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The pursuit of novel therapeutics is a complex and resource-intensive endeavor marked by significant challenges, including high costs and low success rates. In response, drug repositioning strategies leverage existing FDA-approved compounds to predict their efficacy across diverse diseases. Peptidyl arginine deiminase 4 (PAD4) plays a pivotal role in protein citrullination, a process implicated in the autoimmune pathogenesis of rheumatoid arthritis (RA). Targeting PAD4 has thus emerged as a promising therapeutic approach. This study employs computational and enzyme inhibition strategies to identify potential PAD4-targeting compounds from a library of FDA-approved drugs. In silico docking analyses validated the binding interactions and orientations of screened compounds within PAD4's active site, with key residues such as ASP350, HIS471, ASP473, and CYS645 participating in crucial hydrogen bonding and van der Waals interactions. Molecular dynamics simulations further assessed the stability of top compounds exhibiting high binding affinities. Among these compounds, Saquinavir (SQV) emerged as a potent PAD4 inhibitor, demonstrating competitive inhibition with a low IC50 value of 1.21 ± 0.04 µM. In vitro assays, including enzyme kinetics and biophysical analyses, highlighted significant changes in PAD4 conformation upon SQV binding, as confirmed by circular dichroism spectroscopy. SQV induced localized alterations in PAD4 structure, effectively occupying the catalytic pocket and inhibiting enzymatic activity. These findings underscore SQV's potential as a therapeutic candidate for RA through PAD4 inhibition. Further validation through in vitro and in vivo studies is essential to confirm SQV's therapeutic benefits in autoimmune diseases associated with dysregulated citrullination., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Acquired immunodeficiency syndrome (AIDS) is one of the most challenging infectious diseases to treat on a global scale. Understanding the mechanisms underlying the development of drug resistance is necessary for novel therapeutics. HIV subtype C is known to harbor mutations at critical positions of HIV aspartic protease compared to HIV subtype B, which affects the binding affinity. Recently, a novel double-insertion mutation at codon 38 (L38HL) was characterized in HIV subtype C protease, whose effects on the interaction with protease inhibitors are hitherto unknown. In this study, the potential of L38HL double-insertion in HIV subtype C protease to induce a drug resistance phenotype towards the protease inhibitor, Saquinavir (SQV), was probed using various computational techniques, such as molecular dynamics simulations, binding free energy calculations, local conformational changes and principal component analysis. The results indicate that the L38HL mutation exhibits an increase in flexibility at the hinge and flap regions with a decrease in the binding affinity of SQV in comparison with wild-type HIV protease C. Further, we observed a wide opening at the binding site in the L38HL variant due to an alteration in flap dynamics, leading to a decrease in interactions with the binding site of the mutant protease. It is supported by an altered direction of motion of flap residues in the L38HL variant compared with the wild-type. These results provide deep insights into understanding the potential drug resistance phenotype in infected individuals., Competing Interests: The authors declare no conflicts of interest.

Human immunodeficiency virus-1 (HIV-1) protease is one of the important targets in AIDS therapy. The majority of HIV infections are caused due to non-B subtypes in developing countries. The co-occurrence of mutations along with naturally occurring polymorphisms in HIV-1 protease cause resistance to the FDA approved drugs, thereby posing a major challenge in the treatment of antiretroviral therapy. In this work, the resistance mechanism against SQV due to active site mutations G48V and V82F in CRF01_AE (AE) protease was explored. The binding free energy calculations showed that the direct substitution of valine at position 48 introduces a bulkier side chain, directly impairing the interaction with SQV in the binding pocket. Also, the intramolecular hydrogen bonding network of the neighboring residues is altered, indirectly affecting the binding of SQV. Interestingly, the substitution of phenylalanine at position 82 induces conformational changes in the 80's loop and the flap region, thereby favoring the binding of SQV. The V82F mutant structure also maintains similar intramolecular hydrogen bond interactions as observed in AE-WT.Communicated by Ramaswamy H. Sarma.

Hypothesis: The aim of this study was the development of nanostructured lipid carriers (NLCs) decorated with a polycationic cell-penetrating peptide (CPP). A coating with polyphosphates (PP) enables charge conversion at target cells being triggered by the membrane bound enzyme intestinal alkaline phosphatase (IAP)., Experiments: The CPP, stearyl-nona-L-arginine (R9SA) was obtained by solid phase synthesis. Formed nanocarriers were characterized regarding size, polydispersity index, zeta potential and charge conversion in the presence of IAP and on Caco-2 cells. The BCS class IV drug saquinavir (SQV) was loaded into NLCs in different concentrations. Mucus diffusion ability of the NLCs was evaluated by the rotating tube method. Furthermore, cellular uptake was evaluated on Caco-2 cells and endosomal escape properties were investigated using erythrocytes., Findings: All NLCs were obtained in a size range between 146 nm and 152 nm and a polydispersity index of 0.2. Incubation of PP coated PP-R9SA-NLCs with IAP led to a charge conversion from -41.8 mV to 6.4 mV (Δ48.2 mV). After four hours of incubation with IAP, phosphate release reached a plateau, indicating a faster polyphosphate cleavage than on Caco-2. Drug load and encapsulation efficiency of SQV was obtained up to 80.6% and 46.5 µg/mg. Mucus diffusion was increasing in the following rank order: R9SA-NLCs < blank NLCs < PP-R9SA-NLCs. R9SA-NLCs and PP-R9SA-NLCs increased the cellular uptake 15.6- and 13.2-fold, respectively, compared to the control NLCs. Erythrocytes interaction study revealed enhanced endosomal escape properties for R9SA-NLCs and PP-R9SA-NLCs when incubated with IAP., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

There is a need of computational tools to rank bRo5 drug candidates in the very early phases of drug discovery when chemical matter is unavailable. In this study, we selected three compounds: (a) a Ro5 drug (Pomalidomide), (b) a bRo5 orally available drug (Saquinavir), and (c) a polar PROTAC (CMP 98) to focus on computational access to physicochemical properties. To provide a benchmark, the three compounds were first experimentally characterized for their lipophilicity, polarity, IMHBs, and chameleonicity. To reproduce the experimental information content, we generated conformer ensembles with conformational sampling and molecular dynamics in both water and nonpolar solvents. Then we calculated Rgyr, 3D PSA, and IMHB number. An innovative pool of strategies for data analysis was then provided. Overall, we report a contribution to close the gap between experimental and computational methods for characterizing bRo5 physicochemical properties.

Developing novel drug molecules against HIV is a scientific quest necessitated by development of drug resistance against used drugs. We report comparative results of molecular dynamics simulation studies on 11 structural analogues of Saquinavir (SQV) - against HIV-protease that were earlier examined for pharmacodynamic and pharmacokinetic properties. We reported analogues S1, S5 and S8 to qualify the ADMET criterion and may be considered as potential lead molecules. In this study the designed molecules were successively docked with native HIV-protease at AutoDock. Docking scores established relative goodness of the 11 analogues against the benchmark for Saquinavir. The docked complexes were subjected to molecular dynamics simulation studies using GROMACS 4.6.2. Four parameters viz. H-bonding, RMSD, Binding energy and Protein-Ligand Distance were used for comparative analyses of the analogues relative to Saquinavir. The comparison and analysis of the results are indicative that analogues S8, S9 and S1 are promising candidates among all the analogues studied. From our earlier work and present study it is evident that among the three S8 and S1 qualify the ADMET criterion and between S1 and S8, the analogue S8 shows more target efficacy and specificity over S1 and have better molecular dynamics simulation results. Thus, of the 11 de novo Saquinavir analogues, the S8 appears to be the most promising candidate as lead molecule for HIV-protease inhibitor and is best suited for testing under biological system. Further validation of the proposed lead molecules through wet lab studies involving antiviral assays however is required.Communicated by Ramaswamy H. Sarma.

Drug-resistance-associated mutation in essential proteins of the viral life cycle is a major concern in anti-retroviral therapy. M46I, a non-active site mutation in HIV-1 protease has been clinically associated with saquinavir resistance in HIV patients. A 100 ns molecular dynamics (MD) simulation and MM-PBSA calculations were performed to study the molecular mechanism of M46I-mutation-based saquinavir resistance. In order to acquire deeper insight into the drug-resistance mechanism, the flap curling, closed/semi-open/open conformations, and active site compactness were studied. The M46I mutation significantly affects the energetics and conformational stability of HIV-1 protease in terms of RMSD, RMSF, Rg, SASA, and hydrogen formation potential. This mutation significantly decreased van der Waals interaction and binding free energy (∆G) in the M46I-saquinavir complex and induced inward flap curling and a wider opening of the flaps for most of the MD simulation period. The predominant open conformation was reduced, but inward flap curling/active site compactness was increased in the presence of saquinavir in M46I HIV-1 protease. In conclusion, the M46I mutation induced structural dynamics changes that weaken the protease grip on saquinavir without distorting the active site of the protein. The produced information may be utilized for the discovery of inhibitor(s) against drug-resistant HIV-1 protease.

The aim of this study was to evaluate the effect of lipid digestion on the permeability and absorption of orally administered saquinavir (SQV), a biopharmaceutics classification system (BCS) class IV drug, in different lipid-based formulations. Three LBFs were prepared: a mixed short- and medium-chain lipid-based formulation (SMCF), a medium-chain lipid-based formulation (MCF), and a long-chain lipid-based formulation (LCF). SQV was loaded into these LBFs at 26.7 mg/g. To evaluate the pharmacokinetics of SQV in vivo , drug-loaded formulations were predispersed in purified water at 3% w/w and orally administered to rats. A low dose (0.8 mg/rat) was employed to limit confounding effects on drug solubilization, and consistent with this design, presolubilization of SQV in the LBFs did not increase in vivo exposure compared to a control suspension formulation. The areas under the plasma concentration-time curve were, however, significantly lower after administration of SQV as MCF and LCF compared to SMCF. To evaluate the key mechanisms underpinning absorption, each LBF containing SQV was digested, and the flux of SQV from the digests across a dialysis membrane was evaluated in in vitro permeation experiments. This study revealed that the absorption profiles were driven by the free concentration of SQV and that this varied due to differences in SQV solubilization in the digestion products generated by LBF digestion. The apparent first-order permeation rate constants of SQV ( k app,total ) were estimated by dividing the flux of SQV in the dialysis membrane experiments by the concentration of total SQV on the donor side. k app,total values strongly correlated with in vivo AUC. The data provide one of the first studies of the effect of digestion products on the free concentration of a drug in the GI fluid and oral absorption. This simple permeation model may be a useful tool for the evaluation of the impact of lipid digestion on apparent drug permeability from lipid-based formulations. These effects should be assessed alongside, and in addition to, the more well-known effects of lipids on enhancing intestinal solubilization of poorly water-soluble drugs.

Aims: To predict potential drugs for COVID-19 by using molecular docking for virtual screening of drugs approved for other clinical applications., Background: SARS-CoV-2 is the betacoronavirus responsible for the COVID-19 pandemic. It was listed as a potential global health threat by the WHO due to high mortality, high basic reproduction number, and lack of clinically approved drugs and vaccines. The genome of the virus responsible for COVID-19 has been sequenced. In addition, the three-dimensional structure of the main protease has been determined experimentally., Objective: To identify potential drugs that can be repurposed for treatment of COVID-19 by using molecular docking based virtual screening of all approved drugs., Methods: A list of drugs approved for clinical use was obtained from the SuperDRUG2 database. The structure of the target in the apo form, as well as structures of several target-ligand complexes, were obtained from RCSB PDB. The structure of SARS-CoV-2 Mpro determined from X-ray diffraction data was used as the target. Data regarding drugs in clinical trials for COVID-19 was obtained from clinicaltrials.org. Input for molecular docking based virtual screening was prepared by using Obabel and customized python, bash, and awk scripts. Molecular docking calculations were carried out with Vina and SMINA, and the docked conformations were analyzed and visualized with PLIP, Pymol, and Rasmol., Results: Among the drugs that are being tested in clinical trials for COVID-19, Danoprevir and Darunavir were predicted to have the highest binding affinity for the Main protease (Mpro) target of SARS-CoV-2. Saquinavir and Beclabuvir were identified as the best novel candidates for COVID-19 therapy by using Virtual Screening of drugs approved for other clinical indications., Conclusion: Protease inhibitors approved for treatment of other viral diseases have the potential to be repurposed for treatment of COVID-19., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Among targets selected for studies aimed at identifying potential inhibitors against COVID-19, SARS-CoV2 main proteinase (M pro ) is highlighted. M pro is indispensable for virus replication and is a promising target of potential inhibitors of COVID-19. Recently, monomeric SARS-CoV2 M pro , drug repurposing, and docking methods have facilitated the identification of several potential inhibitors. Results were refined through the assessment of dimeric SARS-CoV2 M pro , which represents the functional state of enzyme. Docking and molecular dynamics (MD) simulations combined with molecular mechanics/generalized Born surface area (MM/GBSA) studies indicated that dimeric M pro most significantly impacts binding affinity tendency compared with the monomeric state, which suggests that dimeric state is most useful when performing studies aimed at identifying drugs targeting M pro . In this study, we extend previous research by performing docking and MD simulation studies coupled with an MM/GBSA approach to assess binding of dimeric SARS-CoV2 M pro to 12 FDA-approved drugs (darunavir, indinavir, saquinavir, tipranavir, diosmin, hesperidin, rutin, raltegravir, velpatasvir, ledipasvir, rosuvastatin, and bortezomib), which were identified as the best candidates for the treatment of COVID-19 in some previous dockings studies involving monomeric SARS-CoV2 M pro . This analysis identified saquinavir as a potent inhibitor of dimeric SARS-CoV2 M pro ; therefore, the compound may have clinical utility against COVID-19. Graphical abstract.

Background: The COVID-19 has now been declared a global pandemic by the World Health Organization. There is an emergent need to search for possible medications., Method: Utilization of the available sequence information, homology modeling, and in slico docking a number of available medications might prove to be effective in inhibiting the SARS-CoV-2 two main drug targets, the spike glycoprotein, and the 3CL protease., Results: Several compounds were determined from the in silico docking models that might prove to be effective inhibitors for SARS-CoV-2. Several antiviral medications: Zanamivir, Indinavir, Saquinavir, and Remdesivir show potential as and 3CL PRO main proteinase inhibitors and as a treatment for COVID-19., Conclusion: Zanamivir, Indinavir, Saquinavir, and Remdesivir are among the exciting hits on the 3CL PRO main proteinase. It is also exciting to uncover that Flavin Adenine Dinucleotide (FAD) Adeflavin, B2 deficiency medicine, and Coenzyme A, a coenzyme, may also be potentially used for the treatment of SARS-CoV-2 infections. The use of these off-label medications may be beneficial in the treatment of the COVID-19., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This study investigated the dissolution behavior of BCS class II ionizable weak base Saquinavir and its mesylate salt in the multi-compartment transfer setup employing different composition of dissolution media. The dissolution behavior of Saquinavir was studied by using a two-compartment transfer model representing the transfer of drug from the stomach (donor compartment) to the upper intestine (acceptor compartment). Various buffers like phosphate, bicarbonate, FaSSIF, and FeSSIF were employed. The dissolution was also studied in the concomitant presence of the additional solute, i.e., Quercetin. Further, the dissolution profiles of Saquinavir and its mesylate salt were simulated by GastroPlus TM , and the simulated dissolution profiles were compared against the experimental ones. The formation of in situ HCl salt and water-soluble amorphous phosphate aggregates was confirmed in the donor and acceptor compartments of the transfer setup, respectively. As the consequence of the lower solubility product of HCl salt of Saquinavir, the solubility advantage of mesylate salt was vanished leading to the lower than the predicted dissolution in the acceptor compartment. However, the formation of water-soluble aggregates in the presence of the phosphate salts was observed leading to the higher than the predicted dissolution of the free base in the transfer setup. Interestingly, the formation of such water-soluble aggregates was found to be hindered in the concomitant presence of an ionic solute resulting in the lower dissolution rates. The in situ generation of salts and aggregates in the transfer model lead to the inconsistent prediction of dissolution profiles by GastroPlus TM .

Garlic has been used as a traditional medicine to treat various diseases. Garlic reduces the risk of some diseases. This protective effect is due to the organosulfur compounds of garlic. The aim of this study was to investigate the inhibition effects of garlic-derived compounds on human immunodeficiency virus type 1 (HIV-1) and as the most important anti-HIV-1 medicine. The activation of saquinavir is believed to be the principal mechanism behind the protective effects of HIV-1. Our theoretical calculations are performed for blood phase by using the density functional theory for the main compounds of garlic. The chemical activity and solubility of ajoene and the mainly derived compounds of garlic as theoretical calculations are important for the medical research comparing with the other compounds of the garlic. The theoretical calculations have helped us to determine which active ingredient of the garlic having inhibition effects on HIV-1 and saquinavir., (© 2018 Wiley Periodicals, Inc.)

Retroviral protease inhibitors (RPIs) such as lopinavir (LP) and saquinavir (SQ) are active against Plasmodium parasites. However, the exact molecular target(s) for these RPIs in the Plasmodium parasites remains poorly understood. We hypothesised that LP and SQ suppress parasite growth through inhibition of aspartyl proteases. Using reverse genetics approach, we embarked on separately generating knockout (KO) parasite lines lacking Plasmepsin 4 (PM4), PM7, PM8, or DNA damage-inducible protein 1 (Ddi1) in the rodent malaria parasite Plasmodium berghei ANKA. We then tested the suppressive profiles of the LP/Ritonavir (LP/RT) and SQ/RT as well as antimalarials; Amodiaquine (AQ) and Piperaquine (PQ) against the KO parasites in the standard 4-day suppressive test. The Ddi1 gene proved refractory to deletion suggesting that the gene is essential for the growth of the asexual blood stage parasites. Our results revealed that deletion of PM4 significantly reduces normal parasite growth rate phenotype (P = 0.003). Unlike PM4&#95;KO parasites which were less susceptible to LP and SQ (P = 0.036, P = 0.030), the suppressive profiles for PM7&#95;KO and PM8&#95;KO parasites were comparable to those for the WT parasites. This finding suggests a potential role of PM4 in the LP and SQ action. On further analysis, modelling and molecular docking studies revealed that both LP and SQ displayed high binding affinities (-6.3 kcal/mol to -10.3 kcal/mol) towards the Plasmodium aspartyl proteases. We concluded that PM4 plays a vital role in assuring asexual stage parasite fitness and might be mediating LP and SQ action. The essential nature of the Ddi1 gene warrants further studies to evaluate its role in the parasite asexual blood stage growth as well as a possible target for the RPIs., Competing Interests: The authors have declared that no competing interests exist.

FISLE-412 is the first reported small molecule peptidomimetic that neutralizes anti-dsDNA autoantibodies associated with systemic lupus erythematosus (SLE) pathogenesis. FISLE-412 is a complex small molecule that involves a challenging synthesis scheme, but has attractive pharmacological activities as a potential small molecule therapeutic in lupus. Therefore, we initiated a Structure-Activity Relationship study to simplify the complexity of FISLE-412. We synthesized a small library of mimetopes around the FISLE-412 structure and identified several analogues which could neutralize anti-DNA lupus antibodies in vitro and ex vivo. Our strategies reduced the structural complexity of FISLE-412 and provide important information that may guide development of potential autoantibody-targeted lupus therapeutics., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lipid-polymer hybrid nanoparticles (NPs) are advantageous for drug delivery. However, their intracellular trafficking mechanism and relevance for oral drug absorption are poorly understood. In this study, self-assembled core-shell lipid-polymer hybrid NPs made of poly(lactic-co-glycolic acid) (PLGA) and various lipids were developed to study their differing intracellular trafficking in intestinal epithelial cells and their relevance for oral absorption of a model drug saquinavir (SQV). Our results demonstrated that the endocytosis and exocytosis of hybrid NPs could be changed by varying the kind of lipid. A glyceride mixture (hybrid NPs-1) decreased endocytosis but increased exocytosis in Caco-2 cells, whereas the phospholipid (E200) (hybrid NPs-2) decreased endocytosis but exocytosis was unaffected as compared with PLGA nanoparticles. The transport of hybrid NPs-1 in cells involved various pathways, including caveolae/lipid raft-dependent endocytosis, and clathrin-mediated endocytosis and macropinocytosis, which was different from the other groups of NPs that involved only caveolae/lipid raft-dependent endocytosis. Compared with that of the reference formulation (nanoemulsion), the oral absorption of SQV-loaded hybrid NPs in rats was poor, probably due to the limited drug release and transcytosis of NPs across the intestinal epithelium. In conclusion, the intracellular processing of hybrid NPs in intestinal epithelia can be altered by adding lipids to the NP. However, it appears unfavorable to use PLGA-based NPs to improve oral absorption of SQV compared with nanoemulsion. Our findings will be essential in the development of polymer-based NPs for the oral delivery of drugs with the purpose of improving their oral absorption., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background and Objectives: Although lipid-based drug delivery systems have gained much importance in recent years due to their ability to improve the solubility and bioavailability of poorly soluble drugs, compartmental pharmacokinetic analyses have not been extensively explored. The oral pharmacokinetics of commercial liquid formulation and a developed semisolid system containing saquinavir mesylate (SQVM) were compared in Beagle dogs. A compartmental analysis after intravenous bolus administration of this drug (1 mg/kg) was also performed., Method: Pharmacokinetic profiles were analyzed using both non-compartmental and compartmental approaches. Plasma concentration of the drug was determined by high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS)., Results: The disposition curve of SQVM given intravenously was better described by a three-compartment model. In contrast, plasma profiles obtained following the oral administration were fitted to a two-compartment model with lag time due to the fact that the distribution phase was masked by the absorption phase in these formulations., Conclusion: The proposed semisolid lipid system was found to be a promising formulation for commercial purposes given the similarity of SQVM absorption rate to that from the commercial liquid formulation.

Aim: Saquinavir (SQV) is the first protease inhibitor for the treatment of HIV infection, but with poor solubility. The aim of this study was to prepare a colloidal nanocrystal suspension for improving the oral absorption of SQV., Methods: SQV nanocrystals were prepared using anti-solvent precipitation-high pressure homogenization method. The nanocrystals were characterized by a Zetasizer and transmission electron microscopy (TEM). Their dissolution, cellular uptake and transport across the human colorectal adenocarcinoma cell line (Caco-2) monolayer were investigated. Bioimaging of ex vivo intestinal sections of rats was conducted with confocal laser scanning microscopy. Pharmacokinetic analysis was performed in rats administered nanocrystal SQV suspension (50 mg/kg, ig), and the plasma SQV concentrations were measured with HPLC., Results: The SQV nanocrystals were approximately 200 nm in diameter, with a uniform size distribution. The nanocrystals had a rod-like shape under TEM. The dissolution, cellular uptake, and transport across a Caco-2 monolayer of the nanocrystal formulation were significantly improved compared to those of the coarse crystals. The ex vivo intestinal section study revealed that the fluorescently labeled nanocrystals were located in the lamina propria and the epithelium of the duodenum and jejunum. Pharmacokinetic study showed that the maximal plasma concentration (Cmax) was 2.16-fold of that for coarse crystalline SQV suspension, whereas the area under the curve (AUC) of nanocrystal SQV suspension was 1.95-fold of that for coarse crystalline SQV suspension., Conclusion: The nanocrystal drug delivery system significantly improves the oral absorption of saquinavir.

Although anti-HIV-1 protease drugs nelfinavir (NFV) and saquinavir (SQV) share common functional groups, D30N is a major resistance mutation against NFV but remains susceptible to SQV. We have determined the crystal structure of D30N mutant-tethered HIV-1 protease in complex with SQV to 1.79 Å resolution. Structural analysis showed that SQV forms two direct hydrogen bonds with the main chain atoms of the residues Asp29 and Asp30 that are not observed in the D30N-NFV complex. Apart from maintaining these two main chain hydrogen bonds, the P2-asparagine of SQV forms an additional hydrogen bond to the mutated side chain of the residue 30. These could be the reasons why D30N is not a drug resistance mutation against SQV. This structure supports the previous studies showing that the interactions between a potential inhibitor and backbone atoms of the enzyme are important to maintain potency against drug-resistant HIV-1 protease., (© 2014 John Wiley & Sons A/S.)

Modulation of CYP3A and/or P-gp function by several excipients has been reported. However, relatively few studies have investigated their effects in humans. Therefore, the aim of this clinical study was to clarify the effects of Cremophor EL on the inhibition of CYP3A and P-gp in the human small intestine. Eight healthy Japanese subjects received an oral dose of saquinavir (2 mg, substrate of P-gp/CYP3A) or fexofenadine (50 μg, substrate of P-gp) without or with Cremophor EL (720 mg and 1440 mg). Significant increases in Cmax (1.3-fold) and AUC0-24 (1.6-fold) were observed for fexofenadine when administered with 1440 mg of Cremophor EL. In contrast, a significant decrease was observed for saquinavir when administered with 720 mg of Cremophor EL. The equilibrium dialysis experiment was performed to investigate the micellar interaction between Cremophor EL and drugs. The equilibrium dialysis study showed that saquinavir was far extensively entrapped into the micelles. The reduced concentration of free saquinavir by entrapping in micelles was considered to cause the reduction of systemic exposure for saquinavir. In conclusion, this clinical study suggests that Cremophor EL at least inhibits P-gp in the human small intestine., (Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Purpose: Intestinal lymphatic transport of specific lipophilic drugs offers therapeutic advantages and maximises oral bioavailability. The aims of this study were; to compare intestinal lymphatic transport of a range of drugs and to investigate the influence of cyclosporine A on the mechanism/extent of lymphatic transport., Methods: Caco2 cells and an anaesthetised mesenteric lymphatic cannulated rat model were used for in vitro and in vivo studies. Lymphatic transport of three lipophilic drugs was directly compared in a long chain fatty acid formulation. In addition, the impact of cyclosporine A on triglyceride turnover was evaluated in vivo and in vitro., Results: The extent of intestinal lymphatic transport in rats was positively correlated with drug solubility in triglyceride and negatively correlated with drug aqueous solubility. Cyclosporine A displayed non-linear lymphatic transport kinetics and reduced intestinal lymph triglyceride. In vitro experiments indicated that the cellular processes affected were intracellular lipid processing and/or lipid secretion., Conclusions: The linear correlations obtained using a range of lipophilic drugs confirm that the simplified approach of determining aqueous or triglyceride drug solubility is useful in predicting the extent of lymphatic transport. In vitro experiments correlated with in vivo observations, demonstrating the usefulness of the Caco-2 model for mechanistic investigations.

A useful method was developed for the synthesis of active esters by palladium-catalyzed alkoxycarbonylation of (hetero)aromatic bromides. The protocol was general for a range of oxygen nucleophiles including N-hydroxysuccinimide (NHS), pentafluorophenol (PFP), hexafluoroisopropyl alcohol (HFP), 4-nitrophenol, and N-hydroxyphthalimide. A high functional group tolerance was displayed, and several active esters were prepared with good to excellent isolated yields. The protocol was extended to access an important synthetic precursor to the HIV-protease inhibitor, saquinavir, by formation of an NHS ester followed by acyl substitution.

Objective: Saquinavir (SQV) is a US-FDA approved HIV protease inhibitor (HPI) for HIV cure. The purpose of the present investigation was to develop and characterize the anticancer potential of the SQV-loaded folic acid (FA) conjugated PEGylated and non-PEGylated poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) (SQV-Fol-PEG-PLGA and SQV-Fol-PLGA) employing PC-3 (human prostate) and MCF-7 (human breast) cancer cell lines., Materials and Methods: Developed NPs were characterized by IR, NMR, DSC, XRD, size, charge and further tested for drug loading and cellular uptake properties., Result: The entrapment efficiency was found to be 56 ± 0.60 and 58 ± 0.80 w/v for SQV-Fol-PEG-PLGA and SQV-PLGA NPs, respectively. The obtained results of SQV-Fol-PEG-PLGA showed enhanced cytotoxicity and cellular uptake and were most preferentially taken up by the cancerous cells via folate receptor-mediated endocytosis (RME) mechanism. At 260 µM concentration, SQV-PLGA NPs and SQV-Fol-PEG-PLGA NPs showed 20%, 20% and 23% cell growth inhibition in PC-3 cells, respectively whereas in MCF-7 cells it was 12%, 15% and 14% cell growth inhibition, respectively., Conclusions: Developed targeted SQV-Fol-PEG-PLGA NPs were superior anticancer potential as compared to non-targeted SQV-PLGA NPs. Thus, these targeted NPs provide another option for anticancer drug delivery scientists.

An ionic liquid-based dispersive liquid-liquid microextraction followed by RP-HPLC determination of the most commonly prescribed protease inhibitor, saquinavir, in rat plasma was developed and validated. The effects of different ionic liquids, dispersive solvents, extractant/disperser ratio and salt concentration on sample recovery and enrichment were studied. Among the ionic liquids investigated, 1-butyl-3-methylimidazolium hexafluorophosphate was found to be most effective for extraction of saquinavir from rat serum. The recovery was found to be 95% at an extractant/disperser ratio of 0.43 using 1-butyl-3-methylimidazolium hexafluorophosphate and methanol as extraction and dispersive solvents. The recovery was further enhanced to 99.5% by addition of 5.0% NaCl. A threefold enhancement in detection and quantification limits was achieved, at 0.01 and 0.03 µg/mL, compared with the conventional protein precipitation method. A linear relationship was observed in the range of 0.035-10.0 µg/mL with a correlation coefficient (r(2) ) of 0.9996. The method was validated and applied to study pharmacokinetics of saquinavir in rat serum., (Copyright © 2014 John Wiley & Sons, Ltd.)

Saquinavir mesylate (SM) is a protease inhibitor with activity against human immunodeficiency virus type 1 (HIV-1) and is available in tablet form, which has three major problems. First, the drug undergoes extensive first pass metabolism. Second, the drug has a poor aqueous solubility. And third, it has low GIT permeability and absorption. These constrains lead to decrease oral bioavailability (4% only) and administration of large doses which increase the incidence of occurrence of the side effects. The aim of this research was to utilize nanotechnology to formulate (SM) into a nasal in situ nanosized microemulsion gel (NEG) to provide a solution for the previously mentioned problems. The solubility of (SM) in various oils, surfactants, and cosurfactants was estimated. Pseudo-ternary phase diagrams were developed and various nanosized microemulsion (NE) were prepared, and subjected to characterization, stability study, and droplet size measurements. Gellan gum was used as an in situ gelling agent. The gel strength, critical ionic concentration, gelation characteristics, in vitro release, and ex vivo nasal permeation were determined. The pharmacokinetic study was carried out in rabbits. Stable NEs were successfully developed with a droplet size range of 25-61 nm. A NEG composed of 17.5% Labrafac PG, 33% Labrasol, and 11% Transcutol HP successfully provided the maximum in vitro and ex vivo permeation, and enhanced the bioavailability in the rabbits by 12-fold when compared with the marketed tablets. It can be concluded that the nasal NEG is a promising novel formula for (SM) that has higher nasal tissue permeability and enhanced systemic bioavailability., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This study was performed to investigate the impact of pharmaceutical excipients commonly used for lymphatic transport on in vitro drug association with chylomicrons (CM). A CM association study was conducted using saquinavir solubilized in four different pharmaceutical excipients. We observed a linear relationship between saquinavir solubility and drug association, suggesting that the solubility of saquinavir in excipients is a key determinant for successful lymphatic delivery. Broadly, these results suggest that excipients with good solubilization properties may be advantageous for enhancing lymphatic drug delivery.

The main objectives of the present study were (i) to evaluate the effect of the mucus layer on saquinavir-loaded nanostructured lipid carriers (SQV-NLCs) uptake and (ii) to evaluate the mucopenetrating properties of dextran-protamine (Dex-Prot) coating on NLCs as per SQV permeability enhancement. Three different NLC formulations differing on particle size and surfactant content were obtained and coated with Dex-Prot complexes. SQV permeability was then evaluated across Caco-2 cell monolayers (enterocyte-like model) and Caco-2/HT29-MTX cell monolayers (mucus model). In the Caco-2 monolayers, Dex-Prot-NLCs increased up to 9-fold SQV permeability in comparison to uncoated nanoparticles. In the Caco-2/HT29-MTX monolayers, Dex-Prot-NLCs presenting a surface charge close to neutrality significantly increased SQV permeability. Hence, Dex-Prot complex coating is a promising strategy to ensure successful nanoparticle mucus-penetration, and thus, an efficient nanoparticle oral delivery. To our knowledge, this is the first time that Dex-Prot coating has been described as a nanoparticle muco-penetration enhancer across the intestinal mucus barrier., (Copyright © 2014 Elsevier B.V. All rights reserved.)

P-glycoprotein (P-gp) is involved in the transport of xenobiotic compounds and responsible for the decrease of the drug accumulation in multi-drug-resistant cells. In this investigation we compare several docking algorithms in order to find the conditions that are able to discriminate between P-gp binders and nonbinders. We built a comprehensive dataset of binders and nonbinders based on a careful analysis of the experimental data available in the literature, trying to overcome the discrepancy noticeable in the experimental results. We found that Autodock Vina flexible docking is the best choice for the tested options. The results will be useful to filter virtual screening results in the rational design of new drugs that are not expected to be expelled by P-gp.

Cholesterol-mediated cationic solid lipid nanoparticles (CSLNs) were formulated with esterquat 1 (EQ 1) and stearylamine as positively charged external layers on hydrophobic internal cores of cacao butter. These CSLNs were employed to deliver saquinavir (SQV) to the brain. The permeability of SQV across the blood-brain barrier (BBB) using SQV-loaded CSLNs (SQV-CSLNs) was estimated with an in vitro model of a monolayer of human brain-microvascular endothelial cells (HBMECs) regulated by human astrocytes. The results revealed that the average diameter of SQV-CSLNs diminished when the weight percentage of cholesterol and EQ 1 increased. The morphological images indicated a uniform size of SQV-CSLNs with compact lipid structure. In addition, an increasing weight percentage of cholesterol and EQ 1 enhanced the zeta potential of SQV-CSLNs. The fluorescent staining demonstrated that HBMECs could internalize SQV-CSLNs. An increase in the weight percentage of cholesterol and EQ 1 also promoted the uptake of SQV-CSLNs by HBMECs. Moreover, a high content of cholesterol and EQ 1 in SQV-CSLNs increased the BBB permeability of SQV. The cholesterol-mediated SQV-CSLNs can be an efficacious drug delivery system for brain-targeting delivery of antiviral agents., (© 2013 American Institute of Chemical Engineers.)

Solid dispersions of saquinavir mesylate containing either Gelucire® 44/14 or poly(ethylene glycol) (PEG) 4000, or mixtures of each carrier with Tween 80 or polyvinyl pyrrolidone (PVP) K30 were prepared in order to enhance the drug dissolution rate. These systems were prepared by the melting method and characterized by X-ray powder diffraction, microscopical techniques, and Raman spectroscopy aiming to establish a relationship between physicochemical and dissolution properties under different cooling conditions. Modifications in degree of crystalline order/disorder over time were observed in preparations with both carriers. Overall, formulations cooled and stored at -20 °C showed less variation in dissolution rates than those at 25 °C. Although Tween 80 has enhanced the known self-emulsifying properties of Gelucire® 44/14, its combination with PEG 4000 displayed miscibility problems. The addition of PVP K30 was not the most effective approach in enhancing the dissolution in early steps; however, the drug dissolution was stable after 7 days of storage at 25 °C. The combination of PEG 4000 and PVP K30 maintained the dissolution properties for 60 and 90 days at 25 °C/95% relative humidity and 40 °C/75% (f₂ values >50), respectively.

The aims of this work were (i) to evaluate the potential of nanostructured lipid carriers (NLCs) as a tool to enhance the oral bioavailability of poorly soluble compounds using saquinavir (SQV), a BCS class IV drug and P-gp substrate as a model drug, and (ii) to study NLC transport mechanisms across the intestinal barrier. Three different NLC formulations were evaluated. SQV transport across Caco-2 monolayers was enhanced up to 3.5-fold by NLCs compared to SQV suspension. M cells did not enhance the transport of NLCs loaded with SQV. The size and amount of surfactant in the NLCs influenced SQV's permeability, the transcytosis pathway and the efflux of SQV by P-gp. An NLC of size 247 nm and 1.5% (w/v) surfactant content circumvented P-gp efflux and used both caveolae- and clathrin-mediated transcytosis, in contrast to the other NLC formulations, which used only caveolae-mediated transcytosis. By modifying critical physicochemical parameters of the NLC formulation, we were thus able to overcome the P-gp drug efflux and alter the transcytosis mechanism of the nanoparticles. These findings support the use of NLCs approaches for oral delivery of poorly water-soluble P-gp substrates., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Application of cationic solid lipid nanoparticles (CSLNs), comprising complex internal matrix and lipid-regulated external surface, is an intriguing issue in current bionanotechnology. This study presents dissolution kinetics of saquinavir (SQV) from CSLNs with cholesterol-mediated esterquat 1 (EQ 1) and biocompatibility of SQV-loaded CSLNs with human brain-microvascular endothelial cells (HBMECs). CSLNs with SQV in lipid cores containing cholesterol were dissolved and incubated with HBMECs. The results revealed that an increase in the weight percentage of EQ 1 reduced the entrapment efficiency of SQV. In addition, the entrapment efficiency of SQV enhanced, when the weight percentage of cholesterol increased from 0% to 25% (w/w). The reverse was true when cholesterol increased from 0% to 75% (w/w). The dissolution profiles demonstrated that the mediation of cholesterol favored the sustained release of SQV. When the weight percentage of EQ 1 increased, the viability of HBMECs enhanced. An increase in the weight percentage of cholesterol, however, reduced the viability of HBMECs. The innovated CSLNs containing cholesterol can be effective in controlled release of SQV without inducing significant endothelial toxicity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The goal of this study was to develop and characterize an intravaginal nanomedicine for the active targeted delivery of saquinavir (SQV) to CD4(+) immune cells as a potential strategy to prevent or reduce HIV infection. The nanomedicine was formulated into a vaginal gel to provide ease in self-administration and to enhance retention within the vaginal tract. SQV-encapsulated nanoparticles (SQV-NPs) were prepared from poly(lactic-co-glycolic acid) (PLGA) and conjugated to antihuman anti-CD4 antibody. Antibody-conjugated SQV-NPs (Ab-SQV-NPs) had an encapsulation efficiency (EE%) of 74.4% + 3.7% and an antibody conjugation efficiency (ACE%) of 80.95% + 1.10%. Over 50% of total loaded SQV was released from NPs over 3 days. NPs were rapidly taken up by Sup-T1 cells, with more than a twofold increase in the intracellular levels of SQV when delivered by Ab-SQV-NPs in comparison to controls 1 hour post-treatment. No cytotoxicity was observed when vaginal epithelial cells were treated for 24 hours with drug-free Ab-NPs (1,000 μg/mL), 1% HEC placebo gel (200 mg/mL), or 1% HEC gel loaded with drug-free Ab-NPs (5 mg NPs/g gel, 200 mg/mL of gel mixture). Overall, we described an intravaginal nanomedicine that is nontoxic and can specifically deliver SQV into CD4(+) immune cells. This platform may demonstrate potential utility in its application as postexposure prophylaxis for the treatment or reduction of HIV infection, but further studies are required.

We have examined the influence of the nitric oxide (NO)-modified anti-inflammatory drug (S,R)-3-phenyl-4,5-dihydro-5-isoxasole acetic acid (VGX-1027) named GIT-27NO or the NO-modified antiviral drug saquinavir (Saq) named Saq-NO on two colon cancer cell lines, mouse CT26CL25 and human HCT116. The effects of the drugs on cell viability, apoptosis, proliferation, and metastatic potential were analyzed. The release of NO and oxygen and nitrogen species was also determined. The efficacy of the drugs was evaluated in vivo in BALB/c mice injected with CT26CL25 cells. Both agents suppressed the growth of colon cancer cells in vitro and reduced tumor volume in syngeneic BALB/c mice. However, their mechanisms of action were different because GIT-27NO released larger amounts of nitrite than Saq-NO in cell cultures and its antitumor action depended on the intracellular NO release inside the cells. On the contrary, Saq-NO released barely detectable amounts of NO and its antitumor action was NO-independent. In fact, cotreatment with an NO-peroxynitrite scavenger revealed that GIT-27NO but not Saq-NO acts through peroxynitrite-mediated cell destruction. At the cellular level, GIT-27NO prevalently induced proapoptotic signals followed by caspase-dependent apoptosis. In contrast, Saq-NO blocked cell proliferation, changed the adhesive, migratory, and invasive properties of the cells, and decreased metastatic potential in vivo. In conclusion, differences in NO release and oxidative stress generation between GIT-27NO and Saq-NO resulted in different mechanisms that caused cell death.

In an approach to overcome biological barriers mediated by P-glycoprotein (P-gp) and cytochrome P450 3A (CYP3A), a series of stereoisomeric valine-valine prodrugs of saquinavir (SQV) were synthesized and investigated with respect to affinity for efflux pump P-gp, and resistance to oxidative and hydrolytic enzymes. Cellular uptake and bidirectional transport in Caco-2 cells indicated that all peptide SQV conjugates can bypass P-gp-mediated efflux significantly, regardless of stereochemistry in promoieties. In comparison with D-configuration, L-configuration was favored for the interaction between prodrugs and rat hepatic CYP3A enzymes, and resulted in a relatively rapid clearance by CYP3A. Elimination half-life of SQV in rat liver microsomes was prolonged dramatically by sevenfold to 40-fold because of the prodrug modification with the rank order of D-valine-D-valine-SQV > D-valine-L-valine-SQV > L-valine-D-valine-SQV > L-valine-L-valine-SQV > D-valine-SQV > L-valine-SQV > SQV. Results of hydrolysis studies performed in rat intestinal homogenates and plasma indicated that prodrugs attached with D-valine exhibited significantly reduced biodegradation. In conclusion, the enhanced transepithelial accumulation and enzymatic stability observed by SQV peptide prodrug modification are found to be stereoselective. Specific stereoisomeric dipeptide prodrugs with optimized metabolic stability can be employed to improve oral bioavailability of SQV., (Copyright © 2012 Wiley Periodicals, Inc.)

Although anti-retroviral therapy is the most efficient disease management strategy for HIV-AIDS, its applications are limited by several factors including the low bioavailability and first pass metabolism of the drugs. Nanocarriers such as liposomes have been developed to circumvent some of these problems. We report here preparation of novel liposome formulations for efficient delivery of anti-retroviral drugs to mammalian cells in culture. The liposomes were prepared and surface was modified using poly (ethylene glycol). Encapsulation efficiency of the anti-retroviral drug saquinavir was found to be approximately 33% and also exhibited sustained release of the drug. Although PEGylated liposomes were more stable in protein-supplemented media, had better colloidal stability and exhibited lesser sonochemical stability due to lower cavitation threshold. The cell viability studies using Jurkat T-cells revealed that the PEGylated liposomes loaded with saquinavir were less cytotoxic as compared to the non-PEGylated liposomes or free drug confirming the potential of the liposomes as a sustained drug-release system. The drug delivery potential of the liposomes loaded with Alexa flour 647 was evaluated using Jurkat T-cells and flow cytometry showing uptake upto 74%. Collectively, our data demonstrate efficient targeting of mammalian cells using novel liposome formulations with insignificant levels of cytotoxicity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Purpose: To predict and determine whether the protease inhibitors (PIs) nelfinavir, amprenavir, atazanavir, ritonavir, and saquinavir could serve as metabolic inhibitors of the human CES1 (hCES1) using both molecular modeling techniques and in vitro inhibition assays., Methods: Initially, a molecular modeling approach was utilized to predict whether the selected PIs could serve as hCES1 inhibitors. The inhibitory effects of these PIs on hCES1 activity were then further evaluated utilizing previously established in vitro assay., Results: Pharmacophore and 2D-QSAR modeling predicted that nelfinavir would serve as a potent hCES1 inhibitor. This hypothesis was validated by in vitro hCES1 inhibition studies. Other PIs (amprenavir, atazanavir, ritonavir, saquinavir) were evaluated and also shown to be hCES1 inhibitors in vitro, although substantially less potent relative to nelfinavir., Conclusion: Computational molecular modeling is a valid approach to identify potential hCES1 inhibitors as candidates for further assessment using validated in vitro techniques. DDIs could occur when nelfinavir is co-administered with drugs metabolized by hCES1.

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.)

Saquinavir is an anti-retroviral drug with very low oral bioavailability (e.g. 0.7-4.0%) due to its affinity toward efflux transporters (P-gp) and metabolic enzymes (CYP3A4). The aim of this study was to characterize the effects of high-energy ball milling on saquinavir solid-state characteristics and aqueous solubility for the design of effective buccal drug delivery systems. The solubility of saquinavir mesylate was evaluated in simulated saliva before and after milling for 1, 3, 15, 30, 50, and 60 h. To elucidate changes in crystallinity and long-range structure in the drug, analyses of the milled powders were performed using XRD, ATR-IR, DSC/TGA, BET surface area, EDX and SEM. In addition, the effects of milling time on saquinavir solubility were statistically correlated using repeated measures ANOVA. Results of this study indicate that the milling of saquinavir mesylate produces nanoporous particles with unique surface structures, thermal properties, and increased aqueous solubility. Optimal milling time occurred at 3h and corresponded to a 9-fold solubility enhancement in simulated saliva. Thermal analysis revealed only a slight decrease in melting point (T(m)) from 242 °C to 236 °C after 60 h milling. XRD diffractograms indicate a gradual crystalline-to-amorphous transition with some residual crystallinity remaining after 60 h milling time. Unstable polymorphic structures appeared between 15 and 30 h which were converted to more stable isomorphs at 60 h. Aggregate formation also seems to occur after 15 h but no metal contamination of the drug was observed during the milling process as determined by EDX analysis. In conclusion, high-energy ball milling may be a method of choice for improving the solubility of saquinavir and facilitating novel drug formulations design., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.)

Poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) with surface poly-(γ-glutamic acid) (γ-PGA) were applied to enhance the transport of saquinavir (SQV) across the blood-brain barrier (BBB). PLGA NPs encapsulated SQV and grafted with γ-PGA to form drug carriers (γ-PGA/SQV-PLGA NPs) for crossing through a monolayer of human brain-microvascular endothelial cells (HBMECs) regulated with human astrocytes. The results revealed that a lower molecular weight of γ-PGA yielded a higher grafting efficiency of γ-PGA on PLGA NPs. In addition, γ-PGA with a low molecular weight accelerated the dissolution of SQV from γ-PGA/SQV-PLGA NPs. A higher grafting efficiency (more didecyl dimethylammonium bromide) and a lower molecular weight of γ-PGA increased the permeability of SQV across the BBB, in general. When the grafting efficiency was 85.2% at 6kDa of γ-PGA, γ-PGA/SQV-PLGA NPs reached about 6 times the permeability of free SQV (the maximal permeability). γ-PGA could also promote the endocytosis of NPs and expression of ornithine decarboxylase by HBMECs. γ-PGA/SQV-PLGA NPs are efficacious nanoparticulate carriers in delivering antiretroviral drug across the BBB., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although, the antiviral activity, tolerability and convenience of protease inhibitors have improved significantly in recent years, toxicity-associated adverse events including diarrhea, lipid alterations, disturbance of glucose homeostasis and liver enzyme elevations still remain a major concern during treatment of HIV-1 patients. We have recently shown that the covalent attachment of the NO moiety to the HIV-1 protease inhibitor saquinavir (Saq-NO) reduces its toxicity. In this study, we evaluated in vitro the anti-HIV activity of Saq-NO vs. its parental compound Saq. Site directed mutants with the most frequently identified Saq associated resistance mutations and their combinations were generated on proviral AD8-based backbones. Phenotypic assays were conducted using wild type clinical isolates and fully replicating recombinant viruses with Saq and Saq-NO in parallel on purified CD4+ T cells. The following recombinant viruses were generated and tested: L33F, M46I, G48V, I54V, I84V + L90M, M46I + L90M, G48V + L90M, M46I + I54V + L90M, L33F + M46I + L90M. The fold change resistance compared to the wild type viruses was between 1.3 and 7 for all single mutants, between 3.4 and 20 for double mutants and between 16.7 and 28.5 for viruses carrying three mutations for both compounds. The results clearly demonstrate that Saq-NO maintains an anti-HIV-1 profile very similar to that of Saq. The possibility to reduce Saq associated side effects and to increase the concentration of the drug in vivo may allow a higher and possibly more effective dosage of Saq-NO in HIV-1-infected patients and to increase the genetic barrier of this PI thus impairing the selection of resistant clones., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In the search for a successful HIV microbicide, many poorly water-soluble antiviral agents are currently being investigated. Unfortunately, solubility and precipitation issues may limit intravaginal concentrations and thus availability of these agents upon application of an aqueous gel formulation. In the present study, we evaluated the in vitro precipitation behavior of the HIV protease inhibitor saquinavir in vaginal and seminal fluid simulants (VFS and SFS). Despite its limited solubility, the mesylate salt of saquinavir enables formulation of sufficiently high concentrations (2.5 mM, i.e. ca. 10(5)-fold in vitro IC(50) values) in a standard aqueous vehicle. While saquinavir stays in solution upon dilution with VFS, SFS induces precipitation of saquinavir, resulting in a 5-fold reduced availability and antiviral potency. Inclusion of the solubilizing excipients polyethylene glycol 1000 (12%) and hydroxypropyl-β-cyclodextrin (2.5%) was required to avoid saquinavir precipitation in SFS and to restore the antiviral potency of the formulation. This study illustrates the importance of identifying solubility and precipitation issues of microbicide candidates in biorelevant media and provides a simple in vitro procedure to implement this evaluation in early microbicide development., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In order to improve oral absorption, a novel prodrug of saquinavir (Saq), ascorbyl-succinic-saquinavir (AA-Su-Saq) targeting sodium dependent vitamin C transporter (SVCT) was synthesized and evaluated. Aqueous solubility, stability and cytotoxicity were determined. Affinity of AA-Su-Saq towards efflux pump P-glycoprotein (P-gp) and recognition of AA-Su-Saq by SVCT were studied. Transepithelial permeability across polarized MDCK-MDR1 and Caco-2 cells were determined. Metabolic stability of AA-Su-Saq in rat liver microsomes was investigated. AA-Su-Saq appears to be fairly stable in both DPBS and Caco-2 cells with half lives of 9.65 and 5.73 h, respectively. Uptake of [(3)H]Saquinavir accelerated by 2.7 and 1.9 fold in the presence of 50 μM Saq and AA-Su-Saq in MDCK-MDR1 cells. Cellular accumulation of [(14)C]AA diminished by about 50-70% relative to control in the presence of 200 μM AA-Su-Saq in MDCK-MDR1 and Caco-2 cells. Uptake of AA-Su-Saq was lowered by 27% and 34% in the presence of 5mM AA in MDCK-MDR1 and Caco-2 cells, respectively. Absorptive permeability of AA-Su-Saq was elevated about 4-5 fold and efflux index reduced by about 13-15 fold across the polarized MDCK-MDR1 and Caco-2 cells. Absorptive permeability of AA-Su-Saq decreased 44% in the presence of 5mM AA across MDCK-MDR1 cells. AA-Su-Saq was devoid of cytotoxicity over the concentration range studied. AA-Su-Saq significantly enhanced the metabolic stability but lowered the affinity towards CYP3A4. In conclusion, prodrug modification of Saq through conjugation to AA via a linker significantly raised the absorptive permeability and metabolic stability. Such modification also caused significant evading of P-gp mediated efflux and CYP3A4 mediated metabolism. SVCT targeted prodrug approach can be an attractive strategy to enhance the oral absorption and systemic bioavailability of anti-HIV protease inhibitors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Saquinavir (SQV) is a weak base compound, whose solubility is strongly influenced by pH variations. Thus, in the present work, we thought it worthy of interest to investigate in-depth the combined effect of pH control and cyclodextrin (CyD) complexation on SQV solubilization. Phase-solubility studies were performed by adding excess drug to buffered (pH from 1.1 to 7.4) aqueous solutions containing increasing concentrations of Methyl-Beta-CyD (M-β-CyD) in order to evaluate the role of the unionized species of SQV in improving solubility by CyD complexation and to be able to select the most suitable conditions for optimizing drug solubilization. Our study reveals that the integrated approach of pH adjustment and CyD complexation can be successfully used for improving the CyD solubilizing power towards an ionizable drug such as SQV, thus allowing a smaller quantity of CyD to solubilize a given amount of drug, offering clear economic and technologic advantages as well. When biopharmaceutics of the optimized cyclodextrin-based formulation of SQV was studied in Wistar rats after intravenous and oral administrations, we found that inclusion of SQV into M-β-CyD could dramatically improve its oral bioavailability and decrease the variation of its oral pharmacokinetics. Compared to the control, the presence of M-β-CyD significantly increased the area under the plasma concentration-time curve (439.7±161.35 to 2312.03±159.53, p<0.01) and the peak plasma concentration (117.24±35.77 to 1347.88±276.76, p<0.01) of orally administered SQV. The modulating effect of M-β-CyD on the bidirectional transport of SQV was also investigated using a modified Ussing chamber system. The results demonstrated that the enhancing effect of M-β-CyD on the oral bioavailability of SQV is due not only to its solubilizing effect on SQV but also, at least in part, to the inhibitory effect of M-β-CyD on the P-glycoprotein (P-gp) mediated efflux of SQV in the gastrointestinal tract. The present results suggest that M-β-CyD is particularly useful in designing oral preparations of SQV with an enhanced bioavailability and a reduced variability in absorption., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The structural and kinetic effects of amprenavir (APV), a clinical HIV protease (PR) inhibitor, were analyzed with wild-type enzyme and mutants with single substitutions of V32I, I50V, I54V, I54M, I84V and L90M that are common in drug resistance. Crystal structures of the APV complexes at resolutions of 1.02-1.85 Å reveal the structural changes due to the mutations. Substitution of the larger side chains in PR(V32I) , PR(I54M) and PR(L90M) resulted in the formation of new hydrophobic contacts with flap residues, residues 79 and 80, and Asp25, respectively. Mutation to smaller side chains eliminated hydrophobic interactions in the PR(I50V) and PR(I54V) structures. The PR(I84V)-APV complex had lost hydrophobic contacts with APV, the PR(V32I)-APV complex showed increased hydrophobic contacts within the hydrophobic cluster and the PR(I50V) complex had weaker polar and hydrophobic interactions with APV. The observed structural changes in PR(I84V)-APV, PR(V32I)-APV and PR(I50V)-APV were related to their reduced inhibition by APV of six-, 10- and 30-fold, respectively, relative to wild-type PR. The APV complexes were compared with the corresponding saquinavir complexes. The PR dimers had distinct rearrangements of the flaps and 80's loops that adapt to the different P1' groups of the inhibitors, while maintaining contacts within the hydrophobic cluster. These small changes in the loops and weak internal interactions produce the different patterns of resistant mutations for the two drugs., (© 2010 The Authors Journal compilation © 2010 FEBS.)

Antiretroviral drugs are ineffective at treating viral infection in the brain because they cannot freely diffuse across the blood-brain barrier (BBB). Therefore, HIV-1 viral replication persists in the central nervous system (CNS) and continues to augment the neuropathogenesis process. Nanotechnology can play a pivotal role in HIV-1 therapeutics as it can increase drug solubility, enhance systemic bioavailability, and at the same time offer multifunctionality. Moreover, following conjugation with transferrin (Tf), these drug-loaded nanoformulations can permeate across biological barriers such as the blood brain barrier (BBB) via a receptor mediated transport mechanism. In the current study, we have stably incorporated the antiviral drug, Saquinavir, within Tf-conjugated quantum rods (QRs), which are novel nanoparticles with unique optical properties. We have evaluated the transversing ability of the QR-Tf-Saquinavir nanoformulation across an in vitro model of BBB. In addition, we have analyzed the subsequent antiviral efficacy of this targeted nanoformulation in HIV-1 infected peripheral blood mononuclear cells (PBMCs), which are cultured on the basolateral end of the in vitro BBB model. Our results show a significant uptake of QR-Tf-Saquinavir by brain microvascular endothelial cells (BMVECs), which constitute the BBB. In addition, we observed a significant enhancement in the transversing capability of QR-Tf-Saquinavir across the BBB, along with a marked decrease in HIV-1 viral replication in the PBMCs. These observations indicate that drug-loaded nanoparticles can deliver therapeutics across the BBB. These results highlight the potential of this nanoformulation in the treatment of Neuro-AIDS and other neurological disorders.

The mutation G48V in HIV-1 protease is a major resistance mutation against the drug saquinavir. Recently, G48V mutation is found to co-exist with the mutation C95F in AIDS patients treated with saquinavir. We report here the three-dimensional crystal structure of G48V/C95F tethered HIV-1 protease/saquinavir complex. The structure indicates following as the possible causes of drug resistance: (1) loss of direct van der Waals interactions between saquinavir and enzyme residues PHE-53 and PRO-1081, (2) loss of water-mediated hydrogen bonds between the carbonyl oxygen atoms in saquinavir and amide nitrogen atoms of flap residues 50 and 1050, (3) changes in inter-monomer interactions, which could affect the energetics of domain movements associated with inhibitor-binding, and (4) significant reduction in the stability of the mutant dimer. The present structure also provides a rationale for the clinical observation that the resistance mutations C95F/G48V/V82A occur as a cluster in AIDS patients., ((c) 2010 Elsevier Inc. All rights reserved.)

A series of new thienyl ring containing analogues of nelfinavir and saquinavir with different substitution patterns were synthesized from suitable enantiopure diols. Their inhibitory activity against wild type recombinant HIV-1 protease was evaluated. In general thienyl groups spaced from the core by a methylene group gave products showing IC(50) in the nanomolar range, irrespective of the type and the substitution pattern of the heterocycle. The range of activity of the two most active compounds is substantially maintained or even increased against two commonly selected mutants, under drug pressure, such as V32I and V82A.

In the two decades since AZT was first approved for clinical use in 1987, 24 additional antiretroviral agents have been approved. They include 7 nucleoside analogs, a nucleotide analog and 4 non-nucleoside reverse transcriptase inhibitors, 10 protease inhibitors, 2 entry inhibitors and an integrase inhibitor. More than 20 investigational agents are currently being studied in clinical trials. Highly active antiretroviral therapy (HAART), which involves a combination of anti-HIV-1 drugs, is extremely effective in suppressing HIV-1 replication and increasing CD4+ number and results in substantial reductions in HIV-1-related morbidity and mortality. In last 20 years, much has been learned about resistance to antiretroviral drugs, drug interactions and metabolic complications of antiviral drug use. Drugs are now selected on the basis of resistance tests and on the risk of specific drug complications in individual patients. As a result, decisions about the therapy of HIV/AIDS have become personalized and are made on a patient-by-patient basis. With appropriate medical management, a person with HIV-1 now has the possibility of a nearly normal life expectancy.