Purpose: The purpose of this study was to determine the influence of out-of-pocket prescription and healthcare costs on adherence to guideline recommended statins, angiotensin converting enzyme (ACE) inhibitors/angiotensin receptor blockers (ARB), and beta-blockers (BB) used for secondary prevention of coronary heart disease and the associations of adherence with cardiovascular mortality in community-dwelling Medicare beneficiaries ≥ 65 years. Methods: Data from the 2004, 2005, and 2006 Medicare Current Beneficiary Survey (MCBS) was utilized to conduct a retrospective, cross-sectional (i.e., multiple cohort) study. Dependent variables of interest included adherence to statins, ACE/ARBs or BBs, and all-cause mortality, with out-of-pocket (OOP) costs, and adherence to these medications the primary independent variables of interest in these models. Adherence was analyzed as a binary variable with ≥ 80 percent annual adherence the threshold utilized in primary analyses. Total OOP prescription costs for all medications and total OOP healthcare costs borne by the beneficiary were reported. Complex survey design-specified logistic regression with sampling weights was the main statistical analysis used. Sensitivity analyses on adherence thresholds and subgroups were additionally conducted. Results: A significant positive relationship between total OOP prescription costs and statin adherence was identified across observation years in the primary models. Similar relationships were noted for ACE/ARBs and BB in 2004, and ACE/ARBs in 2005. No significant association between adherence and total OOP healthcare costs was indicated in the primary models. Mortality could not be used as a clinical outcome of interest due to limitations with the data. Thus, acute coronary syndrome (ACS) events were used as the clinical outcome. At the ≥ 80 percent threshold, no significant reductions in ACS events were reported. However, various sensitivity analyses did suggest significant reductions in ACS events with ACE/ARBs. Additionally, significantly higher risk of ACS was noted when BB adherence thresholds were reduced to ≥ 60 percent. Conclusions: OOP prescription costs are a significant factor influencing adherence to these medications used for secondary prevention of CAD/MI in Medicare beneficiaries. Continuing to monitor how these costs impact adherence and ultimately outcomes will be critical, particularly given policy changes such as Medicare Part-D.

C2H2-zinc fingers (ZFs) are commonly found in transcription factors that code for nearly 3% of gene products in the human genome. ZF proteins are commonly involved in gene regulation during development, cell differentiation, and tumor suppression. Each "finger" is a domain composed of approximately 30 amino acids. Since the discovery of these domains over 25 years ago, several groups have contributed to the structural and biochemical knowledge to understand their DNA-binding properties. Taking advantage of the simplicity of manipulating the DNA-binding potential of a ZF, the technology has now evolved to make sequence-specific Zinc Finger Nucleases (ZFNs), Artificial Transcription Factors (ATFs), Zinc Finger Recombinases, and DNA detection tools. ZFPs have been used for various applications, ranging from regulating genes by ZF-ATFs to manipulating genomes in diverse organisms. ZFNs have remarkably revolutionized the field of genome engineering. ZFN-modified T-cells have now advanced into human clinical trials for cell-based therapies as a treatment against HIV. Despite the advances in the ZFN technology, one of the challenges in the field is obtaining effective ZFNs using publicly available tools. The traditional method of synthesizing custom ZF arrays was using modular assembly (MA). In this method, preselected ZFs from publicly available one-finger archives can be assembled modularly to make long arrays. MA of ZFNs provides a rapid method to create proteins that can recognize a broad spectrum of DNA sequences. However, three- and four-finger arrays often fail to produce active nucleases. The low success rate of MA ZF arrays was attributed to the fact that they suffer from finger-finger incompatibility referred to as context-dependent effects. However, we hypothesized that the low affinity of MA arrays was the limiting factor. The work presented in this dissertation describes our efforts at addressing these fundamental methodological challenges. We developed the Extended Modular Assembly method that overcomes the limitations of both the previous Modular Assembly. We performed a systematic investigation of number and composition of modules on ZFN activity and analyzed ZFN specificity both in vitro and in vivo. Our current experiments apply the ZFNs produced by our method to study the role of genetic variation in human disease.

OBJECTIVE:To compare and contrast patients that receive treatment following an osteoporotic fracture to those patients that do not. METHODS:Data were taken from the Medical Expenditures Panel Survey (MEPS). Subjects who reported a wrist, vertebral, or hip fracture were identified. Prescription data were assessed for these subjects and two groups were identified: those who received treatment following a fracture and those who did not. RESULTS:The final sample consisted of n=129 subjects. Of these subjects, only 38% received treatment following an osteoporotic fracture. The only variable showing significant effects on treatment were type of insurance coverage. There was evidence of a relationship for other variables: race, inability to obtain necessary prescription medicines, family income, vertebral fracture and patient's perceived health.CONCLUSIONS: Overall treatment rates following a fracture remain low. Substantial efforts should be made to close the gap between guideline recommendations and clinical practice.

Background: Alzheimer’s disease (AD) is the most common form of dementia and is characterized by a progressive loss of memory, judgment, and thinking in older adults. The current treatment is cholinesterase inhibitors, which increase acetylcholine at the synapse. Medications with anticholinergic (AC) activity are given for a variety reasons including for the treatment of comorbid conditions or side effects of cholinesterase inhibitors (ChEIs). These drugs inhibit acetylcholine in the brain. Studies have shown the detrimental outcomes of using AC medications with ChEIs in older adults. Moreover, older patients take more medications and have an increased risk of developing AC toxicity as these effects are additive. The association between AC burden with functional, cognitive, and behavioral outcomes bears further evaluation. Methods: This study is a retrospective observational study that investigated the effect of AC medications on function, cognition, and behavior. Data was collected from charts on dementia patients who resided at Piedmont Geriatric Hospital. Descriptive statistics and GEE regression were performed using MS Excel 2007 and SPSS 18.0. Results: There were a total of 83 subjects included in this study with a median age of 77 years old and with a median length of stay of 536 days. 33.7% of the patients were taking cognitive-enhancing medications. The analysis found that AC burden was not a significant predictor of functional, cognitive or behavioral decline. Conclusion: The minimal amount of literature on this association, suggests that AC burden may have negative consequences on function, cognition and behavior in dementia patients. The study results provided inconclusive evidence about the association of AC burden on poorer functional, cognitive and behavioral outcomes. Future research in this field is needed to determine if there is a true association between worsening outcomes and AC burden.

ix Objectives: To quantify polypharmacy and assess the socio-economic predictors of medication use and expenditure in diabetics. Methods: This study analyzed adult diabetes patients using a nationally representative sample in Medical Expenditure Panel Survey in 2006. Top ten most highly utilized drug classes were identified. Descriptive statistics were used to portray the patients’ medication utilization and spending. Generalized linear models were conducted to assess the socio-economic variants in drug use and spending. Results: On average, a diabetes patient had 45 prescriptions in 2006, for total annual spending of $3,161. A diabetes patient used drugs from 3.43 classes within top ten drug classes. Races and insurance coverage are associated with drug use and spending, holding other factors constant. Conclusion: Diabetes patients use multiple classes of drugs. Insurance coverage and races are related with drug spending and utilization. More research is needed to evaluate the potential risks of drug-drug interactions due to polypharmacy.

This research explored quantitative relationships (QSPKR) between different molecular descriptors and pertinent, systemic PK properties for 14 calcium channel blockers (CCB). Physicochemical properties (PC) such as molecular weight (MW), molar volume (MV), calculated logP (clogP), pKa, calculated logD7.4 (clogD), % ionized at pH 6.3 and pH 7.4, hydrogen bond donors (HBD), hydrogen bond acceptors (HBA), and number of rotatable bonds (nRot) were chosen as possible predictor variables for systemic PK properties for CCB, obtained from pertinent literature, assessing the PK of CCB after intravenous administration to healthy humans. All PC properties and molecular descriptors were computed using ACD-solubility/DB 12.01. Total body clearance (CLtot), steady-state volume of distribution (Vdss), total area under the plasma concentration-time profile (AUCoo), terminal half-life (t1/2), and fraction of drug excreted unchanged in urine (fe), if available, were obtained or derived from original references, exclusively from IV studies that administered CCB to healthy human volunteers. Several articles focused on drug interactions with grapefruit juice or the impact of renal/hepatic dysfunction, and in such cases, data from the healthy control group were used. Each study was evaluated for study design, PK sampling schedule, bioanalytical and PK analysis methods before inclusion into the final database. The assumption of linear systemic PK was verified by assessing AUCoo versus (IV) dose. Plasma protein binding information was collected from in-vitro experiments to obtain the fraction unbound in plasma (fu). Unbound volume of distribution at a steady state (Vdssu), unbound total (CLtotu), renal (CLrenu), and non-renal clearance (CLnonrenu) were estimated and compared with the relevant physiological references for Vdssu (plasma volume, blood volume, extracellular and intracellular spaces, total body water and body weight) and for the unbound clearances (liver blood flow, renal plasma flow, and glomerular filtration rate, GFR). Final PK property values were obtained by averaging across available studies. The distribution of both PC and PK properties were evaluated, and correlation matrices amongst PC properties were constructed to assess for collinearity. If two PC descriptors were found to be collinear, i.e. r, ≥ 0.8, only one of them was used in the final univariate analysis. Finally, univariate linear regression of all PK variables versus each molecular descriptor was performed; any relationship with p<0.05 and r2≥0.30 was considered to be statistically significant. The PC properties of the final 14 CCB were reasonably normally distributed with few exceptions. Overall, CCBs are small (MW range of 316-496 Da), basic and lipophilic (logD7.4 range of 1.5-5.1) molecules. On the other hand, for the PK properties, the distributions were found to be skewed with high standard deviations. Thus, all PK variables (except fu) were log-transformed. Although CCB are mostly highly plasma protein bound (fu range of 0.2-20%), they are characterized by extensive extravascular tissue distribution (Vdss range of 0.6-20.4 l/kg) and high, mainly metabolic, clearance (CLtot range of 3.7-131.7 ml/min/kg). Clevidipine is the only CCB undergoing extensive, extra-hepatic ester hydrolysis, responsible for the highest CLtot value. Urinary excretion for CCB is negligible. Amlodipine is a PK outlier due to its high Vdss (20.4 l/kg) and low CLtot (6.9 ml/min/kg, due to low hepatic extraction) with fu of 2%. Therefore, the final QSPKR analysis was performed including, as well as excluding amlodipine. Excluding amlodipine, the relationship between fu and logD7.4 was negative and significant (r2 of 0.4, n=12). The relationships between CLtotu, CLnonrenu and CLrenu and logD7.4 were found to be positive and significant (r2 between 0.6-0.7, n=3-12); none of the other PC variables affected any of the clearance terms. Although the relationship between Vdssu and logD7.4 was not significant (r2 of 0.25, n=12), it showed the expected positive slope. In fact, after removing bepridil (the remaining outlier in Vdssu), the relationship with logD7.4 became statistically significant (r2=0.46, n=11). The QSPKR obtained in this study for CCB, with logD7.4 being the main PC determinant for systemic PK properties, were similar to those previously reported for opioids, β-adrenergic receptor ligands and benzodiazepines. However, slope estimates for the relationships of CLnonrenu and CLtotu as a function of logD7.4 for CCB were higher compared to these previously studied compounds, which showed higher sensitivity, most likely as a result of their higher lipophilicity. Overall, lipophilicity measured as logD7.4 was found to be a statistically significant and plausible PC determinant for the biologically relevant systemic PK properties for CCB and other classes of drugs.

Heparin is a highly sulfated glycosaminoglycan with potent anticoagulant, antimetastatic, and anti-inflammatory effects. Polymeric and polyanionic nature of heparin makes dosing and side effects a nightmare for healthcare professionals. Our laboratory has proposed appropriately designed, small, highly sulfated aromatic molecules as potential mimetics of heparin. These easier-to-synthesize small molecules have been shown to possess interesting pharmacological and improved toxicological profiles. However, the detection and characterization of these highly sulfated molecules is challenging. A robust RP-IP UPLC-MS method was developed to successfully retain, resolve and quantify sulfated non-saccharide GAG mimetics without the requirement of pre- or post- column derivatization. Comparative analysis reveals intricate dependence of resolution and ionization on the structure of ion-pairing agents. This is the first report showing systematic use of MS cone voltage to fingerprint sulfated GAG mimetics, perhaps eliminating the need for tandem MS techniques.

Background. Allometric scaling (AS) is widely used in predicting human clearance (CL) based on animal data. Substantial prediction errors have been commonly observed and various modifications to AS have not provided a broad reliable improvement. In this study, an extensive data set was assembled including animal and human systemic CL and physiochemical properties. The allometric exponents were calculated based on multiple species AS and single-species AS methods. The correlations between the allometic exponents and physiochemical properties were evaluated in an attempt to find covariates that may explain the inter-compound variability in the allometric exponents. Lastly, the statistical approaches in analyzing the allometric function were evaluated with the collected data. Methods. 1- A nonlinear mixed effect modeling (MEM) approach was performed to investigate the central tendency and distribution of AS exponents as well as to identify whether there are any correlations between the allometric exponent, and coefficient, with the physicochemical and drug metabolism and pharmacokinetics (DMPK) properties of the compounds. 2- Single-species AS was performed to estimate the single-species AS exponent distributions and their corresponding central tendencies. The correlation between the estimated single-species AS exponents and the physicochemical and DMPK properties of the compounds were also examined. 3- The methodologies of log-log transformation followed by linear regression (LL-LR) and direct nonlinear regression methods (NLS) with different weighting schemes on the AS power function were investigated. The central tendency and distribution of the allometric exponents were evaluated and compared across methods. Furthermore, the human CL prediction performance was evaluated among methods. Results. The estimated central tendency and distribution of AS exponents from the nonlinear MEM as well as the single-species AS approaches were consistent with literature reports. There were no significant correlations identified between the estimated AS exponents and the physicochemical or DMPK properties. The methods of LL-LR and the NLS with 1/w2 weighting (variance weighted by CL2 during the variance minimization process) results in the most similar allometric exponent with central tendency around 0.668 and provided the best human CL prediction among methods investigated. Conclusion. The knowledge gained in this work by extensive modeling and simulations contributed to a better understanding of the variability in AS exponents and better practice in performing AS in human CL prediction

Lopinavir (LPV) is a potent protease inhibitor specific for HIV-1. However, LPV has poor placental penetration due to substrate activity for efflux transporter by P-glycoprotein (P-gp). Since fatty acid transporters are highly expressed in the placenta during pregnancy, we designed fatty acid ester prodrug of lopinavir as substrates of fatty acid transporter in order to improve their uptake into placenta. Seven dicarboxylic acid esters of lopinavir have been made in our lab. The structures were characterized by 1H-NMR, 13C-NMR, LC-MS/MS, HRMS, IR and melting points. After making the prodrugs, an LC-MS/MS method with high specificity and sensitivity, as well as simultaneous quantitative analyses of lopinavir and SLPV, GLPV and DLPV in the BeWo cells methanol extraction was established and validated. The uptake of prodrugs (SLPV, GLPV and DLPV) in the BeWo cells was then determined. GLPV has the highest uptake followed by SLPV and then DLPV. The results suggest that the carbon length of the promoiety may have a positive relationship with the uptake. Ideal prodrugs should be stable before they reach placenta and can be hydrolyzed in the placenta and/or in fetal plasma. We did a series of stability and hydrolysis studies in human tissue fractions. The results showed that GLPV and SLPV were very stable in HIC, HLC and human adult plasma. DLPV was stable in HIC, HLC, but can be hydrolyzed in human adult plasma. GLPV and SLPV cannot be hydrolyzed in either human placenta or fetal plasma, while DLPV can be hydrolyzed in both human placenta and fetal plasma. Anti-HIV activities study of prodrugs was also conducted. The results showed that the EC50 of three prodrugs (GLPV, SLPV and DLPV) are 0.86 μM, 0.84 μM and 0.05 μM, which are much lower than 50 μM (The active drug criteria for this assay). It suggests that prodrugs have apparently anti-HIV activity. DLPV has comparable apparent anti-HIV activity to LPV (<0.02 μM). After incubation with CEM-SS cells for 6 days, almost half of DLPV was hydrolyzed into LPV. Therefore, the high anti-HIV potent of DLPV may be due to the anti-HIV activity of generated LPV.

Background: Despite the numerous pharmaceutical applications of alcohols andglycols, the interspecies differences with respect to their pharmacokinetics (PK) arepoorly understood. The aim of this research was to use in-vivo and in-vitro approaches to compare and model the PK characteristics across various species.Methods: Appropriate published in-vivo studies (in different species) foralcohols and glycols were carefully selected. PK analysis was performed using (a) noncompartmental analysis and (b) compartmental modeling to estimate relevant dose-independent PK parameters. Next, six alcohols (methanol, ethanol, 1 -propanol, 1 -butanol, 1-hexanol, and 1-octanol), two glycols (ethylene glycol and propylene glycol)and one secondary alcohol (2-propanol) were examined as in-vitro substrates for equine ADH using a UV spectrophotometric assay to evaluate the effect of molecular structure. Furthermore, in-vitro metabolism of ethanol and propylene glycol was also characterized in hepatic cytosolic fractions from rat, rabbit, dog and human and in-vitro in-vivo correlation for the hepatic disposition parameters was assessed. Finally, allometric scaling relationships for ethanol and propylene glycol PK parameters (in-vivo and in-vitro) were developed and validated.Results: Alcohols and glycols exhibited nonlinear PK due to saturable hepaticmetabolism in all species. The reported in-vivo data were well described by oneltwo compartment PK models with parallel saturable metabolism and first-order renalexcretion. In-vitro equine ADH experiments revealed differences in affinity andturnover between the substrates: Enzyme affinity (1/Km) and in-vitro intrinsic clearance (CLintin-vitro) correlated positively with logP values; glycols showed lower CLintin-vitro values than straight-chain alcohols. In-vitro hepatic cytosol studies yielded acceptable in-vivo predictions for the metabolic clearance (CLmet) of ethanol and propylene glycol in the rat, dog and human, but not the rabbit. Vdss, Vmax, CLintin-vitro, CLmet scaled allometrically across species with similar powers for both ethanol and propylene glycol, and good agreement between in-vivo and in-vitro scaling was noted. The allometric scaling models gave excellent predictions when externally validated against in-vivo concentration-time data. Conclusions: The present research demonstrates .the successful application of amodeling-based approach to elucidate interspecies relationships for alcohols andglycols, compounds which exhibit nonlinear PK and mainly low hepatic extractionbehavior. The in-vitro experimental systems have been used successfully forcharacterizing alcohol/glycol metabolism and predicting in-vivo disposition.