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Human immunodeficiency virus 1 (HIV-1) infection is a global health issue since neither a cure nor a vaccine is available. However, the highly active antiretroviral therapy (HAART) has improved the life expectancy for patients with acquired immunodeficiency syndrome (AIDS). Nucleoside reverse transcriptase inhibitors (NRTIs) are in almost all HAART and target reverse transcriptase (RT), an essential enzyme for the virus. Even though NRTIs are highly effective, they have limitations caused by RT resistance. The main mechanisms of RT resistance to NRTIs are discrimination and excision. Understanding the molecular mechanisms for discrimination and excision are essential to develop more potent and selective NRTIs. Using protein X-ray crystallography, we determined the first crystal structure of RT in its post-catalytic state in complex with emtricitabine, (-)FTC or stavudine (d4T). Our structural studies provide the framework for understanding how RT discriminates between NRTIs and natural nucleotides, and for understanding the requirement of (-)FTC to undergo a conformation change for successful incorporation by RT. The crystal structure of RT in post-catalytic complex with d4T provides a "snapshot" for considering the possible mechanism of how RT develops resistance for d4T via excision. The findings reported herein will contribute to the development of next generation NRTIs.

SARS-CoV-2, a member of the coronavirus family, is responsible for the current COVID-19 worldwide pandemic. We previously demonstrated that five nucleotide analogues inhibit the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), including the active triphosphate forms of Sofosbuvir, Alovudine, Zidovudine, Tenofovir alafenamide and Emtricitabine. We report here the evaluation of a library of nucleoside triphosphate analogues with a variety of structural and chemical features as inhibitors of the RdRps of SARS-CoV and SARS-CoV-2. These features include modifications on the sugar (2' or 3' modifications, carbocyclic, acyclic, or dideoxynucleotides) or on the base. The goal is to identify nucleotide analogues that not only terminate RNA synthesis catalyzed by these coronavirus RdRps, but also have the potential to resist the viruses' exonuclease activity. We examined these nucleotide analogues for their ability to be incorporated by the RdRps in the polymerase reaction and to prevent further incorporation. While all 11 molecules tested displayed incorporation, 6 exhibited immediate termination of the polymerase reaction (triphosphates of Carbovir, Ganciclovir, Stavudine and Entecavir; 3'-OMe-UTP and Biotin-16-dUTP), 2 showed delayed termination (Cidofovir diphosphate and 2'-OMe-UTP), and 3 did not terminate the polymerase reaction (2'-F-dUTP, 2'-NH 2 -dUTP and Desthiobiotin-16-UTP). The coronaviruses possess an exonuclease that apparently requires a 2'-OH at the 3'-terminus of the growing RNA strand for proofreading. In this study, all nucleoside triphosphate analogues evaluated form Watson-Crick-like base pairs. The nucleotide analogues demonstrating termination either lack a 2'-OH, have a blocked 2'-OH, or show delayed termination. Thus, these nucleotide analogues are of interest for further investigation to evaluate whether they can evade the viral exonuclease activity. Prodrugs of five of these nucleotide analogues (Cidofovir, Abacavir, Valganciclovir/Ganciclovir, Stavudine and Entecavir) are FDA-approved medications for treatment of other viral infections, and their safety profiles are well established. After demonstrating potency in inhibiting viral replication in cell culture, candidate molecules can be rapidly evaluated as potential therapies for COVID-19., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Human Immunodeficiency Virus (HIV) damages the immune system and leads to the life-threatening acquired immunodeficiency syndrome (AIDS). Despite the advances in the field of antiretroviral treatment, HIV remains a major public health challenge. Nucleosides represent a prominent chemotherapeutic class for treating viruses, however their cellular uptake, kinase-mediated activation and catabolism are limiting factors. Herein, we report the synthesis and in vitro evaluation of stavudine (d4T) ProTides containing polyfluorinated aryl groups against two strains; HIV-1 (IIIB) and HIV-2 (ROD). ProTide 5d containing a meta-substituted pentafluorosulfanyl (3-SF 5 ) aryl group showed superior antiviral activity over the parent d4T and the nonfluorinated analogue 5a. ProTide 5d has low nanomolar antiviral activity; (IC 50  = 30 nM, HIV-1) and (IC 50  = 36 nM, HIV-2) which is over tenfold more potent than d4T. Interestingly, ProTide 5d showed a significantly high selectivity indices with SI = 1753 (HIV-1) and 1461 (HIV-2) which is more than twice that of the d4T. All ProTides were screened in wild type as well as thymidine kinase deficient (TK - ) cells. Enzymatic activation of ProTide 5d using carboxypeptidase Y enzyme and monitored using both 31 P and 19 F NMR is presented., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Stavudine (d4T, 2',3'-didehydro-2',3'-dideoxythymidine) was one of the first chain-terminating nucleoside analogs used to treat HIV infection. We present the first structure of the active, triphosphate form of d4T (d4TTP) bound to a catalytic complex of HIV-1 RT/dsDNA template-primer. We also present a new strategy for disulfide (S-S) chemical cross-linking between N 6 of a modified adenine at the second overhang base to I63C in the fingers subdomain of RT. The cross-link site is upstream of the duplex-binding region of RT, however, the structure is very similar to published RT structures with cross-linking to Q258C in the thumb, which suggests that cross-linking at either site does not appreciably perturb the RT/DNA structures. RT has a catalytic maximum at pH 7.5. We determined the X-ray structures of the I63C-RT/dsDNA/d4TTP cross-linked complexes at pH 7, 7.5, 8, 8.5, 9, and 9.5. We found small (~0.5 Å), pH-dependent motions of the fingers subdomain that folds in to form the dNTP-binding pocket. We propose that the pH-activity profile of RT relates to this motion of the fingers. Due to side effects of neuropathy and lipodystrophy, use of d4T has been stopped in most countries, however, chemical modification of d4T might lead to the development of a new class of nucleoside analogs targeting RNA and DNA polymerases., (© 2018 The Protein Society.)

The concentration of antiretroviral drugs in wastewater treatment plants (WWTP) effluents and surface waters of many countries has increased significantly due to their widespread use for HIV treatment. In this study, the removal of stavudine and zidovudine under UV 254 photolysis or UV 254 /H 2 O 2 was investigated in a microcapillary film (MCF) photoreactor, using minimal water samples quantities. The UV 254 quantum yield of zidovudine, (2.357 ± 0.0589)·10 -2  mol ein -1 (pH 4.0-8.0), was 28-fold higher that the yield of stavudine (8.34 ± 0.334)·10 -4  mol ein -1 (pH 6.0-8.0). The second-order rate constant k OH,i of reaction of hydroxyl radical with the antiretrovirals (UV 254 /H 2 O 2 process) were determined by kinetics modeling: (9.98 ± 0.68)·10 8  M -1  s -1 (pH 4.0-8.0) for zidovudine and (2.03 ± 0.18)·10 9  M -1  s -1 (pH 6.0-8.0) for stavudine. A battery of ecotoxicological tests (i.e. inhibition growth, bioluminescence, mutagenic and genotoxic activity) using bacteria (Aliivibrio fischeri, Salmonella typhimurium), crustacean (Daphnia magna) and algae (Raphidocelis subcapitata) revealed a marked influence of the UV dose on the ecotoxicological activity. The UV 254 /H 2 O 2 treatment process reduced the ecotoxicological risk associated to direct photolysis of the antiretrovirals aqueous solutions, but required significantly higher UV 254 doses (≥2000 mJ cm -2 ) in comparison to common water UV disinfection processes., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

The objectives of this research work were to develop optimized nanoparticulate formulations of poly (d,l-lactic-co-glycolic acid) (PLGA) (85:15) with an anti-AIDS drug stavudine and to evaluate their in-vitro uptake by the macrophages and hepatotoxicity in-vivo. Nanoparticles were prepared by nanoprecipitation method based on a factorial design with varying parameters such as the amounts of polymer and stabilizer used. Physicochemical characterizations such as drug-excipient interaction, surface morphology, particle size, and zeta potential measurements were carried out. The best formulation was selected and tagged with fluorescein isothiocyanate (FITC) for cellular uptake study of the formulation. In-vitro uptake of nanoparticles by macrophages was carried out. Formulation-induced hepatotoxicity was assessed by analyzing some serum hepatotoxic parameters and hepatic histology following 10-day treatment in comparison with the free drug. Nanoparticles exhibited smooth surface with particle size 84-238 nm, high entrapment efficiency (approx 85%), and negative surface charge. Formulations showed a sustained drug release pattern over the study period. In-vitro uptake study by macrophages exhibited a time-dependent profile. In-vivo studies on rats showed improvement in the serum parameters and maintenance of the integrity of the hepatic architecture indicating decreased hepatotoxicity with the formulations as compared to the free drug. The experimental results showed a positive outcome in the development of antiretroviral drug carrier exhibiting sustained drug release, macrophage-targeted delivery characteristics, and having reduced hepatoxicity. This could be beneficial for the management of early stage of HIV infection besides reducing the drug load for effective treatment, thereby offering an attractive option in AIDS therapy.

The human immunodeficiency virus (HIV) continues to be a global pandemic and there is an urgent need for innovative treatment. Immune cells represent a major target of virus infection, but are also therapeutic targets. Currently, no antiretroviral therapy targets macrophages, which function as portal of entry and as major long-term deposit of HIV. It has been shown before that human macrophages efficiently internalize gold nanoparticles, a fact which might be used to target them with drug-nanoparticle conjugates. Here, the authors use gold nanocarriers to facilitate delivery of stavudine, a widely used antiretroviral drug, to primary human macrophages. Using an ease-of-use coupling method, a striking potentiation of stavudine intake by macrophages using gold nanocarriers is shown. Further, the carriers induce a specific subtype of proinflammatory activation indicative for antiviral activity of macrophages, which suggests promising novel treatment options for HIV., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

When thymidine was treated with hypobromous acid (HOBr) in 100 mM phosphate buffer at pH 7.2, two major product peaks appeared in the HPLC chromatogram. The products in each peak were identified by NMR and MS as two isomers of 5-hydroxy-5,6-dihydrothymidine-6-phosphate (a novel compound) and two isomers of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol) with comparable yields. 5-Hydroxy-5,6-dihydrothymidine-6-phosphate was relatively stable, and decomposed with a half-life of 32 h at pH 7.2 and 37°C generating thymidine glycol. The results suggest that 5-hydroxy-5,6-dihydrothymidine-6-phosphate in addition to thymidine glycol may have importance for mutagenesis by the reaction of HOBr with thymine residues in nucleotides and DNA.

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.

The use of pronucleotides to circumvent the well-known drawbacks of nucleotide analogs has played a significant role in the area of antiviral and anticancer drug delivery. Several motifs have been designed to mask the negative charges on the phosphorus moiety of either nucleoside monophosphates or nucleoside phosphonates, in order to increase their hydrophobicity and allow entry of the compound into the cell. Among them the bis-amidate analogs, having two identical amino acids as masking groups through a P-N bond, represent a more recent approach for the delivery of nucleotide analogs, endowed with antiviral or anticancer activity. Different synthetic strategies are commonly used for preparing phosphorodiamidates of nucleosides. In this protocol, we would like to focus on the description of the synthetic methodology that in our hand gave the best results using 2'-3'-didehydro-2'-3'-dideoxythymidine (d4T, Stavudine) as model nucleoside. A second strategy for preparing diamidates of nucleoside phosphonates will be reported using {[2-(6-amino-9 H-purin-9-yl)ethoxy]methyl}phosphonic acid (PMEA, adefovir) as model substrate., (Copyright © 2015 John Wiley & Sons, Inc.)

An efficient, stereoselective synthesis of 4'-Ed4T is demonstrated. The synthesis is highlighted by a regioselective TMSOTf-mediated acetal opening, a Claisen rearrangement to set the key 4'-stereocenter as well as the olefin, and a one-pot nonaflation/elimination to deliver the alkyne moiety. The synthesis proceeds in eight steps from 5-methyluridine and occurs in 37% overall yield.

Background: To improve in vitro antiviral activity and selectivity of stavudine (d4T), a range of its bi-functional prodrugs, 5'-O-myristoylated derivatives, have been synthesized., Methods: Stavudine 5'-O-myristoylated esters were synthesized using modified Parang's procedure. The cytotoxicity and anti-HIV activity was evaluated in the established MT-4 cell line. The level of p24 protein in culture medium was assayed, and EC50 and EC90 values were determined., Results: Excellent anti-HIV activity was obtained for stavudine derivatives 2',3'-didehydro-2',3'-dideoxy-5'-O-(11-thioethylundecanoyl) thymidine, 2',3'-didehydro-2',3'-dideoxy-5'-O-(12-thioethyldodecanoyl) thymidine and 5'-O-(12-azidododecanoyl)-2',3'-didehydro-2',3'-dideoxythymidine with C10 and C11 alkyl chains bearing thioethyl- and azido- substituents. These prodrugs were more potent than the parent stavudine, as is clear from their EC50 values: 2',3'-didehydro-2',3'-dideoxy-5'-O-(11-thioethylundecanoyl) thymidine (R=CO(CH2)10SC2H5, EC50 0.06 μM), 2',3'-didehydro-2',3'-dideoxy-5'-O-(12-thioethyldodecanoyl) thymidine (R=CO(CH2)11SC2H5, EC50 0.09 μM) and 5'-O-(12-azidododecanoyl)-2',3'-didehydro-2',3'-dideoxythymidine (R=CO(CH2)11N3, EC50 0.06 μM), while 50% cytotoxic concentration was >16.65 μM, >7.5 μM and >18.53 μM, respectively., Conclusions: Overall data demonstrate that compounds 2',3'-didehydro-2',3'-dideoxy-5'-O-(11-thioethylundecanoyl) thymidine, 2',3'-didehydro-2',3'-dideoxy-5'-O-(12-thioethyldodecanoyl) thymidine and 5'-O-(12-azidododecanoyl)-2',3'-didehydro-2',3'-dideoxythymidine are very potent and selective anti-HIV agents and could be useful in treatment of HIV infections of the central nervous system.

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.

The binding of stavudine (STV) to two human blood proteins [human hemoglobin (HHb) and human serum albumin (HSA)] was studied in vitro under simulated physiological conditions by spectroscopic methods viz., fluorescence, UV absorption, resonance light scattering, synchronous fluorescence, circular dichroism (CD) and three-dimensional fluorescence. The binding parameters of STV-blood protein were determined from fluorescence quenching studies. Stern-Volmer plots indicated the presence of static quenching mechanism in the interaction of STV with blood proteins. The values of n close to unity indicated that one molecule of STV bound to one molecule of blood protein. The binding process was found to be spontaneous. Analysis of thermodynamic parameters revealed the presence of hydrogen bond and van der Waals forces between protein and STV. Displacement experiments indicated the binding of STV to Sudlow's site I on HSA. Secondary structures of blood proteins have undergone changes upon interaction with STV as evident from the reduction of α-helices (from 46.11% in free HHb to 38.34% in STV-HHb, and from 66.44% in free HSA to 52.26% in STV-HSA). Further, the alterations in secondary structures of proteins in the presence of STV were confirmed by synchronous and 3D-fluorescence spectral data. The distance between the blood protein (donor) and acceptor (STV) was found to be 5.211 and 5.402 nm for STV-HHb and STV-HSA, respectively based on Föster's non-radiative energy transfer theory. Effect of some metal ions was also investigated. The fraction of STV bound to HSA was found to be 87.8%.

The interaction between stavudine, a nucleoside reverse transcriptase inhibitor and human serum albumin (HSA), was investigated by fluorescence quenching technique and isothermal titration calorimetry (ITC). A good linearity of albumin fluorescence quenching in the presence of stavudine was determined. Analyzing these data we obtained for the dissociation constant the value K(d)=(18.18 ± 0.46) × 10(-5)M. However, due to contradictory results obtained in ITC experiments, we checked the fluorescence quenching data for the inner-filter effect, the main confounding factor in the observed quenching. Based on the UV-vis absorption data we have corrected the observed fluorescence intensities and concluded, in accordance with ITC results, that stavudine binding to HSA is negligible and the observed quenching effect is entirely caused by a failure to correct for the inner-filter effect., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A novel and efficient method for the preparation of antiviral nucleoside 5'-H-phosphonamidates has been developed. The oxidization of the H-phosphonamidate intermediates with iodine and sulfur afforded nucleoside 5'-phosphoramidates and 5'-thiophosphoramidates in high yields.

The interaction of an anti-HIV drug, stavudine (STV) with calf thymus deoxyribonucleic acid (DNA) was investigated employing acridine orange (AO) as a fluorescence probe. Spectroscopic investigations revealed the intercalative mode of binding of STV to DNA. The analysis of fluorescence data indicated the presence of static quenching mechanism between STV and DNA. Thermodynamic parameters indicated the presence of van der Waals forces in addition to intercalative mode of binding. CD data revealed the partial B → A conformational transition of DNA upon intercalative mode of binding with STV.

Branched sugar nucleosides have attracted much attention due to their biological activities. We have demonstrated that epoxysugar nucleosides serve as versatile precursor for the stereo-defined synthesis of these nucleoside derivatives on the basis of its ring opening with organoaluminum or organosilicon reagents. In this review article, novel methods for the synthesis of nucleoside analogues branched at the 1' and 4'-position will be described. During this study, we could discover an anti-HIV agent, 4'-ethynylstavudine (Festinavir). Festinavir showed more potent anti-HIV activity than the parent compound stavudine (d4T). Other significant properties of Festinavir are as follows: 1) much less toxic to various cells and also to mitochondorial DNA synthesis than d4T, 2) better substrate for human thymidine kinase than d4T, 3) resistant not only to chemical glycosidic bond cleavage but also to catabolism by thymidine phosphorylase, 4) the activity improves in the presence of a major mutation, K103N, associated with resistance to non-nucleoside reverse transcriptase inhibitors. Detailed profile of the antiviral activities, biology and pharmacology of Festinavir are also described.

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., (Copyright © 2012 John Wiley & Sons, Ltd.)

Methyl-substituted cycloSal-pronucleotides of d4TMP were synthesized with high diastereoselectivities in satisfying chemical yields. The individual diastereomers were tested against HIV-1 and HIV-2 infected wild-type CEM/0 and HIV-2 infected thymidine kinase deficient CEM cells. All diastereomers tested showed significant antiviral activity in CEM/0 and strong activity in CEM/TK(-) cell cultures. The antiviral activities were strongly dependent on the chirality at the phosphate group and the position of the methyl-group(s) in the cycloSal moiety. In CEM/TK(-) cell cultures the difference in antiviral potency was found to be 7- to 20-fold. The stability of each diastereomer was studied in aqueous phosphate buffer and in CEM/0 cell extracts. Large differences in the half-lives were found. A comparison of the relative lipophilicity of the methyl-substituted cycloSal triesters was performed based on the retention times obtained by reversed phase HPLC. The results obtained clearly confirm the importance of a diastereoselective synthesis of cycloSal-pronucleotides.

This study investigates the capability of methylmethacrylate-sulfopropylmethacrylate (MMA-SPM) nanoparticles (NPs) with grafted RMP-7 (RMP-7/MMA-SPM NPs) to deliver stavudine (D4T), delavirdine (DLV), and saquinavir (SQV) across the blood-brain barrier (BBB). The permeability coefficients of the three drugs across the BBB were evaluated by a co-culture model containing human brain-microvascular endothelial cells and human astrocytes. An increase in the concentration of ammonium persulfate (APS), the polymerization initiator, enhanced the particle size of drug-loaded RMP-7/MMA-SPM NPs. When the concentration of APS was 0.6%, the average particle diameter was smaller than 50 nm. These spherical drug carriers were uniform in size and displayed a dominant topography of discrete hillocks and deep pits in deposited film. Smaller RMP-7/MMA-SPM NPs yielded a larger drug loading efficiency. The order of drug in the loading efficiency and in the particle uptake was, respectively, D4T>DLV>SQV and D4T>SQV>DLV. Endocytosis of RMP-7/MMA-SPM NPs and tight junction mediation can improve the permeability of D4T, DLV, and SQV across the BBB., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: We describe the development, evaluation, and comparison of colloidal gold-loaded, poly(d,l-lactic-co-glycolic acid)-based nanoparticles containing anti-acquired immunodeficiency syndrome drug stavudine and uptake of these nanoparticles by macrophages in vitro., Methods: WE USED THE FOLLOWING METHODS IN THIS STUDY: drug-excipient interaction by Fourier transform infrared spectroscopy, morphology of nanoparticles by field-emission scanning electron microscopy, particle size by a particle size analyzer, and zeta potential and polydispersity index by a zetasizer. Drug loading and in vitro release were evaluated for formulations. The best formulation was incorporated with fluorescein isothiocyanate. Macrophage uptake of fluorescein isothiocyanate nanoparticles was studied in vitro., Results: Variations in process parameters, such as speed of homogenization and amount of excipients, affected drug loading and the polydispersity index. We found that the drug was released for a prolonged period (over 63 days) from the nanoparticles, and observed cellular uptake of stavudine nanoparticles by macrophages., Conclusion: Experimental nanoparticles represent an interesting carrier system for the transport of stavudine to macrophages, providing reduced required drug dose and improved drug delivery to macrophages over an extended period. The presence of colloidal gold in the particles decreased the drug content and resulted in comparatively faster drug release.

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., (Copyright © 2011 Wiley-Liss, Inc.)

Solid lipid nanoparticles (SLNs) with complex internal phase were fabricated for formulating stavudine (D4T), delavirdine (DLV), and saquinavir (SQV). The lipids including Compritol 888 ATO, tripalmitin, and cacao butter were stabilized by L-α-phospatidylcholine, cholesteryl hemisuccinate, and taurocholate to form SLNs. The results revealed that the morphology of SLNs was spheroidal with shallow surface pits. An increase in the weight percentage of Compritol 888 ATO increased the average diameter of D4T-entrapping SLNs and decreased that of DLV- and SQV-entrapping SLNs. Preservation at 4°C over 6 weeks slightly enhanced the size of SLNs. For a specific drug, an increase in the entrapment efficiency enlarged the nanocarriers. The order of drug in the average particle diameter and in the entrapment efficiency was SQV>DLV>D4T, in general. In addition, the dissolution of the three drugs from SLNs showed the characteristics of sustained release. The order of drug in the cumulative release percentage was D4T>DLV>SQV. SLNs containing Compritol 888 ATO, tripalmitin, and cacao butter are efficient in carrying antiretroviral agents for medicinal application., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The production of stavudine-loaded solid lipid nanoparticles (SLN) for intravenous injection was scaled up from lab scale (40 g) to medium scale (10 kg) and large scale (20/60 kg). The SLN were produced by high pressure homogenization of stavudine lipid melt dispersed in hot surfactant solution (pre-emulsion) applying 800 bar pressure. Employed were piston-gap homogenizers with increasing capacity (APV Gaulin products LAB 40, LAB 60 and Gaulin 5.5, and Avestin C50), using them in the continuous (circulation) and discontinuous mode. Size analysis was performed by photon correlation spectroscopy (PCS), laser diffractometry and light microscopy. At lab scale a PCS size of 53 nm was obtained. At the same pressure, all homogenizers on larger scale yielded a size in the range of the lab scale product (35-70 nm). Differences were found in the size as a function of circulation time (size increase or size reduction with time) and the number of cycles required (1 or 5) for the optimal product. The stavudine SLN formulation (2% lipid content, high surfactant to lipid ratio) showed a different behavior to conventional higher concentrated SLN suspensions or nanoemulsions (10% or 20% lipid/oil, low surfactant to lipid ratio). In general, smallest sizes were obtained in the discontinuous mode after just one homogenization cycle. The continuous production mode was only efficient with a 10 kg batch size using the LAB 60. In addition, the long-term stability over 1 year was monitored at refrigeration, room temperature and at 40°C to assess a potential effect of the homogenizer type on stability. All batches at room temperature and below were stable, only a negligible increase in size was observed., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Wiley-Liss, Inc.)

Present research investigates the formulation of stavudine loaded biodegradable microspheres from different grades of Poly (D, L Lactide-co-glycolide) as a depot system for parenteral delivery. Prolonged release of stavudine facilitates reduction in symptoms of HIV infection and delay AIDS progression by reducing viral load to undetectable levels. Microspheres were prepared from PLGA 85:15 and PLGA 50:50 (RESOMER(®) 505H) by solvent evaporation technique with different drug/polymer ratios (1:4, 1:10, 1:20, 1:50, 1:100) and a polymer solution/vehicle ratio of 1:2. The effects of various formulation variables like polymer type and concentration, surfactant concentration and drug to polymer ratio on the characteristics of microspheres were evaluated. All thirteen formulations of microspheres were evaluated for yield, entrapment efficiency, particle size and In vitro release studies. Microspheres were characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffraction (XRD), residual solvent analysis and confocal laser scanning microscopy (CLSM). Microspheres showed excellent surface topography with uniform distribution and structural integrity of the drug. Resulting microspheres showed the maximum entrapment efficiency of 68.0 ± 1.62% and mean particle diameter below 100µ. Drug release kinetics data were obtained from various kinetic models and best explained by "Higuchi Kinetic" i.e. drug release was largely governed by diffusion through water-filled pores in the matrix. Korsmeyer-Peppas equation depicted that drug release mechanism is anomalous transport, i.e. diffusion as well as polymer relaxation. Drug release from microspheres exhibited the characteristic release pattern of a monolithic matrix system with a maximum of 80-90% drug release in 6-8 weeks demonstrating the feasibility of prolonged delivery of stavudine using biodegradable microspheres for parenteral depot system.

Comprehensive conformational analysis of 2',3'-didehydro-2',3'-dideoxythymidine (d4T), also known as anti-AIDS drug stavudine, has been performed for the first time at the MP2/6-311++G(d,p)//DFT B3LYP/6-31++G(d,p) level of the theory. It was established that d4T energy landscape contained 19 local minima, which corresponded to stable conformers. Eight types of specific intramolecular interactions, which govern the d4T conformational properties, were identified, namely: O5'H-O2, C1'H'-O2, C6H-O5', C6H-O4', C5'H1'-O2, C5'H2'-O2, C6H-H1'C5', C2'-O2. The obtained results confirm the actual point of view that d4T biological activity is, most likely, connected with termination of the DNA chain synthesis in the 5'-3' direction. Thus, d4T competes with canonical thymidine in binding an active site of HIV-1 reverse transcriptase.

Single amino acid deletions in the β3-β4 hairpin loop of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been identified in heavily treated patients. The deletion of Asp-67 together with mutations T69G and K70R (Δ67 complex) are usually associated with thymidine analog resistance mutations (TAMs) (e.g. M41L, T215Y, etc.) while the deletion of Thr-69 (Δ69) is rarely found in isolates containing TAMs. Here, we show that the complex Δ67/T69G/K70R enhances ATP-dependent phosphorolytic activity on primers terminated with 3'-azido-3'-deoxythymidine (AZT) or 2',3'-didehydro-2',3'-dideoxythymidine (d4T) both in the presence or absence of TAMs (i.e. M41L/T215Y), while Δ69 (or the complex S68G/Δ69/K70G) antagonize the effects of TAMs in ATP-mediated excision. These effects are consistent with AZT susceptibility data obtained with recombinant HIV-1 bearing the relevant RTs. Molecular dynamics studies based on models of wild-type HIV-1 RT and mutant Δ69, Δ67/T69G/K70R, and D67N/K70R RTs support a relevant role for Lys/Arg-70 in the excision reaction. In Δ69 RT, the side chain of Lys-70 locates away from the putative pyrophosphate binding site. Therefore, its participation in interactions required for the excision reaction is unlikely. Our theoretical studies also suggest a role for Lys-219 in thymidine analog excision/discrimination. However, pre-steady-state kinetics revealed only minor differences in selectivity of AZT-triphosphate versus dTTP between deletion-containing RTs and their homologous enzymes having the K219E mutation. K219E reduced both ATP- and pyrophosphate-mediated excision of primers terminated with thymidine analogues, only when introduced in RTs bearing Δ69 or S68G/Δ69/K70G, providing further biochemical evidence that explains the lack of association of Δ69 and TAMs in HIV-1 isolates.

A number of 5'-O-fatty acyl derivatives of 2',3'-didehydro-2',3'-dideoxythymidine (stavudine, d4T) were synthesized and evaluated for anti-HIV activities against cell-free and cell-associated virus, cellular cytotoxicity, and cellular uptake studies. The conjugates were found to be more potent than d4T. Among these conjugates, 5'-O-12-azidododecanoyl derivative of d4T (2), displaying EC(50) = 3.1-22.4 μM, showed 4- to 9-fold higher activities than d4T against cell-free and cell-associated virus. Cellular uptake studies were conducted on CCRF-CEM cell line using 5(6)-carboxyfluorescein derivatives of d4T attached through β-alanine (9) or 12-aminododecanoic acid (10) as linkers. The fluorescein-substituted analog of d4T with long chain length (10) showed 12- to 15-fold higher cellular uptake profile than the corresponding analog with short chain length (9). These studies reveal that conjugation of fatty acids to d4T enhances the cellular uptake and anti-HIV activity of stavudine., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This paper presents the development and validation of an improved method for the simultaneous analysis of lamivudine (LVD), stavudine (STV) and nevirapine (NVP) using high-performance thin-layer chromatography (HPTLC) with densitometric detection. Separation was performed on silica gel 60F(254) plates. The mobile phase is comprised of ethylacetate, methanol, toluene and concentrated ammonia (38.7:19.4:38.7:3.2, v:v:v:v). Detection wavelength was 254 nm. The R(f) values were 0.24±0.03, 0.38±0.04 and 0.69±0.04 (n=8) for LVD, STV and NVP, respectively. An F-test indicated that calibration graphs were adequately linear at the evaluated concentration ranges. The pooled %RSD for repeatability of the percentage amount recovered for LVD, STV and NVP were found to be 0.62, 0.54, and 0.79, and the pooled %RSD for time-different intermediate precision were 1.66, 1.27 and 1.21. The percentage recoveries for the trueness were 99.2%±1.5 for LVD, 98.6%±1.5 for STV and 99.3%±1.7 for NVP (n=3). Most factors evaluated in the robustness test were found to have an insignificant effect on the selected responses at 95% confidence level. This method was successfully used to analyze fixed-dose tablets samples of LVD, STV and NVP., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Several amino acid phosphorodiamidate derivatives of d4T as anti-HIV prodrugs were synthesized and investigated using electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)). A novel methyl group migration in gas phase was observed in ESI-MS(2) of the sodium adducts of amino acid methyl ester of phosphorodiamidates of 2',3'-didehydro-2',3'-dideoxythymidine (d4T). The proposed structures of the rearrangement ions were confirmed by high resolution tandem mass spectrometry. A possible mechanism involving the pentacoordinate phosphoric-carboxylic phosphate anhydride was proposed, in which a seven-membered ring intermediate was formed by coordination with the metal ion between the phosphoryl group and carbonyl oxygen atom. Thus, the intrinsic properties of phosphoryl group might be the key factors responsible for this migration.

The first diastereoselective synthesis of aryloxy phosphoramidate prodrugs of 3'-deoxy-2',3'-didehydrothymidine monophosphate (d4TMP) is reported. In our approach, (S)-4-isopropylthiazolidine-2-thione 1 was used as a chiral auxiliary to introduce the stereochemistry at the phosphorus atom. In the last step of the developed reaction sequence, the nucleoside analogue d4T was introduced to a stereochemically pure phosphordiamidate which led to the formation of the almost diastereomerically pure phosphoramidate prodrugs 8a-d (≥95% de). As expected, the individually prepared diastereomers of the phosphoramidate prodrugs showed significant differences in the antiviral activity. Moreover, the difference was strongly dependent on the aryl substituent attached to the phosphoramidate moiety.

Preparation and spectroscopic properties of novel boron-containing derivatives of anti-HIV agent stavudine are presented, The new compounds, (5'-O-(4,4,5,5-tetramethyl-1,3,2-dioxaboronate)-2'-3'-didehydro-2'-3'-dideoxythymidine and 5'-O-(dihydroxyboronate)-2'-3'-didehydro-2'-3'-dideoxythymidine), were prepared by direct reaction between stavudine and reagents containing BH moieties - pinacolborane and borane-dimethylsulfide complexes, respectively. The boron coordination equilibrium of those compounds was analyzed by water titration monitored by NMR. Results of the DFT calculations and NMR experiments pointed to structural and electronic similarity of tetrahedral boron complexes to phosphate group., (2009 Elsevier Inc. All rights reserved.)

A comparative theoretical conformational analysis of the tautomerism of the four most stable conformers of the antiviral analogue D4T (stavudine) and natural thymidine (Thy) nucleosides was carried out, by using the B3LYP and MP2 quantum chemical methods. The calculated structure data and energy values of the tautomers were compared with the T1 keto form and with the results determined for the thymine molecule. For each conformer, only two stable enol forms, T3 and T5, were obtained when considering different positions of hydrogen around the base. Tautomer T3 always appears more stable than T5, and more stable in the natural nucleoside Thy than in thymine or in D4T.The effect of water on the tautomers was estimated by using an explicit number of up to five water molecules to surround the nucleoside, and also by Tomasi's polarized continuum model (PCM). A total of about 200 clusters were optimised and the geometrical parameters and energies discussed. The water net differs in the three tautomers of D4T and Thy. Depending on the nature of the tautomers, cyclic, distributed water molecules, or clustered structures are formed. The deformation and interaction counterpoise (CP)-corrected energies between the nucleoside and water molecules were determined. The relative stabilities of all tautomers were established. The microhydrated environment stabilizes remarkably the enol forms more than the canonical keto one, although this one continues being the most stable. The changes in the intramolecular H-bondings and in the total atomic charges were discussed. Intramolecular H-bonds being stronger in D4T than in Thy could indicate a higher molecular flexibility for Thy.

Val(75) of HIV-1 reverse transcriptase (RT) plays a role in positioning the template nucleotide +1 during the formation of the ternary complex. Mutations, such as V75M and V75A, emerge in patients infected with HIV-1 group M subtype B and group O variants, after failing treatment with stavudine (d4T) and other nucleoside RT inhibitors. V75I is an accessory mutation of the Q151M multidrug resistance complex of HIV-1 RT and is rarely associated with thymidine analogue resistance mutations (TAMs). In vitro, it confers resistance to acyclovir. TAMs confer resistance to zidovudine (AZT) and d4T by increasing the rate of ATP-mediated excision of the terminal nucleotide monophosphate (primer unblocking). In a wild-type HIV-1 group O RT sequence context, V75A and V75M conferred increased excision activity on d4T-terminated primers, in the presence of PP(i). In contrast, V75I decreased the PP(i)-mediated unblocking efficiency on AZT and d4T-terminated primers, in different sequence contexts (i.e. wild-type group M subtype B or group O RTs). Interestingly, in the sequence context of an excision-proficient RT (i.e. M41L/A62V/T69SSS/K70R/T215Y), the introduction of V75I led to a significant decrease of its ATP-dependent excision activity on AZT-, d4T-, and acyclovir-terminated primers. The excision rate of d4T-monophosphate in the presence of ATP (3.2 mm) was about 10 times higher for M41L/A62V/T69SSS/K70R/T215Y than for the mutant M41L/A62V/T69SSS/K70R/V75I/T215Y RT. The antagonistic effect of V75I with TAMs was further demonstrated in phenotypic assays. Recombinant HIV-1 containing the M41L/A62V/T69SSS/K70R/V75I/T215Y RT showed 18.3- and 1.5-fold increased susceptibility to AZT and d4T, respectively, in comparison with virus containing the M41L/A62V/T69SSS/K70R/T215Y RT.

The objective of this study was to synthesize derivatives of the anti-HIV drug stavudine (d4T) with more favourable physicochemical properties for transdermal delivery in an effort to increase transdermal penetration of stavudine and thus reduce the severe side effects associated with the dose-dependent oral therapy. The synthesis, hydrolytic stability, and in vitro human skin permeation flux of a series of novel methoxypoly(ethylene glycol) (MPEG) carbonates of stavudine are reported. The carbonates were synthesized in a two-step process by coupling the MPEG promoiety of various chain lengths to C-5' of d4T. In kinetic studies the carbonates proved to be markedly stable in weakly acidic phosphate medium (pH 5.0) with half-lives ranging from 16 to 58 days. The aqueous solubility increased as the ethylene oxide chain lengthened. However, there was no significant increase in the estimated solubility in octanol. In vitro in the phosphate buffer (200 mM; pH 5.0) almost all carbonates permeate the human skin. However, the most effective penetrant, the derivative with 3 ethylene oxide units in the side chain, exhibited a flux of 26.1 nmol/cm(2)/h as compared to 59.15 nmol/cm(2)/h of the parent drug stavudine. Thus, no permeation enhancement was observed during this study.

Stavudine is a nucleoside reverse transcriptase inhibitor active against HIV, and is known to exist in two polymorphic forms designated as forms I and II, and a hydrate form III. An amorphous solid of stavudine was successfully prepared and characterized during this investigation. A comprehensive evaluation of the stability of this amorphous solid showed that the amorphous solid transforms to either form II (anhydrous) or form III (hydrate) when exposed to temperature, in the absence or presence of moisture, respectively. The amorphous-to-hydrate transformation occurred at relatively low RH (>32%) and led to the formation of crystal aggregates of the hydrated form. Steady state growth rate analyses also showed that the amorphous-to-crystalline transformation occurs at a greater rate in the presence of moisture, compared to the transformation at the same temperature in a dry environment. Crystal growth studies showed that it is possible to stabilize the amorphous solid of stavudine against crystal transformations in the absence of moisture by coating it with poly(methyl methacrylate). However, this polymer coating could not prevent crystal growth from the amorphous solid during exposure to moisture.

The synthesis of a new family of D4T analogues is described to study the influence of pyrazinone base on antiretroviral power. Substitution of 3H by methyl or n-decyl increases the lipophilic character and may facilitate diffusion across cell membranes. The compounds were characterized by (1)H NMR and infrared spectroscopy. Antiviral (HIV-1) properties of these compounds were examined.

The study of the dissociation of the protonated molecular species [M+H](+) and selected fragment ions allowed proposals for the main fragmentation pathways of the title compounds. The main fragments are formed by expelling a molecule of thymine, thymidine (d4T) or isopropyl. The most striking feature of the tandem mass (MS/MS) spectra is the cleavage of C-CO bonds between N-methylpyrrole and carbonyl groups in the presence of the amidine. Electrospray ionization is proven to be a good method for the structural characterization and identification of these kinds of compounds., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

A comparative theoretical analysis on the effect of the solvent on the molecular structure and energetics of the most stable conformers of the nucleoside analogue D4T (stavudine) and of the natural nucleoside thymidine (Thy) was carried out. Solvent effects were considered using the Tomasi's polarized continuum model (PCM) and including a variable number (1-13) of explicit water molecules surrounding the nucleoside in order to simulate the first hydration shell. More than 200 cluster structures with water were analyzed. B3LYP and MP2 quantum chemical methods were used. The CP-corrected interaction energies for D4T and water molecules were computed. For cases where literature data are available, the computed values were in good agreement with previous experimental and theoretical studies. In the isolated state, conformer I (anti-gg-gg) appears the most stable for D4T molecule and conformer II (anti-gg-gt) for Thy molecule. In D4T with eight water molecules, conformer II changes to conformer I. Thus, conformer I seems preferred when water molecules are situated in the first hydration shell. However, in hydrated thymidine conformer Ia (anti-gg-tg) is the more stable one, and the first hydration shell is more extended than in D4T molecule. The effect of the hydration on the total atomic charges and intermolecular distances were also discussed. Several general conclusions on hydrogen bonds network and involved interaction energies were underlined.

In our attempt to further develop the cycloSal pronucleotide concept, we report on 5-(1-acetoxyvinyl)-cycloSal-d4TMPs as a new type of enzyme-activated pronucleotides. These compounds were converted into 5-acetyl-cycloSal-d4TMPs by (carboxy)esterase cleavage inside the cells. The enzymatic reaction led to the formation of a strong electron-withdrawing substituent that strongly accelerates the chemical hydrolysis of the cycloSal nucleotide to give d4TMP. For some cycloSal-d4TMPs a separation into the diastereomers was achieved. The absolute configuration was assigned by correlation of circular dichroism spectra with similar compounds. Most of the compounds showed complete retention of antiviral activity in TK-deficient CEM/TK(-) cells, which proves the TK-bypass potential of this approach. Interestingly, (S(P))-isomers of cycloSal phosphate triesters showed better antiviral activity in HIV-2-infected thymidine-kinase deficient CEM/TK(-) cells than their (R(P))-counterparts.

Recently we reported on conceptually new enzymatically activated cycloSal-pronucleotides. Now, we developed this concept further with new compounds of this type. The basic idea is fast intracellular cleavage of a functionalized group at the cycloSal residue that results in a rapid delivery of the nucleotide and thus an intracellular enrichment of the nucleotide. The introduction of a higher alkylated acylal group, the di- iso-butyryloxymethyl group, to the aromatic ring led to the expected higher stability of these prodrugs against enzymatic cleavage but also entailed surprisingly a decrease in hydrolysis stabilities and solubility problems. For some compounds, a separation of the two diastereomeric forms ( R P or S P) was achieved. By X-ray structure analysis, the absolute configuration at the P-atom was assigned. For all separated diastereomers the ( S P) form showed better antiviral activity than the ( R P) form.

Nucleoside-based antiviral drugs have been synthesized using imidazolium-based ionic liquids as reaction medium. The ionic liquids were proved to be better solvents for all the nucleoside in terms of solubility and reaction medium as compared to conventional molecular solvents.

2',3'-Didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a recently discovered nucleoside reverse transcriptase (RT) inhibitor, exhibits 5- to 10-fold-higher activity against human immunodeficiency virus type 1 (HIV-1) and less cytotoxicity than does its parental compound d4T (stavudine). Using steady-state kinetic approaches, we have previously shown that (i) 4'-ethynyl-d4T triphosphate (4'-Ed4TTP) inhibits HIV-1 RT more efficiently than d4TTP does and (ii) its inhibition efficiency toward the RT M184V mutant is threefold less than that toward wild-type (wt) RT. In this study we used pre-steady-state kinetic approaches in an attempt to understand its mechanism of inhibition. With wt and the M184V mutant RTs, 4'-Ed4TTP has three- to fivefold-lower K(d) (dissociation constant) values than d4TTP, while d4TTP has up to eightfold-higher K(d) values than dTTP. Inhibition is more effective in DNA replication with RNA template than with DNA template. In general, the M184V mutant exhibits poorer binding for all three nucleoside triphosphates than does wt RT. The structural basis for the lower binding affinity of d4TTP than of dTTP could be the lack of hydrogen bonds from the missing 3'-hydroxyl group in d4TTP to the backbone amide of Y115 and also to the side chain of Q151. The structural basis for the higher binding affinity of 4'-Ed4TTP than of d4TTP could be the additional binding of the 4'-ethynyl group in a preformed hydrophobic pocket by A114, Y115, M184, F160, and part of D185.

The objective of the present study was to coprocess 2 active pharmaceutical ingredients (APIs), nevirapine (NVP) and stavudine (STV), by spray drying technique to overcome the respective problems of poor solubility and poor content uniformity. The coprocessed product (NVP-STV CP) and untreated APIs were characterized by powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), particle size, surface area analysis, compressibility, and solubility. Coprocessing enhanced NVP solubility by approximately 1.5 fold and provided uniform distribution of low-dose STV in the formulation composite. Phase solubility studies elucidated the mechanism of enhanced NVP solubility. The coprocessed product was stable under accelerated stability conditions of 40 degrees C/75% relative humidity (RH) for 3 months. The coprocessed product was formulated into 3 drug fixed dose combination (FDC) tablets with lamivudine (LMV), which gave an enhanced in vitro NVP drug release compared with the control formulation. Spray drying as a coprocessing technique optimally utilized the individual components of the antiretroviral FDC tablets and synergistically enhanced the performance attributes.