Protease Inhibitors, Saquinavir and Darunavir, Inhibit Oligodendrocyte Maturation: Implications for Lysosomal Stress.

Despite the introduction of antiretroviral (ARV) therapy (ART), approximately 30-50% of people living with human immunodeficiency virus-1 (HIV-1) will develop a spectrum of measurable neurocognitive dysfunction, collectively called HIV-associated neurocognitive disorder (HAND). While the clinical manifestations of HAND have changed with the advent of ART, certain pathological features have endured, including white matter alterations and dysfunction. The persistence of white matter alterations in the post-ART era suggests that ARV drugs themselves may contribute to HAND pathology. Our group has previously demonstrated that two ARV compounds from the protease inhibitor (PI) class, ritonavir and lopinavir, inhibit oligodendrocyte maturation and myelin protein production. We hypothesized that other members of the PI class, saquinavir and darunavir, could also negatively impact oligodendrocyte differentiation. Here we demonstrate that treating primary rat oligodendrocyte precursor cells with therapeutically relevant concentrations of either ARV drug results in a concentration-dependent inhibition of oligodendrocyte maturation in vitro. Furthermore, we show that acidifying endolysosomal pH via a mucolipin transient receptor potential channel 1 (TRPML1) agonist provides protection against saquinavir- and darunavir-induced inhibition of oligodendrocyte maturation. Moreover, our findings suggest, for the first time, an imperative role of proper endolysosomal pH in regulating OL differentation, and that therapeutic targeting of endolysosomes may provide protection against ARV-induced oligodendrocyte dysregulation. Graphical Abstract Treatment of primary rat oligodendrocyte precursor cells with therapeutically relevant concentrations of either antiretroviral compound of the protease inhibitor class, darunavir or saquinavir, results in a concentration-dependent inhibition of oligodendrocyte maturation in vitro. Additionally, in darunavir or saquinavir-treated cultures we observed a concentration-dependent decrease in the number of acidic lysosomes, via immunostaining with LysoTracker Red, compared with vehicle-treated cultures. Finally, we showed that acidifying endolysosomal pH via a mucolipin transient receptor potential channel 1 (TRPML1) agonist provides protection against saquinavir- or darunavir-induced inhibition of oligodendrocyte maturation. Our findings suggest, for the first time, a critical role of proper endolysosomal pH in regulating OL differentation, and that therapeutic targeting of endolysosomes may provide protection against antiretroviral-induced oligodendrocyte dysregulation.

Saquinavir Induced Suicidal Death of Human Erythrocytes.

BACKGROUND/AIMS: The antiretroviral protease inhibitor saquinavir is used for the treatment of HIV infections. Effects of saquinavir include induction of apoptosis, the suicidal death of nucleated cells. Saquinavir treatment may further lead to anemia. In theory, anemia could result from accelerated erythrocyte loss by enhanced suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress with increase of reactive oxygen species (ROS) and ceramide. The present study explored, whether and how saquinavir induces eryptosis. METHODS: To this end, flow cytometry was employed to estimate erythrocyte volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, ROS abundance from DCFDA fluorescence and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to saquinavir significantly decreased forward scatter (≥ 5 µg/ml), significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly increased Fluo3-fluorescence (15 µg/ml), significantly increased DCFDA fluorescence (15 µg/ml), but did not significantly modify ceramide abundance. The effect of saquinavir on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. CONCLUSIONS: Saquinavir triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation and Ca2+ entry.

Multimodal nanoparticles as alignment and correlation markers in fluorescence/soft X-ray cryo-microscopy/tomography of nucleoplasmic reticulum and apoptosis in mammalian cells.

Correlative fluorescence and soft X-ray cryo-microscopy/tomography on flat sample holders is perfectly suited to study the uncompromised physiological status of adherent cells at its best possible preservation by imaging after fast cryo-immobilization. To understand the mechanism by which herpesviruses induce nucleoplasmic reticulum, i.e. invaginations of the nuclear envelope, during their egress from the host cell nucleus, morphologically similar structures found in laminopathies and after chemical induction were investigated as a potentially more easily accessible model system. For example, anti-retroviral protease inhibitors like Saquinavir also induce invaginations of the nuclear membranes. With the help of newly designed multimodal nanoparticles as alignment and correlation markers, and by optimizing fluorescence cryo-microscopy data acquisition, an elaborate three-dimensional network of nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated rabbit kidney cells expressing a fluorescently labeled inner nuclear membrane protein. In part of the protease inhibitor-treated samples, nuclei exhibited dramatic ultrastructural changes indicative of programmed cell death/apoptosis. This unexpected observation highlights another unique feature of soft X-ray microscopy, i.e. high absorption contrast information not relying on labeled cellular components, at a 3D resolution of approximately 40 nm (half-pitch) and through a sample thickness of several micrometers. These properties make it a valuable part of the cell biology imaging toolbox to visualize the cellular ultrastructure in its completeness.

Novel intravaginal nanomedicine for the targeted delivery of saquinavir to CD4+ immune cells.

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.

Drug synergy of tenofovir and nanoparticle-based antiretrovirals for HIV prophylaxis.

BACKGROUND: The use of drug combinations has revolutionized the treatment of HIV but there is no equivalent combination product that exists for prevention, particularly for topical HIV prevention. Strategies to combine chemically incompatible agents may facilitate the discovery of unique drug-drug activities, particularly unexplored combination drug synergy. We fabricated two types of nanoparticles, each loaded with a single antiretroviral (ARV) that acts on a specific step of the viral replication cycle. Here we show unique combination drug activities mediated by our polymeric delivery systems when combined with free tenofovir (TFV). METHODOLOGY/PRINCIPAL FINDINGS: Biodegradable poly(lactide-co-glycolide) nanoparticles loaded with efavirenz (NP-EFV) or saquinavir (NP-SQV) were individually prepared by emulsion or nanoprecipitation techniques. Nanoparticles had reproducible size (d ∼200 nm) and zeta potential (-25 mV). The drug loading of the nanoparticles was approximately 7% (w/w). NP-EFV and NP-SQV were nontoxic to TZM-bl cells and ectocervical explants. Both NP-EFV and NP-SQV exhibited potent protection against HIV-1 BaL infection in vitro. The HIV inhibitory effect of nanoparticle formulated ARVs showed up to a 50-fold reduction in the 50% inhibitory concentration (IC50) compared to free drug. To quantify the activity arising from delivery of drug combinations, we calculated combination indices (CI) according to the median-effect principle. NP-EFV combined with free TFV demonstrated strong synergistic effects (CI50 = 0.07) at a 1∶50 ratio of IC50 values and additive effects (CI50 = 1.05) at a 1∶1 ratio of IC50 values. TFV combined with NP-SQV at a 1∶1 ratio of IC50 values also showed strong synergy (CI50 = 0.07). CONCLUSIONS: ARVs with different physicochemical properties can be encapsulated individually into nanoparticles to potently inhibit HIV. Our findings demonstrate for the first time that combining TFV with either NP-EFV or NP-SQV results in pronounced combination drug effects, and emphasize the potential of nanoparticles for the realization of unique drug-drug activities.

Saquinavir inhibits early events associated with establishment of HIV-1 infection: potential role for protease inhibitors in prevention.

The maturation of newly formed human immunodeficiency virus type 1 (HIV-1) virions is a critical step for the establishment of productive infection. We investigated the potential of saquinavir (SQV), a protease inhibitor (PI) used in highly active antiretroviral therapy (HAART), as a candidate microbicide. SQV inhibited replication of clade B and clade C isolates in a dose-dependent manner in all cellular models tested: PM-1 CD4 T cells, peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages (MDMs), and immature monocyte-derived dendritic cells (iMDDCs). SQV also inhibited production of infectious virus in cervical, penile, and colorectal explants cocultured with T cells. Moreover, SQV demonstrated inhibitory potency against trans infection of T cells by in vitro-derived dendritic cells and by primary dendritic cells that emigrate from penile and cervical tissue explants. No cellular or tissue toxicity was detected in the presence of SQV, suggesting that this drug could be considered for development as a component of an effective microbicide, capable of blocking viral maturation and transmission of HIV-1 at mucosal surfaces.

Preparation and performance evaluation of saquinavir laden cationic submicron emulsions.

The main purpose of the present study was to investigate different cationic submicron emulsions as potential delivery for oral administration. Different submicron emulsion based formulations were prepared by standard procedures incorporating Chitosan, stearylamine, and protamine as charge inducer. Saquinavir (SQ) laden emulsions were characterized in terms of globule size, zeta potential, entrapment efficiency, release profile, cytotoxicity, LDH release, and stability studies. The prepared formulations were stable in terms of mean globule size, drug content, and tended to retain their cationic charge. Pay load efficiency was found to be pretty high (approximately 95-99%) in various formulations prepared. Sustained release phenomenon was more prominent in the case of chitosan emulsions (CE) followed by stearylamine emulsion (SE), Protamine emulsion (PE), and then plain emulsion (E) containing no charge inducer. The total amounts of drug released in 24 hr from CE, SE, PE, and E were 46%, 52%, 56%, and 62%, respectively. The induction of positive charge in emulsions resulted in enhanced absorption of drug through intestinal membrane. The apparent permeability coefficient through the intestinal sac was in the order of CE > SE > PE > E. The permeation flux of SQ through CE (1.0 microg/min) was more than twice compared to plain emulsion (0.46 microg/min) while it was almost three times (0.3 microg/min) compared to control. However, protamine based emulsion didn't confer significant improvement in absorption when compared to plain emulsion formulation. By this study it can be concluded that induction of positive charge on submicron emulsions can be effective for improving oral absorption of drug safely, as it is evinced with low LDH release into the medium when intestinal tissue is treated with submicron emulsion.

The antitumor properties of a nontoxic, nitric oxide-modified version of saquinavir are independent of Akt.

Application of the HIV protease inhibitor saquinavir (Saq) to cancer chemotherapy is limited by its numerous side effects. To overcome this toxicity, we modified the original compound by covalently attaching a nitric oxide (NO) group. We compared the efficacy of the parental and NO-modified drugs in vitro and in vivo. The novel compound saquinavir-NO (Saq-NO) significantly reduced the viability of a wide spectrum of human and rodent tumor cell lines at significantly lower concentration than the unmodified drug. In contrast to Saq, Saq-NO had no effect on the viability of primary cells and drastically reduced B16 melanoma growth in syngeneic C57BL/6 mice. In addition, at the equivalent of the 100% lethal dose of Saq, Saq-NO treatment caused no apparent signs of toxicity. Saq-NO blocked the proliferation of C6 and B16 cells, up-regulated p53 expression, and promoted the differentiation of these two cell types into oligodendrocytes or Schwann-like cells, respectively. Although it has been well documented that Saq decreases tumor cell viability by inhibiting Akt, the anticancer properties of Saq-NO were completely independent of the phosphatidylinositol 3-kinase/Akt signaling pathway. Moreover, Saq-NO transiently up-regulated Akt phosphorylation, delivering a protective signal that could be relevant for primary cell protection and the absence of drug toxicity in vivo. It was unlikely that released NO was independently responsible for these drug effects because Saq-NO treatment increased intracellular and secreted NO levels only slightly. Rather, the chemical modification seems to have produced a qualitatively new chemical entity, which may have a unique mode of action against cancer cells.

HIV-1 protease inhibitor induced oxidative stress suppresses glucose stimulated insulin release: protection with thymoquinone.

The highly active anti-retroviral therapy (HAART) regimen has considerably reduced the mortality rate in HIV-1 positive patients. However, long-term exposure to HAART is associated with a metabolic syndrome manifesting cardiovascular dysfunction, lipodystrophy, and insulin resistance syndrome (IRS). The inclusion of HIV-1 protease inhibitors (PIs) in HAART has been linked to the induction of IRS. Although several molecular mechanisms of PI-induced effects on insulin action have been postulated, the deleterious effects of PIs on insulin production by pancreatic beta-cells have not been fully investigated and therapeutic strategies to ameliorate insulin dysregulation at this level have not been targeted. The present study showed that exposure to several different PIs, nelfinavir (5-10 microM), saquinavir (5-10 microM) and atazanavir (8-20 microM), decreases glucose stimulated insulin secretion from rat pancreatic beta-cells (INS-1). Nelfinavir significantly increased reactive oxygen species (ROS) generation and suppressed cytosolic, but not mitochondrial superoxide dismutase (SOD) levels. Nelfinvair also decreased both glutathione and ATP and increased UCP2 levels in these cells. Simultaneous treatment with thymoquinone (TQ) (2.5 microM), an active ingredient of black seed oil, significantly inhibited the effect of nelfinavir on augmented ROS production and suppressed SOD levels. Both TQ and black seed oil exposure increased glucose stimulated insulin secretion and ameliorated the suppressive effect of nelfinavir. The present findings imply a direct role of ROS in PI induced deleterious effects on pancreatic beta-cells. Our findings also suggest that TQ may be used as a potential therapeutic agent to normalize the dysregulated insulin production observed in HAART treated patients.

Clearance and clearance inhibition of the HIV-1 protease inhibitors ritonavir and saquinavir in sandwich-cultured rat hepatocytes and rat microsomes.

The metabolism and active transport of ritonavir and saquinavir were studied using sandwich-cultured rat hepatoyctes and rat liver microsomes. For ritonavir four comparable metabolites were observed in the sandwich-culture and in microsomes. For saquinavir eight metabolites were observed in sandwich-culture and 14 different metabolites in microsomes. Ketoconazole did not affect the metabolism of ritonavir in sandwich-culture or microsomes and slightly inhibited the metabolism of saquinavir in sandwich-culture. This inhibition resulted in a different metabolite profile for saquinavir in microsomes. Ritonavir had a pronounced inhibiting effect on the metabolism of saquinavir and affected the hydroxylation of 6beta-testosterone negatively. In the active transport studies, cyclosporin A and PSC833 enhanced the metabolism of ritonavir, suggesting that ritonavir is normally excreted into the bile canaliculi. Verapamil, showed no effect on the metabolism of ritonavir. The intrinsic clearance was estimated at 1.65 and 67.5 microl/min/1 x 10(6) cells and the hepatic metabolism clearance at 0.017 and 6.83ml/min/SRW for ritonavir and saquinavir respectively. In conclusion, for saquinavir the metabolism rate and the amount of metabolites produced was higher than for ritonavir. Ritonavir had a strong inhibitory effect on the metabolism of saquinavir and seemed to be excreted into the bile.

Vascular endothelial toxicity induced by HIV protease inhibitor: evidence of oxidant-related dysfunction and apoptosis.

HIV-protease inhibitor (HIV-PI) drugs are critical for highly active antiretroviral therapy (HAART) efficacy, but several recent reports have suggested that metabolic and/or cardiovascular toxicities are associated with these drugs. Given the importance of the HIV-PI drug class and the widespread and chronic use of these agents in an expanding patient population, further understanding of this potential drug toxicity is imperative. Here, we investigated a role for direct endothelial toxicity induced by saquinavir (SAQ), the first HIV-PI drug marketed in the United States and still an important component of HAART therapies. In initial studies using isolated vascular tissues, we observed selective impairment of endothelium-dependent vasodilation with no effect on contractile responses. Subsequent studies using human endothelial cells in culture at clinically relevant concentrations (5 and 10 microM, 2-48 h) demonstrated concentration-dependent increases in cell death, mainly via apoptosis rather than necrosis (determined via Annexin-V positive membrane labeling). Live cell imaging also demonstrated increased intracellular oxidant production (as measured by DCF fluorescence), which could be abrogated by incubation with the antioxidant N-acetylcysteine (NAC). NAC also prevented SAQ- induced apoptotic cell death. These data demonstrate that SAQ has direct toxicological effects on endothelial cells, and that the toxicity apparently involves apoptotic pathway activation via reactive oxygen and/or nitrogen species.

Mitochondrial membrane hyperpolarization hijacks activated T lymphocytes toward the apoptotic-prone phenotype: homeostatic mechanisms of HIV protease inhibitors.

A decrease of mitochondrial membrane potential has been hypothesized to be a marker of apoptotic cells, including activated T lymphocytes. It was recently demonstrated that HIV protease inhibitors, independently from any viral infection, can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis. To analyze the mechanisms underlying these effects, a specific study was undertaken in both resting and activated human PBL exposed to either receptor (e.g., anti-Fas)- or nonreceptor (e.g., radiation)-mediated apoptotic stimuli. T cell activation was found to be accompanied by a significant increase in mitochondrial membrane potential, or hyperpolarization, which was undetectable in resting cells. We also detected apoptotic hindering by HIV protease inhibitors only in activated T lymphocytes. This was apparently due to the ability of these drugs to block activation-associated mitochondria hyperpolarization, which, in turn, was paralleled by an impairment of cell cycle progression. Remarkably, protease inhibitors also prevented zidovudine-mediated mitochondrial toxicity. Finally, HIV-infected cells from naive patients behaved identically to activated T cells, displaying hyperpolarized mitochondria, while lymphocytes from patients under highly active antiretroviral therapy (which included HIV protease inhibitors) seemed to react as resting cells. Altogether these results clearly indicate that the hyperpolarization state of mitochondria may represent a prerequisite for the sensitization of lymphocytes to the so-called activation-induced cell death. They also suggest that HIV protease inhibitors, by interfering with induction of the mitochondrial hyperpolarization state, can result in cell survival even independent of any viral infection.

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.