Examining pharmacodynamic and pharmacokinetic properties of eleven analogues of saquinavir for HIV protease inhibition.

HIV is one of the most lethal viral diseases in the human population. Patients often suffer from drug resistance, which hampers HIV therapy. Eleven different structural analogues of saquinavir (SQV), designed using ChemSketch™ and named S1 through S11, were compared with SQV with respect to their pharmacodynamic and pharmacokinetic properties. Pharmacokinetic predictions were carried out using AutoDock, and molecular docking between macromolecule HIV protease (PDB ID: 3IXO) and analogues S1 - S11 as ligands was performed. Analogues S1, S3, S4, S9 and S11 had lower binding scores when compared with saquinavir, whereas that of analogue S5 was similar. Pharmacokinetic predictions made using ACDilab2, including the Lipinski profile, general physical features, absorption, distribution, metabolism and excretion parameters, and toxicity values, for the eleven analogues and SQV suggested that S1 and S5 are pharmacodynamically and pharmacokinetically robust molecules that could be developed and established as lead molecules after in vitro and in vivo studies.

HIV-protease inhibitors for the treatment of cancer: Repositioning HIV protease inhibitors while developing more potent NO-hybridized derivatives?

The possible use of HIV protease inhibitors (HIV-PI) as new therapeutic option for the treatment of cancer primarily originated from their success in treating HIV-related Kaposi's sarcoma (KS). While these findings were initially attributed to immune reconstitution and better control of oncogenic viral infections, the number of reports on solid tumors, KS, lymphoma, fibrosarcoma, multiple myeloma and prostate cancer suggest other mechanisms for the anti-neoplastic activity of PIs. However, a major drawback for the possible adoption of HIV-PIs in the therapy of cancer relies on their relatively weak anticancer potency and important side effects. This has propelled several groups to generate derivatives of HIV-PIs for anticancer use, through modifications such as attachment of different moieties, ligands and transporters, including saquinavir-loaded folic acid conjugated nanoparticles and nitric oxide (NO) derivatives of HIV-PIs. In this article, we discuss the current preclinical and clinical evidences for the potential use of HIV-PIs, and of novel derivatives, such as saquinavir-NO in the treatment of cancer.

Cytotoxic and immune-sensitizing properties of nitric oxide-modified Saquinavir in iNOS-positive human melanoma cells.

We have recently shown that covalent attachment of the NO moiety to the HIV protease inhibitor Saquinavir (Saq) produced a qualitatively new chemical entity, named Saquinavir-NO (Saq-NO), with enhanced anticancer properties and reduced toxicity. In this study we evaluated the impact of Saq-NO on the growth of A375 human melanoma cells, as a prototype of NO-dependent cancer model. The novel compound strongly affected the in vitro and in vivo progression of A375 melanoma cell growth. The mechanism of antimelanoma action comprised dual drug activity-induction of apoptotic cell death and acquisition of melanoma cell responsiveness to TRAIL. Saq-NO-triggered apoptosis was dependent on transient AKT up-regulation and reduced pERK and iNOS expression that were observed within the first 12 h of exposure to the drug. Thereafter, however, Saq-NO up-regulated both iNOS transcription and NO endogenous synthesis and sensitized A375 cells to TRAIL. Furthermore, reduced YY1 expression was observed after 24 h of Saq-NO exposure, which correlated with increased expression of DR5. The biological relevance of this complex and powerful action of Saq-NO was consistent with the marked drug-induced inhibition of the growth of A375 xenotransplants in nude mice.

Stereocontrolled synthesis and biological activity of two diastereoisomers of the potent HIV-1 protease inhibitor saquinavir.

A general enantioselective synthesis of new syn-hydroxyethylamine isosteres has been developed. The approach, based on the controlled opening of functionalized optically active 2,3-epoxy amines, can be conveniently used for the preparation of new peptidomimetics with various residues. Finally the total synthesis of two diastereoisomer analogues of HIV-Protease inhibitor Saquinavir has been achieved and their biological activity evaluated.

Intestinal absorption of novel-dipeptide prodrugs of saquinavir in rats.

Saquinavir (SQV) was the first human immuno-virus-1 (HIV-1) protease inhibitor approved by FDA. However, P-glycoprotein (P-gp), an efflux pump limits its oral and brain bioavailabilities. The objective of this study is to investigate whether prodrug modification of SQV to dipeptide prodrugs Valine-Valine-Saquinavir (Val-Val-SQV) and Glycine-Valine-Saquinavir (Gly-Val-SQV) targeting intestinal peptide transporter can enhance intestinal permeability of SQV by circumventing P-gp mediated efflux. Single pass intestinal perfusion experiments in rat jejunum were performed to calculate the absorption rate constant and intestinal permeability of SQV, Val-Val-SQV and Gly-Val-SQV. Equimolar concentration (25 microM) of SQV, Val-Val-SQV and Gly-Val-SQV were employed in the perfusion studies. Perfusion experiments were also carried out in the presence of cyclosporine (10 microM) and glycyl-sarcosine (20 mM). Absorption rate constants in rat jejunum (ka) for SQV, Val-Val-SQV and Gly-Val-SQV were found to be 14.1+/-3.4x10(-3), 65.8+/-4.3x10(-3), and 25.6+/-5.7x10(-3) min(-1), respectively. Enhanced absorption of Val-Val-SQV and Gly-Val-SQV relative to SQV can be attributed to their translocation by the peptide transporter in the jejunum. Significant permeability enhancement of SQV across rat jejunum was observed in the presence of cyclosporine 10 microM (P-gp inhibitor). However, permeability of Val-Val-SQV was unchanged in the presence of cyclosporine suggesting lack of any interaction of the prodrug with efflux pump. Intestinal absorption of Val-Val-SQV was significantly inhibited in the presence of gly-sar indicating the involvement of peptide transporter in intestinal absorption. In conclusion, peptide transporter targeted prodrug modification of P-gp substrates could lead to shielding of these drug molecules from efflux pumps.

Saquinavir-NO inhibits IL-6 production in macrophages.

Covalent attachment of the nitric oxide (NO) moiety to the HIV protease inhibitor Saquinavir (Saq) produced a new chemical entity, named Saquinavir-NO, (Saq-NO) with reduced toxicity and potent immunoregulatory influence on T lymphocytes. In this study, we have compared head-to-head the effects of Saq-NO and Saq on mouse and rat peritoneal macrophage cytokine secretion and NO production upon in vitro, ex vivo and in vivo conditions. The results demonstrate that Saq-NO, but not Saq, potently decreased interleukin (IL)-10, IL-6 and nitrite accumulation and increased the levels of IL-1ß and tumour necrosis factor (TNF) in supernatants of mouse and rat macrophage cultures in vitro. Treatment of mice with Saq-NO, but not Saq, inhibited ex vivo secretion of IL-6 from macrophages. Consistent with these findings, Saq-NO also reduced blood levels of IL-6 in lipopolysaccharide-treated mice. The observed inhibitory influence of Saq-NO on IL-6 generation in macrophages may be involved in the observed antitumour and immunomodulatory effects of the drug.

Saquinavir-NO inhibits S6 kinase activity, impairs secretion of the encephalytogenic cytokines interleukin-17 and interferon-gamma and ameliorates experimental autoimmune encephalomyelitis.

NO-hybridization of the HIV protease inhibitor Saquinavir generates a new chemical entity named Saq-NO, that retains the anti-viral activity and exerts lower toxicity. We show that Saq-NO inhibited the generation of various cytokines in ConA-stimulated unfractionated murine spleen cells and rat lymph nodes stimulated with ConA as well as in purified CD4(+) T cells in vitro and reduced the circulating levels of cytokines in mice challenged with anti-CD3 antibody. Furthermore, Saq-NO reduced IL-17 and IFN-γ production in myelin basic protein (MBP)-specific cells isolated from rats immunized with MBP. These findings translated well into the in vivo setting as Saq-NO ameliorated the course of the disease in two preclinical models of multiple sclerosis. Our results demonstrate that Saq-NO exerts immunomodulatory effects that warrant studies on its application in autoimmune diseases.

Unique antineoplastic profile of Saquinavir-NO, a novel NO-derivative of the protease inhibitor Saquinavir, on the in vitro and in vivo tumor formation of A375 human melanoma cells.

We have recently shown that covalent attachment of the nitric oxide (NO) moiety to the HIV protease inhibitor Saquinavir (Saq) produced a qualitatively new chemical entity, named Saquinavir-NO (Saq-NO), with enhanced anticancer properties and reduced toxicity both in vitro and in vivo. The aim of this study was to address several unanswered questions both on the pharmacological profile of Saq-NO as well as on the in vivo role of NO in the oncogenesis of A375 human melanoma cells. To this end, we have evaluated here the impact of single and combined effects of Saq-NO, Saq, the NO-donor DETA NONOate and the iNOS inhibitor L-NAME on the in vitro as well as in vivo growth of the iNOS positive A375 cells. Our data confirm clear-cut evidence for a strong and powerful anti-melanoma action of Saq-NO that is not duplicable by the combined use of Saq and DETA NONOate. Surprisingly, but also in agreement with the complex and multifaceted role of endogenous NO in A375 cells, both DETA NONOate and L-NAME significantly suppressed the in vivo growth of xenotransplants.

Stereoselective evasion of P-glycoprotein, cytochrome P450 3A, and hydrolases by peptide prodrug modification of saquinavir.

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.

Saquinavir-NO-targeted S6 protein mediates sensitivity of androgen-dependent prostate cancer cells to TRAIL.

We previously reported that the NO-modified form of HIV protease inhibitor Saquinavir (Saq) is a potent antitumoral agent efficient against numerous tumor cell lines in vitro and in vivo. In acute toxicity studies, doses of Saq-NO equivalent to DL100 of the parental drug were completely nontoxic. Beside direct effect on malignant cell growth, Saq-NO sensitizes certain type of cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cell death. In this study, we evaluated the effects of Saq-NO on androgen-dependent prostate cancer LNCaP. Saq-NO inhibited both the growth of LNCaP cells in vitro and in xenograft models. Suppression of tumor growth was accompanied with cell cycle arrest in G 0/G 1 phase and established a persistent inhibition of proliferation. Furthermore, Saq-NO reverted sensitivity of LNCaP cells to TRAIL but not to TNF. Treatment of cells with Saq-NO induced transient upregulation of Akt and ERK1/2. This, however, did not represent the primary mode of action of Saq-NO, as elimination with specific inhibitors did not compromise the chemotherapic efficacy of the drug. However, permanent abrogation of phosphorylation of the S6 protein, which is the downstream target of both signaling pathways, was observed. Diminished S6 phosphorylation was associated with re-established sensitivity to TRAIL and reduction of X-linked inhibitor of apoptosis protein (XIAP). In summary, NO modification of Saq led to a new chemical entity with stronger and more pleiotropic antitumor activity than the parental drug.

The new and less toxic protease inhibitor saquinavir-NO maintains anti-HIV-1 properties in vitro indistinguishable from those of the parental compound saquinavir.

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.

In vitro and in vivo anticancer action of Saquinavir-NO, a novel nitric oxide-derivative of the protease inhibitor saquinavir, on hormone resistant prostate cancer cells.

The NO-derivative of the HIV protease inhibitor saquinavir (Saq-NO) is a nontoxic variant of the parental drug with enhanced anticancer activity on several cell lines. However, it is still unclear whether the p53 status of the target cell might influence the sensitivity to Saq-NO. In this study we evaluated the in vitro and in vivo activity of Saq-NO on the p53-deficient hormone resistant prostate cancer PC-3 cells. We demonstrate that the absence of functional p53 is not essential for the capacity of Saq-NO to reduce prostate cancer cell growth. In contrast to its previously described cytostatic action in B16 and C6 cell lines, Saq-NO exerted cytotoxic effects in PC-3 cells leading to dominant induction of apoptosis and enhanced production of proapoptotic Bim. In addition, differently from saquinavir, Saq-NO restored TRAIL sensitivity that was correlated with increased expression of DR5 independent from ROS/RNS production and YY1 repression. NF-κB activation may be responsible of the Saq-NO induced DR5 expression. Moreover, Saq-NO but not saquinavir, exerted synergistic activity with conventional cytostatic therapy. In agreement with these in vitro studies, Saq-NO inhibited the in vivo growth of PC-3 cells xenotransplants to a greater extent than the parental compound. Taken together, these data indicate that Saq-NO possesses powerful and suitable in vitro and in vivo chemotherapeutic potential to be further studied as a novel drug for the treatment of prostate cancer in the clinical setting.

Targeting SVCT for enhanced drug absorption: synthesis and in vitro evaluation of a novel vitamin C conjugated prodrug of saquinavir.

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.

Synthesis of new thienyl ring containing HIV-1 protease inhibitors: promising preliminary pharmacological evaluation against recombinant HIV-1 proteases.

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.

Anticancer effects of the nitric oxide-modified saquinavir derivative saquinavir-NO against multidrug-resistant cancer cells.

The human immunodeficiency virus (HIV) protease inhibitor saquinavir shows anticancer activity. Although its nitric oxide-modified derivative saquinavir-NO (saq-NO) was less toxic to normal cells, it exerted stronger inhibition of B16 melanoma growth in syngeneic C57BL/6 mice than saquinavir did. Saq-NO has been shown to block proliferation, upregulate p53 expression, and promote differentiation of C6 glioma and B16 cells. The anticancer activity of substances is frequently hampered by cancer cell chemoresistance mechanisms. Therefore, we here investigated the roles of p53 and the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein 1 (BCRP1) in cancer cell sensitivity to saq-NO to get more information about the potential of saq-NO as anticancer drug. Saq-NO exerted anticancer effects in lower concentrations than saquinavir in a panel of human cancer cell lines. Neither p53 mutation or depletion nor expression of P-gp, MRP1, or BCRP1 affected anticancer activity of saq-NO or saquinavir. Moreover, saq-NO sensitized P-gp-, MRP1-, or BCRP1-expressing cancer cells to chemotherapy. Saq-NO induced enhanced sensitization of P-gp- or MRP1-expressing cancer cells to chemotherapy compared with saquinavir, whereas both substances similarly sensitized BCRP1-expressing cells. Washout kinetics and ABC transporter ATPase activities demonstrated that saq-NO is a substrate of P-gp as well as of MRP1. These data support the further investigation of saq-NO as an anticancer drug, especially in multidrug-resistant tumors.

Interaction of dipeptide prodrugs of saquinavir with multidrug resistance protein-2 (MRP-2): evasion of MRP-2 mediated efflux.

Saquinavir (SQV), the first protease inhibitor approved by FDA to treat HIV-1 infection. This drug is a well-known substrate for multidrug resistance protein-2 (MRP-2). The objective of this study was to investigate whether derivatization of SQV to dipeptide prodrugs, valine-valine-saquinavir (Val-Val-SQV) and glycine-valine-saquinavir (Gly-Val-SQV), targeting peptide transporter can circumvent MRP-2 mediated efflux. Uptake and transport studies were carried out across MDCKII-MRP2 cell monolayers to investigate the interaction of SQV and its prodrugs with MRP-2. In situ single pass intestinal perfusion experiments in rat jejunum were performed to calculate intestinal absorption rate constants and permeabilities of SQV, Val-Val-SQV and Gly-Val-SQV. Uptake studies demonstrated that the prodrugs have significantly lower interaction with MRP-2 relative to SQV. Transepithelial transport of Val-Val-SQV and Gly-Val-SQV across MDCKII-MRP2 cells exhibited an enhanced absorptive flux and reduced secretory flux as compared to SQV. Intestinal perfusion studies revealed that synthesized prodrugs have higher intestinal permeabilities relative to SQV. Enhanced absorption of Val-Val-SQV and Gly-Val-SQV relative to SQV can be attributed to their translocation by the peptide transporter in the jejunum. In the presence of MK-571, a MRP family inhibitor, there was a significant increase in the permeabilities of SQV and Gly-Val-SQV indicating that these compounds are probably substrates for MRP-2. However, there was no change in the permeability of Val-Val-SQV with MK-571 indicating lack of any interaction of Val-Val-SQV with MRP-2. In conclusion, peptide transporter targeted prodrug modification of MRP-2 substrates may lead to shielding of these drug molecules from MRP-2 efflux pumps.

Potent HIV protease inhibitors incorporating high-affinity P2-ligands and (R)-(hydroxyethylamino)sulfonamide isostere.

Design and synthesis of a series of very potent nonpeptide HIV protease inhibitors are described. The inhibitors are derived from novel high affinity P2-ligands and (R)-(hydroxyethylamino)sulfonamide isostere.