Development of a novel cell-based assay to monitor the transactivation activity of the HSV-1 protein ICP0

The herpes simplex virus type 1 (HSV-1) immediate-early phosphoprotein infected cell protein 0 (ICP0) is a potent transcriptional activator of viral genes and is required for efficient viral replication and reactivation from latency. However, it is largely unknown what role specific cellular factors play in the transactivator function of ICP0. With the long-term goal of identifying these factors, we developed a cell-based assay in a 96-well format to measure this activity of ICP0. We designed a system using a set of HSV-1 GFP reporter viruses in which the expression of GFP is potently induced by ICP0 in cell culture. The initial feasibility of this system was confirmed over a 24-hour period by fluorescence microscopy. We adapted this assay to a 96-well plate format, quantifying GFP expression with a fluorescence scanner. Our results indicate that the cell-based assay we developed is a valid and effective method for examining the transactivating activity of ICP0. This assay can be used to identify cellular factors that regulate the transactivating activity of ICP0

Utilization of I-domain of LFA-1 to Target Drug and Marker Molecules to Leukocytes

The long-term objective of this project is to utilize the I-domain protein for the α-subunit of LFA-1 to target drugs to lymphocytes by binding to ICAM receptors on the cell surface. The short-term goal is to provide proof-of-concept that I-domain conjugated to small molecules can still bind to and uptake by ICAM-1 on the surface of lymphocytes (i.e., Raji cells). To accomplish this goal, the I-domain protein was labeled with FITC at several lysine residues to produce the FITC-I-domain and CD spectroscopy showed that the FITC-I-domain has a secondary structure similar to that of the parent I-domain. The FITC-I-domain was taken up by Raji cells via receptor-mediated endocytosis and its uptake can be blocked by anti-I-domain mAb but not by its isotype control. Antibodies to ICAM-1 enhance the binding of I-domain to ICAM-1, suggesting it binds to ICAM-1 at different sites than the antibodies. The results indicate that fluorophore modification does not alter the binding and uptake properties of the I-domain protein. Thus, I-domain could be useful as a carrier of drug to target ICAM-1-expressing lymphocytes

Development of a Grp94 inhibitor

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/ja303477g.Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/β (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/β client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein

Understanding medication compliance and persistence from an economics perspective

AbstractObjectivesAn increased understanding of the reasons for noncompliance and lack of persistence with prescribed medication is an important step to improve treatment effectiveness, and thus patient health. Explanations have been attempted from epidemiological, sociological, and psychological perspectives. Economic models (utility maximization, time preferences, health capital, bilateral bargaining, stated preference, and prospect theory) may contribute to the understanding of medication-taking behavior.MethodsEconomic models are applied to medication noncompliance. Traditional consumer choice models under a budget constraint do apply to medication-taking behavior in that increased prices cause decreased utilization. Nevertheless, empiric evidence suggests that budget constraints are not the only factor affecting consumer choice around medicines. Examination of time preference models suggests that the intuitive association between time preference and medication compliance has not been investigated extensively, and has not been proven empirically. The health capital model has theoretical relevance, but has not been applied to compliance. Bilateral bargaining may present an alternative model to concordance of the patient–prescriber relationship, taking account of game-playing by either party. Nevertheless, there is limited empiric evidence to test its usefulness. Stated preference methods have been applied most extensively to medicines use.ResultsEvidence suggests that patients' preferences are consistently affected by side effects, and that preferences change over time, with age and experience. Prospect theory attempts to explain how new information changes risk perceptions and associated behavior but has not been applied empirically to medication use.ConclusionsEconomic models of behavior may contribute to the understanding of medication use, but more empiric work is needed to assess their applicability

Effects of an increase in prescription copayment on utilization of low-sedating antihistamines and nasal steroids

BACKGROUND: Health plans are using 3-tier copayment designs and other methods to control utilization that shifts drug costs to plan members. There is a need to determine the effects of increased member cost sharing on drug utilization and drug costs. OBJECTIVE: To assess the impact of a $10 increase in prescription copayment in a public employer health plan for 2 classes of drugs used for allergic rhinitis. METHODS: Changes in the number of prescriptions dispensed for 2 therapeutic classes low-sedating antihistamines (LSAs) and nasal steroids (NSs) were examined 1 year prior to and 1 year after copayment increase. Relative price effects were measured as arc price elasticity, the ratio of the percent change in prescription utilization over the percent change in price, an indicator of how responsive patients are to the copayment increase. RESULTS: Of 8,643 continuously enrolled health plan beneficiaries, 2,150 patients (24.8$10 increase in copayment per prescription was associated with no statistically significant change in utilization of combined LSA and NS prescriptions, 2.89 per patient in 1998 and 2.94 in 1999 (P = 0.597). Health plan costs for study drugs, unadjusted for inflation, decreased by 16.3% from $86.86 per patient in 1998 to$72.68 in 1999 (P = 0.004). Health plan costs per patient per month (PPPM) for all drugs for the 2,150 allergic rhinitis patients decreased by 13% from $41.33 PPPM in 1998 to$35.93 in 1999 (Pless than0.001), and health plan drug costs for all 8,643 members decreased by 13% from $14.93 per member per month (PMPM) in 1998 to$12.99 in 1999 (Pless than0.001). The actual average copayment increase was $7.23 (a 41$10.98 (71%) for NSs, which was associated with an 11.3% decrease in utilization of NSs and a 10.2% decrease in the number of users of nasals steroids in 1999; the number of nasal steroid prescriptions per patient per year was unchanged at 2.05 in 1999 versus 2.07 in 1998 (P = .842). The combined utilization of LSA and NS prescriptions increased by 8.9% following the increase in copayments for these 2 therapeutically interchangeable drugs for allergic rhinitis. LSA prescriptions were less elastic, with an unadjusted arc elasticity of 0.39, while nasal steroid prescriptions were more responsive to the copayment change, with an unadjusted arc elasticity of .0.22. CONCLUSIONS: An average $10 increase in patient cost sharing per prescription (46.9% copayment increase) was associated with an increase in combined utilization of 2 drug classes used for allergic rhinitis (LSAs and NSs) but no change in the number of prescriptions per patient. Health plan costs decreased significantly for allergic rhinitis drugs, all drugs used by allergic rhinitis patients, and all drugs used by continuously enrolled health plan members. NSs exhibited a greater arc price elasticity compared with low-sedating oral antihistamines