Hybrid Cyclic-Linear Cell-Penetrating Peptides Containing Alternative Positively Charged and Hydrophobic Residues as Molecular Transporters.

The cell membrane properties create a significant obstacle in intracellular delivery of cell-impermeable and negatively charged molecules. Herein, we report the synthesis and biological evaluation of a novel series of hybrid cyclic-linear peptides containing alternative positive and hydrophobic amino acids on the ring and side chain [(RW)5]K(RW)X (X = 1-5) to compare their molecular transporter efficiency. The peptides were synthesized through Fmoc solid-phase peptide synthesis. In vitro cytotoxicity of the peptides showed that the peptides did not exhibit any significant cytotoxicity at the concentration of 10 µM in human leukemia carcinoma cell line (CCRF-CEM), human ovarian adenocarcinoma cells (SK-OV-3), human epithelial embryonic kidney healthy (HEK-293), and human epithelial mammary gland adenocarcinoma cells (MDA-MB-231) after 3 h incubation. The cellular uptake of a fluorescence-labeled phosphopeptide (F'-GpYEEI) and anti-human immunodeficiency virus (HIV) drugs (lamivudine (F'-3TC), emtricitabine (F'-FTC), Stavudine (F'-d4T)), where F' is carboxyfluorescein, was measured in the presence of the peptides in CCRF-CEM and SK-OV-3 cells. Among all peptides, [(RW)5K](RW)5 (10 µM) was the most efficient transporter that improved the cellular uptake of F'-GpYEEI (2 µM) by 18- and 11-fold in CCRF-CEM and SK-OV-3, respectively, compared with F'-GpYEEI alone. Fluorescence-activated cell sorting (FACS) analysis results indicated that the cellular uptake of fluorescence-labeled peptide (F'-[(RW)5K](RW)5) was only partially inhibited by chlorpromazine as an endocytosis inhibitor after 3 h incubation in MDA-MB-231 cells. These data suggest the potential of this series of hybrid cyclic-linear peptides as cell-penetrating peptides and molecular transporters.

Evaluation of the Properties of Encapsulated Stavudine Microparticulate Lipid-based Drug Delivery System in Immunocompromised Wistar Rats.

BACKGROUND: Lipid-based formulations have been confirmed to lower some side effects of drugs and can be tailor-made to offer sustained drug release of drugs with short half-life like stavudine. AIM: This study aimed to evaluate the immunomodulatory properties of stavudine-loaded solid lipid microparticles (SLMs) using immunocompromised Wistar rats. METHODS: The SLMs were formulated by the homogenization method. The optimized batches were used for further in vivo studies. The effect of formulation on the CD4 count and the haematological properties of immunocompromised Wistar rats were studied. RESULTS: The particle size range was 4 -8 µm, EE range was 85-93 % and maximum drug release was observed at 10 h. The CD4 cells increased from 115 ± 3.17 cell/mm3 at day zero to 495 ± 5.64 cell/mm3 at day 14 of treatment and 538 ± 6.31 cell/mm3 at day 21. The red blood cells increased from 2.64 ± 1.58 (x 106/mm3) at day zero to 6.96 ± 3.47 (x 106/mm3) at day 14 and 7.85 ± 3.64 (x 106/mm3) at day 21. PCV increased significantly (p < 0.05) to about 42-50 % at day 21 in the groups that received the SLMs formulations. White blood cells (WBC) also were 12 x 103/mm3, for SLM formulations, while the rats that received plain stavudine exhibited WBC of 9.6 x 103/mm3 at day 21. The histopathological studies revealed that oral stavudine-loaded SLMs had no significant damage to the kidney, liver, spleen and the brain of Wistar rats. CONCLUSION: The formulations exhibited significantly higher immunomodulatory properties than plain stavudine (p<0.05) and showed good properties for once daily oral administration and could be a better alternative to plain stavudine tablets for the management of patients living with HIV.

Application of SRMS 154 as Sustained Release Matrix for the Delivery of Stavudine: In vitro and in vivo Evaluation and Effect of Poloxamer 188 on the Properties of the Tablets.

BACKGROUND: Stavudine is an antiretroviral therapy with so many side effects and has a short half-life of 1.5 h. It degrades to thymine under hydrolytic, oxidative and photolytic conditions hence has major formulation challenges. OBJECTIVES: To formulate sustained release lipid based stavudine and to study the properties of the formulations by in vitro and in vivo methods. METHODS: Stavudine tablets were formulated by moulding using validated tablets moulds. The carrier used were solidified reverse micellar solution (SRMS) made up of varying ratios of hydrogenated palm oil and Phospholipid admixtures. Evaluation tests were carried out on the tablets using both Pharmacopoeial and non Pharmacopoeial test. Drug release was studied in both simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.2). In vivo release was studied using Wistar rats. RESULTS: The results showed that stavudine tablets exhibited weight range of 372 ± 0.14 to 386 ± 0.52 mg, friability ranged from 0.00 to 0.13 % and hardness ranged from 4.27 ± 0.25 to 5.30 ± 0.21 Kgf. Tablets formulated with SRMS 1:2 had erosion time range of 60.80 ± 1.23 to 87.90 ± 2.33 min and was affected significantly by the presence of Poloxamer 188 (p < 0.05). The formulations exhibited T100 % at 10 to13 h in SIF. Stavudine tablets showed the area under the curve (AUC) of 854.0 µg/h/ml, significantly higher than the AUC of the reference (p < 0.05). CONCLUSION: Stavudine SRMS-based tablets had good stability and sustained release properties. Formulations containing 1 % Poloxamer 188 exhibited enhanced in vivo absorption and hence could be used once daily in order to enhance the bioavailability of this drug.

Can We Improve Stavudine's Safety Profile in Children? Pharmacokinetics of Intracellular Stavudine Triphosphate with Reduced Dosing.

Stavudine remains a useful replacement option for treatment for HIV+ children. WHO reduced the adult dose to 30 mg twice daily, which maintains efficacy and lowers mitochondrial toxicity. We explored intracellular stavudine triphosphate levels in children receiving a reduced dose of 0.5 to 0.75 mg/kg of body weight twice daily to investigate whether a similar dose optimization can safely be made. A population pharmacokinetic model was developed to describe the pharmacokinetics of intracellular stavudine triphosphate in 23 HIV+ children and 24 HIV+ adults who received stavudine at 0.5 mg/kg and 20 mg twice daily for 7 days, respectively. Simulations were employed to optimize the pediatric dosing regimen to match exposures in adults receiving the current WHO-recommended dose of 30 mg twice daily. A biphasic disposition model with first-order appearance and disappearance described the pharmacokinetics of stavudine triphosphate. The use of allometric scaling with fat-free mass characterized well the pharmacokinetics in both adults and children, and no other significant effect could be detected. Simulations of 30 mg twice daily in adults predicted median (interquartile range [IQR]) stavudine triphosphate minimum drug concentration (Cmin) and maximum drug concentration (Cmax) values of 13 (10 to 19) and 45 (38 to 53) fmol/106 cells, respectively. Targeting this exposure, simulations in HIV+ children were used to identify a suitable weight-band dosing approach (0.5 to 0.75 mg/kg), which was predicted to achieve median (IQR) Cmin and Cmax values of 13 (9 to 18) and 49 (40 to 58) fmol/106 cells, respectively. Weight-band dosing using a stavudine dose of 0.5 to 0.75 mg/kg is proposed, and it shows comparable exposures to adults receiving the current WHO-recommended dose of 30 mg twice daily. Our pharmacokinetic results suggest that the decreased stavudine dose in children >2 years would have a reduced toxic effect while retaining antiretroviral efficacy.

In vitro-in vivo Pharmacokinetic correlation model for quality assurance of antiretroviral drugs.

INTRODUCTION: The in vitro-in vivo pharmacokinetic correlation models (IVIVC) are a fundamental part of the drug discovery and development process. The ability to accurately predict the in vivo pharmacokinetic profile of a drug based on in vitro observations can have several applications during a successful development process. OBJECTIVE: To develop a comprehensive model to predict the in vivo absorption of antiretroviral drugs based on permeability studies, in vitro and in vivo solubility and demonstrate its correlation with the pharmacokinetic profile in humans. METHODS: Analytical tools to test the biopharmaceutical properties of stavudine, lamivudine y zidovudine were developed. The kinetics of dissolution, permeability in caco-2 cells and pharmacokinetics of absorption in rabbits and healthy volunteers were evaluated. RESULTS: The cumulative areas under the curve (AUC) obtained in the permeability study with Caco-2 cells, the dissolution study and the pharmacokinetics in rabbits correlated with the cumulative AUC values in humans. These results demonstrated a direct relation between in vitro data and absorption, both in humans and in the in vivo model. CONCLUSIONS: The analytical methods and procedures applied to the development of an IVIVC model showed a strong correlation among themselves. These IVIVC models are proposed as alternative and cost/effective methods to evaluate the biopharmaceutical properties that determine the bioavailability of a drug and their application includes the development process, quality assurance, bioequivalence studies and pharmacosurveillance.

INTRODUCCIÓN: Los modelos de correlación In vitro-in vivo (IVIVC) son parte integral del proceso de investigación y desarrollo de fármacos. La capacidad de predecir con exactitud el perfil in vivo a partir de las observaciones in vitro tiene diversas aplicaciones durante el desarrollo exitoso de una formulación. OBJETIVO: Desarrollar un modelo integral para predecir la absorción in vivo de fármacos antirretrovirales con base en estudios de permeabilidad, solubilidad in vitro e in vivo y demostrar su correlación con la farmacocinética en humanos. MÉTODOS: Se desarrollaron y validaron las técnicas bioanalíticas para valorar las propiedades biofarmacéuticas de Estavudina, Lamivudina y Zidovudina. Se evaluó las cineticas de disolución, la permeabilidad en monocapas celulares Caco-2 y la farmacocinética de absorción in vivo en conejos y voluntarios sanos. RESULTADOS: Los valores de AUC acumulados en el sistema de células Caco-2, en la disolución y en el modelo animal, fueron correlacionados con los valores de AUC acumulados en el humano. Con lo anterior se demostró una relación directamente proporcional entre los resultados in vitro con respecto a los obtenidos en la fase de absorción tanto en el humano como en el modelo animal. CONCLUSIONES: Los métodos analíticos y procedimientos aplicados en la IVIVC demostraron las correspondencias directas entre sí, con altos niveles de correlación. Se proponen estos modelos IVIVC como métodos alternativos costo/efectivos para la valoración de las propiedades biofarmacéuticas que determinan la biodisponibilidad, en el desarrollo de productos, en el aseguramiento de la calidad y como pruebas de bioequivalencia en los programas de farmacovigilancia.

In vitro-in vivo Pharmacokinetic correlation model for quality assurance of antiretroviral drugs / Modelo de correlación farmacocinético in vitro-in vivo para el aseguramiento de la calidad de medicamentos antirretrovirales

Introduction: The in vitro-in vivo pharmacokinetic correlation models (IVIVC) are a fundamental part of the drug discovery and development process. The ability to accurately predict the in vivo pharmacokinetic profile of a drug based on in vitro observations can have several applications during a successful development process. Objective: To develop a comprehensive model to predict the in vivo absorption of antiretroviral drugs based on permeability studies, in vitro and in vivo solubility and demonstrate its correlation with the pharmacokinetic profile in humans. Methods: Analytical tools to test the biopharmaceutical properties of stavudine, lamivudine y zidovudine were developed. The kinetics of dissolution, permeability in caco-2 cells and pharmacokinetics of absorption in rabbits and healthy volunteers were evaluated. Results: The cumulative areas under the curve (AUC) obtained in the permeability study with Caco-2 cells, the dissolution study and the pharmacokinetics in rabbits correlated with the cumulative AUC values in humans. These results demonstrated a direct relation between in vitro data and absorption, both in humans and in the in vivo model. Conclusions: The analytical methods and procedures applied to the development of an IVIVC model showed a strong correlation among themselves. These IVIVC models are proposed as alternative and cost/ effective methods to evaluate the biopharmaceutical properties that determine the bioavailability of a drug and their application includes the development process, quality assurance, bioequivalence studies and pharmacosurveillance.

Introducción: Los modelos de correlación In vitro-in vivo (IVIVC) son parte integral del proceso de investigación y desarrollo de fármacos. La capacidad de predecir con exactitud el perfil in vivo a partir de las observaciones in vitro tiene diversas aplicaciones durante el desarrollo exitoso de una formulación. Objetivo: Desarrollar un modelo integral para predecir la absorción in vivo de fármacos antirretrovirales con base en estudios de permeabilidad, solubilidad in vitro e in vivo y demostrar su correlación con la farmacocinética en humanos. Métodos: Se desarrollaron y validaron las técnicas bioanalíticas para valorar las propiedades biofarmacéuticas de Estavudina, Lamivudina y Zidovudina. Se evaluó las cineticas de disolución, la permeabilidad en monocapas celulares Caco-2 y la farmacocinética de absorción in vivo en conejos y voluntarios sanos. Resultados: Los valores de AUC acumulados en el sistema de células Caco-2, en la disolución y en el modelo animal, fueron correlacionados con los valores de AUC acumulados en el humano. Con lo anterior se demostró una relación directamente proporcional entre los resultados in vitro con respecto a los obtenidos en la fase de absorción tanto en el humano como en el modelo animal. Conclusiones: Los métodos analíticos y procedimientos aplicados en la IVIVC demostraron las correspondencias directas entre sí, con altos niveles de correlación. Se proponen estos modelos IVIVC como métodos alternativos costo/efectivos para la valoración de las propiedades biofarmacéuticas que determinan la biodisponibilidad, en el desarrollo de productos, en el aseguramiento de la calidad y como pruebas de bioequivalencia en los programas de farmacovigilancia.

Modified Release Biodegradable Polymeric Microspheres of Stavudine: Cell Viability, Cellular Uptake, Hemolysis Studies and In Vivo Pharmacokinetics.

Family of retroviruses which replicates through the use of the reverse transcriptase enzyme or the enzyme needed to convert RNA to DNA for replication bears Human Immunodeficiency Virus (HIV). It causes irreversible destruction of the immune system leading to the occurrence of opportunistic infections and malignancies. The eradication of HIV is highly unlikely as the cells of the mononuclear phagocyte system (MPS) besides CD4 T lymphocytes are the specific hosts for HIV which need to be targeted even after extended blood plasma profile of antiviral drug to maintain viral suppression and reduced disease progression. Aiming the current goal, biodegradable polymeric microspheres of PLGA 50: 50 (RESOMER(®) 505H) were developed and evaluated. These polymeric particles encapsulating Stavudine (d4T) exhibited nearly 100% cell viability during cytotoxicity studies in comparison to pure d4T. The histological studies have revealed the in vivo biocompatibility while hemolysis studies certified the liability of formulation to be used parenteraly exhibiting no significant hemolytic toxixicty. The in vivo pharmacokinetics has shown the extended drug release from microsphere matrix upto a month. The calculated AUCtotal for d4T loaded polymeric microspheres was found to be 3341.656 µM h/ml; which was nearly 54 times than the total AUC of free d4T injected subcutaneously to the control group of animals; exhibiting the stable d4T concentration in blood avoiding fluctuation of the same indicating decreased probabilities of development of resistance against the treatment. Combination of targeted and subcutaneous administration of d4T will not only provide the stable and extended release of drug but also eradicate the hidden HIV hosted by macrophages. The concomitant regimen will potentially enhance the therapeutic efficacy with patient compliance; renewing new hopes for complete cure and improved quality of life in the AIDS patient.

Effect of reducing the paediatric stavudine dose by half: a physiologically-based pharmacokinetic model.

Owing to significant dose-related toxicity, the adult stavudine dose was reduced in 2007. The paediatric dose, however, has not been reduced. Although the intended paediatric dose is 1 mg/kg twice daily (b.i.d.), the current weight-band dosing approach results in a mean actual dose of 1.23±0.47 mg/kg. Both efficacy and mitochondrial toxicity depend on the concentration of the intracellular metabolite stavudine triphosphate (d4T-TP). We simulated the effect of reducing the paediatric dose to 0.5 mg/kg. A physiologically-based pharmacokinetic model consisting of 13 tissue compartments plus a full ADAM model was used to describe the elimination of stavudine. The volume of distribution at steady-state and apparent oral clearance were simulated and the resulting AUC profile was compared with literature data in adult and paediatric populations. A biochemical reaction model was utilised to simulate intracellular d4T-TP levels for both the standard and proposed reduced paediatric doses. Simulated and observed exposure after oral dosing showed adequate agreement. Mean steady-state d4T-TP for 1.23 mg/kg b.i.d. was 27.9 (90% CI 27.0-28.9) fmol/10(6) cells, 25% higher than that achieved by the 40 mg adult dose. The 0.5 mg/kg dose resulted in d4T-TP of 13.2 (12.7-13.7) fmol/10(6) cells, slightly higher than the adult dose of 20 mg b.i.d. [11.5 (11.2-11.9) fmol/10(6) cells], which has excellent antiviral efficacy and substantially less toxicity. Current paediatric dosing may result in even higher d4T-TP than the original 40 mg adult dose. Halving the paediatric dose would significantly reduce the risk of mitochondrial toxicity without compromising antiviral efficacy.

Pharmacokinetics of nevirapine, stavudine and lamivudine in Indian HIV-infected children receiving generic fixed dose combinations.

OBJECTIVE: To determine the trough and two hour plasma levels of nevirapine, stavudine, and lamivudine when administered in fixed dose combinations (FDC). DESIGN: Cross sectional. SETTING: Tertiary care hospital in Northern India. PARTICIPANTS: 79 HIV-infected children receiving antiretroviral therapy with FDCs for more than month. INTERVENTION: Two-point sampling (0 and 2 hours after the morning dose). OUTCOME MEASURES: Plasma concentrations of all three drugs were simultaneously assayed by liquid chromatography/mass spectroscopy. RESULTS: Majority (77%) of children were receiving fixed dose combination of stavudine, lamivudine, nevirapine in the ratio of 6:30:50 mg. The median (IQR) trough and 2-hour plasma levels (µg/mL) of nevirapine, stavudine and lamivudine were 5.2 (4.0, 6.3) and 7.9 (6.0, 9.7); 0.1 (0.06, 0.16) and 1.1 (0.59, 1.6); 0.1 (0.02, 0.2) and 2.5 (1.4, 3.1), respectively. Very few children had sub-therapeutic plasma drug levels of stavudine (2.5%), lamivudine (7.6%) and nevirapine (10%). Inadequate viral suppression at 6 months follow up was significantly associated with initial high viral load, low CD4 percentage at the time of enrolment in study, and lower doses of lamivudine and stavudine. CONCLUSIONS: The currently available generic pediatric fixed dose antiretroviral combinations in India provide adequate drug exposure in majority of children.

Estimation of intracellular concentration of stavudine triphosphate in HIV-infected children given a reduced dose of 0.5 milligrams per kilogram twice daily.

The antiviral efficacy of stavudine depends on the trough concentration of its intracellular metabolite, stavudine-triphosphate (d4T-TP), while the degree of stavudine's mitochondrial toxicity depends on its peak concentration. Rates of mitochondrial toxicity are high when stavudine is used at the current standard pediatric dose (1 mg/kg twice daily [BID]). Evidence from adult work suggests that half of the original standard adult dose (i.e., 20 mg BID) may be equally effective, with markedly less mitochondrial toxicity. We present a population pharmacokinetic model to predict intracellular d4T-TP concentrations in pediatric HIV-infected patients administered a dose of 0.5 mg/kg BID. Our model predicted that the reduced pediatric dose would result in a trough intracellular d4T-TP concentration above that of the reduced 20-mg adult dose and a peak concentration below that of the 20-mg adult dose. The simulated pediatric intracellular d4T-TP at 0.5 mg/kg BID resulted in median peak and trough values of approximately 23.9 fmol/10(6) cells (95% prediction interval [PI], 14.2 to 41 fmol/10(6) cells) and 14.8 fmol/10(6) cells (95% PI, 7.2 to 31 fmol/10(6) cells), respectively. The peak and trough concentrations resulting from a 20-mg BID adult dose were 28.4 fmol/10(6) cells (95% PI, 17.3 to 45.5 fmol/10(6) cells) and 13 fmol/10(6) cells (95% PI, 6.8 to 28.6 fmol/10(6) cells), respectively. Halving the current standard pediatric dose should therefore not compromise antiviral efficacy, while markedly reducing mitochondrial toxicity.

Stavudine extended release (once-daily, Bristol-Myers Squibb) for the treatment of HIV/AIDS.

INTRODUCTION: Stavudine extended release (d4T XR) was a formulation which tried to solve the two main problems associated with the use of stavudine immediate release (d4T IR). These were twice daily dosing schema at a time when most formulations were long-life allowing once daily dosing; and that the use of d4T IR was associated with long-term toxicity through mitochondrial toxicity clinically expressed as peripheral neuropathy, pancreatitis and above all, lipodystrophy. The link between stavudine exposure and lipodystrophy had a great negative impact on its use in clinical practice. AREAS COVERED: The authors cover the most relevant papers related to the efficacy and safety of d4T XR-based antiretroviral therapy. EXPERT OPINION: The development of d4T XR has only been partially successful with regard to its objectives. Improved pharmacokinetic properties allow its once daily dosing, and although it exhibits less mitochondrial toxicity it is still hampered by its development in a significant proportion of patients. This has caused its use to be almost residual in industrialised countries. As of now, d4T XR has not been made available in developing countries, despite the extended use of the immediate-release formulation. Currently, if there is no other chance of starting combination antiretroviral therapy, d4T XR could play a role in the treatment of HIV infection.

Pharmacokinetics of nevirapine in HIV-infected infants weighing 3 kg to less than 6 kg taking paediatric fixed dose combination tablets.

OBJECTIVES: To evaluate pharmacokinetics of nevirapine, lamivudine and stavudine in HIV-infected Zambian infants receiving fixed dose combination (FDC) antiretroviral tablets (Triomune Baby). DESIGN: Phase I/II study. METHODS: Sixteen HIV-infected children at least 1 month, weighing 3 kg to less than 6 kg were enrolled. Blood was sampled at t = 0, 2, 6 and 12 h after observed intake of one FDC tablet (50 mg nevirapine, 6 mg stavudine, 30 mg lamivudine) 4 weeks after starting treatment. Safety and viral load response over 48 weeks were determined. RESULTS: The median [interquartile range (IQR)] age, body weight and daily nevirapine dose in 15 included children (eight girls) were 4.8 (4.2, 8.4) months, 5.3 (4.3, 5.5) kg and 348 (326 385) mg/m, respectively. The median (IQR) nevirapine area under the concentration-time curve (AUC0-12 h), Cmax and C12 h were 70 (56, 104) h mg/l, 7.5 (6.2, 10) mg/l, and 4.3 (2.9, 6.9) mg/l, respectively. Values were on average higher than reported in adults, but approximately 20% lower than previously reported in children weighing at least 6 kg. Four of 15 (27%) children had a subtherapeutic nevirapine C12 h (defined as <3.0 mg/l) compared to only three of 63 (5%) children weighing at least 6 kg (P = 0.02), whereas children aged less than 5 months [three of six (50%)] may have the highest risk for subtherapeutic nevirapine C12 h (P = 0.24). No association was found between viral load values and nevirapine plasma pharmacokinetic parameters (P > 0.3). Stavudine-lamivudine pharmacokinetic parameters were broadly comparable to heavier children. CONCLUSION: Exposure to nevirapine in African, HIV-infected infants with low body weight taking FDC tablets appears on average to be adequate, but due to large intersubject variability a relatively high proportion had subtherapeutic nevirapine C12 h levels, particularly those aged less than 5 months.

Stavudine concentrations in women receiving postpartum antiretroviral treatment and their breastfeeding infants.

First-line antiretroviral treatment regimens in resource-limited settings used in breastfeeding mothers often include stavudine (d4T). Limited data describing d4T concentrations in breast milk are available. We analyzed d4T concentrations in 52 mother-infant pairs using ultra-performance liquid chromatography-tandem mass spectrometry (lower limit of quantification: 5 ng/mL in plasma, 20 ng/mL in breast milk). Median (interquartile range) d4T concentrations were 86 (36-191) ng/mL in maternal plasma, 151 (48-259) ng/mL in whole milk, 190 (58-296) ng/mL in skim milk, and <5 (<5 to <5) ng/mL in infant plasma. Although d4T is concentrated in breast milk relative to maternal plasma, the infant d4T dose received from breast milk is very small and not clinically significant.

Exploring dried blood spot sampling technique for simultaneous quantification of antiretrovirals: lamivudine, stavudine and nevirapine in a rodent pharmacokinetic study.

A high-performance liquid chromatography/positive ion electrospray tandem mass spectrometry method for the simultaneous quantification of lamivudine, stavudine and nevirapine was developed and validated in dried blood spot (DBS) cards. The analytes were separated using an isocratic mobile phase on a reverse phase column and analyzed by MS/MS in the MRM mode using the respective [M + H]⁺ ions, m/z 230-112 for lamivudine, m/z 225-127 for stavudine, m/z 267-226 for nevirapine, m/z 383-337 for zidovudine (IS). The lower limit of quantification was 1 ng/mL for both lamivudine and stavudine and 10 ng/mL for nevirapine. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The method was successfully applied to quantify them in a rat pharmacokinetic study in whole blood, plasma and DBS cards after a single oral co-administration at the dose of 10, 2 and 13 mg/kg for lamivudine, stavudine and nevirapine, respectively, to male Wistar rats. Following oral administration the pharmacokinetic results in all the matrices are in close agreement. Thus accomplishment of this method would facilitate the ease of collection of clinical samples on DBS cards for lamivudine, stavudine and nevirapine during human clinical trials and therapeutic drug monitoring.

Biowaiver monographs for immediate-release solid oral dosage forms: stavudine.

Literature data relevant to the decision to allow a waiver of in vivo bioequivalence (BE) testing for the approval of immediate-release (IR) solid oral dosage forms containing stavudine (d4T) are reviewed. According to Biopharmaceutics Classification System (BCS), d4T can be assigned to BCS class I. No problems with BE of IR d4T formulations containing different excipients and produced by different manufacturing methods have been reported and, hence, the risk of bioinequivalence caused by these factors appears to be low. Furthermore, d4T has a wide therapeutic index. It is concluded that a biowaiver is appropriate for IR solid oral dosage forms containing d4T as the single active pharmaceutical ingredient (API) provided that (a) the test product contains only excipients present in the IR d4T drug products that have been approved in a number of countries for the same dosage form, and (b) both test product and its comparator are either "very rapidly dissolving" or "rapidly dissolving" with similarity of dissolution profiles demonstrated at pH 1.2, 4.5, and 6.8.

Bioequivalence of dispersed stavudine: opened versus closed capsule dosing.

BACKGROUND: Stavudine, a nucleoside reverse transcriptase inhibitor, is used commonly to treat HIV-infected children in the developing world. The paediatric liquid formulation presents major logistical difficulties in rural and resource-limited areas, and prescribers are frequently forced to employ off-label 'opened capsule' dosing methods using the adult capsule. The South African Department of Health (DoH) has advised that caregivers should be instructed to disperse the contents of an adult capsule in 5 ml water and then withdraw the required dose using a syringe. The bioavailability of stavudine using the opened capsule dosing method has not previously been validated. METHODS: This was a randomized crossover pharmacokinetic study with each subject serving as his/her own control. A total of 28 healthy HIV-negative adult volunteers were randomized on a 1:1 basis to receive one of the two generic preparations typically available in state hospitals. They were then further randomized to receive either intact or opened 30 mg capsules. After 1 week, those who initially received intact capsules, were given opened capsules and vice versa. The capsule dispersion technique used was identical to that prescribed by the DoH. Serial blood samples were collected and plasma stavudine concentrations were assayed by liquid chromatography tandem mass spectrometry. Stavudine pharmacokinetics were analysed using non-compartmental methods and formulations were compared using ANOVA. RESULTS: Plasma drug exposure after stavudine administration as a solution using an opened capsule dosing method was found to be bioequivalent to intact capsule administration. This was true for both generics tested. CONCLUSIONS: The opened capsule dosing technique is bioequivalent to intact capsule dosing for stavudine in HIV-seronegative adults.

Stavudine entrapped lipid nanoparticles for targeting lymphatic HIV reservoirs.

The main objective of present research study was to evaluate the potential of lipid nanoparticles for active delivery of an antiretroviral drug to lymphatic tissues. Stavudine entrapped drug loaded solid lipid nanoparticles (SLNs) were prepared and characterized for a variety of physicochemical parameters such as appearance, particle size, polydispersity index and zeta potential. The targeting potential of the prepared nanoparticles was investigated by carrying out ex vivo cellular uptake studies in macrophages which depicted several times enhanced uptake as compared to pure drug solution. Further, the lymphatic drug levels and organ distribution studies demonstrated efficiency of the developed nanoparticles for prolonged residence in spleenic tissues. Thus it was concluded that stavudine entrapped lipid carriers can be exploited for effective and targeted delivery to cellular and anatomical HIV reservoirs and may ultimately increase the therapeutic safety and reduce side effects.

Optimization of release kinetics from sustained-release formulations using model-independent pharmacokinetic simulation.

In the present work, a single model-independent approach was developed to optimize the release kinetics of drugs from sustained-release formulations, using stavudine (d4T) as a model drug. This approach is based on the pharmacokinetic simulation of drug plasma levels through a semiparametric approach of the input function and on convolution with an empirical polyexponential unit impulse response function. Input functions were evaluated using different zero-order and first-order release constants. Optimum drug release to obtain a specific pharmacokinetic profile was approached using target model-independent pharmacokinetic parameters such as C(max)(SS), C(min)(SS), t(max)(SS), and peak-trough fluctuations. A Monte Carlo simulation was performed to estimate the fractional attainment of d4T plasma concentrations over therapeutic d4T levels. Zero-order (K(0) = 4 mg/h) and first-order (K(1) = 0.05 h(-1)) release constants were optimal for the formulation of sustained-release d4T tablets, plasma concentrations within the therapeutic range being achieved.

Diastereoselective synthesis of cyclosaligenyl-nucleosyl-phosphotriesters.

A diastereoselective synthesis of cycloSal-phosphotriesters (cycloSal=cycloSaligenyl) based on chiral auxiliaries has been developed that allows the synthesis of single diastereomers of the cycloSal-pronucleotides. In previously described synthesis routes, the cycloSal-compounds were always obtained as 1:1 diastereomeric mixtures that could be separated in only rare cases. However, it was shown that the diastereomers have different antiviral activity, toxicity, and hydrolysis stabilities. Here, first a chiral thiazoline derivative was used to prepare nonsubstituted and 5-methyl-cycloSal-phosphotriesters in 48 and ≥95% de (de=diastereomeric excess). However, this approach failed to give the important group of 3-substituted cycloSal-nucleotides. Therefore, two other chiral groups were discovered that allowed the synthesis of (R(P))- and (S(P))-3-methyl-cycloSal-phosphotriesters as well. The antiviral activity was found to be five- to 20-fold different between the two individual diastereomers, which proved the importance of this approach.