Expression, Activity, and Regulation of Phosphorylating Enzymes in Tissues and Cells Relevant to HIV-1 Sexual Transmission.

Phosphorylating enzymes (PEs) are responsible for activating nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) such as tenofovir (TFV) and are critical for their conversion to obtain intracellular antiviral activity. However, there are limited data available regarding the expression of PEs and their activity in the female genital tract. This work compared the messenger RNA (mRNA) expression levels of PEs in human female genital tissue, immune cells, and animal models that are commonly used in human immunodeficiency virus (HIV) research. Furthermore, the effect of contraceptive hormones and proinflammatory cytokines on tenofovir diphosphate (TFV-DP) formation and efficacy in human vaginal, epithelial, and immune cells was also evaluated. We found that human vaginal and ectocervical tissues had similar mRNA expression for seven PEs tested. Polymerase chain reaction results revealed that creatine kinase brain (CKB), mitochondrial creatine kinase 1 (CKMT1), mitochondrial creatine kinase 2 (CKMT2), adenylate kinase AK3L1 (AK4), and nucleoside diphosphate kinase 1 (NME1) exhibited a 10- to 10,000-fold higher expression level in a vaginal epithelial cell line, VK2, compared with CD4+ T cells (p < .05). Medroxyprogesterone acetate (MPA)/progesterone (P4) and IL-1ß/IL-8 treatment resulted in altered TFV-DP levels in VK2 and PM1 cells. MPA and P4 at concentrations above 0.1 µM, as well as IL-1ß and IL-8 at concentrations above 10 ng/mL, significantly decreased HIV-1BaL inhibition in PM1 cells when 1 µM TFV was added. However, this observed effect of hormones and cytokines was abrogated when TFV concentration was raised to 1 mM. These in vitro results elucidate the role of PEs in TFV metabolism and provide information regarding differences in PE tissue expression for animal models commonly used in HIV testing. This information can be applied to better understand and interpret data obtained using these models.

Heart rhythm complexity as predictors for the prognosis of end-stage renal disease patients undergoing hemodialysis.

BACKGROUND: Heart rhythm complexity, a measure of heart rate dynamics and a risk predictor in various clinical diseases, has not been systematically studied in patients with end-stage renal disease. The aim of this study is to investigate the heart rhythm complexity and its prognostic value for mortality in end-stage renal disease patients undergoing hemodialysis. METHODS: To assess heart rhythm complexity and conventional heart rate variability measures, 4-h continuous electrocardiography for a retrospective cohort of 202 ostensibly healthy control subjects and 51 hemodialysis patients with end-stage renal disease were analyzed. Heart rhythm complexity was quantified by the complexity index from the measurement of the multiscale entropy profile. RESULTS: During a follow-up of 13 months, 8 people died in the patient group. Values of either traditional heart rate variability measurements or complexity indices were found significantly lower in patients than those in healthy controls. In addition, the complexity indices (Area 1-5, Area 6-15 and Area 6-20) in the mortality group were significantly lower than those in the survival group, while there were no significant differences in traditional heart rate variability parameters between the two groups. In receiver operating characteristic curve analysis, Area 6-20 (AUC = 0.895, p < 0.001) showed the strongest predictive power between mortality and survival groups. CONCLUSION: The results suggest that heart rhythm complexity is impaired for patients with end-stage renal disease. Furthermore, the complexity index of heart rate variability quantified by multiscale entropy may be a powerful independent predictor of mortality in end-stage renal disease patients undergoing hemodialysis.

[Design and Implementation of Infusion Monitor Software Based on C# Language].

OBJECTIVE: To design an infusion monitor software and implement the establishment of wireless intelligent infusion monitoring system in hospital infusion area combined with the existing platform equipment in the laboratory. METHODS: On the platform of Visual Studio 2013, C# language was used to realize the software development of the upper computer, and the data from infusion monitoring equipment was received through WiFi wireless transmission technology. RESULTS: The upper computer software had a friendly user interface, and it could receive infusion data correctly and display normally. The upper computer software had alarm function and good performance. CONCLUSIONS: Infusion monitor software can realize comprehensive monitoring of multiple infusion monitors and real-time monitor of infusion process. It is easy to operate and carry which is helpful to improve the efficiency and quality of medical care.

The Effect of Commonly Used Excipients on the Epithelial Integrity of Human Cervicovaginal Tissue.

Pharmaceutical excipients are widely used in vaginal drug products. The epithelial integrity of the cervicovaginal tissue is important for HIV-1 prevention. However, the effects of excipients on cervicovaginal epithelium remain unknown. This study aims at assessing the effects of vaginal product excipients on the integrity of human cervicovaginal epithelium and on a lead HIV prevention antiretroviral drug, tenofovir (TFV). In the current study, nine excipients commonly used in vaginal formulations were incubated for 6 h with excised human ectocervical tissue. The effects of the excipients were examined by measuring the transepithelial electrical resistance (TEER), epithelial morphology, paracellular/transcellular permeability, and cell viability. The efficacy of TFV for preventing HIV-1 infection in the ex vivo cultured ectocervix was also tested. We found that disodium ethyl-enediaminetetraacetate (EDTA), sorbic acid, and benzoic acid had no effect on the tissue TEER. Butylated hydroxyanisole, glycerin, propylene glycol, methylparaben, and propylparaben slightly to moderately decreased tissue TEER, whereas citric acid significantly decreased the TEER in a time-dependent manner. Tissue morphology observed post-exposure strongly correlated with TEER data; however, a less strong correlation was observed between paracellular permeability and TEER data after exposure to different excipients. In addition, treatment with EDTA, methylparaben, and propylene glycol at tested levels had no effect on the efficacy of TFV in preventing tissue HIV-1 infection. In conclusion, the combined measurements of TEER, morphology, permeability, and viability using human cervicovaginal tissue represent a clinically relevant platform for safety evaluation of excipients and formulated products for HIV-1 prevention.

Expression, regulation, and function of drug transporters in cervicovaginal tissues of a mouse model used for microbicide testing.

P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance protein 4 (MRP4) are three efflux transporters that play key roles in the pharmacokinetics of antiretroviral drugs used in the pre-exposure prophylaxis of HIV sexual transmission. In this study, we investigated the expression, regulation, and function of these transporters in cervicovaginal tissues of a mouse model. Expression and regulation were examined using real-time RT-PCR and immunohistochemical staining, in the mouse tissues harvested at estrus and diestrus stages under natural cycling or after hormone synchronization. The three transporters were expressed at moderate to high levels compared to the liver. Transporter proteins were localized in various cell types in different tissue segments. Estrous cycle and exogenous hormone treatment affected transporter mRNA and protein expression, in a tissue- and transporter-dependent manner. Depo-Provera-synchronized mice were dosed vaginally or intraperitoneally with (3)H-TFV, with or without MK571 co-administration, to delineate the function of cervicovaginal Mrp4. Co-administration of MK571 significantly increased the concentration of vaginally-administered TFV in endocervix and vagina. MK571 increased the concentration of intraperitoneally-administered TFV in the cervicovaginal lavage and vagina by several fold. Overall, P-gp, Bcrp, and Mrp4 were positively expressed in mouse cervicovaginal tissues, and their expression can be regulated by the estrous cycle or by exogenous hormones. In this model, the Mrp4 transporter impacted TFV distribution in cervicovaginal tissues.

Comparative Expression Analysis of Cytochrome P450 1A1, Cytochrome P450 1B1 and Nuclear Receptors in the Female Genital and Colorectal Tissues of Human and Pigtailed Macaque.

This manuscript summarizes our recent progress in examine the CYP1A1 and CYP1B1 as well as a number of nuclear receptors in the female genital and colorectal tissues of human and pigtailed macaque. Understanding the nuclear receptor mediated regulation of CYP1A1 and 1B1 expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. However, there is a lack of systematic and comparative analysis of the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and clinically relevant animal models. The current study aims to fill this gap. We found CYP1A1, CYP1B1 and a number of nuclear receptors were expressed in the female genital and colorectal tissues of human and macaque. However, the mRNA level and protein localization of these CYP enzymes and NRs depended on the type of tissue examined. Cytochrome P450 (CYP) 1A1 and CYP1B1 activate hormonal and environmental procarcinogens, and are associated with carcinogenesis in female genital and colorectal tissues. Understanding the nuclear receptor (NR) mediated regulation of CYP expression in these tissues is necessary for identifying cancer risk factors and developing CYP1A1/1B1-targeted anti-cancer therapeutics. The study aims to analyze the expression profile of CYP1A1, 1B1 and NRs in the female genital and colorectal tissues of human and pigtailed macaques. We found that compared to the liver, human CYP1A1 mRNA level in the genital and colorectal tissues was significantly lower, while the CYP1B1 level was significantly higher. CYP1A1 protein was mainly localized in the plasma membrane of the uterine and endocervical epithelial cells. The CYP1B1 protein was concentrated in the nucleus of genital and colorectal tissues. Fourteen NRs in the genital tract and 12 NRs in colorectal tissue were expressed at levels similar to or higher than the liver. The expression and localization of CYP1A1, CYP1B1, and NRs in macaque tissues were usually comparable to those of human tissues. In addition, menopause did not significantly alter the ectocervical mRNA levels of CYP1A1, CYP1B1, or NRs.

Drug transporters in tissues and cells relevant to sexual transmission of HIV: Implications for drug delivery.

Efflux and uptake transporters of drugs are key regulators of the pharmacokinetics of many antiretroviral drugs. A growing body of literature has revealed the expression and functionality of multiple transporters in female genital tract (FGT), colorectal tissue, and immune cells. Drug transporters could play a significant role in the efficacy of preventative strategies for HIV-1 acquisition. Pre-exposure prophylaxis (PrEP) is a promising strategy, which utilizes topically (vaginally or rectally), orally or other systemically administered antiretroviral drugs to prevent the sexual transmission of HIV to receptive partners. The drug concentration in the receptive mucosal tissues and target immune cells for HIV is critical for PrEP effectiveness. Hence, there is an emerging interest in utilizing transporter information to explain tissue disposition patterns of PrEP drugs, to interpret inter-individual variability in PrEP drug pharmacokinetics and effectiveness, and to improve tissue drug exposure through modulation of the cervicovaginal, colorectal, or immune cell transporters. In this review, the existing literature on transporter expression, functionality and regulation in the transmission-related tissues and cells is summarized. In addition, the relevance of transporter function for drug delivery and strategies that could exploit transporters for increased drug concentration at target locales is discussed. The overall goal is to facilitate an understanding of drug transporters for PrEP optimization.

Vaginal Microbicide Film Combinations of Two Reverse Transcriptase Inhibitors, EFdA and CSIC, for the Prevention of HIV-1 Sexual Transmission.

PURPOSE: EFdA is a potent nucleoside reverse transcriptase inhibitor (NRTI) with activity against a wide spectrum of wild-type and drug resistant HIV-1 variants. CSIC is a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) with demonstrated anti-HIV properties important for use in topical prevention of HIV transmission. The objective of this study was to develop and characterize film-formulated EFdA and CSIC for use as a female-controlled vaginal microbicide to prevent sexual transmission of HIV. METHODS: Assessments of EFdA- and CSIC-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity studies, compatibility with the normal vaginal Lactobacillus flora and anti-HIV bioactivity evaluations. RESULTS: No significant change in physicochemical properties or biological activity of the combination films were noted during 3 months storage. In vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) of either EFdA or CSIC was several orders of magnitude higher than the 50% effective concentration (EC50) values. Film-formulated EFdA and CSIC combination showed additive inhibitory activity against wild type and drug-resistant variants of HIV. Epithelial integrity studies demonstrated that the combination vaginal film had a much lower toxicity to HEC-1A monolayers compared to that of VCF®, a commercial vaginal film product containing nonoxynol-9. Polarized ectocervical explants showed films with drug alone or in combination were effective at preventing HIV infection. CONCLUSIONS: Our data suggest that vaginal microbicide films containing a combination of the NRTI EFdA and the NNRTI CSIC have potential to prevent HIV-1 sexual transmission.

Expression and localization of p-glycoprotein, multidrug resistance protein 4, and breast cancer resistance protein in the female lower genital tract of human and pigtailed macaque.

Antiretroviral drug absorption and disposition in cervicovaginal tissue is important for the effectiveness of vaginally or orally administered drug products in preexposure prophylaxis (PrEP) of HIV-1 sexual transmission to women. Therefore, it is imperative to understand critical determinants of cervicovaginal tissue pharmacokinetics. This study aimed to examine the mRNA expression and protein localization of three efflux transporters, P-glycoprotein (P-gp), multidrug resistance-associated protein 4 (MRP4), and breast cancer resistance protein (BCRP), in the lower genital tract of premenopausal women and pigtailed macaques. Along the human lower genital tract, the three transporters were moderately to highly expressed compared to colorectal tissue and liver, as revealed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). In a given genital tract segment, the transporter with the highest expression level was either BCRP or P-gp, while MRP4 was always expressed at the lowest level among the three transporters tested. The immunohistochemical staining showed that P-gp and MRP4 were localized in multiple cell types including epithelial cells and vascular endothelial cells. BCRP was predominantly localized in the vascular endothelial cells. Differences in transporter mRNA level and localization were observed among endocervix, ectocervix, and vagina. Compared to human tissues, the macaque cervicovaginal tissues displayed comparable expression and localization patterns of the three transporters, although subtle differences were observed between the two species. The role of these cervicovaginal transporters in drug absorption and disposition warrants further studies. The resemblance between human and pigtailed macaque in transporter expression and localization suggests the utility of the macaque model in the studies of human cervicovaginal transporters.

Short communication: expression of transporters and metabolizing enzymes in the female lower genital tract: implications for microbicide research.

Topical vaginal microbicides have been considered a promising option for preventing the male-to-female sexual transmission of HIV; however, clinical trials to date have not clearly demonstrated robust and reproducible effectiveness results. While multiple approaches may help enhance product effectiveness observed in clinical trials, increasing the drug exposure in lower genital tract tissues is a compelling option, given the difficulty in achieving sufficient drug exposure and positive correlation between tissue exposure and microbicide efficacy. Since many microbicide drug candidates are substrates of transporters and/or metabolizing enzymes, there is emerging interest in improving microbicide exposure and efficacy through local modulation of transporters and enzymes in the female lower genital tract. However, no systematic information on transporter/enzyme expression is available for ectocervical and vaginal tissues of premenopausal women, the genital sites most relevant to microbicide drug delivery. The current study utilized reverse transcriptase polymerase chain reaction (RT-PCR) to examine the mRNA expression profile of 22 transporters and 19 metabolizing enzymes in premenopausal normal human ectocervix and vagina. Efflux and uptake transporters important for antiretroviral drugs, such as P-gp, BCRP, OCT2, and ENT1, were found to be moderately or highly expressed in the lower genital tract as compared to liver. Among the metabolizing enzymes examined, most CYP isoforms were not detected while a number of UGTs such as UGT1A1 were highly expressed. Moderate to high expression of select transporters and enzymes was also observed in mouse cervix and vagina. The implications of this information on microbicide research is also discussed, including microbicide pharmacokinetics, the utilization of the mouse model in microbicide screening, as well as the in vivo functional studies of cervicovaginal transporters and enzymes.

Formulation and delivery of microbicides.

The development of pre-exposure prophylactics or microbicide products for the reduction or elimination of the sexual transmission of HIV has numerous challenges or barriers to success. Historically traditional dosage forms such as gels have been developed in the field but more recently controlled release dosage forms such as vaginal rings and novel dosage forms such as polymeric thin films have been studied. Studies have begun to incorporate scientific strategies into the formulation design of microbicide products in order to develop safer and more effective products. In addition advanced drug delivery strategies to overcome barriers to delivery and specific drug targeting methods are being employed. In the present review, a comprehensive discussion of formulation efforts and novel delivery strategies in the field of microbicide product development is presented.