Opioid dependence treatment: options in pharmacotherapy.

The development of effective treatments for opioid dependence is of great importance given the devastating consequences of the disease. Pharmacotherapies for opioid addiction include opioid agonists, partial agonists, opioid antagonists, and alpha-2-adrenergic agonists, which are targeted toward either detoxification or long-term agonist maintenance. Agonist maintenance therapy is currently the recommended treatment for opioid dependence due to its superior outcomes relative to detoxification. Detoxification protocols have limited long-term efficacy, and patient discomfort remains a significant therapy challenge. Buprenorphine's effectiveness relative to methadone remains a controversy and may be most appropriate for patients in need of low doses of agonist treatment. Buprenorphine appears superior to alpha-2 agonists, however, and office-based treatment with buprenorphine in the USA is gaining support. Studies of sustained-release formulations of naltrexone suggest improved effectiveness for retention and sustained abstinence; however, randomized clinical trials are needed.

Using Acceptance and Commitment Therapy during Methadone Dose Reduction: Rationale, Treatment Description, and a Case Report.

Many clients who undergo methadone maintenance (MM) treatment for heroin and other opiate dependence prefer abstinence from methadone. Attempts at methadone detoxification are often unsuccessful, however, due to distressing physical as well as psychological symptoms. Outcomes from a MM client who voluntarily participated in an Acceptance and Commitment Therapy (ACT) - based methadone detoxification program are presented. The program consisted of a 1-month stabilization and 5-month gradual methadone dose reduction period, combined with weekly individual ACT sessions. Urine samples were collected twice weekly to assess for use of illicit drugs. The participant successfully completed the program and had favorable drug use outcomes during the course of treatment, and at the one-month and one-year follow-ups. Innovative behavior therapies, such as ACT, that focus on acceptance of the inevitable distress associated with opiate withdrawal may improve methadone detoxification outcomes.

Studies of the mechanism of daunorubicin carcinogenesis: Modulation by vitamin E

Daunorubicin (DNR) is an anthracycline antibiotic used as a cancer chemotherapeutic agent. However, it causes mammary adenocarcinomas in female Sprague-Dawley (SD) rats. Vitamin E (E) has been found to reduce DNR carcinogenicity. I investigated the mechanism of DNR carcinogenicity and its interaction with E in SD rats by studying DNR-DNA adduct formation and the influence of E status on DNR clearance and free radical producing and detoxifying enzymes.^ The hypothesis was that DNR exerts its tumorigenic effect via free radicals generated during redox cycling and production of reactive intermediates capable of forming DNA adducts. E was postulated to act as a protective agent through a combination of its antioxidant property, modulation of drug clearance and levels of free radical producing and detoxifying enzymes.^ DNA adduct formation was measured by the nuclease P1 $\sp{32}$P-post labeling assay. In vitro, DNR was activated by rat liver microsomes and either NADPH or cumene hydrogen peroxide (CuOOH). Rat liver DNA incubated with this mixture formed two adducts when the cofactor was NADPH and three adducts when CuOOH was used. In vivo, SD rats were treated with i.v. doses of DNR. No detectable DNR-DNA adducts were formed in liver or mammary DNA in vivo, although there was an intensification of endogenous DNA adducts.^ Groups, 1, 2, 3 and 4 of weanling female SD rats were fed 0, 100, 1,000 and 10,000 mg $\alpha$-tocopheryl acetate/kg diet respectively. A comparison of Groups 1 and 4 showed no effect of E status on clearance of 10 mg tritiated DNR/kg body weight over 72 hours. However, liver cleared DNR at a faster rate than mammary epithelial cells (MEC).^ Xanthine oxidase, which catalyzes DNR redox cycling, was significantly decreased in liver and MEC of rats in group 4 compared to groups 1, 2, and 3. Detoxifying enzymes were not dramatically affected by E supplementation. Quinone reductase in MEC was significantly increased in group 4 compared to other groups. Overall, the liver had higher levels of free radical detoxifying enzymes compared to MEC.^ These data support a role of free radicals in DNR carcinogenicity because (1) endogenous DNA adducts formed due to free radical insult are further intensified by DNR treatment in vivo, (2) MEC, the specific target of DNR carcinogenicity, cannot rapidly clear DNR and have a lower free radical detoxifying capability than liver, (3) E supplementation caused lowering of free radical generating potential via xanthine oxidase, and increased DNR detoxification due to elevation of quinone reductase in MEC. ^

Genetic mechanisms of Na+, K+-ATPase regulation in hypertension

The spontaneously hypertensive rat (SHR) is a model of essential hypertension. During the early development of hypertension, the SHR demonstrates increased proximal tubule (PT) Na+ reabsorption. I hypothesized that the increased PT Na+ reabsorption exhibited by the young SHR was due to altered sub-cellular distribution of Na+, K +-ATPase compared to the normotensive Wistar Kyoto (WKY). The hypothesis is supported, herein, by observations of greater Na+, K +-ATPase α 1 abundance in PT plasma membrane and lower abundance in late endosomes of 4wk SHR despite no difference in total PT α 1 abundance. There is a greater amount of Ser-18 unphosphorylated α 1 in the 4wk SHR PT. Total PT Na+, K+-ATPase γ abundance is greater in SHR at 4wk and 16wk but γ abundance in plasma membrane is greater only at 4wk. The phosphatase, calcineurin, was chosen for study because it is involved in the stimulation of Na+, K +-ATPase. No difference in calcineurin coding sequence, expression, or activity was observed in SHR. Gene expression arrays were next used to find candidate genes involved in the regulation of Na+, K +-ATPase. The first candidate analyzed was soluble epoxide hydrolase (sEH). The gene encoding sEH (EPHX2) showed lower expression in SHR. There was also a reduction in sEH protein abundance but there was no correlation between protein abundance and blood pressure in F2 progeny. Two EPHX2 alleles were identified, an ancestral allele and a variant allele containing four polymorphisms. sEH activity was greater in animals carrying the variant allele but the inheritance of the variant allele did not correlate with blood pressure. Gene expression arrays also led to the examination of genes involved in redox balance/Na+, K+-ATPase regulation. A pattern of lower expression of genes involved in reactive radical detoxification in SHR was discerned. Six transcription factor binding sites were identified that occurred more often in these genes. Three transcription factors that bind to the HNF1 site were expressed at lower levels in SHR. This points to the HNF1 transcriptional complex as an important trans-acting regulator of a wide range of genes involved in altered redox balance in SHR. ^

Hepatitis C and human immunodeficiency virus infections in injecting drug users in drug treatment centers in Vietnam

Background. Injecting drug users (IDUs) are at risk of infection with Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV). Independently, each of these viruses is a serious threat to health, with HIV ravaging the body’s immune system, and HCV causing cirrhosis, liver cancer and liver failure. Co-infection with HIV/HCV weakens the response to antiretroviral therapy in HIV patients. IDUs with HIV/HCV co-infection are at a 20 times higher risk of having liver-related morbidity and mortality than IDUs with HIV alone. In Vietnam, studies to ascertain the prevalence of HIV have found high rates, but little is known about their HCV status. ^ Purpose. To measure the prevalence of HCV and HIV infection and identify factors associated with these viruses among IDUs at drug treatment centers in northern Vietnam. ^ Methods. A cross-sectional study was conducted from November 2007 to February 2008 with 455 injecting drug users aged 18 to 39 years, admitted no more than two months earlier to one of four treatment centers in Northern Vietnam (Hatay Province) (response rate=95%). Participants, all of whom had completed detoxification and provided informed consent, completed a risk assessment questionnaire and had their blood drawn to test for the presence of antibody-HCV and antibody-HIV with enzyme immuno assays. Univariate and multivariable logistic regression models were utilized to explore the strength of association using HIV, HCV infections and HIV/HCV co-infection as outcomes and demographic characteristics, drug use and sexual behaviors as factors associated with these outcomes. Unadjusted and adjusted odds ratios and 95% confidence intervals were calculated. ^ Results. Among all IDU study participants, the prevalence of HCV alone was 76.9%, HIV alone was 19.8%. The prevalence of HIV/HCV co-infection was 92.2% of HIV-positive and 23.7% of HCV-positive respondents. No sexual risk behaviors for lifetime, six months or 30 days prior to admission were significantly associated with HCV or HIV infection among these IDUs. Only duration of injection drug use was independently associated with HCV and HIV infection, respectively. Longer duration was associated with higher prevalence. Nevertheless, while HCV infection among IDUs who reported being in their first year of injecting drugs were lower than longer time injectors, their rates were still substantial, 67.5%. ^ Compared with either HCV mono-infection or HIV/HCV non-infection, HIV/HCV co-infection was associated with the length of drug injection history but was not associated with sexual behaviors. Higher education was associated with a lower prevalence of HIV/HCV co-infection. When compared with HIV/HCV non-infection, current marriage was associated with a lower prevalence of HIV/HCV co-infection. ^ Conclusions. HCV was prevalent among IDUs from 18 to 39 years old at four drug treatment centers in northern Vietnam. Co-infection with HCV was predominant among HIV-positive IDUs. HCV and HIV co-infection were closely associated with the length of injection drug history. Further research regarding HCV/HIV co-infection should include non-injecting drug users to assess the magnitude of sexual risk behaviors on HIV and HCV infection. (At these treatment centers non-IDUs constituted 10-20% of the population.) High prevalence of HCV prevalence among IDUs, especially among HIV-infected IDUs, suggests that drug treatment centers serving IDUs should include not only HIV prevention education but they should also include the prevention of viral hepatitis. In addition, IDUs who are HIV-positive need to be tested for HCV to receive the best course of therapy and achieve the best response to HIV treatment. These data also suggest that because many IDUs get infected with HCV in the first year of their injection drug career, and because they also engaged in high risk sexual behaviors, outreach programs should focus on harm reduction, safer drug use and sexual practices to prevent infection among drug users who have not yet begun injecting drugs and to prevent further spread of HCV, HIV and co-infection. ^

Mutagenicity study of the urinary metabolites of 2-aminonaphthalene

The mutagenicity study of the urinary metabolites of 2-aminonaphthalene was conducted to determine whether differences in metabolism between different acetylator phenotypes could account for a proposed mechanism of bladder carcinogenesis. This required the use of fast and slow acetylator rabbits with phenotypic similarities to humans. In the absence of available slow acetylators, it was necessary to inhibit fast acetylators. The proposed mechanism was that slow acetylators were at greater potential risk of bladder carcinogenesis due to low rates of acetylation, a detoxification mechanism for certain aromatic amines. The alternate metabolic pathway will be hydroxylation. The fast acetylators were proposed to exhibit lower risk of bladder carcinogenicity as a result of higher acetylation rates and less mutagenic metabolites.^ This hypothesis was approached by determining from in vitro mutagenicity assays with Salmonella typhimurium strains TA98 and TA100 whether different metabolites were mutagenic. The acetylation rate of each rabbit and a suitable method of acetylation inhibition were determined through oral exposure to dapsone and the acetylation inhibitor, K-p-aminosalicylic acid. Residues of dapsone and its acetylated metabolite were extracted from blood samples and analyzed by ultra-violet spectrometry using standard curves for each metabolite. The urine samples were concentrated on XAD-2 resin and analyzed both as whole urine concentrates and as isolated metabolites from spots on high performance thin layer chromatography plates. The major isolated spots were identified and quantified through extraction and analysis by high performance liquid chromatography when possible.^ Acetylation rate determination and inhibition were successfully demonstrated in rabbits. Significant mutagenicity was noted for several critical metabolites. None of the mutagenic metabolites were detected in higher concentration in the inhibited acetylators and thus, no clear relationship of metabolite concentration to bladder carcinogenesis was evident for the compounds analyzed. There was some evidence that the inhibitor may have affected critical enzyme systems other than acetylation alone. This would account for the lower concentrations of mutagenic hydroxylated compounds observed. ^

SYSTEMIC TOXICITY IN RODENTS FOLLOWING ACUTE AND SUBCHRONIC INHALATION OF FORMALDEHYDE (FLOW CYTOMETRY, CELL CYCLE KINETICS, ALKALINE ELUTION)

Systemic toxicity was evaluated in Sprague-Dawley (SD) rats and A-strain mice exposed to HCHO inhalation at 0, 0.5, 3, or 15 ppm for six hours/day, five days/week for up to 24 weeks. Toxicity was measured by flow cytometry to detect changes in cell cycle RNA and DNA content and by alkaline elution to detect DNA protein cross-link (DPC) formation.^ A G(,2)M block was detected in SD rat marrow following one week of exposure to 0.5, 3, or 15 ppm HCHO, but this block did not persist. No effect was noticed in mouse marrow. Only a minimal increase in RNA content was detected in rat or mouse marrow while exfoliated lung cells showed a significant increase in RNA activity after one week of exposure.^ Acute exposure in SD rats for four hours/day for one or three days at 150 ppm showed an increase in RNA activity in exfoliated lung cells but not in the marrow after one day. On the third day, dead cells were detected in exfoliated lung cells.^ In alkaline elution studies, no DPC were detected in marrow of SD rats after 24 weeks exposure up to 15 ppm. During acute exposures, a dose response relationship was detected in SD rat exfoliated lung cells which yielded cross-linking factors of 0.954, 1.237, and 1.417 following a four hour exposure to 15, 50, or 150 ppm, respectively. No DPC were detected in the marrow at 150 ppm. In vitro exposures to HCHO of CHO and SHE cells and rat marrow cells revealed the production of DPC and DNA-DNA cross-links.^ Cytoxan treatment of SD rats was used to provide positive controls for flow cytometry and alkaline elution. A drastic reduction in RNA content and cycling cells occurred one day following treatment. After four days, RNA content was greatly increased; and on day eleven the marrow had regenerated. DPCs were detected in both the marrow and the exfoliated lung cells.^ The lack of significant responses in SD rats and A-strain mice below 15 ppm HCHO is explainable by host defense mechanisms. Apparently, the mucociliary apparatus and enzymatic detoxification are sufficient to reduce systemic toxicity to low level concentrations of formaldehyde. ^

Signaling pathways in the transcriptional and post-translational regulation of the human glutathione S -transferase P1 gene

The human glutathione S-transferase P1 (GSTP1) protein is an endogenous inhibitor of c-jun N-terminal kinases (JNKs) and an important phase II detoxification enzyme. ^ Recent identification of a cAMP response element (CRE) in the 5 ′-region of the human GSTP1 gene and several putative phosphorylation sites for the Ser/Thr protein kinases, including, cAMP-dependent protein kinases (PKAs), protein kinases C (PKCs), and JNKs in the GSTP1 protein raised the possibility that signaling pathways may play an important role in the transcriptional and post-translational regulation of GSTP1 gene. This study examined (a) whether the signaling pathway mediated by CAMP, via the GSTP1 CRE, is involved in the transcriptional regulation of the GSTP1 gene, (b) whether signaling pathways mediated by the Ser/Thr protein kinases (PKAs, PKCs, and JNKs) induce post-translational modification, viz. phosphorylation of the GSTP1 protein, and (c) whether such phosphorylation of the GSTP1 protein alters its functions in metabolism and in JNK signaling. ^ The first major finding in this study is the establishment of the human GSTP1 gene as a novel CAMP responsive gene in which transcription is activated via an interaction between PKA activated CRE binding protein-1 (CREB-1) and the CRE in the 5′-regulatory region. ^ The second major finding in this study is the observation that the GSTP1 protein undergoes phosphorylation and functionally activated by second messenger-activated protein kinases, PKA and PKC, in tumor cells with activated signaling pathways. Following phosphorylation by PKA or PKC, the catalytic activity of the GSTP1 protein was significantly enhanced, as indicated by a decrease in its Km (2- to 3.6-fold) and an increase in Kcat/ Km (1.6- to 2.5-fold) for glutathione. Given the frequent over-expression of GSTP1 and the aberrant PKA/PKC signaling cascade observed in tumors, these findings suggest that phosphorylation of GSTP1 may contribute to the malignant progression and drug-resistant phenotype of these tumors. ^ The third major finding in this study is that the GSTP1 protein, an inhibitor of JNKs, undergoes significant phosphorylation in tumor cells with activated JNK signaling pathway and in those under oxidative stress. Following phosphorylation by JNK, the ability of GSTP1 to inhibit JNK downstream function, i.e. c-jun phosphorylation, was significantly enhanced, suggesting a feedback mechanism of regulation of JNK-mediated cellular signaling. (Abstract shortened by UMI.) ^