Pharmacokinetics of acute and sub-chronic aripiprazole in P-glycoprotein deficient mice

Background

P-glycoprotein (P-gp), an efflux transporter localized in the blood-brain barrier, limits the access of multiple xenobiotics to the central nervous system (CNS). For the new antipsychotic aripiprazole and its active metabolite dehydroaripiprazole differences in disposition in blood and brain were investigated after acute and sub-chronic administration in a P-gp knockout mouse model.

Methods

Serum and brain concentrations of both drugs were measured at several time points 1-24 h after i.p. injection of 10 mg/kg aripiprazole and after 11 days of sub-chronic administration in several tissues. Moreover, the expression of P-gp was determined by Western blot analysis after sub-chronic administration of the drug.

Results

In both wild type and abcb1ab (−/−) mice concentration of aripiprazole in brain were up to 9 fold higher than in serum. For dehydroaripiprazole the mean brain to serum ratios were below two. Brain to serum concentrations of both substances were significantly higher after acute and sub-chronic administration in connection to the expression of P-gp indicated by higher levels in abcb1ab (−/−) mice especially for dehydroaripiprazole.Sub-chronic aripiprazole treatment in WT animals had no effect on P-gp expression in the blood-brain barrier.

Conclusions

Aripiprazole and, even more pronounced its active metabolite dehydroaripiprazole could be identified as substrates of P-gp. The efflux transporter P-gp must therefore be considered as a relevant factor that contributes to wanted or unwanted clinical effects in patients treated with aripiprazole.     

COX-2 inhibition controls P-glycoprotein expression and promotes brain delivery of phenytoin in chronic epileptic rats

Epileptic seizures drive expression of the blood-brain barrier efflux transporter P-glycoprotein via a glutamate/cyclooxygenase-2 mediated signalling pathway. Targeting this pathway may represent an innovative approach to control P-glycoprotein expression in the epileptic brain and to enhance brain delivery of antiepileptic drugs.Therefore, we tested the effect of specific cyclooxygenase-2 inhibition on P-glycoprotein expression in two different status epilepticus models. Moreover, the impact of a cyclooxygenase-2 inhibitor on expression of the efflux transporter and on brain delivery of an antiepileptic drug was evaluated in rats with recurrent spontaneous seizures.The highly selective cyclooxygenase-2 inhibitors SC-58236 and NS-398 both counteracted the status epilepticus-associated increase in P-glycoprotein expression in the parahippocampal cortex and the ventral hippocampus. In line with our working hypothesis, a sub-chronic 2-week treatment with SC-58236 in the chronic epileptic state kept P-glycoprotein expression at control levels. As described previously, enhanced P-glycoprotein expression in chronic epileptic rats was associated with a significant reduction in the brain penetration of the antiepileptic drug phenytoin. Importantly, the brain delivery of phenytoin was significantly enhanced by sub-chronic cyclooxygenase-2 inhibition in rats with recurrent seizures.In conclusion, the data substantiate targeting of cyclooxygenase-2 in the chronic epileptic brain as a promising strategy to control the expression levels of P-glycoprotein despite recurrent seizure activity. Cyclooxygenase-2 inhibition may therefore help to increase concentrations of antiepileptic drugs at the target sites in the epileptic brain. It needs to be further evaluated whether the approach also enhances efficacy.     

Evaluation of the role of P-glycoprotein in the uptake of paroxetine, clozapine, phenytoin and carbamazapine by bovine retinal endothelial cells

Expression of the drug transport proteins, including P-glycoprotein (Pgp), in the brain vascular endothelium represents a challenge for the effective delivery of drugs for the treatment of several central nervous system (CNS) disorders including depression, schizophrenia and epilepsy. It has been hypothesized that Pgp plays a major role in drug efflux at the blood-brain barrier, and may be an underlying factor in the variable responses of patients to CNS drugs. However, the role of Pgp in the transport of many CNS drugs has not been directly demonstrated. To explore the role of Pgp in drug transport across an endothelial cell barrier derived from the central nervous system, the expression and activity of Pgp in bovine retinal endothelial cells (BRECs) and the effects of representative CNS drugs on Pgp activity were examined. Significant Pgp expression in BRECs was demonstrated by western analyses, and expression was increased by treatment of the cells with hydrocortisone. Intracellular accumulation of the well-characterized Pgp-substrate Taxol was markedly increased by the non-selective transporter inhibitor verapamil and the Pgp-selective antagonist PGP-4008, demonstrating that Pgp is active in these endothelial cells. In contrast, neither verapamil nor PGP-4008 affected the intracellular accumulation of [3H]paroxetine, [14C]phenytoin, [3H]clozapine or [14C]carbamazapine, indicating that these drugs are not substrates for Pgp. Paroxetine, clozapine and phenytoin were shown to be Pgp inhibitors, while carbamazapine did not inhibit Pgp at any concentration tested. These results indicate that Pgp is not likely to modulate patient responses to these drugs.     

In vitro and in vivo models of celiac disease

ABSTRACT

Introduction: Human in vitro blood-brain barrier (BBB) models could be important tools for studying BBB development, maintenance and regulation. However, our capacity to obtain information from these models is still limited in part because only in recent years have (i) these models been derived from non-brain cell sources (e.g. stem cells), (ii) microfluidic systems been developed to recapitulate aspects of BBB physiology and (iii) new insights into the molecular and cellular mechanisms of BBB diseases (e.g. Huntington´s, Allan-Herndon-Dudley Syndrome) been described.

Area covered: This article reviews the technological advances in the derivation of human cells from the neurovascular unit using stem cells and the creation of personalized BBB models generated from patients with neurodegenerative diseases. It also reviews the scientific advances generated from in vitro BBB models.

Expert opinion: The recent technological advances in the derivation of human cells from the neurovascular unit from stem cells as well as in the generation of BBB-on-a-chip that recapitulate in vitro part of the BBB physiology are significant to generate more robust BBB models; however, a considerable effort is still needed to validate the potential of these models to recapitulate the in vivo cellular and molecular mechanisms, in particular regarding BBB function in health and disease.     

In vitro P-glycoprotein activity does not completely explain in vivo efficacy of novel centrally effective oxime acetylcholinesterase reactivators

Novel-substituted phenoxyalkyl pyridinium oxime acetylcholinesterase (AChE) reactivators (US patent 9,227,937) that showed convincing evidence of penetration into the brains of intact rats were developed by our laboratories. The oximes separated into three groups based on their levels of brain AChE reactivation following exposure of rats to the sarin surrogate nitrophenyl isopropyl methylphosphonate (NIMP). P-glycoprotein (P-gp) is a major blood–brain barrier (BBB) transporter and requires ATP for efflux. To determine if P-gp affinity screening could be used to reduce animal use, we measured in vitro oxime-stimulated ATPase activity to see if the in vivo reactivation efficacies related to the oximes’ functions as P-gp substrates. High efficacy oximes were expected to be poor P-gp substrates, thus remaining in the brain longer. The high efficacy oximes (24–35% brain AChE reactivation) were worse P-gp substrates than the low efficacy oximes (0–7% brain AChE reactivation). However, the oxime group with medium in vivo reactivation of 10–17% were even worse P-gp substrates than the high efficacy group so their reactivation ability was not reflected by P-gp export. The results suggest that in vitro P-gp ATPase activity can remove the low efficacy oximes from in vivo testing, but is not sufficient to differentiate between the top two tiers.     

In vitro and ex vivo evidence for modulation of P-glycoprotein activity by progestins

The well known gender-related differences in drug action may partly be explained by changes in activity and expression of drug metabolising enzymes, but also by modulation of active drug transport systems (e.g. P-glycoprotein, Pgp) by sexual steroids, which is yet not well investigated. Because many women are using hormones (e.g. as oral contraceptives) we investigated the influence of different synthetic progestins on Pgp activity. Pgp inhibition of progesterone, medroxyprogesterone, chlormadinone, cyproterone, levonorgestrel, norethisterone, desogestrel, and norgestimate was measured in vitro in two Pgp over-expressing cell lines (L-MDR1, P388/dx cells) and the corresponding parental cell lines by means of calcein assay, and ex vivo in human peripheral blood mononuclear cells (PBMCs) by rhodamine123 efflux. For most progestins tested, concentrations needed to double baseline fluorescence (f2) in L-MDR1 cells were similar to that of the potent Pgp inhibitor quinidine, whereas levonorgestrel and norethisterone did not reach f2. The results in P388/dx cells essentially confirmed our findings in L-MDR1 cells. Additionally, Pgp inhibitory activity of all progestins tested was also shown ex vivo in PBMCs. The potent Pgp inhibition by several synthetic progestins in vitro and ex vivo suggests that such an interaction might be clinically relevant despite generally low plasma concentrations of progestins. The results may be of particular importance for Pgp substrates, such as protease inhibitors and chemotherapeutic agents, for which intracellular concentrations are critical.     

Novel flavonoid-based biodegradable nanoparticles for effective oral delivery of etoposide by P-glycoprotein modulation: an in vitro,ex vivo and in vivo investigations

Abstract

A receptor level interaction of etoposide with P-glycoprotein (P-gp) and subsequent intestinal efflux has an adverse effect on its oral absorption. The present work is aimed to enhance the bioavailability of etoposide by co-administering it with quercetin (a P-gp inhibitor) in dual-loaded polymeric nanoparticle formulation. Poly-lactic-co-glycolic acid (PLGA) nanoparticles were optimized for various parameters like o/w phase volume ratio, poly-vinyl alcohol concentration, PLGA concentration and sonication time. The cytotoxicity studies (MTT assay) revealed a 9- and 11-fold decrease in the IC 50 values for etoposide-loaded nanoparticles (ENP) and etoposide?+?quercetin dual-loaded nanoparticles (EQNP) when compared to that of free etoposide, respectively, and the results were further supported by florescent-activated cell sorter studies. The confocal imaging of the intestinal sections treated with ENP and EQNP containing fluorescent probe (rhodamine) showed the superiority of the EQNP to permeate deeper. Furthermore, pharmacokinetic studies on rats revealed that EQNP exhibited a 2.4-fold increase in bioavailability of etoposide than ENP with no quercetin. The developed loaded nanoparticles have the high potential to enhance the bioavailability of the etoposide and sensitize the resistant cells.     

The Changes of P-glycoprotein Activity by Interferon-γ and Tumor Necrosis Factor-α in Primary and Immortalized Human Brain Microvascular Endothelial Cells

The purpose of this study was to investigate the modification of expression and functionality of the drug transporter P-glycoprotein (P-gp) by tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) at the blood-brain barrier (BBB). We used immortalized human brain microvessel endothelial cells (iHBMEC) and primary human brain microvessel endothelial cells (pHBMEC) as in vitro BBB model. To investigate the change of p-gp expression, we carried out real time PCR analysis and Western blotting. To test the change of p-gp activity, we performed rhodamin123 (Rh123) accumulation study in the cells. In results of real time PCR analysis, the P-gp mRNA expression was increased by TNF-α or IFN-γ treatment for 24 hr in both cell types. However, 48 hr treatment of TNF-α or IFN-γ did not affect P-gp mRNA expression. In addition, co-treatment of TNF-α and IFN-γ markedly increased the P-gp mRNA expression in both cells. TNF-α or IFN-γ did not influence P-gp protein expression whatever the concentration of cytokines or duration of treatment in both cells. However, P-gp expression was increased after treatments of both cytokines together in iHBMEC cells only compared with untreated control. Furthermore, in both cell lines, TNF-α or IFN-γ induced significant decrease of P-gp activity for 24 hr treatment. And, both cytokines combination treatment also decreased significantly P-gp activity. These results suggest that P-gp expression and function at the BBB is modulated by TNF-α or/and IFN-γ. Therefore, the distribution of P-gp depending drugs in the central nervous system can be modulated by neurological inflammatory diseases.     

In vivo diminution by chelators of snake venom-provoked hemorrhage and in vitro inhibition of proteolytic activity

Topical application of alcohol-soluble metal-complexing agents, particularly α-mercapto-β-(2-furyl)acrylic acid (MFA)1, diminished the severity of hemorrhages caused by s.c. injection to mice of thirteen hemorrhagic pit viper venoms and two viper venoms. Metal dependency of the alkaline protease activity ranged from 0 to 100% for these venoms, plus one non-hemorrhagic pit viper venom; MFA completely inhibited this activity in six pit viper and two viper venoms. The presence of zinc-dependent proteases in venoms of Crotalus atrox and Bitis arietans was indicated. Less compelling evidence suggested the presence of zinc metalloproteases in at least 11 of 14 other venoms. The potencies of hemorrhagic and alkaline protease activities were not related in the venoms and we did not investigate if metalloproteases were the targets of chelator antagonism of hemorrhage development. Topically active chelators may be useful as adjunctive therapy in some snake envenomations.     

In vitro inhibition and enhancement of liver microsomal S-777469 metabolism by long-chain fatty acids and serum albumin: insight into in vitro and in vivo discrepancy of metabolite formation in humans

1.?It was previously demonstrated that 10% of S-777469, a cannabinoid receptor 2 selective agonist, is metabolized to its carboxylic acid metabolite (S-777469 5-carboxylic acid, 5-CA) in humans in vivo, while the formation of 5-CA is extremely low in human cryopreserved hepatocytes and liver microsomes (HLMs). In this study, factors causing the different metabolite formation rates of S-777469 in vitro and in vivo were investigated.

2.?Formation of 5-CA and S-777469 5-hydroxymethyl (5-HM), a precursor metabolite of 5-CA, was catalyzed by CYP2C9. Arachidonic acid, α-linolenic acid, oleic acid and myristic acid, which have been reported to exist in liver microsomes, inhibited S-777469 oxidation by CYP2C9, but serum albumin enhanced this reactions.

3.?The IC₅₀ values of these fatty acids for 5-CA formation from 5-HM were lower than those of 5-HM formation from S-777469. Serum albumin extensively enhanced 5-CA formation from 5-HM in comparison to 5-HM formation from S-777469.

4.?CYP2C9 was the enzyme responsible for S-777469 oxidation in human livers. The suppressive effects of several fatty acids and enhancing action of serum albumin in vitro are likely to be the causal factors for the apparently different rates of in vitro and in vivo metabolite formation of S-777469.     

In vivo and in vitro inhibition of cholinesterase by methyl-1 (S methyl phosphoryl-3) imidazolium (MSPI), a model of an “instantly” aged phosphorylated enzyme

Methyl-1 (S-methylphosphoryl-3) imidazolium (MSPI) was approximately equipotent with DFP in producing in mice a lethal intoxication and in inhibiting in vitro cholinesterases of electric-eel, brain and plasma mice. In vivo, a lethal dose of MSPI inhibited plasma but not brain ChE. The intoxication by MSPI was not cured by the combination of Atropine and TMB₄ or of diazepam with Atropine and (or) TMB₄ and the cholinesterase inhibition was not reversed by TMB₄ in vivo nor in vitro in the conditions where DFP intoxication or ChE inhibition were.