Pharmacokinetics, safety, and tolerability of BMS-986263, a lipid nanoparticle containing HSP47 siRNA, in participants with hepatic impairment.

BMS-986263 is a retinoid-conjugated lipid nanoparticle delivering small interfering RNA designed to inhibit synthesis of HSP47 protein, a collagen-specific chaperone protein involved in fibrosis development. This is a phase I, open-label, two-part study evaluating pharmacokinetics and safety of BMS-986263 in participants with hepatic impairment (HI). Part 1 (n = 24) of this study enrolled two cohorts with mild and moderate HI and a separate cohort of age- and body mass index (BMI)-matched participants with normal hepatic function. Part 2 enrolled eight participants with severe HI and eight age- and BMI-matched participants with normal hepatic function. All participants received a single intravenous 90 mg BMS-986263 infusion. Compared with normal-matched participants, geometric mean area under the plasma concentration-time curve time zero to the time of the last quantifiable concentration (AUC(0-T) ) and AUC from zero to infinity (AUC(INF) ) of HSP47 siRNA were similar in participants with mild HI and 34% and 163% greater in those with moderate and severe HI, respectively, whereas the maximum plasma concentration was ~25% lower in mild and moderate HI groups but 58% higher in the severe HI group than in the normal group. Adverse events were reported by two of eight, four of eight, and three of eight participants with mild, moderate, or severe HI, respectively; none were reported in the normal-matched group. Overall, single-dose BMS-986263 was generally safe and well-tolerated and dose adjustment is not considered necessary for participants with mild or moderate HI. Although available data do not indicate that dose adjustment should be performed in patients with severe HI; the optimal posology of BMS-986263 in patients with severe HI may be determined later in its clinical development when additional data to establish exposure-safety/efficacy relationship becomes available.

Design and conduct considerations for studies in patients with hepatic impairment.

Despite the liver being the primary site for clearance of xenobiotics utilizing a myriad of mechanisms ranging from cytochrome P450 enzyme pathways, glucuronidation, and biliary excretion, there is a dearth of information available as to how the severity of hepatic impairment (HI) can alter drug absorption and disposition (i.e., pharmacokinetics [PK]) as well as their efficacy and safety or pharmacodynamics (PD). In general, regulatory agencies recommend conducting PK studies in subjects with HI when hepatic metabolism/excretion accounts for more than 20% of drug elimination or if the drug has a narrow therapeutic range. In this tutorial, we provide an overview of the global regulatory landscape, clinical measures for hepatic function assessment, methods to stage HI severity, and consequently the impact on labeling. In addition, we provide an in-depth practical guidance for designing and conducting clinical trials for patients with HI and on the application of modeling and simulation strategies in lieu of dedicated trials for dosing recommendations in patients with HI.

Overview of Clinical Pharmacology Packages of New Drug Applications Approved for the Treatment of Rare Diseases.

There are more than 7000 rare diseases affecting approximately 30 million people in the United States. More than 90% of these diseases lack approved therapies. Several challenges face the development of "orphan drugs", such as the small populations of patients, high development costs, and long development timelines. This study evaluates clinical pharmacology assessments conducted during the development of drugs to treat rare diseases approved by the United States Food and Drug Administration in 2020 and 2021. Thirty-nine new drug applications (NDAs) have been identified and the associated regulatory reviews, approved labels, and approval letters were reviewed. Approximately, 95%, 74%, and 77% of these submissions contained at least one type of drug-drug interaction, the effect of organ impairment (hepatic and renal) on drug exposure, and QT liability assessment, respectively. Modeling and simulation approaches were utilized to address many clinical pharmacology questions, with population pharmacokinetic analyses used extensively in the evaluation of the effect of organ impairment on drug exposure and with physiologically based pharmacokinetic analyses used mainly in assessing drug interaction risks. In general, the clinical pharmacology packages in the NDAs of orphan drugs are not optimal and more work is needed to obtain a complete clinical pharmacology package at the time of initial approval to ensure the safe and effective use of these drugs across the spectrum of the target patient population. This study provides insights into the clinical pharmacology studies needed for drugs to treat rare diseases and would help both the regulators and drug developers to identify challenges and opportunities in conducting clinical pharmacology assessments for drugs developed to treat rare diseases.

Trends in FDA Transporter-Based Post-Marketing Requirements and Commitments Over the Last Decade.

Characterizing interactions between new molecular entities (NMEs) and drug transporters is a critical element of drug development that helps in assessing potential transporter-based drug-drug interactions (DDIs). However, not all NME new drug applications (NDAs) include a full characterization of NMEs transporter-based DDIs, which necessitates the issuance of post-marketing requirement (PMR)/post-marketing commitment (PMC) by the US Food and Drug Administration (FDA) to characterize these potential interactions. The objective of this analysis is to identify trends in transporter-based PMRs/PMCs issued by the FDA between 2012 and 2021. A decrease in the number of transporter-based PMRs/PMCs was observed from 2012 to 2016 and an increasing trend in the number of PMRs/PMCs was observed after 2017. The majority of these transporter-based PMRs/PMCs requested clinical evaluation (48%), some requested in vitro assessment (38%), and 2.5% requested modeling and simulation assessment. Most of the PMRs/PMCs requested evaluation of NMEs as perpetrator with the efflux transporters, P-gp and/or BCRP (53%). Forty-eight percent of the PMRs/PMCs were fulfilled with 67% resulted in labeling updates. On average, 2.5 years were needed for the information related to PMRs/PMCs to show in NMEs labeling. In conclusion, this analysis highlights the increased emphasis from the FDA on proper characterization of transporter-based DDI and call for the need of early characterization of NMEs-transporters interaction before initial NDA approval.

FDA Approval Summary: Nivolumab in Combination with Ipilimumab for the Treatment of Unresectable Malignant Pleural Mesothelioma.

On October 2, 2020, FDA approved nivolumab with ipilimumab as first-line treatment for adult patients with unresectable malignant pleural mesothelioma (MPM). The approval was based on results from Study CA209743 (CHECKMATE-743), an open-label trial of patients with MPM randomized to receive nivolumab and ipilimumab for up to 2 years (n = 303) or six cycles of chemotherapy with cisplatin or carboplatin plus pemetrexed (n = 302). Overall survival (OS) was improved for patients who received nivolumab and ipilimumab, with a median OS of 18.1 months [95% confidence interval (CI), 16.8-21.5] compared with 14.1 months (95% CI: 12.5-16.2; HR, 0.74; 95% CI, 0.61-0.89; P = 0.002), for patients who received chemotherapy. The magnitude of benefit was larger for patients with non-epithelioid versus epithelioid histology. Additional clinical pharmacology data support an alternative dosing regimen of nivolumab than evaluated in the trial, which will reduce the number of required treatment visits. This application was reviewed under FDA's Project Orbis, in collaboration with Australia's Therapeutic Goods Administration, Switzerland's Swissmedic, Health Canada, and Brazil's National Health Surveillance Agency or ANVISA (Agência Nacional de Vigilância Sanitária). Nivolumab and ipilimumab is the first drug regimen approved by FDA for MPM since 2004.

Characterization of a commercially available line of iPSC hepatocytes as models of hepatocyte function and toxicity for regulatory purposes.

It has recently become possible to produce hepatocytes from human induced pluripotent stem cells (iPSC-heps), which may offer some advantages over primary human hepatocytes (Prim-heps) in the regulatory environment. The aim of this research was to assess similarities and differences between commercially available iPSC-heps and Prim-heps in preliminary assays of drug metabolism, hepatotoxicity, and drug transport. Hepatocytes were either cultured in collagen-coated 96-well plates (Prim-heps and 2d-iPSC-heps) or in ultra-low adhesion plates as spheroids (3d-iPSC-heps). 3d-iPSC-heps were used to enhance physiological cell-cell contacts, which is essential to maintain the phenotype of mature hepatocytes. Cytochrome P450 (CYP) 3A4, CYP1A2, and CYP2B6 activity levels were evaluated using fluorescent assays. Phase II metabolism was assessed by HPLC measurement of formation of glucuronides and sulfates of 4-methylumbelliferone, 1-naphthol, and estradiol. The toxicity of acetaminophen, amiodarone, aspirin, clozapine, tacrine, tamoxifen, and troglitazone was monitored using a luminescent cell viability assay. Canaliculi formation was monitored by following the fluorescence of 5,6-carboxy-2',7'-dichlorofluorescein diacetate. All culture models showed similar levels of basal CYP3A4, CYP1A2 and CYP2B6 activity. However, while Prim-heps showed a vigorous response to CYP inducing agents, 2d-iPSC-heps showed no response and 3d-iPSC-heps displayed an inconclusive response. 2d-iPSC-heps showed reduced, yet appreciable, glucuronide and sulfate formation compared to Prim-heps. All culture models showed similar activity in tests of hepatotoxicity, with Prim-heps generally being more sensitive. All models formed canaliculi capable of transporting carboxy-2',7'-dichlorofluorescein. The iPSC-heps appear to be useful for toxicity and transport studies, but metabolic activity is not optimum, and metabolism studies would benefit from a more mature model.

FDA Approval Summary: Gilteritinib for Relapsed or Refractory Acute Myeloid Leukemia with a FLT3 Mutation.

On November 28, 2018, the FDA approved gilteritinib (Xospata; Astellas), a small-molecule FMS-like tyrosine kinase 3 (FLT3) inhibitor, for treatment of relapsed or refractory acute myeloid leukemia with a FLT3 mutation as detected by an FDA-approved test. In the ADMIRAL study, patients were randomized 2:1 to receive gilteritinib or standard chemotherapy and stratified by response to first-line treatment and intensity of prespecified chemotherapy. Efficacy was established on interim analysis on the basis of complete remission (CR) + CR with partial hematologic recovery (CRh) rate, duration of CR + CRh, and conversion from transfusion dependence to transfusion independence in 138 patients in the gilteritinib arm. With median follow-up of 4.6 months [95% confidence interval (CI), 2.8-15.8 months] at interim analysis, the CR + CRh rate was 21% (95% CI, 15%-29%), median duration of CR + CRh was 4.6 months (range, 0.1-15.8+), and conversion from transfusion dependence to transfusion independence was 31%. Revised labeling approved on May 29, 2019 included the results of the final analysis, showing an improvement in overall survival (OS) with gilteritinib compared with chemotherapy (HR, 0.64; 95% CI, 0.49-0.83; one-sided P = 0.0004; median OS, 9.3 vs. 5.6 months). The OS benefit was observed in both high and low chemotherapy intensity subgroups. Labeling includes a boxed warning for differentiation syndrome and warnings for posterior reversible encephalopathy syndrome, QT prolongation, pancreatitis, and embryo-fetal toxicity. Safe use requires frequent monitoring of electrocardiograms and blood chemistries. Assessments of long-term safety are pending.

In vitro UGT1A1 inhibition by tyrosine kinase inhibitors and association with drug-induced hyperbilirubinemia.

PURPOSE: Hyperbilirubinemia has been observed in patients treated with tyrosine kinase inhibitor (TKI) drugs. Therefore, it would be beneficial to understand whether there is a relationship between inhibition of uridine-5'-diphosphate glucuronosyltransferase (UGT) 1A1 activity and observed bilirubin elevations in TKI drug-treated patients. UGT1A1 is responsible for the glucuronidation of bilirubin which leads to its elimination in the bile. METHODS: To examine this question, an in vitro glucuronidation assay was developed to determine the inhibitory effect of TKI drugs employing human liver microsomes (HLM) with varying UGT1A1 activity. Utilizing ß-estradiol as the UGT1A1 probe substrate, 20 TKI drugs were evaluated at concentrations that represent clinical plasma levels. Adverse event reports were searched to generate an empirical Bayes geometric mean (EGBM) score for clinical hyperbilirubinemia with the TKI drugs. RESULTS: Erlotinib, nilotinib, regorafenib, pazopanib, sorafenib and vemurafenib had IC50 values that were lower than their clinical steady-state Cmax concentrations. These TKI drugs had high incidences of hyperbilirubinemia and higher EBGM scores. The IC50 values and Cmax/IC50 ratios correlated well with EBGM scores for hyperbilirubinemia (P < 0.005). For the TKI drugs with higher incidence of hyperbilirubinemia in Gilbert syndrome patients, who have reduced UGT1A1 activity, six of eight had smaller ratios in the low UGT1A1 activity microsomes than the wild-type microsomes for drugs, indicating greater sensitivity to the drugs in this phenotype. CONCLUSIONS: These results suggest that in vitro UGT1A1 inhibition assays have the potential to predict clinical hyperbilirubinemia.

Challenges with the precise prediction of ABC-transporter interactions for improved drug discovery.

INTRODUCTION: Given that membrane efflux transporters can influence a drug's pharmacokinetics, efficacy and safety, identifying potential substrates and inhibitors of these transporters is a critical element in the drug discovery and development process. Additionally, it is important to predict the inhibition potential of new drugs to avoid clinically significant drug interactions. The goal of preclinical studies is to characterize a new drug as a substrate or inhibitor of efflux transporters. Areas covered: This article reviews preclinical systems that are routinely utilized to determine whether a new drug is substrate or inhibitor of efflux transporters including in silico models, in vitro membrane and cell assays, and animal models. Also included is an examination of studies comparing in vitro inhibition data to clinical drug interaction outcomes. Expert opinion: While a number of models are employed to classify a drug as an efflux substrate or inhibitor, there are challenges in predicting clinical drug interactions. Improvements could be made in these predictions through a tier approach to classify new drugs, validation of preclinical assays, and refinement of threshold criteria for clinical interaction studies.

The development of biological therapies for neurological diseases: moving on from previous failures.

INTRODUCTION: Although years of research have expanded the use of biologics for several clinical conditions, such development has not yet occurred in the treatment of neurological diseases. With the advancement of biologic technologies, there is promise for these therapeutics as novel therapeutic approaches for neurological diseases. Areas covered: In this article, the authors review the therapeutic potential of different types of biologics for the treatment of neurological diseases. Preclinical and clinical studies that investigate the efficacy and safety of biologics in the treatment of neurological diseases, namely Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson disease, multiple sclerosis, and stroke, were reviewed. Moreover, the authors describe the key challenges in the development of therapeutically safe and effective biologics for the treatment of neurological diseases. Expert opinion: Several biologics have shown promise in the treatment of neurological diseases. However, the complexity of the CNS, as well as a limited understanding of disease progression, and restricted access of biologics to the CNS has limited successful development. Therefore, more research needs to be conducted to overcome these hurdles before developing effective and safe biologics for neurological diseases. The emergence of new technologies for the design, production and delivery of biologics will accelerate translating biologics to the clinic.

Effect of mouse strain as a background for Alzheimer's disease models on the clearance of amyloid-ß.

Novel animal models of Alzheimer's disease (AD) are relentlessly being developed and existing ones are being fine-tuned; however, these models face multiple challenges associated with the complexity of the disease where most of these models do not reproduce the full phenotypical disease spectrum. Moreover, different AD models express different phenotypes that could affect their validity to recapitulate disease pathogenesis and/or response to a drug. One of the most important and understudied differences between AD models is differences in the phenotypic characteristics of the background species. Here, we used the brain clearance index (BCI) method to investigate the effect of strain differences on the clearance of amyloid ß (Aß) from the brains of four mouse strains. These mouse strains, namely C57BL/6, FVB/N, BALB/c and SJL/J, are widely used as a background for the development of AD mouse models. Findings showed that while Aß clearance across the blood-brain barrier (BBB) was comparable between the 4 strains, levels of LRP1, an Aß clearance protein, was significantly lower in SJL/J mice compared to other mouse strains. Furthermore, these mouse strains showed a significantly different response to rifampicin treatment with regard to Aß clearance and effect on brain level of its clearance-related proteins. Our results provide for the first time an evidence for strain differences that could affect ability of AD mouse models to recapitulate response to a drug, and opens a new research avenue that requires further investigation to successfully develop mouse models that could simulate clinically important phenotypic characteristics of AD.

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.

Astrocytes drive upregulation of the multidrug resistance transporter ABCB1 (P-Glycoprotein) in endothelial cells of the blood-brain barrier in mutant superoxide dismutase 1-linked amyotrophic lateral sclerosis.

The efficacy of drugs targeting the CNS is influenced by their limited brain access, which can lead to complete pharmacoresistance. Recently a tissue-specific and selective upregulation of the multidrug efflux transporter ABCB1 or P-glycoprotein (P-gp) in the spinal cord of both patients and the mutant SOD1-G93A mouse model of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that prevalently kills motor neurons has been reported. Here, we extended the analysis of P-gp expression in the SOD1-G93A ALS mouse model and found that P-gp upregulation was restricted to endothelial cells of the capillaries, while P-gp expression was not detected in other cells of the spinal cord parenchyma such as astrocytes, oligodendrocytes, and neurons. Using both in vitro human and mouse models of the blood-brain barrier (BBB), we found that mutant SOD1 astrocytes were driving P-gp upregulation in endothelial cells. In addition, a significant increase in reactive oxygen species production, Nrf2 and NFκB activation in endothelial cells exposed to mutant SOD1 astrocytes in both human and murine BBB models were observed. Most interestingly, astrocytes expressing FUS-H517Q, a different familial ALS-linked mutated gene, also drove NFκB-dependent upregulation of P-gp. However, the pathway was not dependent on oxidative stress but rather involved TNF-α release. Overall, these findings indicated that nuclear translocation of NFκB was a converging mechanism used by endothelial cells of the BBB to upregulate P-gp expression in mutant SOD1-linked ALS and possibly other forms of familial ALS. GLIA 2016 GLIA 2016;64:1298-1313.

Effects of Genetic Polymorphism in CYP3A4 and CYP3A5 Genes on Tacrolimus Dose Among Kidney Transplant Recipients.

INTRODUCTION: This study aimed to evaluate the effects of single nucleotide polymorphisms CYP3A4*1B and CYP3A5*3 on tacrolimus dose requirement among kidney transplant recipients. MATERIALS AND METHODS: Blood levels of tacrolimus were measured using microparticle enzyme immunoassay. Genotyping analysis utilized specific polymerase chain reaction-restriction fragment length polymorphism methods for 137 kidney transplant recipients. RESULTS: The median tacrolimus dose was significantly lower in the CYP3A4*1/*1 carriers (0.06 mg/kg/d; range, 0.007 mg/kg/d to 0.17 mg/kg/d) as compared to the CYP3A4*1B/*1B carriers (0.1 mg/kg/d; range, 0.03 mg/kg/d to 0.22 mg/kg/d; P = .001). Patients with at least 1 CYP3A5*1 wild-type allele required higher median doses of tacrolimus (median, 0.08 mg/kg/d; range, 0.03 mg/kg/d to 0.22 mg/kg/d) as compared to the CYP3A5*3 carriers (median, 0.05 mg/kg/d; range, 0.007 mg/kg/d  to 0.17 mg/kg/d; P = .002). CONCLUSIONS: This study showed that tacrolimus dose requirement is lower in Jordanian kidney transplant recipients compared to other populations. Moreover, we found a correlation between genetic variations in CYP3A4 and CYP3A5 enzymes and tacrolimus blood levels among our kidney transplant recipients.

Oleocanthal enhances amyloid-ß clearance from the brains of TgSwDI mice and in vitro across a human blood-brain barrier model.

Numerous clinical and preclinical studies have suggested several health promoting effects for the dietary consumption of extra-virgin olive oil (EVOO) that could protect and decrease the risk of developing Alzheimer's disease (AD). Moreover, recent studies have linked this protective effect to oleocanthal, a phenolic secoiridoid component of EVOO. This protective effect of oleocanthal against AD has been related to its ability to prevent amyloid-ß (Aß) and tau aggregation in vitro, and enhance Aß clearance from the brains of wild type mice in vivo; however, its effect in a mouse model of AD is not known. In the current study, we investigated the effect of oleocanthal on pathological hallmarks of AD in TgSwDI, an animal model of AD. Mice treatment for 4 weeks with oleocanthal significantly decreased amyloid load in the hippocampal parenchyma and microvessels. This reduction was associated with enhanced cerebral clearance of Aß across the blood-brain barrier (BBB). Further mechanistic studies demonstrated oleocanthal to increase the expression of important amyloid clearance proteins at the BBB including P-glycoprotein and LRP1, and to activate the ApoE-dependent amyloid clearance pathway in the mice brains. The anti-inflammatory effect of oleocanthal in the brains of these mice was also obvious where it was able to reduce astrocytes activation and IL-1ß levels. Finally, we could recapitulate the observed protective effect of oleocanthal in an in vitro human-based model, which could argue against species difference in response to oleocanthal. In conclusion, findings from in vivo and in vitro studies provide further support for the protective effect of oleocanthal against the progression of AD.

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.

Regulation of ABC efflux transporters at blood-brain barrier in health and neurological disorders.

The strength of the blood-brain barrier (BBB) in providing protection to the central nervous system from exposure to circulating chemicals is maintained by tight junctions between endothelial cells and by a broad range of transporter proteins that regulate exchange between CNS and blood. The most important transporters that restrict the permeability of large number of toxins as well as therapeutic agents are the ABC transporters. Among them, P-gp, BCRP, MRP1 and MRP2 are the utmost studied. These efflux transporters are neuroprotective, limiting the brain entry of neurotoxins; however, they could also restrict the entry of many therapeutics and contribute to CNS pharmacoresistance. Characterization of several regulatory pathways that govern expression and activity of ABC efflux transporters in the endothelium of brain capillaries have led to an emerging consensus that these processes are complex and contain several cellular and molecular elements. Alterations in ABC efflux transporters expression and/or activity occur in several neurological diseases. Here, we review the signaling pathways that regulate expression and transport activity of P-gp, BCRP, MRP1 and MRP2 as well as how their expression/activity changes in neurological diseases. This article is part of a Special Issue entitled SI: Neuroprotection.

Orlistat limits cholesterol intestinal absorption by Niemann-pick C1-like 1 (NPC1L1) inhibition.

The known mechanism by which orlistat decreases the absorption of dietary cholesterol is by inhibition of intestinal lipases. The aim of this study was to investigate the ability of orlistat to limit cholesterol absorption by inhibition of the cholesterol transport protein Niemann-Pick C1-like 1 (NPC1L1) as another mechanism of action. In situ rat intestinal perfusion studies were conducted to study the effect of orlistat on jejunal cholesterol absorption. Inhibition kinetic parameters were calculated from in vitro inhibition studies using Caco2 and NPC1L1 transfected cell lines. The in situ studies demonstrated that intestinal perfusion of orlistat (100µM) was able to reduce cholesterol absorption by three-fold when compared to control (i.e. in the absence of orlistat, P<0.01). In vitro studies using Caco2 cells demonstrated orlistat to reduce the cellular uptake of cholesterol by 30%. Additionally, orlistat reduced the cellular uptake of cholesterol in dose dependent manner in NPC1L1 transfected cell line with an IC50=1.2µM. Lineweaver-Burk plot indicated a noncompetitive inhibition of NPC1L1 by orlistat. Beside the already established mechanism by which orlistat reduces the absorption of cholesterol, we demonstrated for the first time that orlistat limits cholesterol absorption by the inhibition of NPC1L1 transport protein.

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.

Mixed oligomers and monomeric amyloid-ß disrupts endothelial cells integrity and reduces monomeric amyloid-ß transport across hCMEC/D3 cell line as an in vitro blood-brain barrier model.

Senile amyloid plaques are one of the diagnostic hallmarks of Alzheimer's disease (AD). However, the severity of clinical symptoms of AD is weakly correlated with the plaque load. AD symptoms severity is reported to be more strongly correlated with the level of soluble amyloid-ß (Aß) assemblies. Formation of soluble Aß assemblies is stimulated by monomeric Aß accumulation in the brain, which has been related to its faulty cerebral clearance. Studies tend to focus on the neurotoxicity of specific Aß species. There are relatively few studies investigating toxic effects of Aß on the endothelial cells of the blood-brain barrier (BBB). We hypothesized that a soluble Aß pool more closely resembling the in vivo situation composed of a mixture of Aß40 monomer and Aß42 oligomer would exert higher toxicity against hCMEC/D3 cells as an in vitro BBB model than either component alone. We observed that, in addition to a disruptive effect on the endothelial cells integrity due to enhancement of the paracellular permeability of the hCMEC/D3 monolayer, the Aß mixture significantly decreased monomeric Aß transport across the cell culture model. Consistent with its effect on Aß transport, Aß mixture treatment for 24h resulted in LRP1 down-regulation and RAGE up-regulation in hCMEC/D3 cells. The individual Aß species separately failed to alter Aß clearance or the cell-based BBB model integrity. Our study offers, for the first time, evidence that a mixture of soluble Aß species, at nanomolar concentrations, disrupts endothelial cells integrity and its own transport across an in vitro model of the BBB.