Development and validation of RP-HPLC method for simultaneous estimation of docetaxel and ritonavir in PLGA nanoparticles.

OBJECTIVES: The main objective of the present study was to develop and validate simple, precise, sensitive and accurate RP-HPLC method for the simultaneous estimation of docetaxel (DTX) and ritonavir (RTV) in PLGA nanoparticles (PLGA-NPs). METHODS: The DTX and RTV co-loaded PLGA-NPs were developed by the nanoprecipitation technique. The RP-HPLC method was developed by using (Agilent Compact LC-1220) and Zorbax Eclipse plus C18 column (150×4.6mm, 3.5µm, Agilent). Finally, the developed method was validated according to the international conference on harmonization (ICH) guidelines. RESULTS: The chromatographic separations of DTX and RTV with good resolutions have been achieved by using the mobile phase Acetonitrile: Water (60:40 v/v) containing 0.1% v/v of orthophosphoric acid at a flow rate of 1.0mL/min, injection volume of 25µL, and at 239nm wavelengths. The validated method found to be linear in the range of 0.001-100µg/mL for DTX and RTV. Detection and quantification limits for DTX were found to be 0.7 and 2.31µg/mL respectively and for RTV it is 0.3 and 2.87µg/mL respectively. The % RSD was found to be less than 2% revealing the precision of the developed method. Besides, the recovery rate was observed close to 100% for both the drugs confirming the accuracy of the method. Minor alterations in the chromatographic conditions have revealed robustness and ruggedness of the developed method. CONCLUSION: The developed analytical method is simple, precise, sensitive, and reproducible which can be used for the simultaneous estimation of DTX and RTV in the PLGA-NPs.

Single and multiple dose pharmacokinetics of maraviroc in saliva, semen, and rectal tissue of healthy HIV-negative men.

BACKGROUND: Antiretroviral pharmacology in seminal plasma (SP) and rectal tissue (RT) may provide insight into antiretroviral resistance and the prevention of sexual transmission of human immunodeficiency virus (HIV). Saliva may be of utility for noninvasively measuring adherence. METHODS: A pharmacokinetic study was performed in 12 HIV-negative men receiving maraviroc 300 mg twice daily for 8 days. Seven time-matched pairs of blood plasma (BP) and saliva samples were collected over 12 h on day 1 (PK1) and days 7 and 8 (PK2). One RT sample from each subject was collected during PK1 and PK2. Two SP samples were collected from each subject during PK1, and 6 SP samples were collected from each subject during PK2. RESULTS: SP AUCs were ∼50% lower than BP. However, protein binding in SP ranged from 4% to 25%, resulting in protein-free concentrations >2-fold higher than BP. RT AUCs were 7.5- to 26-fold higher than BP. Maraviroc saliva AUCs were ∼70% lower than BP, but saliva concentrations correlated with BP (r(2) = 0.58). CONCLUSIONS: More pharmacologically available maraviroc was found in SP than BP. High RT concentrations are promising for preventing rectal HIV acquisition. Saliva correlation with BP suggests that this may be useful for monitoring adherence. CLINICAL TRIALS REGISTRATION: NCT00775294.

Concentrations of tenofovir and emtricitabine in saliva: implications for preexposure prophylaxis of oral HIV acquisition.

To prevent acquisition of HIV through oral sex, drugs used for preexposure prophylaxis (Prep) need to diffuse in saliva. We measured tenofovir (TFV) and emtricitabine (FTC) concentrations simultaneously in the plasma and saliva of 41 HIV-infected patients under stable antiretroviral treatment. Mean ratios of saliva/plasma concentration were 3% (±4%) and 86.9% (±124%) for TFV and FTC, respectively. Tenofovir disoproxil fumarate (TDF) should be used in combination with FTC to prevent oral acquisition of HIV.

Efficacy of silk fibroin biomaterial vehicle for in vivo mucosal delivery of Griffithsin and protection against HIV and SHIV infection ex vivo.

INTRODUCTION: The majority of new HIV infections occur through mucosal transmission. The availability of readily applicable and accessible platforms for anti-retroviral (ARV) delivery is critical for the prevention of HIV acquisition through sexual transmission in both women and men. There is a compelling need for developing new topical delivery systems that have advantages over the pills, gels and rings, which currently fail to guarantee protection against mucosal viral transmission in vulnerable populations due to lack of user compliance. The silk fibroin (SF) platform offers another option that may be better suited to individual circumstances and preferences to increase efficacy through user compliance. The objective of this study was to test safety and efficacy of SF for anti-HIV drug delivery to mucosal sites and for viral prevention. METHODS: We formulated a potent HIV inhibitor Griffithsin (Grft) in a mucoadhesive silk fibroin (SF) drug delivery platform and tested the application in a non-human primate model in vivo and a pre-clinical human cervical and colorectal tissue explant model. Both vaginal and rectal compartments were assessed in rhesus macaques (Mucaca mulatta) that received SF (n = 4), no SF (n = 7) and SF-Grft (n = 11). In this study, we evaluated the composition of local microbiota, inflammatory cytokine production, histopathological changes in the vaginal and rectal compartments and mucosal protection after ex vivo SHIV challenge. RESULTS: Effective Grft release and retention in mucosal tissues from the SF-Grft platform resulted in protection against HIV in human cervical and colorectal tissue as well as against SHIV challenge in both rhesus macaque vaginal and rectal tissues. Mucoadhesion of SF-Grft inserts did not cause any inflammatory responses or changes in local microbiota. CONCLUSIONS: We demonstrated that in vivo delivery of SF-Grft in rhesus macaques fully protects against SHIV challenge ex vivo after two hours of application and is safe to use in both the vaginal and rectal compartments. Our study provides support for the development of silk fibroin as a highly promising, user-friendly HIV prevention modality to address the global disparity in HIV infection.

Cariogenic and erosive potential of the medication used by HIV-infected children: pH and sugar concentration.

OBJECTIVE: The aim of this work was to analyze pH and sugar concentration in seven antiretroviral and three antibacterial medications frequently prescribed to HIV infected paediatric patients. METHOD: Sugars (sucrose, glucose, lactose and fructose) and pH were measured from every one of ten medications with different serial numbers in two samples. The pH was determined by a previously calibrated digital pHmeter (Beckman). Analysis of free sugars was performed using thin-layer chromatography (TLC). The pH results and the amount of sugar originated from the two samples in each lot were added. The arithmetic mean of these results were computed. RESULTS: Two antiretrovirals (Zidovudin and Abacavir Sulphate) had pH below critical level (3.55 and 3.93, respectively). All three antibacterials analyzed had pH above 5.5, and one of them (Azithromycin) had the highest pH level of the ten medications examined (9.28). Sugar was present in seven out of 10 of the medications analyzed. The antibacterials contained the highest concentration of sucrose, ranging from 40% to 54%. Glucose was found in one of the ten, sucrose was present in seven of them and none showed lactose. Fructose was not observed with the technique used. CONCLUSIONS: A number of medications frequently used by HIV-infected children may cause a significant risk of both caries and dental erosion.

One-by-one imprinting in two eccentric layers of hollow core-shells: Sequential electroanalysis of anti-HIV drugs.

Double layered one-by-one imprinted hollow core-shells@ pencil graphite electrode was fabricated for sequential sensing of anti-HIV drugs. For this, two eccentric layers were developed on the surface of vinylated silica nanospheres to obtain double layered one-by-one imprinted solid core-shells. This yielded hollow core-shells on treatment with hydrofluoric acid. The modified hollow core-shells (single layered dual imprinted) evolved competitive diffusion of probe/analyte molecules. However, the corresponding double layered one-by-one imprinted hollow core-shells (outer layer imprinted with Zidovudine, and inner layer with Lamivudine) were found relatively better owing to their bilateral diffusions into molecular cavities, without any competition. The entire work is based on differential pulse anodic stripping voltammetry at double layered one-by-one imprinted hollow core-shells. This resulted in indirect detection of electro inactive targets with limits of detection as low as 0.91 and 0.12 (aqueous sample), 0.94 and 0.13 (blood serum), and 0.99 and 0.20 ng mL-1 (pharmaceutics) for lamivudine and zidovudine, respectively in anti-HIV drug combination.

Characterization of silicone elastomer vaginal rings containing HIV microbicide TMC120 by Raman spectroscopy.

Silicone elastomer vaginal rings are currently being pursued as a controlled-release strategy for delivering microbicidal substances for the prevention of heterosexual transmission of HIV. Although it is well established that the distribution of drugs in delivery systems influences the release characteristics, in practice the distribution is often difficult to quantify in-situ. Therefore, the aim of this work was to determine whether Raman spectroscopy might provide a rapid, non-contact means of measuring the concentrations of the lead candidate HIV microbicide TMC120 in a silicone elastomer reservoir-type vaginal ring. Vaginal rings loaded with TMC120 were manufactured and sectioned before either Raman mapping an entire ring cross-section (100 microm resolution) or running line scans at appropriate time intervals up to 30 h after manufacture. The results demonstrated that detectable amounts of TMC120, above the silicone elastomer saturation concentration, could be detected up to 1 mm into the sheath, presumably as a consequence of permeation and subsequent reprecipitation. The extent of permeation was found to be similar in rings manufactured at 25 and 80 degrees C.

Direct measurement of in-vivo vaginal microbicide levels of PRO 2000 achieved in a human safety study.

OBJECTIVES: To directly measure the cervico-vaginal lavage (CVL) and plasma PRO 2000 concentrations achieved by vaginal dosing. DESIGN: A sub-study of a prospective randomized, double-blind phase 1 trial of a candidate vaginal microbicide, PRO 2000 gel. METHODS: Thirty-six sexually abstinent women self-administered 4% PRO 2000 gel, 0.5% PRO 2000 gel or placebo gel twice on day 0 and then once daily for a further 12 days. RESULTS: There was no evidence of systemic absorption of PRO 2000. PRO 2000 concentrations in CVL exceeded 25 microg/ml in all women in both the 4 and 0.5% groups at 2 h post-first dose, and in 10 of 12 of the women in the 4% gel group compared with five of 12 of women in the 0.5% group at 12 h post-seventh dose. Single use of both 4 and 0.5% PRO 2000 gels was therefore associated with levels of PRO 2000 in CVL that would be capable of preventing HIV infection in vitro, although the 4% gel gave a greater margin of excess. Levels substantially in excess of the target concentration were present 12 h after repeated dosing in twice as many 4% gel recipients compared with 0.5% gel recipients. CONCLUSIONS: Both PRO 2000 gel strengths provided satisfactory in-vivo HIV inhibitory concentrations. However, our observations show that higher concentrations of PRO 2000 are likely to provide a greater margin of potential efficacy in the context of sexual intercourse provided safety issues are equivalent for differing concentrations of the agent.

Evaluation of saliva as an alternative matrix for monitoring plasma Zidovudine, Lamivudine and nevirapine concentrations in Rwanda.

Saliva may provide interesting advantages as matrix for compliance measurements, pharmacokinetic studies and therapeutic drug monitoring in resource limited countries. We investigated the feasibility of using saliva for compliance monitoring of zidovudine (ZDV), lamivudine (3TC) and nevirapine (NVP) in 29 HIV-1 infected patients from Rwanda. ZDV, 3TC and NVP drug levels were quantified by an LC/MS-MS method in plasma and stimulated saliva samples and compared using Bland-Altman analysis. Seven patients demonstrated undetectable saliva ZDV levels while five out of these seven also showed no 3TC salivary concentrations. For the other samples, we observed a good agreement between salivary and plasma concentrations of each antiretroviral drug. A significant relation between the difference in saliva and plasma ZDV concentrations and the average ZDV concentration in the two matrices was deduced as follow: y = -380.15 + 1.79 x. The log saliva and plasma concentration difference of both 3TC and NVP was consistent across the range of average log concentration. Overall, we showed large agreement limits suggesting a wide inter patient variability that may result to non-reliable plasma level predictions from saliva drug measurements. Therefore, our results indicate that saliva may serve as a valuable tool only for NVP compliance testing because of its high salivary concentration.

Determination of salivary efavirenz by liquid chromatography coupled with tandem mass spectrometry.

A novel and robust screening method for the determination of the non-nucleoside reverse transcriptase inhibitor, efavirenz (EFV), in human saliva has been developed and validated based on high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). Sample preparation of the saliva involved solid-phase extraction (SPE) on C18 cartridges. The analytes were separated by high performance liquid chromatography (Phenomenex Kinetex C18, 150mm×3mm internal diameter, 2.6µm particle size) and detected with tandem mass spectrometry in electrospray positive ionization mode with multiple reaction monitoring. Gradient elution with increasing methanol (MeOH) concentration was used to elute the analytes, at a flow-rate of 0.4mL/min. The total run time was 8.4min and the retention times for the internal standard (reserpine) was 5.4min and for EFV was 6.5min. The calibration curves showed linearity (r(2), 0.989-0.992) over the concentration range of 3.125-100µg/L. Mean intra- and inter-assay relative standard deviation, accuracy, mean extraction recovery, limit of detection (LOD) and limit of quantification (LOQ) were 0.46-9.43%, 80-120%, 60% (±7.95), 1.84 and 6.11µg/L respectively. The working range was defined as 6.25-100µg/L. This novel LC-MS/MS assay is suitable for reliable detection of low EFV concentrations in saliva and can be used as a screening tool for monitoring EFV compliance.

Novel conducting polymer functionalized with metal-cyclam complex and its sensor application: development of azidothymidine drug sensor.

Functionalization of polyanthranilic acid (PAA) a self-doped conducting polymer with Co(II) metal complex has been reported and is used in the development of azidothymidine drug sensor. For the first time synthesis of a new type of polymer complex of Co(II)-cyclam macrocyclic ligand (1,4,8,11-tetraazacyclotetradecane) with carboxylated polymer (as a second ligand) has been successfully accomplished and discussed in the present paper. The interaction of Co(II)-cyclam complex with polyanthranilic acid has been studied in solution phase using UV-vis spectra. Further, the formation and growth study of mixed ligand complex is carried out using electrochemical quartz crystal microbalance. The characterization of solid mixed ligand complex Co(II)-cyclam-polyanthranilic acid (Co(II)-Cy-PAA) has been carried out for its structural, thermal and electrochemical properties using various techniques viz. FT-IR, SEM, ESR, DSC, impedance and electrochemical techniques. Electrochemical study shows the potential of mixed ligand complex towards catalytic and sensor applications. The mixed ligand complex for the first time is used in the development of "azidothymidine" anti-HIV drug sensor. The analysis of azidothymidine is known over hanging drop mercury electrode using electroreduction technique, however, for the first time its analysis is reported over graphite paste electrode modified with newly synthesized mixed ligand complex. Azidothymidine is quantified in wide range of concentration with 1 microM detection limit over modified graphite paste electrode, which shows potential to develop users friendly (non-toxic and simple) gadgets and low cost screen printed electrodes.

Comparison of human immunodeficiency virus type 1-specific inhibitory activities in saliva and other human mucosal fluids.

Several human mucosal fluids are known to possess an innate ability to inhibit human immunodeficiency virus type 1 (HIV-1) infection and replication in vitro. This study compared the HIV-1 inhibitory activities of several mucosal fluids, whole, submandibular/sublingual (sm/sl), and parotid saliva, breast milk, colostrum, seminal plasma, and cervicovaginal secretions, from HIV-1-seronegative donors by using a 3-day microtiter infection assay. A wide range of HIV-1 inhibitory activity was exhibited in all mucosal fluids tested, with some donors exhibiting high levels of activity while others showed significantly lower levels. Colostrum, whole milk, and whole saliva possessed the highest levels of anti-HIV-1 activity, seminal fluid, cervicovaginal secretions, and sm/sl exhibited moderate levels, and parotid saliva consistently demonstrated the lowest levels of HIV-1 inhibition. Fast protein liquid chromatography gel filtration studies revealed the presence of at least three distinct peaks of inhibitory activity against HIV-1 in saliva and breast milk. Incubation of unfractionated and fractionated whole saliva with antibodies raised against human lactoferrin (hLf), secretory leukocyte protease inhibitor (SLPI), and, to a lesser extent, MG2 (high-molecular-weight mucinous glycoprotein) reduced the HIV-1 inhibitory activity significantly. The results suggest that hLf and SLPI are two key components responsible for HIV-1 inhibitory activity in different mucosal secretions. The variation in HIV inhibitory activity between the fluids and between individuals suggests that there may be major differences in susceptibility to HIV infection depending both on the individual and on the mucosal fluid involved.

Long-term, controlled release of the HIV microbicide TMC120 from silicone elastomer vaginal rings.

OBJECTIVES: The feasibility of providing prolonged and controlled release of the experimental non-nucleoside reverse transcriptase inhibitor TMC120 from a silicone vaginal ring in quantities sufficient to maintain a vaginal concentration offering protection against heterosexual HIV transmission was investigated. METHODS: Core-type, silicone elastomer vaginal rings containing TMC120 were manufactured, and in vitro release studies performed under sink conditions. The experimental release data, as determined by HPLC, were correlated with estimates of vaginal TMC120 concentrations required to inhibit HIV replication. RESULTS: Continuous, zero-order release of TMC120 from core-type vaginal rings was observed in vitro over a 71 day period, equivalent to 136 microg/day. The release rate is predicted to maintain vaginal concentrations of the antiretroviral in the range of several orders of magnitude in excess of reported HIV inhibitory concentration values. CONCLUSIONS: Continuous and prolonged zero-order release of TMC120 from a silicone vaginal ring device at quantities predicted to prevent HIV infection was observed.

Therapeutic drug monitoring for patients with HIV infection: Children's National Medical Center, Washington DC experience.

This paper provides a brief overview of therapeutic drug monitoring (TDM) in patients with HIV infection. The manuscipt not only focuses on an update on TDM in HIV infection but also provides the latest information on tandem mass spectrometric methods for antiretroviral drug quantification. Also discussed are the preliminary new and original data on free antiretroviral drug measurement in both plasma and saliva.

Salivary secretory leukocyte protease inhibitor is associated with reduced transmission of human immunodeficiency virus type 1 through breast milk.

Secretory leukocyte protease inhibitor (SLPI), a protein found in saliva, breast milk, and genital secretions, is capable of inhibiting human immunodeficiency virus (HIV) type 1 in vitro. The aim of this study was to determine whether SLPI in infant saliva provides protection against mother-to-child HIV-1 transmission. In total, 602 saliva specimens were collected from 188 infants at birth and at ages 1, 3, and 6 months. Infants' median salivary SLPI concentrations were higher at birth than at 6 months (341 vs. 219 ng/mL; P=.001). There was no association between SLPI concentration and HIV-1 transmission overall. However, among 122 breast-fed infants who were HIV-1 uninfected at 1 month, higher salivary SLPI levels were associated with a decreased risk of HIV-1 transmission through breast milk (hazard ratio, 0.5; 95% confidence interval, 0.3-0.9; P=.03). These results suggest that SLPI plays an important role in reducing HIV-1 transmission through breast milk.

Quantitative determination of (-)-2'-deoxy-3'-thiacytidine (lamivudine) in human plasma, saliva and cerebrospinal fluid by high-performance liquid chromatography with ultraviolet detection.

A high-performance liquid chromatographic method for the quantitative determination of the HIV reverse transcriptase inhibitor lamivudine ((-)-2'-deoxy-3'-thiacytidine, 3TC, Epivir) in human plasma, saliva and cerebrospinal fluid is described. Lamivudine was extracted from samples using silica extraction columns prior to reversed-phase high-performance liquid chromatography with ultraviolet detection at 270 nm. The method has been validated over the range of 10 (lower limit of quantitation) to 5000 ng/ml using a 0.5-ml sample volume. Between-day and within-day precisions ranged from 3.5 to 9.0%. The assay has been used for the quantitative analysis of lamivudine in plasma and cerebrospinal fluid of HIV-1 infected patients.