Application of synthetic membranes in establishing bio-predictive IVPT for testosterone transdermal gel.

The current study investigated the use of synthetic membranes in developing a bio-predictive in vitro permeation testing (IVPT) method for 1.62% testosterone gel. The IVPT studies were carried out using both Franz (FC), and Flow-through (FTC) diffusion cells. The experimental variables included the type of synthetic membranes (hydrophilic polyamide nylon, polysulfone tuffryn and STRAT-M (SM) membrane) and the type of receiver media (phosphate buffer containing various concentrations of sodium lauryl sulfate). In vivo drug release rates were obtained from published reports for 1.62% testosterone gel applied to either abdominal area (treatment group A), upper arms/shoulders (treatment group B), or alternating between abdomen and arms/shoulders (treatment group C). The in vitro-in vivo correlations were established using GastroPlus software. The best IVPT method was selected based on establishing point-to-point correlation with the in vivo data of treatment group A with minimal prediction errors (%PE) of AUC0-24 and Cmax. The results showed that the IVPT method which employed the FTC diffusion system, SM membrane and phosphate buffer without surfactant established the best IVIVR model with a correlation coefficient (R2) of 0.9966 and an exponential function of Y = (1.35)5 × X3.6. The in vivo data obtained from treatment group A and B was used for internal validation of the prediction model. The validation data was acceptable, with %PE of less than 10% for both AUC0-24 and Cmax. In conclusion, these results suggest that bio-predictive IVPT methods for testosterone gels may be developed using synthetic membranes and diffusion apparatus by varying the composition of the receiver medium.

Drug recrystallization in drug-in-adhesive transdermal delivery system: A case study of deteriorating the mechanical and rheological characteristics of testosterone TDS.

This study investigated the effects of drug recrystallization on the in vitro performance of testosterone drug-in-adhesive transdermal delivery system (TDS). Six formulations were prepared with a range of dry drug loading in the adhesive matrix from 1% to 10% w/w with the aim of generating TDS with various levels of drug crystals. We visually quantified the amount of crystals in TDS by polarized light microscopy. The effect of drug recrystallization on adhesion, tackiness, cohesive strength, viscoelasticity, drug release, and drug permeation through human cadaver skin were evaluated for these TDS samples. The Optical images showed no crystals in 1% and 2% testosterone TDSs; however, the amount of crystals increased by increasing testosterone loading from 4 to 10%. A proportional and significant decrease (p < 0.05) in tack, peel, and shear strength of the adhesive matrix with increasing amount of crystals in TDS was observed. The drug crystals resulted in a proportional deterioration of the viscoelastic properties of the adhesive matrix. The 2% testosterone TDS showed faster drug release rate when compared to 1% testosterone TDS. The increase in drug loading from 2% to 4% w/w slightly increased the cumulative amount of testosterone released. Further increase in drug loading in TDS to 6, 8, and 10% was nonsignificant (p > 0.05) to affect the drug release and permeation. In conclusion, this study demonstrated that the extent of drug recrystallization can be quantitatively correlated with the deterioration of performance characteristics of TDS products.

Excess glutamate secreted from astrocytes drives upregulation of P-glycoprotein in endothelial cells in amyotrophic lateral sclerosis.

In amyotrophic lateral sclerosis (ALS), upregulation in expression and activity of the ABC transporter P-glycoprotein (P-gp) driven by disease advancement progressively reduces CNS penetration and efficacy of the ALS drug, riluzole. Post-mortem spinal cord tissues from ALS patients revealed elevated P-gp expression levels in endothelial cells of the blood-spinal cord barrier compared to levels measured in control, non-diseased individuals. We recently found that astrocytes expressing familial ALS-linked SOD1 mutations regulate expression levels of P-gp in endothelial cells, which also exhibit a concomitant, significant increase in reactive oxygen species production and NFκB nuclear translocation when exposed to mutant SOD1 astrocyte conditioned media. In this study, we found that glutamate, which is abnormally secreted by mutant SOD1 and sporadic ALS astrocytes, drives upregulation of P-gp expression and activity levels in endothelial cells via activation of N-Methyl-D-Aspartic acid (NMDA) receptors. Surprisingly, astrocyte-secreted glutamate regulation of endothelial P-gp levels is not a mechanism shared by all forms of ALS. C9orf72-ALS astrocytes had no effect on endothelial cell P-gp expression and did not display increased glutamate secretion. Utilizing an optimized in vitro human BBB model consisting of patient-derived induced pluripotent stem cells, we showed that co-culture of endothelial cells with patient-derived astrocytes increased P-gp expression levels and transport activity, which was significantly reduced when endothelial cells were incubated with the NMDAR antagonist, MK801. Overall, our findings unraveled a complex molecular interplay between astrocytes of different ALS genotypes and endothelial cells potentially occurring in disease that could differentially impact ALS prognosis and efficacy of pharmacotherapies.

Blood-Brain Barrier Driven Pharmacoresistance in Amyotrophic Lateral Sclerosis and Challenges for Effective Drug Therapies.

The blood-brain barrier (BBB) is essential for proper neuronal function, homeostasis, and protection of the central nervous system (CNS) microenvironment from blood-borne pathogens and neurotoxins. The BBB is also an impediment for CNS penetration of drugs. In some neurologic conditions, such as epilepsy and brain tumors, overexpression of P-glycoprotein, an efflux transporter whose physiological function is to expel catabolites and xenobiotics from the CNS into the blood stream, has been reported. Recent studies reported that overexpression of P-glycoprotein and increase in its activity at the BBB drives a progressive resistance to CNS penetration and persistence of riluzole, the only drug approved thus far for treatment of amyotrophic lateral sclerosis (ALS), rapidly progressive and mostly fatal neurologic disease. This review will discuss the impact of transporter-mediated pharmacoresistance for ALS drug therapy and the potential therapeutic strategies to improve the outcome of ALS clinical trials and efficacy of current and future drug treatments.

Oleocanthal ameliorates amyloid-ß oligomers' toxicity on astrocytes and neuronal cells: In vitro studies.

Extra-virgin olive oil (EVOO) has several health promoting effects. Evidence have shown that EVOO attenuates the pathology of amyloid-ß (Aß) and improves cognitive function in experimental animal models, suggesting it's potential to protect and reduce the risk of developing Alzheimer's disease (AD). Available studies have linked this beneficial effect to oleocanthal, one of the active components in EVOO. The effect of oleocanthal against AD pathology has been linked to its ability to attenuate Aß and tau aggregation in vitro, and enhance Aß clearance from the brains of wild-type and AD transgenic mice in vivo. However, the ability of oleocanthal to alter the toxic effect of Aß on brain parenchymal cells is unknown. In the current study, we investigated oleocanthal effect on modulating Aß oligomers (Aßo) pathological events in neurons and astrocytes. Our findings demonstrated oleocanthal prevented Aßo-induced synaptic proteins, SNAP-25 and PSD-95, down-regulation in neurons, and attenuated Aßo-induced inflammation, glutamine transporter (GLT1) and glucose transporter (GLUT1) down-regulation in astrocytes. Aßo-induced inflammation was characterized by interleukin-6 (IL-6) increase and glial fibrillary acidic protein (GFAP) upregulation that were reduced by oleocanthal. In conclusion, this study provides further evidence to support the protective effect of EVOO-derived phenolic secoiridoid oleocanthal against AD pathology.

Amylin Enhances Amyloid-ß Peptide Brain to Blood Efflux Across the Blood-Brain Barrier.

Findings from Alzheimer's disease (AD) mouse models showed that amylin treatment improved AD pathology and enhanced amyloid-ß (Aß) brain to blood clearance; however, the mechanism was not investigated. Using the Tg2576 AD mouse model, a single intraperitoneal injection of amylin significantly increased Aß serum levels, and the effect was abolished by AC253, an amylin receptor antagonist, suggesting that amylin effect could be mediated by its receptor. Subsequent mechanistic studies showed amylin enhanced Aß transport across a cell-based model of the blood-brain barrier (BBB), an effect that was abolished when the amylin receptor was inhibited by two amylin antagonists and by siRNA knockdown of amylin receptor Ramp3. To explain this finding, amylin effect on Aß transport proteins expressed at the BBB was evaluated. Findings indicated that cells treated with amylin induced LRP1 expression, a major receptor involved in brain Aß efflux, in plasma membrane fraction, suggesting intracellular translocation of LRP1 from the cytoplasmic pool. Increased LRP1 in membrane fraction could explain, at least in part, the enhanced uptake and transport of Aß across the BBB. Collectively, our findings indicated that amylin induced Aß brain to blood clearance through amylin receptor by inducing LRP1 subcellular translocation to the plasma membrane of the BBB endothelium.

High-Throughput Screening for Identification of Blood-Brain Barrier Integrity Enhancers: A Drug Repurposing Opportunity to Rectify Vascular Amyloid Toxicity.

The blood-brain barrier (BBB) is a dynamic interface that maintains brain homeostasis and protects it from free entry of chemicals, toxins, and drugs. The barrier function of the BBB is maintained mainly by capillary endothelial cells that physically separate brain from blood. Several neurological diseases, such as Alzheimer's disease (AD), are known to disrupt BBB integrity. In this study, a high-throughput screening (HTS) was developed to identify drugs that rectify/protect BBB integrity from vascular amyloid toxicity associated with AD progression. Assessing Lucifer Yellow permeation across in-vitro BBB model composed from mouse brain endothelial cells (bEnd3) grown on 96-well plate inserts was used to screen 1280 compounds of Sigma LOPAC®1280 library for modulators of bEnd3 monolayer integrity. HTS identified 62 compounds as disruptors, and 50 compounds as enhancers of the endothelial barrier integrity. From these 50 enhancers, 7 FDA approved drugs were identified with EC50 values ranging from 0.76-4.56 µM. Of these 7 drugs, 5 were able to protect bEnd3-based BBB model integrity against amyloid toxicity. Furthermore, to test the translational potential to humans, the 7 drugs were tested for their ability to rectify the disruptive effect of Aß in the human endothelial cell line hCMEC/D3. Only 3 (etodolac, granisetron, and beclomethasone) out of the 5 effective drugs in the bEnd3-based BBB model demonstrated a promising effect to protect the hCMEC/D3-based BBB model integrity. These drugs are compelling candidates for repurposing as therapeutic agents that could rectify dysfunctional BBB associated with AD.

Role of P-glycoprotein in mediating rivastigmine effect on amyloid-ß brain load and related pathology in Alzheimer's disease mouse model.

Recently, we showed that rivastigmine decreased amyloid-ß (Aß) brain load in aged rats by enhancing its clearance across the blood-brain barrier (BBB) via upregulation of P-glycoprotein (P-gp) and low-density lipoprotein receptor-related protein 1 (LRP1). Here, we extend our previous work to clarify P-gp role in mediating rivastigmine effect on Aß brain levels and neuroprotection in a mouse model of Alzheimer's disease (AD) that expresses different levels of P-gp. APPSWE mice were bred with mdr1a/b knockout mice to produce littermates that were divided into three groups; APP(+)/mdr1(+/+), APP(+)/mdr1(+/-) and APP(+)/mdr1(-/-). Animals received rivastigmine treatment (0.3mg/kg/day) or vehicle for 8weeks using Alzet osmotic mini-pumps. ELISA analysis of brain homogenates for Aß showed rivastigmine treatment to significantly decrease Aß brain load in APP(+)/mdr1(+/+) by 25% and in APP(+)/mdr1(+/-) mice by 21% compared to their vehicle treated littermates, but not in APP(+)/mdr1(-/-) mice. In addition, rivastigmine reduced GFAP immunostaining of astrocytes by 50% and IL-1ß brain level by 43% in APP(+)/mdr1(+/+) mice, however its effect was less pronounced in P-gp knockout mice. Moreover, rivastigmine demonstrated a P-gp expression dependent neuroprotective effect that was highest in APP(+)/mdr1(+/+)>APP(+)/mdr1(+/-)>APP(+)/mdr1(-/-) as determined by expression of synaptic markers PSD-95 and SNAP-25 using Western blot analysis. Collectively, our results suggest that P-gp plays important role in mediating rivastigmine non-cholinergic beneficial effects, including Aß brain load reduction, neuroprotective and anti-inflammatory effects in the AD mouse models.

Extra-virgin olive oil attenuates amyloid-ß and tau pathologies in the brains of TgSwDI mice.

Extra-virgin olive oil (EVOO) is one of the main elements of Mediterranean diet. Several studies have suggested that EVOO has several health promoting effects that could protect from and decrease the risk of Alzheimer's disease (AD). In this study, we investigated the effect of consumption of EVOO-enriched diet on amyloid- and tau-related pathological alterations that are associated with the progression of AD and cerebral amyloid angiopathy (CAA) in TgSwDI mice. Feeding mice with EVOO-enriched diet for 6months, beginning at an age before amyloid-ß (Aß) accumulation starts, has significantly reduced total Aß and tau brain levels with a significant improvement in mouse cognitive behavior. This reduction in brain Aß was explained by the enhanced Aß clearance pathways and reduced brain production of Aß via modulation of amyloid-ß precursor protein processing. On the other hand, although feeding mice with EVOO-enriched diet for 3months, beginning at an age after Aß accumulation starts, showed improved clearance across the blood-brain barrier and significant reduction in Aß levels, it did not affect tau levels or improve cognitive functions of TgSwDI mouse. Collectively, results of this study suggest that the long-term consumption of EVOO-containing diet starting at early age provides a protective effect against AD and its related disorder CAA.

Age-Related Decline in Brain and Hepatic Clearance of Amyloid-Beta is Rectified by the Cholinesterase Inhibitors Donepezil and Rivastigmine in Rats.

In Alzheimer's disease (AD), accumulation of brain amyloid-ß (Aß) depends on imbalance between production and clearance of Aß. Several pathways for Aß clearance have been reported including transport across the blood-brain barrier (BBB) and hepatic clearance. The incidence of AD increases with age and failure of Aß clearance correlates with AD. The cholinesterase inhibitors (ChEIs) donepezil and rivastigmine are used to ease the symptoms of dementia associated with AD. Besides, both drugs have been reported to provide neuroprotective and disease-modifying effects. Here, we investigated the effect of ChEIs on age-related reduced Aß clearance. Findings from in vitro and in vivo studies demonstrated donepezil and rivastigmine to enhance (125)I-Aß40 clearance. Also, the increase in brain and hepatic clearance of (125)I-Aß40 was more pronounced in aged compared to young rats, and was associated with significant reduction in brain Aß endogenous levels determined by ELISA. Furthermore, the enhanced clearance was concomitant with up-regulation in the expression of Aß major transport proteins P-glycoprotein and LRP1. Collectively, our findings that donepezil and rivastigmine enhance Aß clearance across the BBB and liver are novel and introduce an additional mechanism by which both drugs could affect AD pathology. Thus, optimizing their clinical use could help future drug development by providing new drug targets and possible mechanisms involved in AD pathology.

Tacrine sinusoidal uptake and biliary excretion in sandwich-cultured primary rat hepatocytes.

PURPOSE. The knowledge of hepatic disposition kinetics of tacrine, a first cholinesterase inhibitor was approved by FDA for the treatment of Alzheimer's disease (AD), would help to understand its hepatotoxicity, its therapeutic effect, and improve the management of patients with AD. The current study aims to characterize tacrine hepatic transport kinetics and study the role of organic cation transporters (OCTs), P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP2) in tacrine sinusoidal uptake and biliary excretion. METHODS. Modulation of tacrine hepatic uptake and efflux, biliary excretion index (BEI%), were performed in sandwich-cultured primary rat hepatocytes (SCHs) using transporters inhibitors. Conformation of the integrity of SCHs model was established by capturing images with light-contrast and fluorescence microscopy. RESULTS. Tacrine uptake in SCHs was carrier-mediated process and saturable with apparent Km of 31.5±9.6 µM and Vmax of 908±72 pmol/min/mg protein. Tetraethyl ammonium (TEA), cimetidine and verapamil significantly reduced tacrine uptake with more pronounced effect observed with verapamil which caused 3-fold reduction in tacrine uptake, indicating role for OCTs. Tacrine has a biliary excretion in SCHs with maximum BEI% value of 22.9±1.9% at 10 min of incubation. Addition of MK571 and valspodar decreased the BEI% of tacrine by 40 and 60% suggesting roles for canalicular MRP2 and P-gp, respectively. CONCLUSIONS. Our results show that in addition to metabolism, tacrine hepatic disposition is carrier-mediated process mediated by sinusoidal OCTs, and canalicular MRP2 and P-gp.

In vitro investigation of amyloid-ß hepatobiliary disposition in sandwich-cultured primary rat hepatocytes.

Failure in amyloid-ß (Aß) systemic clearance across the liver has been suggested to play a role in Aß brain accumulation and thus to contribute largely to the pathology of Alzheimer's disease (AD). The purpose of this study was to characterize in vitro the transport mechanisms of Aß40 across the liver using sandwich-cultured primary rat hepatocytes (SCHs) and to determine its biliary clearance (CL(bile)) and biliary excretion index (BEI%). ¹²5I-Aß40 BEI% was time dependent and reached steady state at 30 minutes, with an average value of 29.8% and a CL(bile) of 1.47 ml/min per kilogram of body weight. The role of low-density lipoprotein receptor-related protein-1 (LRP1) in mediating the basolateral uptake of ¹²5I-Aß40 in SCHs was assessed using receptor-associated protein (RAP, 2 µM). A significant reduction in ¹²5I-Aß40 BEI% and CL(bile) with RAP was observed, demonstrating a major contribution of LRP1 in mediating hepatic uptake of intact ¹²5I-Aß40 via transcytosis. Furthermore, activity studies suggested a lower role of receptor for advanced glycation end products (RAGE) in ¹²5I-Aß40 hepatic uptake. Verapamil (50 µM) and valspodar (20 µM) significantly reduced ¹²5I-Aß40 BEI%, indicating a role for P-glycoprotein (P-gp) in the biliary excretion of ¹²5I-Aß40 in SCHs. LRP1- and P-gp-mediated ¹²5I-Aß40 biliary excretion was inducible and increased BEI% by 26% after rifampicin pretreatment. In conclusion, our findings demonstrated that besides LRP1, P-gp and, to a lesser extent, RAGE are involved in ¹²5I-Aß40 hepatobiliary disposition and support the use of enhancement of Aß hepatic clearance via LRP1 and P-gp induction as a novel therapeutic approach for the prevention and treatment of AD.

Experimental models for predicting drug absorption and metabolism.

INTRODUCTION: For orally administered drugs, their intestinal absorption and hepatic metabolism are key players for determining a drug's systemic bioavailability and thus therapeutic effect. Drug absorption and metabolism are both complicated processes, with many physicochemical and physiological factors involved. Understanding the contribution of each of these processes is essential in regulating a drug's level in the bloodstream and in maintaining its optimum therapeutic outcome and safety. Several animal models have been established for studying the intestine and liver as barriers to drug delivery and systemic bioavailability. AREAS COVERED: This review provides an overview of available animal and cell-based models that have been used to characterize and predict drug intestinal absorption and hepatic clearance in humans. Among the most commonly used models to study drug intestinal absorption are in-vitro Caco-2 cells, in situ rat intestinal perfusion and the in vivo animal models. On the other hand, hepatic clearance is mostly studied using in vitro techniques. The authors also review in silico approaches which play significant role during early pharmaceutical research. EXPERT OPINION: The current models developed have greatly contributed to our knowledge of drug interactions with physiological factors, while also useful in the prediction of drug intestinal absorption and metabolism. However, much work remains in this area for the successful extrapolation of in vitro-in vivo data and in furthering the development of reliable and accurate models.