Serine Protease Inhibitors Block Invasion of Host Cells by Toxoplasma gondii

We investigated the effect of protease inhibitors on the asexual development of the protozoan parasite Toxoplasma gondii. Among the inhibitors tested only two irreversible serine protease inhibitors, 3,4-dichloroisocoumarin and 4-(2-aminoethyl)-benzenesulfonyl fluoride, clearly prevented invasion of the host cells by specifically affecting parasite targets in a dose-dependent manner, with 50% inhibitory concentrations between 1 and 5 and 50 and 100 microM, respectively. Neither compound significantly affected parasite morphology, basic metabolism, or gliding motility within the range of the experimental conditions in which inhibition of invasion was demonstrated. No partial invasion was observed, meaning that inhibition occurred at an early stage of the interaction. These results suggest that at least one serine protease of the parasite is involved in the invasive process of T. gondii.     

Kinetics of Antiviral Activity and Intracellular Pharmacokinetics of Human Immunodeficiency Virus Type 1 Protease Inhibitors in Tissue Culture

We have examined the kinetics of the inhibition of human immunodeficiency virus type 1 (HIV-1) particle infectivity by protease inhibitors (PIs) in cell culture, using either transfected HeLa cells or infected peripheral blood mononuclear cells (PBMCs) as producers of infectious virions. Both the kinetics of the initiation of antiviral activity after addition of the PIs to these cultures and the kinetics of restoration of virion infectivity after removal of the PIs from the treated cultures were examined. We found that the kinetics of initiation of particle infectivity inhibition produced by a high extracellular concentration (5 microM) of the inhibitors were similar for all five inhibitors tested: loss of particle infectivity was perceptible as early as 1 h after the initiation of PI treatment and increased gradually thereafter. By contrast, the durability of this antiviral effect following removal of the drug from the culture varied dramatically according to the drug studied. In transfected HeLa cells, saquinavir and nelfinavir exerted the most prolonged inhibition, with the half-lives of their antiviral activities being greater than 24 h, while ritonavir exerted an intermediate length of inhibition (18 h) and indinavir and amprenavir exerted a reproducibly shorter length of inhibition (5 h). For all five tested PIs, these kinetics were significantly faster in PBMCs than in HeLa cells. The striking differences in antiviral kinetics observed among the different PIs appear mostly due to differences in their intracellular concentrations and/or rates of cellular clearance. Our observations, although limited to tissue culture conditions, may help delineate the cellular parameters of the antiviral activities of HIV-1 PIs and further optimize the efficiencies of these antiretrovirals in vivo.     

Plasma Tenofovir,Emtricitabine, and Rilpivirine and Intracellular Tenofovir Diphosphate and Emtricitabine Triphosphate Pharmacokinetics following Drug Intake Cessation

Pharmacokinetic (PK) data describing a prolonged time course of antiretrovirals in plasma and peripheral blood mononuclear cells (PBMCs) are important for understanding and managing late or missed doses and to assess the appropriateness of compounds for preexposure prophylaxis (PrEP). This study aimed to evaluate the PK of coformulated tenofovir disoproxil fumarate (DF), emtricitabine, and rilpivirine in plasma and of the intracellular (IC) anabolites tenofovir diphosphate (TFV-DP) and emtricitabine triphosphate (FTC-TP) in healthy volunteers up to 9 days after drug cessation. Individuals received daily tenofovir DF-emtricitabine-rilpivirine (245/200/25 mg) for 14 days. Drug intake was stopped, and serial sampling occurred prior to the final dose and up to 216 h (9 days) after stopping drug intake. Concentrations were quantified and PK parameters calculated. Eighteen volunteers completed the study. The terminal elimination plasma half-lives for tenofovir and emtricitabine over 216 h (geometric mean [90% confidence interval]) were higher than those seen over 0 to 24 h (for tenofovir, 31 h [27 to 40 h] versus 13.3 h [12.5 to 15.1 h]; for emtricitabine, 41 h [36 to 54 h] versus 6.4 h (5.9 to 7.6 h]). Model-predicted IC half-lives (0 to 168 h) were 116 h (TFV-DP) and 37 h (FTC-TP). The plasma rilpivirine concentration at 216 h was 4.5 ng/ml (4.2 to 6.2 ng/ml), and half-lives over 0 to 216 h and 0 to 24 h were 47 h (41 to 59 h) and 35 h (28 to 46 h), respectively. These data contribute to our understanding of drug behavior following treatment interruption; however, adherence to therapy should be promoted. Validated plasma and IC target concentrations are necessary to allow interpretation with respect to sustained virus suppression or HIV prevention. (The trial was conducted in accordance with the Declaration of Helsinki [EudraCT 2012-002781-13].)     

Antimalarial Synergy of Cysteine and Aspartic Protease Inhibitors

It has been proposed that the Plasmodium falciparum cysteine protease falcipain and aspartic proteases plasmepsin I and plasmepsin II act cooperatively to hydrolyze hemoglobin as a source of amino acids for erythrocytic parasites. Inhibitors of each of these proteases have potent antimalarial effects. We have now evaluated the antimalarial effects of combinations of cysteine and aspartic protease inhibitors. When incubated with cultured P. falciparum parasites, cysteine and aspartic protease inhibitors exhibited synergistic effects in blocking parasite metabolism and development. The inhibitors also demonstrated apparent synergistic inhibition of plasmodial hemoglobin degradation both in culture and in a murine malaria model. When evaluated for the treatment of murine malaria, a combination of cysteine and aspartic protease inhibitors was much more effective than higher concentrations of either compound used alone. These results support a model whereby plasmodial cysteine and aspartic proteases participate in the degradation of hemoglobin, and they suggest that combination antimalarial therapy with inhibitors of the two classes of proteases is worthy of further study.Malaria is one of the most important infectious diseases in the world. Infections with Plasmodium falciparum, the most virulent human malaria parasite, are responsible for hundreds of millions of illnesses and over a million deaths per year (22). A major reason for the continued severity of the worldwide malaria problem is the increasing resistance of malaria parasites to available drugs (12). Thus, it is important to identify new targets for antimalarial therapy and to evaluate new modes of therapy directed against these targets.Potential new targets for antimalarial chemotherapy include parasite enzymes required for the degradation of hemoglobin. Erythrocytic malaria parasites degrade hemoglobin in an acidic food vacuole to provide amino acids for parasite protein synthesis (reviewed in references 5 and 16). The food vacuole of P. falciparum contains the cysteine protease falcipain and the aspartic proteases plasmepsin I and plasmepsin II (7, 8, 15). Each of these proteases degrades hemoglobin in vitro, and it has been proposed that the enzymes act in a concerted manner to hydrolyze globin to small peptides or free amino acids (5, 16). In a number of in vitro studies, inhibitors of both cysteine and aspartic proteases had potent effects against cultured malaria parasites (1, 4, 11, 14, 15, 17, 18, 20). In an in vivo study utilizing a murine malaria model, a peptidyl cysteine protease inhibitor cured Plasmodium vinckei-infected mice (14). However, high doses of this inhibitor (200 to 400 mg/kg of body weight/day) were required for a pronounced antimalarial effect.As cysteine and aspartic proteases appear to act cooperatively to degrade hemoglobin, and as inhibitors of both classes of proteases have antimalarial effects, it may be appropriate to use combinations of inhibitors to treat malaria. Such combination therapy might improve efficacy and also slow the development of resistance to new agents. We now report an evaluation of the in vitro and in vivo antimalarial effects of combinations of peptidyl cysteine and aspartic protease inhibitors. These combinations had strong, apparently synergistic inhibitory effects on plasmodial development and hemoglobin degradation in both cultured parasites and in a murine malaria model.     

Antimalarial Asexual Stage-Specific and Gametocytocidal Activities of HIV Protease Inhibitors

The stage-specific antimalarial activities of a panel of antiretroviral protease inhibitors (PIs), including two nonpeptidic PIs (tipranavir and darunavir), were tested in vitro against Plasmodium falciparum. While darunavir demonstrated limited antimalarial activity (effective concentration [EC₅₀], >50 μM), tipranavir was active at clinically relevant concentrations (EC₅₀, 12 to 21 μM). Saquinavir, lopinavir, and tipranavir preferentially inhibited the growth of mature asexual-stage parasites (24 h postinvasion). While all of the PIs tested inhibited gametocytogenesis, tipranavir was the only one to exhibit gametocytocidal activity.The global distributions of HIV and malaria overlap in many regions of the world (reviewed in reference 17). Although data on the number of individuals with both diseases are unavailable, rates of coinfection are likely to be high (7). Furthermore, coinfection often leads to severe disease (4, 12, 19, 20). While the effects of antiretroviral therapy on the outcome of malaria infection are not understood, defining these interactions is important (11, 13, 16, 18). Understanding the antimalarial activities of the antiretroviral protease inhibitors (PIs) (reviewed in reference 17), for example, may lead to treatment recommendations that improve clinical outcomes and may also result in the identification of a new antimalarial drug target.Current data suggest that PIs kill malaria parasites by inhibiting one or more of the six nondigestive vacuole plasmepsins (reviewed in reference 17). In the present study we investigated the stage-specific effects of the PIs on asexual- and sexual-stage Plasmodium falciparum parasites in order to help define the antimalarial target(s) of these drugs and to help guide partner drug choices in the field. To gain additional structure-activity data and information that may be relevant for coinfected individuals we also examined the activities of the nonpeptidic PIs tipranavir (Aptivus) and darunavir (Prezista), new-generation PIs that are active against HIV-1 strains resistant to first generation PIs (9).The antimalarial activities of saquinavir, lopinavir, ritonavir, tipranavir, darunavir, and chloroquine (diphosphate salt; Sigma) were determined as described previously (18). Concentrations required to achieve 10, 50, and 90% growth inhibition (± the standard error [SE]) were determined by nonlinear regression curve fitting. Each assay was performed in triplicate on at least two separate occasions. Stage-specific growth inhibition assays were performed on synchronized parasite cultures (8) at 0 (ring), 24 (trophozoite), and 36 h (schizont) postsynchronization. Cultures were washed post-drug exposure, resuspended in drug-free medium, and seeded into tissue culture plates containing 0.5 μCi/well [³H]hypoxanthine for 40 h. Incorporation of [³H]hypoxanthine was compared to that in vehicle controls.Drug-induced effects on gametocytogenesis were examined using Pfs16-GFP parasites (3) as previously described (14). Assays were performed in triplicate on three separate occasions. The antigametocyte activities of selective PIs were also determined using Pfs16-GFP parasites (3). In these assays gametocytes were sorted from parasite cultures, seeded into microtiter plates (1,000 gametocytes and 5% hematocrit), and exposed to drugs or controls for 48 h. Hydroethidine was used to assess viability. The number of viable gametocytes in test cultures after treatment was compared to controls, and results were analyzed by one-way analysis of variance. Assays were performed in triplicate on two separate occasions.Tipranavir was active against all parasite lines tested, including the chloroquine-resistant line Dd2 (50% effective concentration [EC₅₀], 21±2 μM) and three chloroquine-sensitive lines (3D7, EC₅₀ of 20±2 μM; D10, EC₅₀ of 12±2 μM; Pfs16-GFP, EC₅₀ of 18±4 μM). These EC₅₀s are all below the C_min to C_max range (60 to 185 μM) for this drug in humans (6). Darunavir also inhibited the growth of P. falciparum. However, the EC₅₀ (Dd2 EC₅₀ of 70 μM) (data not shown) for this drug was well above clinically achievable levels (C_min to C_max, 0.7 to 12.4 μM) (5). The EC₅₀s of saquinavir (D10 EC₅₀, 3 ± 1 μM; 3D7 EC₅₀, 3 ± 3 μM; Pfs16-GFP EC₅₀, 5 ± 3 μM), lopinavir (D10 EC₅₀, 2 ± 1 μM; 3D7 EC₅₀, 2 ± 3 μM; Pfs16-GFP EC₅₀, 3 ± 1 μM), ritonavir (D10 EC₅₀, 3 ± 1 μM; 3D7 EC₅₀, 3 ± 1 μM; Pfs16-GFP EC₅₀, 5 ± 1 μM) and chloroquine (D10 EC₅₀, 23 ± 1 nM; 3D7 EC₅₀, 25 ± 6 nM; Pfs16-GFP EC₅₀, 23 ± 3 nM) were comparable between parasite lines and similar to previously published values for 3D7 (1, 2).Saquinavir, lopinavir, and tipranavir demonstrated significantly greater growth inhibition (P < 0.05) against trophozoite and schizont stages in comparison to ring stages (Fig. ​(Fig.1).1). Similar results were obtained for the drug-resistant Dd2 P. falciparum line (data not shown). Chloroquine was used as a control and, as expected, trophozoite stages were more sensitive to this drug than either ring or schizont stages (Fig. ​(Fig.1).1). Each of the four PIs tested also reduced the number of gametocytes produced in vitro (Fig. ​(Fig.2A).2A). The reduction in gametocytes was dose dependent and statistically significant (P < 0.01) when cultures were exposed to EC₉₀ levels of ritonavir and all concentrations of tipranavir (Fig. ​(Fig.2A).2A). Tipranavir was also able to directly kill gametocytes (Fig. ​(Fig.2B)2B) (P < 0.01). While saquinavir, ritonavir, and lopinavir reduced the numbers of live gametocytes in a dose-dependent fashion, these data did not reach statistical significance (Fig. ​(Fig.2B)2B) (P > 0.05).Open in a separate windowFIG. 1.In vitro stage-specific activities of PIs against P. falciparum asexual parasites. Erythrocytes infected with P. falciparum line D10 at ring (filled circles) trophozoite (open circles), or schizont (filled squares) stages were exposed to saquinavir (SQV; 40 μM), tipranavir (TPV; 150 μM), lopinavir (LPV; 20 μM), and chloroquine (CHQ; 50 nM) for 1, 2, 4, 6, or 8 h, as described in the text. Data are presented as percent growth inhibition (+SE) compared to vehicle controls (taken as 100% growth). Each assay was repeated twice in triplicate.Open in a separate windowFIG. 2.Activities of selected PIs in gametocyte and gametocyte induction inhibition assays. (A) In vitro antigametocytogenesis activities of selected PIs as determined using transgenic Pfs16-GFP P. falciparum parasites. (B) Activities of PIs against Pfs16-GFP P. falciparum gametocytes. All drugs were assessed at their EC₁₀, EC₅₀, and EC₉₀ values as determined by [³H]hypoxanthine incorporation against asexually replicating parasites. Bars with * indicate significant changes compared to vehicle control wells (P < 0.01). Pfs16-GFP gametocytes, unlike Pfs16-GFP asexual-stage parasites, express GFP-tagged Pfs16 (3).Evidence suggesting that PIs may be beneficial to HIV and malaria parasite-coinfected individuals is mounting. In addition to possessing antiretroviral activities these drugs also inhibit the growth of malaria parasites (1, 13, 15, 18). In the present study we have extended these data by demonstrating that tipranavir can inhibit the growth of malaria parasites at clinically relevant concentrations (6). Although additional studies, including those examining pharmacokinetic drug interactions and the effects of increased plasma proteins on the activity of tipranavir, are needed (6), these data further indicate that PIs are likely to be beneficial during HIV-malaria coinfection. The antigametocyte activity demonstrated by tipranavir adds an additional dimension to this observation, suggesting that this drug may also have an impact on malaria transmission. These data are novel in that very few antimalarial drugs have antigametocyte activity. Indeed most induce gametocytogenesis in vitro (14) and as a result can perpetuate transmission and the spread of drug-resistant parasites. The observation that none of the PIs induced gametocytogenesis in vitro is significant, given recent malaria eradication goals and the need for tools to achieve this (10).To gain an understanding of how HIV PIs kill P. falciparum we investigated their effects on individual stages of asexual development. Data from these studies indicated that the trophozoite and schizont stages are significantly more sensitive to PIs than ring-stage parasites (Fig. ​(Fig.1).1). Taken together with gametocyte inhibition data these results suggest that the primary target of the PIs is likely to be expressed in both gametocytes and intraerythrocytic parasites. Although expression data (http://plasmodb.org/) require confirmation and the possibility that the PIs might target different proteases in the different parasite stages cannot be ruled out, plasmepsins V, IX, and X appear to be the best candidate targets of these drugs. Future studies investigating these enzymes may identify the antimalarial target of the PIs and help explain the poor antimalarial activity of darunavir. Interestingly, darunavir has a similar structure to amprenavir, another PI with weak antimalarial activity (5, 18).     

Contribution of Plasma Protease Inhibitors to the Inactivation of Kallikrein in Plasma

Although l-inhibitor (l-INH) and α₂-macroglobulin (α₂M) have been reported as the major inhibitors of plasma kallikrein in normal plasma, there is little quantitative support for this conclusion. Thus, we studied the inactivation of purified kallikrein in normal plasma, as well as in plasma congenitally deficient in l-INH, or artificially depleted of α₂M by chemical modification of the inhibitor with methylamine. Under pseudo-first-order conditions, the inactivation rate constant of kallikrein in normal plasma was 0.60 min⁻¹. This rate constant was reduced to 0.35, 0.30, and 0.06 min⁻¹, in plasma deficient respectively in l-INH, α₂M, or both inhibitors. Thus l-INH (42%) and α₂M (50%) were found to be the major inhibitors of kallikrein in normal plasma. Moreover all the other protease inhibitors present in normal plasma contributed only for 8% to the inactivation of the enzyme. To confirm these kinetic results, ¹²⁵I-kallikrein (M_r 85,000) was completely inactivated by various plasma samples, and the resulting mixtures were analyzed by gel filtration on Sepharose 6B CL for the appearance of ¹²⁵I-kallikrein-inhibitor complexes. After inactivation by normal plasma, 52% of the active enzyme were found to form a complex (M_r 370,000) with l-INH, while 48% formed a complex (M_r 850,000) with α₂M. After inactivation by l-INH-deficient plasma, >90% of the active ¹²⁵I-kallikrein was associated with α₂M. A similar proportion of the label was associated with l-INH in plasma deficient in α₂M. After inactivation by plasma deficient in both l-INH and α₂M, ¹²⁵I-kallikrein was found to form a complex of M_r 185,000. This latter complex, which may involve antithrombin III, α₁-protease inhibitor, and/or α₁-plasmin inhibitor, was not detectable in appreciable concentrations in the presence of either l-INH or α₂M, even after the addition of heparin (2 U/ml). These observations demonstrate that l-INH and α₂M are the only significant inhibitors of kallikrein in normal plasma confirming previous predictions based on experiments in purified systems. Moreover, in the absence of either l-INH or α₂M, the inactivation of kallikrein becomes almost entirely dependent on the other major inhibitor.     

Granulocyte Collagenase, Elastase and Plasma Protease Inhibitors in Purulent Sputum

Abstract. Sputum was collected from patients with purulent chronic bronchitis. Immuno-chemical techniques using rabbit antiserum against human granulocyte collagenase and elastase showed the presence of both enzymes. Also the serum protease inhibitors α₁-antitrypsin and α₂-macroglobulin were demonstrated. Their protease inhibiting capacity was saturated. Granulocyte elastase and collagenase occurred not only in complexes with the inhibitors, but also as free enzymes. All sputa showed free proteolytiC., elastolytic and collagenolytic activity. The concentration of collagenase was equal in the sol phase and in the gel phase of the sputa, but most of the elastase was bound to the gel phase.     

Patterns of Oligonucleotide Sequences in Viral and Host Cell RNA Identify Mediators of the Host Innate Immune System

The innate immune response provides a first line of defense against pathogens by targeting generic differential features that are present in foreign organisms but not in the host. These innate responses generate selection forces acting both in pathogens and hosts that further determine their co-evolution. Here we analyze the nucleic acid sequence fingerprints of these selection forces acting in parallel on both host innate immune genes and ssRNA viral genomes. We do this by identifying dinucleotide biases in the coding regions of innate immune response genes in plasmacytoid dendritic cells, and then use this signal to identify other significant host innate immune genes. The persistence of these biases in the orthologous groups of genes in humans and chickens is also examined. We then compare the significant motifs in highly expressed genes of the innate immune system to those in ssRNA viruses and study the evolution of these motifs in the H1N1 influenza genome. We argue that the significant under-represented motif pattern of CpG in an AU context - which is found in both the ssRNA viruses and innate genes, and has decreased throughout the history of H1N1 influenza replication in humans - is immunostimulatory and has been selected against during the co-evolution of viruses and host innate immune genes. This shows how differences in host immune biology can drive the evolution of viruses that jump into species with different immune priorities than the original host.     

Activation of Coagulation Factor V by a Platelet Protease

Factor V must be converted to Factor V_a in order to bind to a high affinity platelet surface site and participate in prothrombin activation. Osterud et al. (10) presented data that suggested that human platelets contain an activated form of Factor V and a Factor V activator. We find that the Factor V released when platelets are disrupted by freezing and thawing or sonication is activated 3- to 10-fold by thrombin as determined by coagulation assay and is therefore stored as the relatively inactive procofactor rather than in the active form Factor V_a.     

Zidovudine Azido-Reductase in Human Liver Microsomes: Activation by Ethacrynic Acid, Dipyridamole, and Indomethacin and Inhibition by Human Immunodeficiency Virus Protease Inhibitors

AZT (zidovudine, 3′-azido-3′-deoxythymidine), although metabolized primarily to AZT-glucuronide, is also metabolized to 3′-amino-3′-deoxythmidine (AMT) by reduction of the azide to an amine. The formation of the myelotoxic metabolite AMT has not been well characterized, but inhibition of AMT formation would be of therapeutic benefit. The aim of this study was to identify compounds that inhibit AMT formation. Using human liver microsomes under anaerobic conditions and [2-¹⁴C]AZT, K_m values of AZT azido-reductase, estimated by radio-thin-layer chromatography, were 2.2 to 3.5 mM (n = 3). Oxygen completely inhibited this NADPH-dependent reduction. Thirteen of the 28 compounds tested inhibited the formation of AMT. In addition to the CYP3A4 inhibitors ketoconazole, fluconazole, indinavir, ritonavir, and saquinavir, metyrapone strongly inhibited AMT formation. An unexpected finding was the more-than-twofold increase in AMT formation in the presence of ethacrynic acid, dipyridamole, or indomethacin. Such activation of toxic metabolite formation would impair drug therapy.     

Small-Molecule Inhibitors of Lethal Factor Protease Activity Protect against Anthrax Infection

Bacillus anthracis, the causative agent of anthrax, manifests its pathogenesis through the action of two secreted toxins. The bipartite lethal and edema toxins, a combination of lethal factor or edema factor with the protein protective antigen, are important virulence factors for this bacterium. We previously developed small-molecule inhibitors of lethal factor proteolytic activity (LFIs) and demonstrated their in vivo efficacy in a rat lethal toxin challenge model. In this work, we show that these LFIs protect against lethality caused by anthrax infection in mice when combined with subprotective doses of either antibiotics or neutralizing monoclonal antibodies that target edema factor. Significantly, these inhibitors provided protection against lethal infection when administered as a monotherapy. As little as two doses (10 mg/kg) administered at 2 h and 8 h after spore infection was sufficient to provide a significant survival benefit in infected mice. Administration of LFIs early in the infection was found to inhibit dissemination of vegetative bacteria to the organs in the first 32 h following infection. In addition, neutralizing antibodies against edema factor also inhibited bacterial dissemination with similar efficacy. Together, our findings confirm the important roles that both anthrax toxins play in establishing anthrax infection and demonstrate the potential for small-molecule therapeutics targeting these proteins.     

Intracellular Antiviral Activity of Low-Dose Ritonavir in Boosted Protease Inhibitor Regimens

Protease inhibitors are largely used for the treatment of HIV infection in combination with other antiretroviral drugs. Their improved pharmacokinetic profiles can be achieved through the concomitant administration of low doses of ritonavir (RTV), a protease inhibitor currently used as a booster, increasing the exposure of companion drugs. Since ritonavir-boosted regimens are associated with long-term adverse events, cobicistat, a CYP3A4 inhibitor without antiviral activity, has been developed. Recently, high intracellular concentrations of ritonavir in lymphocytes and monocytes were reported even when ritonavir was administered at low doses, so we aimed to compare its theoretical antiviral activity with those of the associated protease inhibitors. Intracellular concentrations of ritonavir and different protease inhibitors were determined through the same method. Inhibitory constants were obtained from the literature. The study enrolled 103 patients receiving different boosted protease inhibitors, darunavir-ritonavir 600 and 100 mg twice daily and 800 and 100 mg once daily (n = 22 and 4, respectively), atazanavir-ritonavir 300 and 100 mg once daily (n = 40), lopinavir-ritonavir 400 and 100 mg twice daily (n = 21), or tipranavir-ritonavir 500 and 200 mg twice daily (n = 16). According to the observed concentrations, we calculated the ratios between the intracellular concentrations of ritonavir and those of the companion protease inhibitor and between the theoretical viral protease reaction speeds with each drug, with and without ritonavir. The median ratios were 4.04 and 0.63 for darunavir-ritonavir twice daily, 2.49 and 0.74 for darunavir-ritonavir once daily, 0.42 and 0.74 for atazanavir-ritonavir, 0.57 and 0.95 for lopinavir-ritonavir, and 0.19 and 0.84 for tipranavir-ritonavir, respectively. Therefore, the antiviral effect of ritonavir was less than that of the concomitant protease inhibitors but, importantly, mostly with darunavir. Thus, further in vitro and in vivo studies of the RTV antiviral effect are warranted.     

Evaluation of Misoprostol and Analogs as Inhibitors of T-Cell Activation

Prostaglandin E(2) (PGE(2)) is known to inhibit in vitro T-cell responses to mitogenic and antigenic stimuli. Interaction of PGE(2) with a G protein-coupled receptor activates adenylyl cyclase, leading to cAMP formation and inhibition of interleukin-2 (IL-2) production and T-cell proliferation. Despite these effects, the application of PGE(2) as an anti-inflammatory agent has been compromised by its unfavorable pharmacodynamic and side-effect profile. Because of the potential utility of synthetic analogs as prostaglandin-based therapeutics, we evaluated the effect of misoprostol and over 100 structural analogs on cAMP formation and T-cell activation. Our results indicate that micromolar concentrations of misoprostol and particular analogs elicited a rapid and substantial rise in cAMP levels in human peripheral blood mononuclear cells. Analogs which increased cAMP also suppressed IL-2 production and T-cell growth in vitro, whereas those devoid of suppressive activity weakly induced nucleotide synthesis. Despite extensive chemical alteration of the prostanoid structure, no single analog was superior to misoprostol in inducing cAMP or modulating T-cell activity. Misoprostol and suppressive analogs were also evaluated in vivo in a murine model of antigen-induced T-cell proliferation. Prostaglandins, administered at maximum tolerable doses, were ineffective in blocking a T-cell response to alloantigenic stimulation, whereas cyclosporine and prednisolone were potent inhibitors of this response. Overall, our results indicate that misoprostol and related analogs suppress T-cell activation in vitro but require concentrations 1000-fold greater than the low nanomolar plasma levels achieved with clinical doses of misoprostol. Whether misoprostol analogs of sufficient potency can be developed for pharmacologic attentuation of T-cell activation in vivo remains to be determined.     

In Vivo Effects of Protease Inhibitors on Chickens with Hereditary Muscular Dystrophy

Beginning on day 4 ex ovo, and every 3 d thereafter, genetically dystrophic Line 413 chickens were given intraperitoneal injections (4 mg/kg body wt) of a protease inhibitor, leupeptin, pepstatin, or antipain. Experimental chickens received protease inhibitors dissolved in a water:ethanol:dimethyl sulfoxide solution (50:40:10, vol:vol:vol). Control untreated animals received diluent injections.     

Pharmacokinetic Study of Human Immunodeficiency Virus Protease Inhibitors Used in Combination with Amprenavir

In an open-label, randomized, multicenter, multiple-dose pharmacokinetic study, we determined the steady-state pharmacokinetics of amprenavir with and without coadministration of indinavir, nelfinavir, or saquinavir soft gel formulation in 31 human immunodeficiency virus type 1-infected subjects. The results indicated that amprenavir plasma concentrations were decreased by saquinavir soft gel capsule (by 32% for area under the concentration-time curve at steady state [AUC(ss)] and 37% for peak plasma concentration at steady state [C(max,ss)]) and increased by indinavir (33% for AUC(ss)). Nelfinavir significantly increased amprenavir minimum drug concentration at steady state (by 189%) but did not affect amprenavir AUC(ss) or C(max,ss). Nelfinavir and saquinavir steady-state pharmacokinetics were unchanged by coadministration with amprenavir compared with the historical monotherapy data. Concentrations of indinavir, coadministered with amprenavir, in plasma decreased in both single-dose and steady-state evaluations. The changes in amprenavir steady-state pharmacokinetic parameters, relative to those for amprenavir alone, were not consistent among protease inhibitors, nor were the changes consistent with potential interactions in CYP3A4 metabolism or P-glycoprotein transport. No dose adjustment of either protease inhibitor in any of the combinations studied is needed.     

Recognition of Virus Infection and Innate Host Responses to Viral Gene Therapy Vectors

The innate immune and inflammatory response represents one of the key stumbling blocks limiting the efficacy of viral-based therapies. Numerous human diseases could be corrected or ameliorated if viruses were harnessed to safely and effectively deliver therapeutic genes to diseased cells and tissues in vivo. Recent studies have shown that host cells recognize viruses using an elaborate network of sensor proteins localized at the plasma membrane, in endosomes, or in the cytosol. Three classes of sensors have been implicated in sensing viruses in mammalian cells—Toll-like receptors (TLRs), retinoid acid-inducible gene (RIG)-I-like receptors (RLRs), and nucleotide oligomerization domain (NOD)-like receptors (NLRs). The interaction of virus-associated nucleic acids with these sensor molecules triggers a signaling cascade that activates the principal host defense program aimed to limit or eliminate virus infection and restore tissue homeostasis. In addition, recent data strongly suggest that host cells can mount innate immune responses to viruses without prior recognition of their nucleic acids. To deliver therapeutic genes into the nuclei of diseased cells, viral gene therapy vectors must be efficient at penetrating either the plasma or endosomal membrane. The therapeutic use of high numbers of virus particles disturbs cellular homeostasis, triggering cell damage and stress pathways, or “sensing of modified self”. Accumulating data indicate that the sensing of modified self might represent a powerful framework explaining the innate immune response activation by viral gene therapy vectors.