Sex, drugs, and STIs: Syphilis infection and hepatitis B vaccine compliance among illicit drug users in Houston

Although the association between syphilis infection status and compliance with the hepatitis B virus vaccine has been the focus of investigation, there is a lack of data regarding the association between syphilis infection and HBV vaccine compliance. The author investigated the association between the exposure of syphilis infection and the outcome of HBV vaccine completion, defined as degree of constancy and accuracy with which a patient follows a prescribed regimen. A cohort design was employed using interview and serological data from the Drugs, AIDS, STDs, Hepatitis (DASH) Research Project; analysis was restricted to HIV and HBV seronegative (at baseline), illicit drug users residing in Harris County. Syphilis negative and syphilis positive infection status was determined from the serological data while covariates and outcome information were determined from the DASH Project Questionnaire; enrolled subjects (n=1160) were selected from the data. Association between exposure and outcome was assessed with logistic regression adjusted for data-based confounders. ^ A prevalence of 7% and 71% was found for syphilis and HBV vaccine compliance, respectively. When measuring the actual association between syphilis infection status and HBV vaccine compliance, an odds ratio of 1.49 (95% CI: 0.86, 2.72) was obtained. There was a non-significant association between these two variables. 78% of the study population was syphilis positive and completed the vaccine series compared to 70% of the population that was syphilis negative and received all three doses. This finding confirms that there is a difference between syphilis positive and negative drug users with respect to HBV vaccine compliance. The fact that differences were found in these drug users with respect to vaccine schedule supports the idea that sub-group differences may exist and thus merits further investigation. If these differences are confirmed, it is recommended that STI interventions identify community characteristics of their samples and target populations based on practices specific to that community. ^

Association between ethnicity and treatment status in opioid dependence therapy using methadone and suboxone drugs

Opioids are drugs with opium-like qualities that are either derived from opiates (drugs created from opium, such as morphine or codeine) or chemically produced. In the U.S. opiate abuse and related deaths have been increasing and traditional maintenance treatment has been Methadone with variable success. However, since 2003 synthetic Buprenorphine has been used since it is prescribed daily by physicians in pill form and should improve outcomes. Comparative studies are limited and the effect of ethnicity on treatment outcome is unknown. ^ Data collected at one clinic from December 2005 through May 2009 were used to compare the association between ethnicity and other socioeconomic variables with treatment status, and to identify factors associated with the dropout among participants. Descriptive tables and multiple logistic regression models were used to examine the data on 1,295 total participants. Of the total, 875 participants (68%) were from the Methadone subsample and 420 participants (32%) from the Buprenorphine subsample; only about 15% stayed in treatment. ^ This study showed that with either Methadone or Buprenorphine maintenance therapy, only about 15% participants stay active over 3.5 years. Methadone treated patients that stayed active in treatment were associated with Caucasian ethnicity and were more likely to be employed. With Buprenorphine maintenance treatment only age over 40 years was associated with continuing activity in the program. Further studies that examine the reasons for the high dropout status and the implication of the socioeconomic and ethnic associations found in this data may help to improve treatment outcomes.^

Exploring the use of pharmaceutical patient assistance programs as an option to improve access to and affordability of prescription drugs

Background. Pharmaceutical-sponsored patient assistance programs (PAPs) are charity programs that provide free or reduced-priced medications to eligible patients. PAPs have the potential to improve prescription drug accessibility for patients but currently there is limited information about their use and effectiveness. ^ Objectives and methods. This dissertation described the use of PAPs in the U.S. through the conduct of two studies: (1) a systematic review of primary studies of PAPs from commercially-published and “grey” literature sources; and (2) a retrospective, cross-sectional study of cancer patients' use of PAPs at a tertiary care cancer outpatient center. ^ Results. (1) The systematic review identified 33 studies: 15 evaluated the impact of PAP enrollment assistance programs on patient healthcare outcomes; 7 assessed institutional costs of providing enrollment assistance; 7 surveyed stakeholders; 4 examined other aspects. Standardized mean differences calculated for disease indicator outcomes (most of which were single group, pre-posttest designs) showed significant decreases in glycemic and lipid control, and inconsistent results for blood pressure. Grey literature abstracts reported insufficient statistics for calculations. Study heterogeneity made weighted summary estimates inappropriate. Economic analyses indicated positive financial benefits to institutions providing enrollment assistance (cost) compared to the wholesale value of the medications provided (benefit); analyses did not value health outcomes. Mean quality of reporting scores were higher for observational studies in commercially-published articles versus full text, grey literature reports. (2) The cross-sectional study found that PAP outpatients were significantly more likely to be uninsured, indigent, and < 65 years old than non-PAP patients. Nearly all non-PAP and PAP prescriptions were for non-cancer conditions, either for co-morbidities (e.g., hypertension) or the management of treatment side effects (e.g., pain). Oral chemotherapies from PAPs were significantly more likely to be for breast versus other cancers, and be a newer, targeted versus traditional chemotherapy.^ Conclusions. In outpatient settings, PAP enrollment assistance plus additional medication services (e.g., counseling, reminders, and free samples) is associated with improved disease indicators for patients. Healthcare institutions, including cancer centers, can offset financial losses from uncompensated drug costs and recoup costs invested in enrollment assistance programs by procuring free PAP medications. Cancer patients who are indigent and uninsured may be able to access more outpatient medications for their supportive care needs through PAPs, than for cancer treatment options like oral chemotherapies. Because of the selective availability of drugs through PAPs, there may be more options for newer, oral, targeted chemotherapies for the treatment breast cancer versus other for other cancers.^

INACTIVATION OF MUTAGENIC DRUGS AND THEIR METABOLITES IN THE URINE OF PATIENTS ADMINISTERED ANTINEOPLASTIC THERAPY

Urines from patients administered mutagenic antineoplastic drugs were significantly mutagenic in the Ames assay, and hence may pose a genotoxic hazard to hospital personnel or family members caring for the patient. The urines were tested for mutagenicity in several different strains of Salmonella typhimurium that were uvr positive or negative (TA98, TA100, TA102, UTH8413, UTH8414). The urines were fractionated by high pressure liquid chromatography (HPLC) and the fractions assayed for mutagenicity in the strains in which the whole urine was mutagenic. Only fractions of urines containing the parent compound (cisplatin, doxorubicin, or mitomycin) were mutagenic; no other fraction showed significant mutagenicity. However, urine containing cyclophosphamide had two fractions that were mutagenic. One fraction, the fraction containing cyclophosphamide, required metabolic activation for mutagenicity. The other fraction did not require activation for mutagenicity.^ The chemical and mutagenic stability of these urines at room temperature was assayed over a 14 day period. The parent compound degraded within the first seven days, but the urines remained mutagenic. Cis-platinum was chemically stable in the urine; however, the urine decreased in mutagenicity. The decrease was probably the result of stable ligands binding to the platinum.^ Inactivation methods were developed to reduce the genotoxic hazard. Urine containing cisplatin was inactivated by complexing the cisplatin with diethyldithiocarbamate (DDTC). Oxidation with NaOCl of urines containing mitomycin and doxorubicin (sodium thiosulfate must be added to the doxorubicin urine) results in mutagenic inactivation. Inactivation of urine containing cyclophosphamide requires oxidation with alkaline potassium permaganate and trapping of active degradation products with sodium thiosulfate. Urines containing these drugs can be inactivated, but not always by the same method that inactivates the drug alone in solution. Therefore, in the future development of inactivation methods, both chemical and mutagenic assays are necessary to determine effectiveness. Methods of inactivation of mutagenic excreta developed in this study are both effective and practical. ^

EXPOSURE OF PHARMACY PERSONNEL TO ANTINEOPLASTIC DRUGS

This dissertation consists of two parts: (1) Exposure of pharmacy personnel to antineoplastic drugs. The Salmonella reversion test was used to measure the mutagenic activities of urine concentrates from individuals preparing antineoplastic drugs for intravenous administration. Longitudinal studies were performed in which the total urine produced in 24-hour periods was collected, starting on a Sunday at 7 P.M. after a duty-free weekend and extending over an eight-day period. There was no detectable increase in mutagenic activity in the urine concentrates of three pharmacy administrators who had no contact with these drugs. All six individuals admixing drugs in open-faced, horizontal laminar flow hoods displayed a two-fold increase in mutagenesis by the fourth day with peak values of 2.7 to 24-fold occurring on days five and six, reduced values by day seven with a return to the spontaneous level by day eight. When four of the six positive individuals in the preceding experiment admixed comparable amounts of antineoplastic drugs in a closed-faced, vertical laminar flow hood, no increase in mutagenic activity was detected in their urine concentrates over the eight-day period. (2) Estimate of potential carcinogenic risks of antineoplastic drugs. Excision repair is the major repair system that is involved with the elimination of chemically induced DNA (deoxyribonucleic acid) lesions. This DNA excision repair capability increases in mammalian species with longer life span such as humans. In this study, the effect of functional DNA excision repair on the mutagenesis invoked by 17 antineoplastic drugs was determined by using a Salmonella/Microsome assay which was expanded to include some uvr('+) counterparts of the excisionless (uvrB) tester strains routinely employed. Although extrapolation cannot be made from bacteria to humans, one should be able to make a qualitative comparison as to which antineoplastic drugs are more potentially carcinogenic to humans based on the effects of excision repair on their mutagenesis in bacteria. The tested antineoplastic drugs were divided into three classes: those requiring excision repair for mutagenesis; those producing nonrepairable genetic damage; and those producing mostly repairable premutational DNA lesions. ^

Exposure to nonsteroidal anti-inflammatory drugs and weight loss and hospitalization in non-small cell lung cancer patients

Background. Cancer cachexia is a common syndrome complex in cancer, occurring in nearly 80% of patients with advanced cancer and responsible for at least 20% of all cancer deaths. Cachexia is due to increased resting energy expenditure, increased production of inflammatory mediators, and changes in lipid and protein metabolism. Non-steroidal anti-inflammatory drugs (NSAIDs), by virtue of their anti-inflammatory properties, are possibly protective against cancer-related cachexia. Since cachexia is also associated with increased hospitalizations, this outcome may also show improvement with NSAID exposure. ^ Design. In this retrospective study, computerized records from 700 non-small cell lung cancer patients (NSCLC) were reviewed, and 487 (69.57%) were included in the final analyses. Exclusion criteria were severe chronic obstructive pulmonary disease, significant peripheral edema, class III or IV congestive heart failure, liver failure, other reasons for weight loss, or use of research or anabolic medications. Information on medication history, body weight and hospitalizations was collected from one year pre-diagnosis until three years post-diagnosis. Exposure to NSAIDs was defined if a patient had a history of being treated with NSAIDs for at least 50% of any given year in the observation period. We used t-test and chi-square tests for statistical analyses. ^ Results. Neither the proportion of patients with cachexia (p=0.27) nor the number of hospitalizations (p=0.74) differed among those with a history of NSAID use (n=92) and those without (n=395). ^ Conclusions. In this study, NSAID exposure was not significantly associated with weight loss or hospital admissions in patients with NSCLC. Further studies may be needed to confirm these observations.^

OXIDATIVE STRESS BASED STRATEGIES FOR ENHANCING THE EFFICACY OF HISTONE DEACETYLASE INHIBITORS (HDACi)

Histone deacetylase inhibitors (HDACi) are anti-cancer drugs that primarily act upon acetylation of histones, however they also increase levels of intracellular reactive oxygen species (ROS). We hypothesized that agents that cause oxidative stress might enhance the efficacy of HDACi. To test this hypothesis, we treated acute lymphocytic leukemia cells (ALL) with HDACi and adaphostin (ROS generating agent). The combination of two different HDACi (vorinostat or entinostat) with adaphostin synergistically induced apoptosis in ALL. This synergistic effect was blocked when cells were pre-treated with the caspase-9 inhibitor, LEHD. In addition, we showed that loss of the mitochondrial membrane potential is the earliest event observed starting at 12 h. Following this event, we observed increased levels of superoxide at 16 h, and ultimately caspase-3 activation. Pre-treatment with the antioxidant N-acetylcysteine (NAC) blocked ROS generation and reversed the loss of mitochondrial membrane potential for both combinations. Interestingly, DNA fragmentation and caspase-3 activity was only blocked by NAC in cells treated with vorinostat-adaphostin; but not with entinostat-adaphostin. These results suggest that different redox mechanisms are involved in the induction of ROS-mediated apoptosis. To further understand these events, we studied the role of the antioxidants glutathione (GSH) and thioredoxin (Trx). We found that the combination of entinostat-adaphostin induced acetylation of the antioxidant thioredoxin (Trx) and decreased intracellular levels of GSH. However, no effect on Trx activity was observed in either combination. In addition, pre-treatment with GSH ethyl ester, a soluble form of GSH, did not block DNA fragmentation. Together these results suggested that GSH and Trx are not major players in the induction of oxidative stress. Array data examining the expression of genes involved in oxidative stress demonstrated a differential regulation between cells treated with vorinostat-adaphostin and entinostat-adaphostin. Some of the genes differentially expressed between the combinations include aldehyde oxidase 1, glutathione peroxidase-5, -6, peroxiredoxin 6 and myeloperoxidase. Taken together, these experimental results indicate that the synergistic activity of two different HDACi with adaphostin is mediated by distinct redox mechanisms in ALL cells. Understanding the mechanism involved in these combinations will advance scientific knowledge of how the action of HDACi could be augmented in leukemia models. Moreover, this information could be used for the development of effective clinical trials combining HDACi with other anticancer agents.

NMR structural analysis of cardiac troponin C: Monitoring conformational changes induced by binding calcium, troponin I, and calcium -sensitizing drugs

Contraction of cardiac muscle is regulated through the Ca2+ dependent protein-protein interactions of the troponin complex (Tn). The critical role cardiac troponin C (cTnC) plays as the Ca2+ receptor in this complex makes it an attractive target for positive inotropic compounds. In this study, the ten Met methyl groups in cTnC, [98% 13C ϵ]-Met cTnC, are used as structural markers to monitor conformational changes in cTnC and identify sites of interaction between cTnC and cardiac troponin I (cTnI) responsible for the Ca2+ dependent interactions. In addition the structural consequences that a number of Ca2+-sensitizing compounds have on free cTnC and the cTnC·cTnI complex were characterized. Using heteronuclear NMR experiments and monitoring chemical shift changes in the ten Met methyl 1H-13C correlations in 3Ca2+ cTnC when bound to cTnI revealed an anti-parallel arrangement for the two proteins such that the N-domain of cTnI interacts with the C-domain of cTnC. The large chemical shifts in Mets-81, -120, and -157 identified points of contact between the proteins that include the C-domain hydrophobic surface in cTnC and the A, B, and D helical interface located in the regulatory N-domain of cTnC. TnI association [cTnI(33–80), cTnI(86–211), or cTnI(33–211)] was found also to dramatically reduce flexibility in the D/E central linker of cTnC as monitored by line broadening in the Met 1H- 13C correlations of cTnC induced by a nitroxide spin label, MTSSL, covalently attached to cTnC at Cys 84. TnI association resulted in an extended cTnC that is unlike the compact structure observed for free cTnC. The Met 1H-13C correlations also allowed the binding characteristics of bepridil, TFP, levosimendan, and EMD 57033 to the apo, 2Ca2+, and Ca2+ saturated forms of cTnC to be determined. In addition, the location of drug binding on the 3Ca2+cTnC·cTnI complex was identified for bepridil and TFP. Use of a novel spin-labeled phenothiazine, and detection of isotope filtered NOEs, allowed identification of drug binding sites in the shallow hydrophobic cup in the C-terminal domain, and on two hydrophobic surfaces on N-regulatory domain in free 3Ca2+ cTnC. In contrast, only one N-domain drug binding site exists in 3Ca2+ cTnC·cTnI complex. The methyl groups of Met 45, 60 and 80, which are grouped in a hydrophobic patch near site II in cTnC, showed the greatest change upon titration with bepridil or TFP, suggesting that this is a critical site of drug binding in both free cTnC and when associated with cTnI. The strongest NOEs were seen for Met-60 and -80, which are located on helices C and D, respectively, of Ca2+ binding site II. These results support the conclusion that the small hydrophobic patch which includes Met-45, -60, and -80 constitutes a drug binding site, and that binding drugs to this site will lead to an increase in Ca2+ binding affinity of site II while preserving maximal cTnC activity. Thus, the subregion in cTnC makes a likely target against which to design new and selective Ca2+-sensitizing compounds. ^

A role for p53 in sensing DNA damage and triggering apoptotic responses to anticancer agents

The p53 tumor suppressor protein plays a major role in cellular responses to anticancer agents that target DNA. DNA damage triggers the accumulation of p53, resulting in the transactivation of genes, which induce cell cycle arrest to allow for repair of the damaged DNA, or signal apoptosis. The exact role that p53 plays in sensing DNA damage and the functional consequences remain to be investigated. The main goal of this project was to determine if p53 is directly involved in sensing DNA damage induced by anticancer agents and in mediating down-stream cellular responses. This was tested in two experimental models of DNA damage: (1) DNA strand termination caused by anticancer nucleoside analogs and (2) oxidative DNA damage induced by reactive oxygen species (ROS). Mobility shift assays demonstrated that p53 and DNA-PK/Ku form a complex that binds DNA containing the anticancer nucleoside analog gemcitabine monophosphate in vitro. Binding of the p53-DNA-PK/Ku complex to the analog-containing DNA inhibited DNA strand elongation. Furthermore, treatment of cells with gemcitabine resulted in the induction of apoptosis, which was associated with the accumulation of p53 protein, its phosphorylation, and nuclear localization, suggesting the activation of p53 to trigger apoptosis following gemcitabine induced DNA strand termination. The role of p53 as a DNA damage sensor was further demonstrated in response to oxidative DNA damage. Protein pull-down assays demonstrated that p53 complexes with OGG1 and APE, and binds DNA containing the oxidized DNA base 8-oxoG. Importantly, p53 enhances the activities of APE and OGG1 in excising the 8-oxoG residue as shown by functional assays in vitro. This correlated with the more rapid removal of 8-oxoG from DNA in intact cells with wild-type p53 exposed to exogenous ROS stress. Interestingly, persistent exposure to ROS resulted in the accelerated onset of apoptosis in cells with wild-type p53 when compared to isogenic cells lacking p53. Apoptosis in p53+/+ cells was associated with accumulation and phosphorylation of p53 and its nuclear localization. Taken together, these results indicate that p53 plays a key role in sensing DNA damage induced by anticancer nucleoside analogs and ROS, and in triggering down-stream apoptotic responses. This study provides new mechanistic insights into the functions of p53 in cellular responses to anticancer agents. ^