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.     

Determination of lamivudine and zidovudine permeability using a different ex vivo method in Franz cells

IntroductionThe major processes that control the absorption of orally administered drugs are dissolution and gastrointestinal permeation. These processes depend on two main properties: solubility and permeability. Based on these characteristics, the Biopharmaceutical Classification System (BCS) was proposed as a tool to assist in biowaiver and bioavailability prediction of drugs.MethodsThe purpose of the present study was to evaluate the permeability of lamivudine (3TC) and zidovudine (AZT) using a different ex vivo method in Franz cells. A segment of jejunum was inserted in a Franz cells apparatus, in order to assess drug permeability in the apical–basolateral (A–B) and basolateral–apical (B–A) directions. Each drug was added to the donor chamber, collected from the acceptor chamber and analyzed by HPLC. Fluorescein (FLU) and metoprolol (METO) were used as low and high permeability markers, respectively.ResultsThe apparent permeability (P_app) results for the A–B direction were: P_{app FLU A–B} = 0.54 × 10^? 4 cm·s^? 1, P_{app METO A–B} = 7.99 × 10^? 4 cm·s^? 1, P_{app 3TC A–B} = 4.58 × 10^? 4 cm·s^? 1 and P_{app AZT A–B} = 5.34 × 10^? 4 cm·s^? 1. For the B–A direction, the P_app results were: P_{app FLU B–A} = 0.56 × 10^? 4 cm·s^? 1, P_{app METO B–A} = 0.25 × 10^? 4 cm·s^? 1, P_{app 3TC B–A} = 0.24 × 10^? 4 cm·s^? 1 and P_{app AZT B–A} = 0.19 × 10^? 4 cm·s^? 1.DiscussionFor the A–B direction, the P_app results of fluorescein and metoprolol show low and high permeability, respectively, indicating that the membranes were appropriate for permeability studies. For the A–B direction, the P_app results of 3TC and AZT suggest that these antiretroviral drugs have permeability values close to metoprolol. Nevertheless, for the B–A direction the P_app results do not suggest efflux mechanism for any of the drugs. Thereby, the different ex vivo methods using Franz cells can be successfully applied in drug permeability studies, in particular for drug biopharmaceutical classification.     

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.     

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.     

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.     

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     

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.     

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).     

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.     

Using transdermal iontophoresis to increase granisetron delivery across skin in vitro and in vivo: Effect of experimental conditions and a comparison with other enhancement strategies

The objectives of the study were (i) to investigate the effect of experimental parameters on the iontophoretic transport of granisetron, (ii) to identify the relative contributions of electromigration (EM) and electroosmosis (EO), (iii) to determine the feasibility of delivering therapeutic amounts of drug for the treatment of chemotherapy-induced nausea and vomiting and (iv) to test the in vitro results in a simple animal model in vivo. Preliminary in vitro studies using aqueous granisetron formulations investigating the effect of drug concentration (5, 10, 20 and 40 mM) and current density (0.1, 0.2, 0.3 mA cm^?2) were performed using porcine ear skin. As expected, cumulative delivery in vitro at the 20 and 40 mM concentrations was significantly greater than that at 5 and 10 mM, which were not statistically different (p < 0.05). Increasing the applied current density from 0.1 to 0.3 mA cm^?2 resulted in a ～4.2-fold increase in iontophoretic flux. Furthermore, in the absence of Na⁺ in the formulation, no dependence of iontophoretic flux on drug concentration was reported (at a granisetron concentration of 40 mM, the transport rate was 2.93 ± 0.62 μg cm^?2 min^?1). Co-iontophoresis of acetaminophen was used to show that EM was the predominant transport mechanism accounting for 71–86% of total granisetron delivery. In vivo studies in Wistar rats (40 mM granisetron; application of 0.3 mA cm^?2 for 5 h with Ag/AgCl electrodes and salt bridges) showed an average iontophoretic input rate (k_input) of 0.83 ± 0.26 μg min^?1 and a maximum plasma concentration (C_max) of 0.092 ± 0.004 μg ml^?1. Based on these results and given the known pharmacokinetics, transdermal iontophoresis could achieve therapeutic drug levels for the management of chemotherapy-induced emesis using a reasonably sized (4–6 cm²) patch.     

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.     

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.     

Modulation of ghrelin axis influences the growth of colonic and prostatic cancer cells in vitro

BackgroundThe risk of different cancers seems to be associated with obesity. Moreover, low ghrelin levels observed in obese people may be implicated in cancer development and progression. The aim of this study was to examine the direct effects of both forms of ghrelin (acylated and unacylated) and ghrelin receptor type 1a antagonist (D-Lys-GHRP-6) on the growth of murine colon cancer MC38 and human prostate cancer DU145 cell lines in vitro.MethodsThe cells were cultured for 72 h in the presence of rat or human acylated ghrelin (rG, hG), human unacylated ghrelin (hUAG), D-Lys-GHRP-6 (GHS-RA) applied either alone or jointly. The cell line growth was assessed by the colorimetric Mosmann method.ResultshUAG (10^?6, 10^?7 and 10^?10 M) inhibited MC38 cancer cell growth and, at some concentrations (10^?8, 10^?9, 10^?10 M), enhanced the antineoplastic effect of GHS-RA (10^?4 M). In turn, GHS-RA evoked a biphasic effect on MC38 cancer growth: inhibitory at 10^?4 M and stimulatory at 10^?5 and 10^?6 M. Moreover, GHS-RA at the highest examined concentration (10^?4 M) enhanced the cytostatic effect of FU. Human acylated and unacylated ghrelin and GHS-RA inhibited DU145 cancer growth with moderate and different potencies. A dose-response effect was observed for the inhibitory action of hG together with the synergistic effect of hUAG and GHS-RA.ConclusionThe obtained results indicate an involvement of the ghrelin axis in the growth regulation of colon and prostate cancers and may suggest new therapeutic options for these neoplasms.     

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     

Synthesis and pharmacological properties of new GABA uptake inhibitors

Backgroundγ-Aminobutanoic acid (GABA) is the principal inhibitory neurotransmitter in the mammalian central nervous system. The identification and subsequent development of the GABA transport inhibitors which enhance the GABA-ergic transmission has shown the important role that GABA transporters play in the control of numerous functions of the nervous system. Compounds which inhibit GABA uptake are used as antiepileptic drugs (tiagabine - a selective GAT1 inhibitor), they are also being investigated for other indications, including treatment of psychosis, general anxiety, sleep disorders, drug addiction or acute and chronic pain.MethodsIn this paper, the synthesis of 2-substituted-4-(1,3-dioxoisoindolin-2-ylo)-butanamides and 2-substituted-4-amino-butanoic acids derivatives is described. These compounds were tested in vitro for their ability to inhibit GABA uptake. The inhibitory potency towards murine plasma membrane GABA transporters (mGAT1-4) was performed as [³H]GABA uptake assay based on stably transfected HEK cells. Compound 18, which demonstrated the highest affinity for mGAT1-4 (pIC50 ranged from 4.42 for mGAT1 to 5.07 for mGAT3), was additionally investigated in several behavioral tests in mice.ResultsCompound 18 increased the locomotor activity (14–38%) and had anxiolytic-like properties in the four-plate test (ED₅₀ = 9.3 mg/kg). It did not show analgesic activity in acute pain model, namely the hot plate test, however, it was antinociceptive in the acetic acid-induced writhing test (ED₅₀ = 15.3 mg/kg) and in the formalin model of tonic pain. In the latter assay, it diminished nocifensive behavior in both phases and in the first (neurogenic) phase of this test the obtained ED50 value (5.3 mg/kg) was similar to morphine (3.0 mg/kg).ConclusionCompound 18 exhibited significant anxiolytic-like properties and was antinociceptive in some models of pain in mice. Moreover, it did not impair animals' motor coordination in the chimney test. Some of the described pharmacological activities of compound 18 can be partly explained based on its affinity for plasma membrane GABA transporters.     

Development of a high-throughput electrophysiological assay for the human ether-à-go-go related potassium channel hERG

IntroductionDrug-induced prolongation of the QT interval via block of the hERG potassium channel is a major cause of attrition in drug development. The advent of automated electrophysiology systems has enabled the detection of hERG block earlier in drug discovery. In this study, we have evaluated the suitability of a second generation automated patch clamp instrument, the IonWorks Barracuda, for the characterization of hERG biophysics and pharmacology.MethodsAll experiments were conducted with cells stably expressing hERG. Recordings were made in perforated patch mode either on a conventional patch clamp setup or on the IonWorks Barracuda. On the latter, all recordings were population recordings in 384-well patch plates.ResultsHERG channels activated with a V_1/2 = ? 3.2 ± 1.6 mV (n = 178) on the IonWorks Barracuda versus ? 11.2 ± 6.1 mV (n = 9) by manual patch clamp. On the IonWorks Barracuda, seal resistances and currents were stable (< 30% change) with up to six cumulative drug additions and 1-min incubations per addition. Over 27 experiments, an average of 338 concentration–response curves were obtained per experiment (96% of the 352 test wells on each plate). HERG pharmacology was examined with a set of 353 compounds that included well-characterized hERG blockers. Astemizole, terfenadine and quinidine inhibited hERG currents with IC₅₀ values of 159 nM, 224 nM and 2 μM, respectively (n = 51, 10 and 18). This set of compounds was also tested on the PatchXpress automated electrophysiology system. We determined through statistical methods that the two automated systems provided equivalent results.DiscussionEvaluating drug effects on hERG channels is best performed by electrophysiological methods. HERG activation and pharmacology on the IonWorks Barracuda automated electrophysiology platform were in good agreement with published electrophysiology results. Therefore, the IonWorks Barracuda provides an efficient way to study hERG biophysics and pharmacology.     

Aerodynamic characteristics of a dry powder inhaler at low inhalation flows using a mixing inlet with an Andersen Cascade Impactor

The aerodynamic characteristics of the dose emitted from a dry powder inhaler (DPI) are inhalation flow dependent but have not been determined for low flows. We have designed novel methodology to measure these at <28.3 l min^?1. The original Andersen Cascade Impactor (ACI) designed for use at 60 l min^?1 was adapted to include a mixing inlet (MIXINLET) which allows inhalation flows through the DPI from 5 to 60 l min^?1. The mean fine particle dose (FPD) from a formoterol Turbuhaler using the MIXINLET method at 10, 20, 28.3, 40 and 60 l min^?1 was 0.55, 1.39, 1.80, 2.88 and 5.86 μg and the mass median aerodynamic diameter (MMAD) was 6.6, 6.0, 5.4, 5.1 and 2.8 μm. Similarly, the FPD using the ACI method was 0.13, 0.69, 1.50, 2.48 and 5.42 μg and MMADs were 12.2, 7.4, 5.5, 4.8 and 2.7 μm. The accuracy of the original ACI <28.3 l min^?1 is unknown. The ACI with the mixing inlet allows the determination of the in vitro dose emission properties of DPIs at flows <28.3 l min^?1 whilst maintaining a constant flow through the ACI. This methodology, therefore, can help to focus attention to the lowest inhalation flow required for a DPI.     

The pharmacokinetics of orally administered S-carboxymethyl-l-cysteine in the dog,calf and sheep

The aim of this study was to investigate the feasibility of employing S-carboxymethyl-l-cysteine as a treatment of chronic obstructive pulmonary disease in dogs. To this end the pharmacokinetic parameters of orally administered S-carboxymethyl-l-cysteine were determined in the dog, cow and sheep. Six healthy beagle dogs, six endogenous Greek sheep and four Holstein Fresian calves were orally dosed with 10 mg/kg body weight of S-carboxymethyl-l-cysteine. No significant differences in T_max and T_1/2 were reported between the species. However, significantly higher AUC_(0–last), 21.56 ± 6.67 μg h ml^?1 and AUC_(0–∞), 21.63 ± 6.68 μg h ml^?1 were seen in the dogs compared to the sheep and calves. The calculated V_D was significantly higher in the sheep (10.4 ± 2.7 L kg^?1) and the calves (3.8 ± 0.7 L kg^?1) compared to the dogs (1.0 ± 0.6 L kg^?1). The rank order of increasing C_L was sheep (3.4 ± 2.7 L h^?1 kg^?1) > calves (2.7 ± 0.4 L h^?1 kg^?1) > dogs (0.5 ± 0.2 L h^?1 kg^?1). The result for the dogs was significantly lower that the calculated C_L for the sheep and calves.All these results indicate that the oral administration of S-carboxymethyl-l-cysteine may be useful during the therapeutic management of chronic obstructive pulmonary disease in dogs.     

Investigation of the micellar effect of pluronic P85 on P-glycoprotein inhibition: Cell accumulation and equilibrium dialysis studies

The objective of this study was: (1) to characterize the P-gp inhibitory effect of different concentrations of Pluronic P85 on anti-HIV-1 drug cellular accumulation, and (2) to investigate the relationship between cellular accumulation and free fraction of drug. Cellular accumulation studies in MDCKII-WT and MDCKII-MDR1 cell monolayers showed a biphasic dose response characterized by decline in accumulation at Pluronic concentrations greater than the CMC. This phenomenon was independent of the inhibition of P-gp efflux by Pluronic. Cell-free equilibrium dialysis was used to determine the effect of Pluronic P85 on drug free fraction and the affinity of Pluronic micelles for drug was modeled. Nelfinavir and saquinavir associated extensively with micelles and equilibrium free fractions were low at P85 concentrations above the CMC, with association constants being in the order nelfinavir > saquinavir ? abacavir. Abacavir, a P-gp substrate, showed no association with micelles yet showed a biphasic response in cellular accumulation. These data suggest that, above the CMC, inhibition of P-gp is not affected but rather factors such as micellar trapping could contribute to decreased accumulation. Therefore, the in vitro evaluation of the effect of Pluronic formulations on active transport should take into account both the physicochemical properties of drug and the composition of Pluronic. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4170–4190, 2009     

Baseline health status and quality of life after alcohol treatment for women with alcohol dependence

BackgroundResearch suggests that women with alcohol use disorders (AUDs) experience more severe medical and social consequences from alcohol use compared to men, but little is known about health improvements following alcohol treatment.MethodsThis study sought to characterize the pre-treatment health status of 138 alcohol dependent women enrolled in 12 sessions of female-specific group or individual outpatient treatment and examine the degree to which alcohol treatment might promote positive quality of life changes. Quality of life was assessed using the World Health Organization Quality of Life measure at baseline and 3 months later at the end of treatment.ResultsThe most common health problems at baseline were: smoking cigarettes (34.1%), hypertension (31.2%), obesity (27.5%), arthritis (21.0%), high cholesterol (17.4%), heart problems (8.7%), and a history of cancer (7.2%). Significant improvements across physical, t(117) = 4.67, p < 0.001, d = 0.42; psychological, t(117) = 7.31, p < 0.001, d = 0.62; social, t(117) = 3.18, p = 0.002, d = 0.28; and environmental, t(117) = 2.39, p = 0.018, d = 0.17; quality of life domains were seen after treatment. Percent days abstinent during treatment was positively associated with overall health satisfaction and psychological health at the end of treatment.ConclusionsWomen presenting for outpatient treatment for alcohol use disorders report many comorbid negative health problems. Thus, it is important for both substance use and health care providers to consider the overlap of alcohol use problems and health domains. Furthermore, female-specific cognitive behavioral treatment for alcohol use disorders positively impacted multiple health domains for women, suggesting a potential transdiagnostic intervention to target co-occurring health and substance use problems.