the transport of OMT at 10^?4 M was studied. Moreover, P_app of OMT was determined in the absence/presence of cyclosporine and surfactants at 100?μM to further confirm the relative transport mechanism.

Results The P_{app AP→BL} ranged from (3.040?±?0.23)?×?10^?6 to (3.697?±?0.19)?×?10^?6?cm/s as the concentration varied from 10^?5 to 10^?3 M. OMT showed similar P_app at 4 and 37?°C (p?>?0.05). Increasing the apical pH 7.4 and 8.0 resulted in P_app versus pH 5.0 (p?<?0.01). Furthermore, in the presence of cyclosporine and surfactants including sodium citrate, sodium dodecyl sulphate (SDS) and deoxysodium cholate, P_app was (0.318?±?0.033)?×?10^?5, (0.464?±?0.048)?×?10^?5, (0.897?±?0.115)?×?10^?5 and (1.341?±?0.122)?×?10^?5?cm/s, respectively. In the presence of surfactants, P_app significantly increased up to 1.5–4.3-fold (p?<?0.05).

Discussion and conclusion OMT transport across MDCK cell monolayers was by passive diffusion. Sodium citrate, SDS and deoxysodium cholate serve as excellent absorption enhancers which are useful for the related research improving the oral bioavailability of OMT.