Characterization of envelope glycoprotein gp41 genotype and phenotypic susceptibility to enfuvirtide at baseline and on treatment in the phase III clinical trials TORO-1 and TORO-2.

Enfuvirtide (T-20) is the first entry inhibitor approved for treatment of HIV infection and acts by inhibiting conformational changes in the viral envelope protein gp41 that are necessary for fusion of the virus and host cell membranes. Here we present genotypic and phenotypic data on viral envelopes obtained at baseline (n = 627) and after 48 weeks of enfuvirtide treatment (n = 302) from patients in the TORO (T-20 versus Optimized Regimen Only)-1 and -2 phase III pivotal studies. The amino acid sequence at residues 36-45 of gp41 was highly conserved at baseline except for polymorphism of approximately 16% at position 42. Substitutions within gp41 residues 36-45 on treatment were observed in virus from 92.7% of patients who met protocol defined virological failure criteria and occurred in nearly all cases (98.8%) when decreases in susceptibility to enfuvirtide from baseline of greater than 4-fold were observed. Consistent with previous observations, a wide range of baseline susceptibilities (spanning 3 logs) was observed; however, lower in vitro baseline susceptibility was not significantly associated with a decreased virological response in vivo. Virological response was also independent of baseline coreceptor tropism and viral subtype.

Peroxidation and phospholipase A2 hydrolytic susceptibility of liposomes consisting of mixed species of phosphatidylcholine and phosphatidylethanolamine.

The relationship between lipid peroxidation and phospholipase A2 (PLA2) hydrolytic activity was studied using unilamellar vesicles (liposomes) as model membranes. Hydrolytic specificity was examined using vesicles prepared with pure bovine heart phosphatidylcholine (PC), bovine heart phosphatidylethanolamine (PE), or mixtures of these phospholipids, using two preparative procedures, i.e., sonication or extrusion. Lipid peroxidation was induced by incubating vesicles with cumene hydroperoxide and hematin at 37 degrees C. Determinations of the extent of peroxidation by means of diene conjugate content derived from second derivative spectra or by polarographic measurement of oxygen consumption rates provided a basis for comparing the extent of peroxidation of each phospholipid species to their subsequent hydrolysis by PLA2 (from Crotalus adamanteus). The extent of hydrolysis was determined through the release of arachidonic acid from either PC or PE. The PE distribution among the outer vs. inner leaflet of the membrane bilayer was nearly equal in sonicated vesicles, whereas most of the phospholipid was incorporated into the inner leaflet in extruded vesicles. The proportion of PE found in the inner leaflet progressively increased as the ratio of PE to PC increased in both sonicated and extruded vesicle preparations. Lipid peroxidation had no effect on PE distribution under the conditions examined. There was a clear preference for PC peroxidation for all vesicle compositions tested and PC was preferentially hydrolyzed by PLA2. This effect is proposed to result from a perturbation of membrane structure following peroxidation with assimilation of PC into PLA2-susceptible domains whereas PE peroxidation and hydrolysis is less affected in mixed PC/PE vesicles. Lipid peroxidation imposes an additional hydrolytic susceptibility over the effects exerted through the mixing of these phospholipids which is based on structural changes rather than formation of specific substrates for PLA2.

Ritonavir and saquinavir combination therapy for the treatment of HIV infection.

OBJECTIVE: To evaluate the safety and antiretroviral activity of ritonavir (Norvir) and saquinavir (Invirase) combination therapy in patients with HIV infection. DESIGN: A multicenter, randomized, open-label clinical trial. SETTING: Seven HIV research units in the USA and Canada. PATIENTS: A group of 141 adults with HIV infection, CD4 T lymphocyte counts of 100-500 x 10(6) cells/l, whether treated previously or not with reverse transcriptase inhibitor therapy, but without previous HIV protease inhibitor drug therapy. INTERVENTIONS: After discontinuation of prior therapy for 2 weeks, group I patients were randomized to receive either combination (A) ritonavir 400 mg and saquinavir 400 mg twice daily or (B) ritonavir 600 mg and saquinavir 400 mg twice daily. After an initial safety assessment of group I patients, group II patients were randomized to receive either (C) ritonavir 400 mg and saquinavir 400 mg three times daily or (D) ritonavir 600 mg and saquinavir 600 mg twice daily. Investigators were allowed to add up to two reverse transcriptase inhibitors (including at least one with which the patient had not been previously treated) to a patient's regimen after week 12 for failure to achieve or maintain an HIV RNA level < or = 200 copies/ml documented on two consecutive occasions. MEASUREMENTS: Plasma HIV RNA levels and CD4+ T-lymphocyte counts were measured at baseline, every 2 weeks for 2 months, and monthly thereafter. Safety was assessed through the reporting of adverse events, physical examinations, and the monitoring of routine laboratory tests. RESULTS: The 48 weeks of study treatment was completed by 75% (106/141) of the patients. Over 80% of the patients on treatment at week 48 had an HIV RNA level < or = 200 copies/ml. In addition, intent-to-treat and on-treatment analyses revealed comparable results. Suppression of plasma HIV RNA levels was similar for all treatment arms (mean areas under the curve minus baseline through 48 weeks were-1.9, -2.0, -1.6, -1.8 log10 copies/ml in ritonavir-saquinavir 400-400 mg twice daily, 600-400 mg twice daily, 400-400 mg three times daily, and 600-600 mg twice daily, respectively). Median CD4 T-lymphocyte count rose by 128 x 10(6) cells/l from baseline, with an interquartile range (IQR) of 82-221 x 10(6) cells/l. The most common adverse events were diarrhea, circumoral paresthesia, asthenia, and nausea. Reversible elevation of serum transaminases (> 5 x upper limit of normal) occurred in 10% (14/141) of the patients enrolled in this study and was associated with baseline abnormalities in liver function tests, baseline hepatitis B surface antigen positivity, or hepatitis C antibody positivity (relative risk, 5.0; 95% confidence interval 1.5-16.9). Most moderate or severe elevations in liver function tests occurred in patients treated with ritonavir-saquinavir 600-600 mg twice daily. CONCLUSIONS: Ritonavir 400 mg combined with saquinavir 400 mg twice daily with the selective addition of reverse transcriptase inhibitors was the best-tolerated regimen of four dose-ranging regimens and was equally as active as the higher dose combinations in HIV-positive patients without previous protease inhibitor treatment.

Effect of lipid ozonation products on liposomal membranes detected by Laurdan fluorescence.

We report here the influence of the lipid ozonation products, 1-palmitoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphocholine (PC-aldehyde) and 1-palmitoyl-2[8-(5-octyl-1, 2, 4,-trioxolan-3-yl)- octanoyl]-sn-glycero-3-phosphocholine (PC-Criegee ozonide), on the phase domains of small unilamellar vesicles. (See Scheme 1 for structures.) 6-Lauroyl-2-dimethylaminonaphtalene (Laurdan) fluorescence excitation and emission spectra and generalized polarization measurements allowed us to study how lipid ozonation products affect the phase components of phospholipid membranes. A shift of excitation and emission spectra and a decrease in generalized polarization reveal the presence of a more polar environment surrounding the probe. We find that when either PC-aldehyde or PC-Criegee ozonide are incorporated into a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane, or when the POPC membrane is directly ozonated, a change in polarity of the phospholipid environment occurs that changes the properties of the bilayer. The introduction of more oxygenated and more polar phospholipids creates a more polar environment allowing the deeper penetration of water molecules into the membrane. Water penetration also is facilitated by the membrane disorder-producing effect of the ozonation products. The presence of an increased number of water molecules in the membrane effects the bilayer, altering packing order and cooperatively among fatty acyl chains as well as enhancing membrane fluidity.

Scavenging of peroxynitrite by a phenolic/peroxidase system prevents oxidative damage to DNA.

We examined the ability of horseradish peroxidase (HRP), an analog of human myeloperoxidase, to protect DNA against oxidative damage caused by peroxynitrite in the presence of chlorogenic acid (CGA), a naturally occurring polyphenol. Chlorogenic acid inhibits the formation of single strand breaks in supercoiled pBR322 DNA by acting as a scavenger of peroxynitrite. Horseradish peroxidase markedly enhances the extent of DNA protection by catalyzing the decomposition of peroxynitrite in the presence of CGA. Horseradish peroxidase alone does not inhibit peroxynitrite-induced DNA strand breaks, indicating that CGA is required as an electron donor to regenerate the active enzyme. The apparent second order rate constant for the HRP-mediated oxidation of CGA in the presence of peroxynitrite at pH 6.9 is 3.4 x 10(7) M(-1) s(-1). This high rate suggests that CGA and other dietary polyphenols might efficiently scavenge peroxynitrite in peroxidase-containing systems in vivo.

Beta carotene and its oxidation products have different effects on microsome mediated binding of benzo[a]pyrene to DNA.

The effects of beta-carotene (betaC) and its oxidation products on the binding of benzo[a]pyrene (BaP) metabolites to calf thymus DNA was investigated in the presence of rat liver microsomes. Mixtures of betaC oxidation products (betaCOP) as well as separated, individual betaC oxidation products were studied. One set of experiments, for example, involved the use of the mixture of betaCOP obtained after a 2-h radical-initiated oxidation. For this data set, the incorporation of unoxidized betaC into microsomal membranes caused the level of binding of BaP metabolites to DNA to decrease by 29% over that observed in the absence of betaC; however, the incorporation of the mixture of betaCOP caused the binding of BaP metabolites to DNA to increase 1.7-fold relative to controls without betaC. Two variations of this experiment were studied: (1) When no NADPH was added, betaC decreased the binding of BaP metabolites to DNA by 19%, but the mixture of betaCOP increased binding by 3.3-fold relative to that observed in the absence of betaC. (2) When NADPH was added under near-anaerobic conditions, betaC caused an almost total (94%) decrease in binding whereas betaCOP had no effect on the amount of binding relative to that observed in the absence of betaC. Both betaCOP and cumene hydroperoxide caused BaP metabolites to bind to DNA even when NADPH was omitted from the incubation mixture. Separation of the mixture of betaC oxidation products into fractions by HPLC allowed preliminary testing of individual betaC oxidation products separately; of the various fractions tested, the products tentatively identified as 11,15'-cyclo-12,15-epoxy-11,12,15,15'-tetrahydro-beta-carotene and beta-carotene-5,6-epoxide appeared to cause the largest increase in BaP-DNA binding. Microsomes from rats induced with 3-methylcholanthrene (3MC) or Aroclor 1254 produced different levels of binding in some experimental conditions. We hypothesize that, under some conditions, the incorporation of betaC into microsomal membranes can be protective against P450-catalyzed BaP binding to DNA; however, the incorporation of betaCOP facilitates the formation of BaP metabolites that bind DNA, although only certain P450 isoforms catalyze the binding process.

Competitive reactions of peroxynitrite with 2'-deoxyguanosine and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG): relevance to the formation of 8-oxodG in DNA exposed to peroxynitrite.

We have examined the formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) in reactions of peroxynitrite with 2'-deoxyguanosine (dG) and calf-thymus DNA. Peroxynitrite reacts with dG at neutral pH, but this reaction does not result in the buildup of 8-oxodG. We also do not find any evidence for the formation of 8-oxodG in calf-thymus DNA upon exposure to peroxynitrite. When 8-oxodG is mixed with 1000-fold excess dG and then allowed to react with peroxynitrite, about 50% of the 8-oxodG is destroyed. The preferential reaction of 8-oxodG is also evident when dG in calf-thymus DNA is partially oxidized in an Udenfriend system and then allowed to react with peroxynitrite. We suggest that 8-oxodG is not produced in peroxynitrite-mediated oxidations of dG and DNA or that it is produced but then is rapidly consumed in further reactions with peroxynitrite. Oxidized DNA bases frequently can be more oxidation sensitive than their corresponding progenitors and, therefore, may be present at] low steady-state concentrations and not represent stable markers of oxidative stress status. The importance of the 8-oxodG/peroxynitrite reaction is discussed in relation to the formation of more stable, secondary oxidation products that might be more useful markers of DNA damage.

Safety and tolerability of zalcitabine (ddC) in patients with AIDS or advanced AIDS-related complex in the European expanded access programme.

This international expanded access programme was initiated to provide zalcitabine (o 75 mg three times daily) to patients with AIDS or advanced ARC who had failed, were no longer able to tolerate or were ineligible to receive zidovudine (ZDV). Data are available from 517 patients. No unexpected adverse events occurred during the study with 13.2% of patients discontinuing treatment due to drug-related adverse events. Peripheral neuropathy (PN) was the most common adverse event reported. This was considered to be at least possibly related to zalcitabine in 12.2% of patients, with only 2.3% of patients withdrawing from the study due to zalcitabine-associated PN. Patients with a baseline diagnosis of AIDS and a CD4 count

Effects of melatonin on lipid peroxidation induced by oxygen radicals.

We here report the activity of the neurohormone melatonin (MLT) as a scavenger of free radicals in two different experimental models: (a) linoleic acid peroxidation initiated by different free radical-generating systems and (b) a multilamellar vesicle system composed of dilinoleoylphosphatidylcholine. In system (a) linoleic acid peroxidation, induced by either the water-soluble initiator 2,2'-azobis (2-amidinopropane) dihydrochloride (ABAP) or Fe2+-EDTA addition to 2.6 mM linoleic acid dispersed in SDS-phosphate buffer, was evaluated as the formation of conjugated dienes, measured spectrophotometrically at 236 nm. MLT did not reduce the rate of peroxidation induced by ABAP, but did reduce, in a concentration-dependent fashion, the rate of the reaction activated by Fe2+-EDTA. In system (b) multilamellar vesicles were used as the substrate for lipid peroxidation, initiated by Fe2+-EDTA and determined by means of malonaldehyde (MDA) and 4-hydroxyalkenal (4-HDA) content. MLT was found to be slightly more effective in system (b) than in the dispersed linoleic acid system (see a). These results show that MLT inhibits lipid damage induced by oxygen free radicals. However, MLT is only about one one-hundredth as effective an antioxidant as vitamin E in the micelles system.

Missing data imputation in two phase III trials treating HIV1 infection.

In most longitudinal clinical trials, some patients drop out before the end of the planned follow-up, and, in order to allow an all-patient intent-to-treat analysis to be performed, it is common practice to use some method of imputation to estimate values for missing data. However, different imputation methods may provide different results, and it is essential to investigate the sensitivity of the analysis using different imputation rules. In our analysis of two trials of the new HIV1 fusion inhibitor enfuvirtide, we compared some standard methods of imputing and analyzing HIV1-RNA data with two novel alternatives, to check the robustness of the primary endpoint results. The standard methods were: (1) last-observation-carried-forward, (2) baseline carried forward, and (3) multiple imputation. These were compared with a nearest-neighbour hot-deck method, specifically proposed for imputation of missing HIV1-RNA data, and with a heuristic approach: censored regression analysis of the last-observation-carried-forward. To supplement this analysis of real clinical trial data, we investigated the performance of the same imputation methods on simulated datasets designed to cover a broader range of missing data patterns.

Multiple polyexponentials and quasipolynomials as empirical nonlinear regression models: a case study with HIV viral load data.

Measurements of HIV1-RNA plasma concentrations are an important method of assessing patient response to anti-HIV1 treatment, and in most clinical trials of such treatments HIV1-RNA levels are assessed at regular intervals of time. HIV1-RNA levels in successfully treated patients tend to follow a standard pattern of biphasic decline-a rapid early decline in viral load, followed by a period of slower decline or a steady level. Fitting nonlinear regression models to these patterns of declining HIV1-RNA levels can be of value in comparing different treatment regimes and in predicting treatment outcome. Simple exponential-decline models can give an adequate fit to the typical pattern of HIV1-RNA decline, but we have explored the extent to which curve-fitting can be improved by using two novel nonlinear model forms. Specifically, we describe the fitting of multiple polyexponential and quasipolynomial forms to longitudinal HIV1-RNA plasma data collected in two recent trials of the novel anti-HIV1 treatment Fuzeon. We comment on the practicalities of fitting these nonlinear models, and compare the fit using various criteria.

Impact of human immunodeficiency virus type 1 gp41 amino acid substitutions selected during enfuvirtide treatment on gp41 binding and antiviral potency of enfuvirtide in vitro.

Enfuvirtide (ENF), a novel human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, has potent antiviral activity against HIV-1 both in vitro and in vivo. Resistance to ENF observed after in vitro passaging was associated with changes in a three-amino-acid (aa) motif, GIV, at positions 36 to 38 of gp41. Patients with ongoing viral replication while receiving ENF during clinical trials acquired substitutions within gp41 aa 36 to 45 in the first heptad repeat (HR-1) of gp41 in both population-based plasma virus sequences and proviral DNA sequences from isolates showing reduced susceptibilities to ENF. To investigate their impact on ENF susceptibility, substitutions were introduced into a modified pNL4-3 strain by site-directed mutagenesis, and the susceptibilities of mutant viruses and patient-derived isolates to ENF were tested. In general, susceptibility decreases for single substitutions were lower than those for double substitutions, and the levels of ENF resistance seen for clinical isolates were higher than those observed for the site-directed mutant viruses. The mechanism of ENF resistance was explored for a subset of the substitutions by expressing them in the context of a maltose binding protein chimera containing a portion of the gp41 ectodomain and measuring their binding affinity to fluorescein-labeled ENF. Changes in binding affinity for the mutant gp41 fusion proteins correlated with the ENF susceptibilities of viruses containing the same substitutions. The combined results support the key role of gp41 aa 36 to 45 in the development of resistance to ENF and illustrate that additional envelope regions contribute to the ENF susceptibility of fusion inhibitor-naïve viruses and resistance to ENF.

Trolox inhibits peroxynitrite-mediated oxidative stress and apoptosis in rat thymocytes.

Peroxynitrite is a strong oxidant that reacts with a variety of biomolecules in vivo and in vitro. When rat thymocytes in phosphate buffer are exposed to 25 microM peroxynitrite for 10 min, DNA single strand breaks (SSB) can be detected. These SSB are repaired if the cells are incubated in fresh media at 37 degrees C for 120 min. In addition, DNA protein cross-links and apoptosis are observed 1 and 6 h, respectively, after peroxynitrite exposure. Peroxynitrite mediates the formation of thiobarbituric acid-reactive substances (TBARS) that may be responsible for the DNA-protein crosslinks (DPXL). Both TBARS and DPXL formation are lowered by posttreating the cells immediately after the 10-min exposure to peroxynitrite with Trolox, a water-soluble vitamin E analog. These results suggest that oxidative stress mediated by peroxynitrite can trigger a critical sequence of events ending in programmed cell death and that intracellular oxidation is a component of the apoptosis of thymocytes, since both oxidative processes and apoptosis can be prevented by Trolox. In addition to Trolox, we obtained partial data on three other phenolic antioxidants (3-tert-butyl-4-hydroxyanisole, butylated hydroxytoluene, and 2,6-diisopropylphenol). We find that Trolox and these three phenols similarly protect rat thymocytes from apoptosis mediated by peroxynitrite.

Peroxynitrite causes apoptosis in rat thymocytes.

Peroxynitrite is demonstrated to cause apoptosis in freshly harvested rat thymocytes suspended in phosphate buffer, as shown by the typical ladder pattern of DNA fragmentation. This evidence suggests a new function for peroxynitrite, already known to be a strong oxidant. Peroxynitrite can nick DNA, oxidize cellular thiols and initiate the apoptotic process responsible for genomic degradation.

Activation of phospholipase A2 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes containing lipid ozonation products.

The activation of phospholipase A2 (PLA2) by lipid ozonation products is reported. The principal products from the ozonation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) are 1-palmitoyl-2-[8-[3(5-octyl-1,2,4-trioxolan-3-yl)octanoyl]--sn-gly cero-3- phosphocholine (PC-Criegee ozonide) and 1-palmitoyl-2-(9-oxononanoyl)-sn-glycero-3- phosphocholine (PC-aldehyde). (See Figure 1 for structures.) Here we test the hypothesis that these two compounds are mediators of ozone toxicity. Using POC vesicles, we find that PLA2 recognizes and hydrolyzes PC-Criegee ozonide at the same rate as that of arachidonic acid. Although the PC-aldehyde is not a substrate for PLA2, the enzymatic rate of hydrolysis by PLA2 of unaltered fatty acids incorporated into POPC is enhanced when PC-aldehyde is present in the bilayer. Thus, both PC-Criegee ozonide and PC-aldehyde alter the activity of PLA2, perhaps via an effect on membrane packing order. The capacity of PLA2 to recognize the PC-Criegee, and therefore ozone-induced damage, suggests a detoxification property of the enzyme and also a role in maintaining the structural properties of bilayer membranes that have been altered by exposure to ozone.