Advancing Alzheimer's Disease Treatment: Lessons from CTAD 2018.

The 2018 Clinical Trials on Alzheimer's Disease (CTAD) conference showcased recent successes and failures in trials of Alzheimer's disease treatments. More importantly, the conference provided opportunities for investigators to share what they have learned from those studies with the goal of designing future trials with a greater likelihood of success. Data from studies of novel and non-amyloid treatment approaches were also shared, including neuroprotective and regenerative strategies and those that target neuroinflammation and synaptic function. New tools to improve the efficiency and productivity of clinical trials were described, including biomarkers and machine learning algorithms for predictive modeling.

The future is now: model-based clinical trial design for Alzheimer's disease.

Failures in trials for Alzheimer's disease (AD) may be attributable to inadequate dosing, population selection, drug inefficacy, or insufficient design optimization. The Coalition Against Major Diseases (CAMD) was formed in 2008 to develop drug development tools (DDT) to expedite drug development for AD and Parkinson's disease. CAMD led a process that successfully advanced a clinical trial simulation (CTS) tool for AD through the formal regulatory review process at the US Food and Drug Administration (FDA) and European Medicines Agency (EMA).

Altered expression of sulfotransferases, glucuronosyltransferases and mrp transporters in FVB/mrp1-/- mice.

1. Genetically altered mice increasingly are being used in toxicology and pharmaceutical development. As such, knowledge of the compensatory activity of enzymes is critical when interpreting the results of studies using these animals. 2. The present study examined alterations in hepatic phase I and II enzyme activity, and alterations in phase III (transporter) RNA expression, between FVB mice and mice lacking the multidrug resistance-associated protein 1 (mrp1) gene (FVB/mrp1-/- mice). It was hypothesized that other transporters and phase I and II enzymes would be increased in the FVB/mrp1-/- mice, presumably as a compensatory mechanism. 3. No differences was found in hepatic cytochrome P450 activity between FVB and FVB/mrp1-/- mice, nor were there differences in the amount of total hepatic glutathione or in glutathione S-transferase enzyme activity. 4. However, sulfotransferase activity towards 2-naphthol was significantly increased by 2.6-fold in the FVB/mrp1-/- mice, whereas glucuronosyltransferase activity towards both 4-nitrophenol and testosterone was significantly reduced 1.5-fold. In addition, mrp2 RNA expression was significantly increased by 3.4-fold and mrp5 expression was significantly increased by 1.6-fold in the FVB/mrp1-/- mice. 5. Mice lacking mrp1 have significantly increased hepatic transcription of at least two other ATP-binding cassette transporters, as well as increased 2-naphthol sulfotransferase activity, presumably to compensate for the lack of mrp1.

Expression of multidrug resistance protein-3 (multispecific organic anion transporter-D) in human embryonic kidney 293 cells confers resistance to anticancer agents.

Multidrug resistance-associated protein (MRP)1 and canalicular multispecific organic anion transporter (cMOAT)/MRP2 are ATP-binding cassette (ABC) transporters that confer resistance to natural product cytotoxic drugs. We recently described the complete coding sequences of four human MRP/cMOAT subfamily members and found that, among these proteins, MRP3/MOAT-D is most closely related to MRP1 (58% identity; M. G. Belinsky and G. D. Kruh, Br. J. Cancer, 80: 1342-1349, 1999). In the present study, we sought to determine whether MRP3 is capable of conferring resistance to cytotoxic drugs. To address this question, human embryonic kidney 293 cells were transfected with an MRP3 expression vector, and the drug resistance phenotype of the transfected cells was analyzed. The MRP3-transfected cells displayed approximately 4-fold resistance to etoposide and approximately 2-fold resistance to vincristine, compared with control transfected cells. In addition, approximately 1.7-fold resistance was observed for the antimetabolite methotrexate. Increased resistance was not observed for several other natural product agents, including anthracyclines and Taxol. The MRP-transfected cells exhibited reduced accumulation of radiolabeled etoposide, consistent with the operation of a plasma membrane efflux pump. These results indicate that MRP3 confers resistance to some anticancer agents but that its resistance pattern is distinct from the resistance patterns of other ABC transporters involved in resistance to natural product chemotherapeutic agents.

Characterization of MOAT-C and MOAT-D, new members of the MRP/cMOAT subfamily of transporter proteins.

BACKGROUND: Multidrug resistance-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) are transporter proteins that pump organic anions across cellular membranes and have been linked to resistance to cytotoxic drugs. We previously identified MOAT-B, an MRP/cMOAT-related transporter, by use of a polymerase chain reaction approach. However, analysis of expressed sequence tag (EST) databases indicated that there might be additional MRP/cMOAT-related transporters. To further define the MRP/cMOAT subfamily of transporters, we used EST probes to isolate complementary DNAs for two related transporter proteins, MOAT-C and MOAT-D. METHODS: MOAT-C and MOAT-D expression patterns in human tissues were determined by RNA blot analysis, and chromosomal localization of the genes was determined by fluorescence in situ hybridization. RESULTS: MOAT-C is predicted to encode a 1437-amino-acid protein that, among eukaryotic transporters, is most closely related to MRP, cMOAT, and MOAT-B (about 36% identity). However, MOAT-C is less related to MRP and cMOAT than MRP and cMOAT are to each other (about 48% identity). Like MOAT-B, MOAT-C lacks an N-terminal membrane-spanning domain, indicating that the topology of this protein is similarly distinct from that of MRP and cMOAT. MOAT-D is predicted to encode a 1527-amino-acid protein that is the closest known relative of MRP (about 58% identity). MOAT-D is also highly related to cMOAT (about 47% identity). The presence of an N-terminal membrane-spanning domain indicates that the topology of MOAT-D is quite similar to that of MRP and cMOAT. MOAT-C transcripts are widely expressed in human tissues; however, MOAT-D transcript expression is more restricted. The MOAT-C and MOAT-D genes are located at chromosomes 3q27 and 17q21.3, respectively. CONCLUSIONS: On the basis of amino acid identity and protein topology, the MRP/cMOAT transporter subfamily falls into two groups; the first group consists of MRP, cMOAT, and MOAT-D, and the second group consists of MOAT-B and MOAT-C.

Structure-activity relationships for xenobiotic transport substrates and inhibitory ligands of P-glycoprotein.

The multixenobiotic resistance phenotype is characterized by the reduced accumulation of xenobiotics by cells or organisms due to increased efflux of the compounds by P-glycoprotein (P-gp) or related transporters. An extensive xenobiotic database, consisting primarily of pesticides, was utilized in this study to identify molecular characteristics that render a xenobiotic susceptible to transport by or inhibition of P-gp. Transport substrates were differentiated by several molecular size/shape parameters, lipophilicity, and hydrogen bonding potential. Electrostatic features differentiated inhibitory ligands from compounds not catagorized as transport substrates and that did no interact with P-gp. A two-tiered system was developed using the derived structure-activity relationships to identify P-gp transport substrates and inhibitory ligands. Prediction accuracy of the approach was 82%. We then validated the system using six additional pesticides of which tow were predicted to be P-gp inhibitors and four were predicted to be noninteractors, based upon the structure-activity analyses. Experimental determinations using cells transfected with the human MDR1 gene demonstrated that five of the six pesticides were properly catagorized by the structure-activity analyses (83% accuracy). Finally, structure-activity analyses revealed that among P-gp inhibitors, relative inhibitory potency can be predicted based upon the surface area or volume of the compound. These results demonstrate that P-gp transport substrates and inhibitory ligands can be distinguished using molecular characteristics. Molecular characteristics of transport substrates suggest that P-gp may function in the elimination of hydroxylated metabolites of xenobiotics.

Chronic toxicity of environmental contaminants: sentinels and biomarkers.

Due to the use of a limited number of species and subchronic exposures, current ecological hazard assessment processes can underestimate the chronic toxicity of environmental contaminants resulting in adverse responses of sentinel species. Several incidences where sentinel species have responded to the effects of chronic exposure to ambient levels of environmental contaminants are discussed, including the development of neoplasia in fish, immunosuppression in marine mammals, pseudohermaphrodism in invertebrates, teratogenicity in amphibians, and aberrations in the sexual development of fish and reptiles. Biomarkers of chronic toxicity, including DNA mutations, alterations in specific protein and mRNA levels, and perturbations in metabolism, are presented. The incorporation of appropriate surrogate species and biomarkers of chronic toxicity into standard toxicity characterizations is proposed as a means of significantly refining the ecological hazard assessment process.

Pesticides: multiple mechanisms of demasculinization.

Many pesticides are known to produce reproductive and developmental effects in chronically exposed non-target organisms, including humans. Recent evidence suggests that demasculinization may be an important mechanism responsible for some of these effects. Some pesticides have been shown to interact with the androgen receptor and to act as antagonists, while others have been shown to interact with the estrogen receptor and function as estrogens in both in vitro and in vivo. Many pesticides can also lower serum androgen levels by altering rates of synthesis or metabolism. Given the ubiquity of pesticides in the environment and the multiple mechanisms whereby they can elicit demasculinizing effects, synergy between such compounds may produce clinical endocrine dysfunction at current human exposure levels.

Interaction of structurally diverse pesticides with the human MDR1 gene product P-glycoprotein.

P-glycoprotein (P-gp) is a 170-kDa membrane-bound glycoprotein shown to efflux a wide variety of chemicals, such as chemotherapeutic agents and carcinogens. Experiments were conducted using B16/F10 murine melanoma cells transfected with the human MDR1 gene (B16/hMDR1 cells), which codes for P-gp, to determine whether this transporter may contribute to the cellular efflux of some pesticides. Thirty-eight pesticides representing several classes of compounds were evaluated for their potential to bind to P-gp, as measured by the inhibition of efflux of the P-gp substrate doxorubicin. Carbamate and pyrethroid insecticides exhibited little interaction with P-gp, while many of the organophosphorus and organochlorine pesticides significantly inhibited the efflux of doxorubicin. Pesticides that significantly inhibited the efflux of doxorubicin were then assessed for P-gp-mediated efflux. One pesticide, endosulfan, exhibited slight though significant transport mediated by P-gp. Competition experiments performed with the P-glycoprotein ligand [3H]azidopine demonstrated that the P-gp inhibitory pesticides bound to P-gp. Both lipophilicity and molecular mass were major physical/chemical determinants in dictating pesticide binding to P-gp, with optimum binding occurring with compounds having a log Kow value of 3.6-4.5 and a molecular weight of 391-490 Da. The transport substrate endosulfan possessed optimal binding characteristics. These results demonstrated that many pesticides are capable of binding to P-gp; however, binding does not infer transport.