Effects of HIV and Drugs of Abuse on the Blood-Brain Barrier

Despite effective systemic therapy, HIV-1 infection within the brain results in neuronal degradation and neurocognitive dysfunction. This neurocognitive dysfunction is worsened in the setting of opiate abuse. The central nervous system (CNS) is protected by the blood-brain barrier (BBB), a selective barrier regulating the passage of substances from peripheral circulation into the CNS. The BBB is composed of microvascular endothelial cells encased by basal lamina, pericytes, and perivascular astrocyte endfeet. Intracellular junctional complexes comprising of adherens and tight junctions are located between the endothelial cells and form tight barrier, preventing traffic of compounds between cells (paracellular flux). Clinical and in vitro data suggest that BBB integrity is compromised in HIV infection, which leads to a leaky barrier. Brain microvascular endothelial cells also express efflux transporters that are responsible for the extrusion of substances from the brain back into the blood. P-glycoprotein is a drug efflux transporter involved in the efflux of many antiretroviral drugs and overexpression of P-glycoprotein can limit therapeutic concentrations of substrate drugs within the brain. Additionally, P-glycoprotein expression and/or function may be altered in the setting of HIV infection and in the setting of drug abuse. In order to study the impact of morphine, a commonly used opiate drug of abuse, on drug-efflux proteins at the BBB, we measured the effects of morphine and the HIV-1 protein Tat on P-glycoprotein expression and function. hCMEC/D3 cells, which are human derived brain microvascular endothelial cells, were pre-treated for 24 hours with Tat (100nM), morphine (500nM), or Tat (100nM) + morphine (500nM). P-glycoprotein function was evaluated by measuring intracellular accumulation of the prototypical P-glycoprotein substrate, rhodamine-123. Compared to control, statistically significant increases in cellular accumulation of rhodamine-123 were observed in both the morphine (mean±SEM; 118±6.5%, p\u3c0.05) and Tat+morphine (118 ±13.1%, p\u3c0.05) groups, suggesting decreased efflux activity of P-glycoprotein. Protein expression of P-glycoprotein was measured using western blot analysis. Significant decreases in P-glycoprotein expression was observed in all treatment groups as compared to control; Tat (63±4.2%, p\u3c 0.05), morphine (64±13.5%, p\u3c0.05) and Tat+morphine (69±15.6%, p\u3c0.05). Understanding the factors that influence efflux transporter function and expression in the BBB are crucial in optimizing antiretroviral penetration into the brain, even in the setting of drug abuse.https://scholarscompass.vcu.edu/uresposters/1263/thumbnail.jp

Dynamics of ABC Transporter P-glycoprotein in Three Conformational States.

We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to obtain a comprehensive view of transporter dynamics (85.8% sequence coverage) occurring throughout the multidrug efflux transporter P-glycoprotein (P-gp) in three distinct conformational states: predominantly inward-facing apo P-gp, pre-hydrolytic (E552Q/E1197Q) P-gp bound to Mg+2-ATP, and outward-facing P-gp bound to Mg+2-ADP-VO4-3. Nucleotide affinity was measured with bio-layer interferometry (BLI), which yielded kinetics data that fit a two Mg+2-ATP binding-site model. This model has one high affinity site (3.2 ± 0.3 µM) and one low affinity site (209 ± 25 µM). Comparison of deuterium incorporation profiles revealed asymmetry between the changes undergone at the critical interfaces where nucleotide binding domains (NBDs) contact intracellular helices (ICHs). In the pre-hydrolytic state, both interfaces between ICHs and NBDs decreased exchange to similar extents relative to inward-facing P-gp. In the outward-facing state, the ICH-NBD1 interface showed decreased exchange, while the ICH-NBD2 interface showed less of an effect. The extracellular loops (ECLs) showed reduced deuterium uptake in the pre-hydrolytic state, consistent with an occluded conformation. While in the outward-facing state, increased ECL exchange corresponding to EC domain opening was observed. These findings point toward asymmetry between both NBDs, and they suggest that pre-hydrolytic P-gp occupies an occluded conformation

P-glycoprotein and its role in drug-drug interactions

Efflux transporters such as P-glycoprotein play an important role in drug transport in many organs. In the gut, P-glycoprotein pumps drugs back into the lumen, decreasing their absorption. Drugs which induce P-glycoprotein, such as rifampicin, can reduce the bioavailability of some other drugs. Inhibitors of P-glycoprotein, such as verapamil, increase the bioavailability of susceptible drugs. Many, but not all, of the drugs which are transported by P-glycoprotein are also metabolised by cytochrome P450 3A4. Important substrates of P-glycoprotein include calcium channel blockers, cyclosporin, dabigatran etexilate, digoxin, erythromycin, loperamide, protease inhibitors and tacrolimus. Predicting clinically important interactions is difficult because of interindividual differences in drug transport

Identification and characterization of the binding sites of P-glycoprotein for multidrug resistance-related drugs and modulators

A major problem in cancer treatment is the development of resistance to multiple chemotherapeutic agents in tumor cells. A major mechanism of this multidrug resistance (MDR) is overexpression of the MDR1 product P-glycoprotein, known to bind to and transport a wide variety of agents. This review concentrates on the progress made toward understanding the role of this protein in MDR, identifying and characterizing the drug binding sites of P-glycoprotein, and modulating MDR by P-glycoprotein-specific inhibitors. Since our initial discovery that P-glycoprotein binds to vinblastine photoaffinity analogs, many P-glycoprotein-specific photoaffinity analogs have been developed and used to identify the particular domains of P-glycoprotein capable of interacting with these analogs and other P-glycoprotein substrates. Furthermore, significant advances have been made in delineating the drug binding sites of this protein by studying mutant P-glycoprotein. Photoaffinity labeling experiments and the use of site-directed antibodies to several domains of this protein have allowed the localization of the general binding domains of some of the cytotoxic agents and MDR modulators on P-glycoprotein. Moreover, site-directed mutagenesis studies have identified the amino acids critical for the binding of some of these agents to P-glycoprotein. Furthermore, equilibrium binding assays using plasma membranes from MDR cells and radioactive drugs have aided our understanding of the modes of drug interactions with P-glycoprotein. Based on the available data, a topological model of P-glycoprotein and the approximate locations of its drug binding sites, as well as a proposed classification of multiple drug binding sites of this protein, is presented in this review

Immunohistochemical analysis of P-glycoprotein expression in diverse histological types of epithelial ovarian tumors.

P-glycoprotein is a transmembrane protein which acts as an energy-dependent drug efflux pump for a variety of anti-cancer drugs. The mdr-1 gene which encodes P-glycoprotein was successfully cloned in 1986. To investigate P-glycoprotein expression in diverse ovarian tumors, including benign, low malignant potential and malignant, immunohistochemical study was done using a monoclonal antibody (C 219). Overall, 8 out of the 59 epithelial ovarian tumors (13.6%) expressed P-glycoprotein. It was noted that 5 of the 12 mucinous tumors were found to express P-glycoprotein, while none of the 31 serous tumors were immunohistochemically positive. In 10 malignant ovarian tumors, P-glycoprotein immunostaining was examined both prior to and after chemotherapy. Nine of them did not express any P-glycoprotein before or after chemotherapy. However, one tumor expressed P-glycoprotein after six courses of multidrug resistance-related drug administration. These findings indicate that P-glycoprotein expression is not so common in ovarian tumors, regardless of their malignant potential. Nevertheless, the results suggest a strong association between P-glycoprotein expression and certain histological cell types in epithelial ovarian tumors. It is also possible that P-glycoprotein appears as a result of chemotherapy, but such a phenomenon can not occur unless chemotherapy is administered at high doses for a long period of time.</p

Pharmacokinetics and allometric scaling of antimalarial drugs

Allometric scaling was found as a plausible technique for dose determination in children. Permeability and P-glycoprotein efflux transport of antimalarials were determined using in-vitro Caco-2 cells. Mefloquine showed P-glycoprotein inhibition. Amodiaquine, artesunate and artemisone were not P-glycoprotein substrates or inhibitors. Methylene-blue showed some P-glycoprotein mediated efflux. Permeability was high for amodiaquine and artemisone, medium for mefloquine and artesunate and low for methylene-blue. P-glycoprotein was up-regulated when exposed to dihydroartemisinin/artemisone in combinations with amodiaquine/mefloquine

ERBB and P-glycoprotein inhibitors break resistance in relapsed neuroblastoma through P-glycoprotein

Neuroblastoma is a pediatric tumor of the sympathetic nervous system that most commonly affects infants and young children. It is a disease with very variable outcomes, which range from spontaneous regression to aggressive disease. Particularly problematic are high-risk cases after relapse, with only 10% of patients surviving for longer than five years and chemotherapy resistance making treatment difficult. In this thesis, I examined differences between primary and relapsed patient gene expression data and between chemotherapy-sensitive and chemotherapy-resistant neuroblastoma cell lines to find a vulnerability that can be targeted to overcome chemotherapy resistance and lead to cell death. I further investigated which type of cell death was induced. My analysis of gene expression data from primary and relapsed neuroblastoma patients suggested that the ERBB family of receptor tyrosine kinases, particularly ERBB4, plays a role in relapsed high-risk neuroblastomas. The ERBB family is crucial in development and is well known for its link to cancer. Furthermore, I functionally examined the resistance breaking effect of 15 clinically relevant drugs on a neuroblastoma cell line that was treated to be resistant to the standard-of-care chemotherapeutic vincristine (VCR). Tariquidar, an inhibitor of P-glycoprotein (P-gp), and afatinib, a FDA approved inhibitor of the ERBB family, were the two most efficient drugs in breaking resistance in this screen. P-gp/ABCB1 is a transmembrane transporter that very efficiently eliminates drugs and other xenobiotics from the cell and has long been recognized for its contribution to chemotherapy resistance. Analysing gene expression datasets of more than 50 different neuroblastoma cell lines (primary and relapsed) and more than 160 neuroblastoma patient samples from the pediatric precision medicine platform INFORM (Individualized Therapy For Relapsed Malignancies in Childhood) confirmed a crucial role of P-gp in neuroblastoma resistance at relapse. The ERBB family appeared to play a minor part. I mechanistically investigated chemotherapy resistance through four pairs of vincristine- sensitive/resistant neuroblastoma sublines - each pair with the same genetic background - and two additional high-risk neuroblastoma cell lines. I effectively overcame resistance by the addition of the pan-ERBB family inhibitors afatinib and lapatinib, as well as the P-gp inhibitors tariquidar and verapamil, to VCR treatment, which synergistically reduced viability. Functional analyses of the ERBB downstream pathways, as well as ERBB4 knock-down, showed the resistance breaking effect of afatinib to be unrelated to ERBB signaling and suggested an off-target effect on P-gp. ABCB1 knock-down and analysis of transporter function confirmed that resistance was mediated through P-gp. My analysis of programmed cell death, in particular apoptosis and ferroptosis, in neuroblastoma cell lines showed that apoptosis was consistently induced, while ferroptosis contributed to cell death in the IMR-32 cell line. The combination treatments of VCR with afatinib or tariquidar in VCR-sensitive and VCR-resistant cell lines led to cell death by apoptosis. In summary, this study showed that P-gp is an important player in chemotherapy resistance in high-risk, relapsed neuroblastoma. ERBB4, although upregulated in individual cases or cell lines, did not contribute to resistance, and the sensitizing effect of the ERBB family inhibitor afatinib was due to an off-target effect on P-gp. I further showed that both apoptosis and ferroptosis are induced in neuroblastoma cell lines, with apoptosis being the dominant mechanism of cell death when chemotherapy resistance was broken

Evaluation of QSAR and ligand enzyme docking for the identification of ABCB1 substrates

P-glycoprotein (P-gp) is an efflux pump that belongs to ATP-binding cassette (ABC) transporter family embedded in the membrane bilayer. P-gp is a polyspecific protein that has demonstrated its function as a transporter of hydrophobic drugs as well as transporting lipids, steroids and metabolic products. Its role in multidrug resistance (MDR) and pharmacokinetic profile of clinically important drug molecules has been widely recognised. In this study, QSAR and enzyme-ligand docking methods were explored in order to classify substrates and non-substrates of P-glycoprotein. A set of 123 compounds designated as substrates (54) or non-substrates (69) by Matsson et al., 2009 was used for the investigation. For QSAR studies, molecular descriptors were calculated using ACD labs/LogD Suite and MOE (CCG Inc.). P-glycoprotein structures available in the Protein data bank were used for docking studies and determination of binding scores using MOE software. Binding sites were defined using co-crystallised ligand structures. Three classification algorithms which included classification and regression trees, boosted trees and support vector machine were examined. Models were developed using a training set of 98 compounds and were validated using the remaining compounds as the external test set. A model generated using BT was identified as the best of three models, with a prediction accuracy of 88%, Mathews correlation coefficient of 0.77 and Youden’s J index of 0.80 for the test set. Inclusion of various docking scores for different binding sites improved the models only marginally

Role of p-glycoprotein expression in predicting response to neoadjuvant chemotherapy in breast cancer-a prospective clinical study

BACKGROUND: Neoadjuvant chemotherapy (NACT) is an integral part of multi-modality approach in the management of locally advanced breast cancer. It is vital to predict response to chemotherapy in order to tailor the regime for a particular patient. The prediction would help in avoiding the toxicity induced by an ineffective chemotherapeutic regime in a non-responder and would also help in the planning of an alternate regime. Development of resistance to chemotherapeutic agents is a major problem and one of the mechanisms considered responsible is the expression of 170-k Da membrane glycoprotein (usually referred to as p-170 or p-glycoprotein), which is encoded by multidrug resistance (MDR1) gene. This glycoprotein acts as an energy dependent pump, which actively extrudes certain families of chemotherapeutic agents from the cells. The expression of p-glycoprotein at initial presentation has been found to be associated with refractoriness to chemotherapy and a poor outcome. Against this background a prospective study was conducted using C219 mouse monoclonal antibody specific for p-glycoprotein to ascertain whether pretreatment detection of p-glycoprotein expression could be utilized as a reliable predictor of response to neoadjuvant chemotherapy in patients with breast cancer. PATIENTS AND METHODS: Fifty cases of locally advanced breast cancer were subjected to trucut(® )biopsy and the tissue samples were evaluated immunohistochemically for p-glycoprotein expression and ER, PR status. The response to neoadjuvant chemotherapy was assessed clinically and by using ultrasound after three cycles of FAC regime (cyclophosphamide 600 mg/m(2), Adriamycin 50 mg/m(2), 5-fluorourail 600 mg/m(2 )at an interval of three weeks). The clinical response was correlated with both the pre and post chemotherapy p-glycoprotein expression. Descriptive studies were performed with SPSS version 10. The significance of correlation between tumor response and p-glycoprotein expression was determined with chi square test. RESULTS: A significant relationship was found between the pretreatment p-glycoprotein expression and clinical response. The positive p-glycoprotein expression was associated with poor clinical response rates. When the clinical response was correlated with p-glycoprotein expression, a statistically significant negative correlation was observed between the clinical response and p- glycoprotein expression (p < 0.05). There was another significant observation in terms of development of post NACT p-glycoprotein positivity. Before initiation of NACT, 26 patients (52%) were p-glycoprotein positive and after three cycles of NACT, the positivity increased to 73.5% patients. CONCLUSION: The study concluded that pretreatment p-glycoprotein expression predicts and indicates a poor clinical response to NACT. Patients with positive p-glycoprotein expression before initiation of NACT were found to be poor responders. Thus pretreatment detection of p-glycoprotein expression may be utilized, as a reliable predictor of response to NACT in patients with breast cancer The chemotherapy induced p-glycoprotein positivity observed in the study could possibly explain the phenomenon of acquired chemoresistance and may also serve as an intermediate end point in evaluating drug response particularly if the adjuvant therapy is planned with the same regime

P-Glycoprotein: One Mechanism, Many Tasks and the Consequences for Pharmacotherapy of Cancers

P-glycoprotein or multidrug resistance protein (MDR1) is an adenosine triphosphate (ATP)binding cassette transporter (ABCB1) intensely investigated because it is an obstacle tosuccessful pharmacotherapy of cancers. P-glycoprotein prevents cellular uptake of alarge number of structurally and functionally diverse compounds, including most cancertherapeutics and in this way causes multidrug resistance (MDR). To overcome MDR,and thus improve cancer treatment, an understanding of P-glycoprotein inhibition at themolecular level is required. With this goal in mind, we propose rules that predict whether acompound is a modulator, substrate, inhibitor, or inducer of P-glycoprotein. This new setof rules is derived from a quantitative analysis of the drug binding and transport propertiesof P-glycoprotein. We further discuss the role of P-glycoprotein in immune surveillanceand cell metabolism. Finally, the predictive power of the proposed rules is demonstratedwith a set of FDA approved drugs which have been repurposed for cancer therapy