Liposomal Delivery of Saquinavir to Macrophages Overcomes Cathepsin Blockade by Mycobacterium tuberculosis and Helps Control the Phagosomal Replicative Niches.

Mycobacterium tuberculosis is able to establish a chronic colonization of lung macrophages in a controlled replication manner, giving rise to a so-called latent infection. Conversely, when intracellular bacteria undergo actively uncontrolled replication rates, they provide the switch for the active infection called tuberculosis to occur. Our group found that the pathogen is able to manipulate the activity of endolysosomal enzymes, cathepsins, directly at the level of gene expression or indirectly by regulating their natural inhibitors, cystatins. To provide evidence for the crucial role of cathepsin manipulation for the success of tuberculosis bacilli in their intracellular survival, we used liposomal delivery of saquinavir. This protease inhibitor was previously found to be able to increase cathepsin proteolytic activity, overcoming the pathogen induced blockade. In this study, we demonstrate that incorporation in liposomes was able to increase the efficiency of saquinavir internalization in macrophages, reducing cytotoxicity at higher concentrations. Consequently, our results show a significant impact on the intracellular killing not only to reference and clinical strains susceptible to current antibiotic therapy but also to multidrug- and extensively drug-resistant (XDR) Mtb strains. Altogether, this indicates the manipulation of cathepsins as a fine-tuning strategy used by the pathogen to survive and replicate in host cells.

Short-Term Administration of HIV Protease Inhibitor Saquinavir Improves Skull Bone Healing with Enhanced Osteoclastogenesis.

BACKGROUND: Using immunomodulatory methods to address the challenging issue of craniofacial bone repair may be a potentially effective approach. The protease inhibitor saquinavir has been shown to inhibit the inflammatory response by targeting the toll-like receptor 4/myeloid differentiation primary response complex. Independently, inhibition of toll-like receptor 4 or myeloid differentiation primary response led to enhanced skull bone repair. Therefore, the authors aimed to investigate the effects of saquinavir on skull bone healing. METHODS: The effects of saquinavir on skull bone healing were assessed by means of gene expression, histology, immunohistochemistry, and tomography in a mouse calvarial defect model. Subsequently, the role of saquinavir in cell viability, migration, and osteogenic and osteoclastogenic differentiation was also evaluated in vitro. RESULTS: One-week saquinavir administration improved skull bone healing based on micro-computed tomographic and histomorphometric analyses. Compared to the vehicle control, 1-week saquinavir treatment (1) enhanced osteoclast infiltration (tartrate-resistant acid phosphatase staining) at day 7, but not at days 14 and 28; (2) induced more CD206 + M2 macrophage infiltration, but not F4/80 + M0 macrophages at days 7, 14, and 28; and (3) elevated osteoclastogenic gene RANKL (quantitative polymerase chain reaction) expression and other osteogenic and cytokine expression. Furthermore, in vitro data showed that saquinavir administration did not influence MC3T3-E1 cell migration or mineralization, whereas higher concentrations of saquinavir inhibited cell viability. Saquinavir treatment also enhanced the osteoclastic differentiation of bone marrow-derived precursors, and partially reversed high-mobility group box 1-driven osteoclastogenesis inhibition and elevated proinflammatory cytokine expression. CONCLUSION: The improved skull bone repair following short-term saquinavir treatment may involve enhanced osteoclastogenesis and modulated inflammatory response following skull injury. CLINICAL RELEVANCE STATEMENT: The authors' work demonstrates improved skull bone healing by short-term application of saquinavir, a drug traditionally used in the treatment of acquired immunodeficiency syndrome. As such, saquinavir may be repurposed for skeletal repair.

Drug binding dynamics of the dimeric SARS-CoV-2 main protease, determined by molecular dynamics simulation.

We performed molecular dynamics simulation of the dimeric SARS-CoV-2 (severe acute respiratory syndrome corona virus 2) main protease (Mpro) to examine the binding dynamics of small molecular ligands. Seven HIV inhibitors, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir, were used as the potential lead drugs to investigate access to the drug binding sites in Mpro. The frequently accessed sites on Mpro were classified based on contacts between the ligands and the protein, and the differences in site distributions of the encounter complex were observed among the ligands. All seven ligands showed binding to the active site at least twice in 28 simulations of 200 ns each. We further investigated the variations in the complex structure of the active site with the ligands, using microsecond order simulations. Results revealed a wide variation in the shapes of the binding sites and binding poses of the ligands. Additionally, the C-terminal region of the other chain often interacted with the ligands and the active site. Collectively, these findings indicate the importance of dynamic sampling of protein-ligand complexes and suggest the possibilities of further drug optimisations.

Computer-guided drug repurposing: identification of trypanocidal activity of clofazimine, benidipine and saquinavir.

In spite of remarkable advances in the knowledge on Trypanosoma cruzi biology, no medications to treat Chagas disease have been approved in the last 40 years and almost 8 million people remain infected. Since the public sector and non-profit organizations play a significant role in the research efforts on Chagas disease, it is important to implement research strategies that promote translation of basic research into the clinical practice. Recent international public-private initiatives address the potential of drug repositioning (i.e. finding second or further medical uses for known-medications) which can substantially improve the success at clinical trials and the innovation in the pharmaceutical field. In this work, we present the computer-aided identification of approved drugs clofazimine, benidipine and saquinavir as potential trypanocidal compounds and test their effects at biochemical as much as cellular level on different parasite stages. According to the obtained results, we discuss biopharmaceutical, toxicological and physiopathological criteria applied to decide to move clofazimine and benidipine into preclinical phase, in an acute model of infection. The article illustrates the potential of computer-guided drug repositioning to integrate and optimize drug discovery and preclinical development; it also proposes rational rules to select which among repositioned candidates should advance to investigational drug status and offers a new insight on clofazimine and benidipine as candidate treatments for Chagas disease. One Sentence Summary: We present the computer-guided drug repositioning of three approved drugs as potential new treatments for Chagas disease, integrating computer-aided drug screening and biochemical, cellular and preclinical tests.

Microglial activation decreases retention of the protease inhibitor saquinavir: implications for HIV treatment.

BACKGROUND: Active HIV infection within the central nervous system (CNS) is confined primarily to microglia. The glial cell compartment acts as a viral reservoir behind the blood-brain barrier. It provides an additional roadblock to effective pharmacological treatment via expression of multiple drug efflux transporters, including P-glycoprotein. HIV/AIDS patients frequently suffer bacterial and viral co-infections, leading to deregulation of glial cell function and release of pro-inflammatory mediators including cytokines, chemokines, and nitric oxide. METHODS: To better define the role of inflammation in decreased HIV drug accumulation into CNS targets, accumulation of the antiretroviral saquinavir was examined in purified cultures of rodent microglia exposed to the prototypical inflammatory mediator lipopolysaccharide (LPS). RESULTS: [(3)H]-Saquinavir accumulation by microglia was rapid, and was increased up to two-fold in the presence of the specific P-glycoprotein inhibitor, PSC833. After six or 24 hours of exposure to 10 ng/ml LPS, saquinavir accumulation was decreased by up to 45%. LPS did not directly inhibit saquinavir transport, and did not affect P-glycoprotein protein expression. LPS exposure did not alter RNA and/or protein expression of other transporters including multidrug resistance-associated protein 1 and several solute carrier uptake transporters. CONCLUSIONS: The decrease in saquinavir accumulation in microglia following treatment with LPS is likely multi-factorial, since drug accumulation was attenuated by inhibitors of NF-κß and the MEK1/2 pathway in the microglia cell line HAPI, and in primary microglia cultures from toll-like receptor 4 deficient mice. These data provide new pharmacological insights into why microglia act as a difficult-to-treat viral sanctuary site.

Methylmethacrylate-sulfopropylmethacrylate nanoparticles with surface RMP-7 for targeting delivery of antiretroviral drugs across the blood-brain barrier.

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.

Nicotine and cotinine increases the brain penetration of saquinavir in rat.

Endothelial tight junctions and efflux transporters of the blood-brain barrier (BBB) significantly limit brain accumulation of many drugs, including protease inhibitors such as saquinavir. The cholinergic agonist nicotine is one of the most commonly used drugs in the world and the incidence is even higher in the human immune deficiency virus population (∼ 70%). We examined the ability of nicotine and its primary metabolite cotinine to modify brain uptake of saquinavir in rats. Both nicotine and cotinine at pharmacological concentrations matching those in smokers, increased brain saquinavir uptake by two fold. Co-perfusion with nicotinic receptor antagonists and passive permeability markers showed that the effect was not caused by receptor activation or BBB permeability disruption. Transport inhibition studies demonstrated that brain saquinavir uptake is limited by multiple efflux transporters, P-glycoprotein (P-gp), breast cancer resistance protein and multidrug resistance-associated protein. In situ perfusion and in vitro experiments using a classical P-gp substrate rhodamine 123 linked the effect of nicotine to inhibition of BBB P-gp transport. The effect was confirmed in vivo in chronic 14 day nicotine administration animals. These data suggest nicotine increases antiretroviral drug exposure to brain and may represent a significant in vivo drug-drug interaction at the BBB. Although this may slightly benefit CNS antiretroviral efficacy, it may also expose the brain to potential serious neurotoxicity.

Impact of drug transporters on cellular resistance towards saquinavir and darunavir.

OBJECTIVES: Highly active antiretroviral therapy is complicated by drug-drug interactions and the development of viral resistance. Drug interactions involve transporters that may critically affect the pharmacokinetics of many antiretroviral drugs and contribute to the formation of functional sanctuary sites. We therefore investigated the effect of saquinavir and darunavir on drug transporter expression and functional consequences for cellular resistance towards these compounds. METHODS: Induction of transporters was investigated in LS180 cells over a period of 4 weeks by means of RT-PCR, and for some transporters also at the protein and functional levels. Cellular resistance was measured by growth inhibition assays. RESULTS: Incubation with 10 µM darunavir for 1 week significantly increased mRNA expression of P-glycoprotein (P-gp/MDR1/ABCB1) 3.8-fold and of organic anion-transporting polypeptide 2B1 (SLCO2B1) 1.9-fold. In contrast, 10 µM saquinavir significantly increased mRNA expression of P-gp 5.7-fold, multidrug resistance-associated protein 1 (MRP1/ABCC1) 2.3-fold, MRP2/ABCC2 4.5-fold, MRP3/ABCC3 2.0-fold, MRP4/ABCC4 1.8-fold, MRP5/ABCC5 3.8-fold, breast cancer resistance protein (BCRP/ABCG2) 4.1-fold, SLCO1B1 4.6-fold, SLCO2B1 1.8-fold and SLCO3A1 1.8-fold. P-gp induction was also confirmed at the protein and functional levels. Induction by darunavir caused an increase in cellular resistance towards this compound, as measured in growth inhibition assays; however, saquinavir treatment did not cause reduced sensitivity of cells, indicating unchanged intracellular concentration. Hence, induction by darunavir increased drug efflux and might therefore lead to a suboptimal intracellular concentration of darunavir. CONCLUSIONS: The study revealed substantial induction of several drug transporters by saquinavir and darunavir, possibly leading to decreased efficacy of antiretrovirals and drugs used to treat co-morbidity.

Cultured CD4T cells and primary human lymphocytes express hOATPs: intracellular accumulation of saquinavir and lopinavir.

BACKGROUND AND PURPOSE: Drug efflux tranporters (P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP)) limit the cellular uptake of human immunodeficiency virus protease inhibitors but the contribution of influx transporters in cells that (over)express P-gp or MRP is less clear. Here, we studied the expression of one influx transporter system, human organic anion-transporting polypeptide (hOATP), in some T-cell lines (CEM, CEM(VBL), CEM(E1000)) and in peripheral blood mononuclear cells (PBMCs) and examined the effects of manipulation of influx/efflux transporters on the uptake of saquinavir and lopinavir. EXPERIMENTAL APPROACH: The expression of hOATPs was studied by PCR. We used hOATP substrate or inhibitor (estrone-3-sulphate (E-3-S) or montelukast, respectively) and inhibitors of P-gp (XR9576) and MRP (MK571 and frusemide) to study functional interactions between influx and efflux transporters in the uptake of saquinavir and lopinavir. Lipophilicity of the drugs was measured by octanol/saline partition coefficient. KEY RESULTS: CEM cells, their variants and PBMCs express various hOATP isoforms, with OATP3A1 detected in all of the cells. MK571, XR9576 and frusemide increased the uptake of saquinavir and lopinavir. E-3-S and montelukast reduced the uptake of saquinavir and lopinavir in some, but not all, of the cells. Pretreatment of the cells with MK571, XR9576 or frusemide, followed by E-3-S co-incubation reduced the cellular accumulation of saquinavir and lopinavir. Lopinavir is much more lipophilic than saquinavir. CONCLUSIONS AND IMPLICATIONS: Human OATPs, MRP, P-gp and lipophilicity determine the cellular uptake and retention of saquinavir and lopinavir. These data may have important implications for drug-drug interactions, drug safety and efficacy.

Amodiaquine metabolism is impaired by common polymorphisms in CYP2C8: implications for malaria treatment in Africa.

Metabolism of the antimalarial drug amodiaquine (AQ) into its primary metabolite, N-desethylamodiaquine, is mediated by CYP2C8. We studied the frequency of CYP2C8 variants in 275 malaria-infected patients in Burkina Faso, the metabolism of AQ by CYP2C8 variants, and the impact of other drugs on AQ metabolism. The allele frequencies of CYP2C8*2 and CYP2C8*3 were 0.155 and 0.003, respectively. No evidence was seen for influence of CYP2C8 genotype on AQ efficacy or toxicity, but sample size limited these assessments. The variant most common in Africans, CYP2C8(*)2, showed defective metabolism of AQ (threefold higher K(m) and sixfold lower intrinsic clearance), and CYP2C8(*)3 had markedly decreased activity. Considering drugs likely to be coadministered with AQ, the antiretroviral drugs efavirenz, saquinavir, lopinavir, and tipranavir were potent CYP2C8 inhibitors at clinically relevant concentrations. Variable CYP2C8 activity owing to genetic variation and drug interactions may have important clinical implications for the efficacy and toxicity of AQ.

Synthesis of poly(ethylene glycol)-based saquinavir prodrug conjugates and assessment of release and anti-HIV-1 bioactivity using a novel protease inhibition assay.

Various poly(ethylene glycol)(PEG)-based prodrug conjugates of the HIV-1 protease inhibitor (PI) saquinavir (SQV) were prepared using several types of chemical groups potentially capable of modifying its pharmacokinetic properties. These prodrug conjugates included SQV-cysteine-PEG3400, SQV-cysteine-PEG3400-biotin, SQV-cysteine(R.I.CK-Tat9) [a cationic retro-inverso-cysteine-lysine-Tat nonapeptide]-PEG3400, and SQV-cysteine(R.I.CK(stearate)-Tat9)-PEG3400. SQV was linked to cysteine to form a releasable SQV-cysteine ester bond in all of the conjugates. The amino group of the cysteine moiety provided an attachment site for a slower-degrading amide bond with N-hydroxysuccinimide-activated forms of PEG- and PEG-biotin. Disulfide bonds were used to attach the cationic peptides, R.I.CK-Tat9 and R.I.CK(stearate)-Tat9 to the cysteine moiety in order to provide cell-specific release. An assay was established and validated for measuring the activity of SQV and other protease inhibitors in biological samples. In this assay, cleavage of an internally quenched fluorescent substrate, Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gly-Lys(DABCYL)-Arg by HIV-1 protease was inhibited by SQV in a dose-dependent manner at concentrations of 0.05-0.5 microM. All prodrug conjugates were shown to be inactive in this assay until the ester bond was cleaved and active SQV was released. The prodrug reconversion half-lives in 0.1 N HCl, phosphate-buffered saline (PBS) at pH 7.4 and in spiked plasma at 37 degrees C were 9, 14, and 0.9 h, respectively. The anti-HIV-1 activity (ED(50)) of the PEG-based SQV prodrug conjugates was evaluated in MT-2 cells using an MTT assay. The activity of conjugated SQV was reduced (ED(50) = 900 nM) for the PEG only conjugate, but restored with the addition of biotin (ED(50) = 125 nM), R.I.CK-Tat9 (ED(50) = 15 nM), and R.I.CK(stearate)-Tat9 (ED(50) = 62 nM) as compared to maximum achievable anti-HIV-1 activity (unconjugated SQV, control, ED(50) = 15 nM), suggesting enhanced cellular uptake of conjugates. Cytotoxicity (LD(50)) was assessed for all prodrug conjugates using non-HIV-1 infected cells and was found to be in the micromolar range. The difference between the LD(50) and ED(50) suggests a favorable therapeutic index for the prodrug conjugates. In conclusion, these promising initial results demonstrate that the reconversion of the conjugate prodrugs was complete and that active SQV was released. Since the major delivery advantages of PEG prodrug conjugates can only be observed in vivo, issues of reconversion and elimination half-lives in plasma will have to be further studied in an in vivo model. The current results also demonstrate that the protease inhibition assay is a simple yet effective bioanalytical tool that can be used to assess the release and anti-HIV-1 activity of HIV-1 PIs from their prodrug forms.

P-glycoprotein expression affects the intracellular concentration and antiviral activity of the protease inhibitor saquinavir in a T cell line.

A number of ATP-binding cassette proteins, which function as cellular efflux pumps, are known to be expressed on the membranes of human cells, including CD4-positive lymphocytes. It has also been shown recently that most anti-HIV protease inhibitors (PIs) are first-rate substrates of one of these membrane transporters, P-glycoprotein (Pgp). These findings raise the question of whether Pgp expression could influence HIV replication and/or affect the action of PIs. To gain new insight into this, initially unexpected, phenomenon, a study was undertaken with the aims of investigating whether treatment with saquinavir (SQV) induces Pgp expression in primary or transformed human T cell lines and, primarily, establishing whether Pgp expression could modify both the uptake of SQV and its antiviral action. Pgp expression, mainly measured by reverse transcription-PCR, was found to be variably detectable in healthy individuals, and short or prolonged SQV treatment was unable to induce or increase the expression of Pgp in a lymphoblastoid cell line or in primary lymphocytes derived from these individuals. However, further experiments, performed in a cell line with high Pgp expression (CEM(VBL100) cells) and its parental cell line (CEM cells), demonstrated that over-expression of Pgp reduces the uptake of SQV This result is consistent with the finding that CEM(VBL100) cells are less sensitive to the antiviral activity of SQV, the ID50 value (100 microM) being significantly higher than the corresponding value in parental CEM cells (4 microM).

Interaction of anti-HIV protease inhibitors with the multidrug transporter P-glycoprotein (P-gp) in human cultured cells.

The anti-HIV protease inhibitors represent a new class of agents for treatment of HIV infection. Saquinavir, ritonavir, indinavir, and nelfinavir are the first drugs approved in this class and significantly reduce HIV RNA copy number with minimal adverse effects. They are all substrates of cytochrome P450 3A4, and are incompletely bioavailable. The drug transporting protein, P-glycoprotein (P-gp), which is highly expressed in the intestinal mucosa, could be responsible for the low oral bioavailability of these and other drugs which are substrates for this transporter. To determine whether these protease inhibitors are modulators of P-gp, we studied them in cell lines which do and do not express P-gp. Saquinavir, ritonavir and nelfinavir significantly inhibited the efflux of [3H]paclitaxel and [3H]vinblastine in P-gp-positive cells, resulting in an increase in intracellular accumulation of these drugs. However, similar concentrations of indinavir did not affect the accumulation of these anticancer agents. In photoaffinity labeling studies, saquinavir and ritonavir displaced [3H]azidopine, a substrate for P-gp, in a dose-dependent manner. These data suggest that saquinavir, ritonavir, and nelfinavir are inhibitors and possibly substrates of P-gp. Because saquinavir has a low bioavailability, its interaction with P-gp may be involved in limiting its absorption.

Active apical secretory efflux of the HIV protease inhibitors saquinavir and ritonavir in Caco-2 cell monolayers.

PURPOSE: To investigate in vitro the mechanisms involved in the gastro-intestinal absorption of the HIV protease inhibitor, saquinavir mesylate (Invirase), whose oral bioavailability is low, variable, and significantly increased by co-administration with ritonavir, also an HIV protease inhibitor but with higher oral bioavailability. METHODS: Confluent epithelial layers of human Caco-2 cells mimicking the intestinal barrier. RESULTS: Both saquinavir and ritonavir showed polarized transport through Caco-2 cell monolayers in the basolateral to apical direction (secretory pathway), exceeding apical to basolateral transport (absorptive pathway) by factors of 50-70 and 15-25, respectively. Active efflux was temperature dependent, saturable and inhibited by verapamil and cyclosporin A. Saquinavir and ritonavir decreased each other's secretory permeability and hence elevated their net transport by the absorptive pathway. CONCLUSIONS: Saquinavir and ritonavir are both substrates for an efflux mechanism in the gut, most likely P-glycoprotein, which acts as a counter-transporter for both drugs. Together with sensitivity to gutwall metabolism by cytochrome P-450 3A, this may partially account for the low and variable oral bioavailability of saquinavir in clinical studies and for its increased bioavailability after co-administration with ritonavir.