Emilie Fromentin

Acyclovir is activated into a HIV-1 reverse transcriptase inhibitor in herpesvirus-infected human tissues

For most viruses, there is a need for antimicrobials that target unique viral molecular properties. Acyclovir (ACV) is one such drug. It is activated into a human herpesvirus (HHV) DNA polymerase inhibitor exclusively by HHV kinases and, thus, does not suppress other viruses. Here, we show that ACV suppresses HIV-1 in HHV-coinfected human tissues, but not in HHV-free tissue or cell cultures. However, addition of HHV-6-infected cells renders these cultures sensitive to anti-HIV ACV activity. We hypothesized that such HIV suppression requires ACV phosphorylation by HHV kinases. Indeed, an ACV monophosphorylated prodrug bypasses the HHV requirement for HIV suppression. Furthermore, phosphorylated ACV directly inhibits HIV-1 reverse transcriptase (RT), terminating DNA chain elongation, and can trap RT at the termination site. These data suggest that ACV anti-HIV-1 activity may contribute to the response of HIV/HHV-coinfected patients to ACV treatment and could guide strategies for the development of new HIV-1 RT inhibitors.

Ribonucleoside Triphosphates as Substrate of Human Immunodeficiency Virus Type 1 Reverse Transcriptase in Human Macrophages

We biochemically simulated HIV-1 DNA polymerization in physiological nucleotide pools found in two HIV-1 target cell types: terminally differentiated/non-dividing macrophages and activated/dividing CD4+ T cells. Quantitative tandem mass spectrometry shows that macrophages harbor 22–320-fold lower dNTP concentrations and a greater disparity between ribonucleoside triphosphate (rNTP) and dNTP concentrations than dividing target cells. A biochemical simulation of HIV-1 reverse transcription revealed that rNTPs are efficiently incorporated into DNA in the macrophage but not in the T cell environment. This implies that HIV-1 incorporates rNTPs during viral replication in macrophages and also predicts that rNTP chain terminators lacking a 3′-OH should inhibit HIV-1 reverse transcription in macrophages. Indeed, 3′-deoxyadenosine inhibits HIV-1 proviral DNA synthesis in human macrophages more efficiently than in CD4+ T cells. This study reveals that the biochemical landscape of HIV-1 replication in macrophages is unique and that ribonucleoside chain terminators may be a new class of anti-HIV-1 agents specifically targeting viral macrophage infection.

Bioavailability of the major bioactive diterpenoids in a rosemary extract: Metabolic profile in the intestine, liver, plasma, and brain of Zucker rats

SCOPE Carnosic acid (CA) and derived diterpenes abundant in rosemary extracts (REs) exert anti-obesity effects. The aim of this study was to investigate the bioavailability of these compounds in a rat model of obesity. METHODS AND RESULTS A total of 26 compounds were tentatively identified based on accurate mass information and the isotopic pattern provided by TOF-MS analyzer. The main metabolites detected in the gut content, liver, and plasma were the glucuronide conjugates of CA, carnosol, and rosmanol. Two other metabolites were also identified: CA 12-methyl ether and 5,6,7,10-tetrahydro-7-hydroxyrosmariquinone. All the metabolites were detected as early as 25 min following oral administration. Most of the compounds remained in the intestine, liver, and (or) plasma at substantial concentrations for several hours supporting their potential health benefits in these tissues. We also corroborated the presence of small quantities of CA and detected trace quantities of the main CA metabolites in the brain. Notably, we did not find significant differences in the metabolic profile between lean and obese rats. CONCLUSION We report for the first time a comprehensive profile of metabolites in various organs following the oral consumption of an RE enriched in CA and contribute to establish the potential bioactive molecules.

Inhibition of Gastric Lipase as a Mechanism for Body Weight and Plasma Lipids Reduction in Zucker Rats Fed a Rosemary Extract Rich in Carnosic Acid

Background Rosemary (Rosmarinus officinalis L.) extracts (REs) exhibit hepatoprotective, anti-obesity and anti-inflammatory properties and are widely used in the food industry. REs are rich in carnosic acid (CA) and carnosol which may be responsible for some of the biological activities of REs. The aim of this study was to investigate whether inhibition of lipase activity in the gut may be a mechanism by which a RE enriched in CA (40%) modulates body weight and lipids levels in a rat model of metabolic disorders and obesity. Methods and Principal Findings RE was administered for 64 days to lean (fa/+) and obese (fa/fa) female Zucker rats and body weight, food intake, feces weight and blood biochemical parameters were monitored throughout the study. Lipase activity (hydrolysis of p-nitrophenylbutyrate) was measured in the gastrointestinal tract at the end of the study and the contents of CA, carnosol and methyl carnosate were also determined. Sub-chronic administration of RE moderately reduced body weight gain in both lean and obese animals but did not affect food intake. Serum triglycerides, cholesterol and insulin levels were also markedly decreased in the lean animals supplemented with RE. Importantly, lipase activity was significantly inhibited in the stomach of the RE-supplemented animals where the highest content of intact CA and carnosol was detected. Conclusions Our results confirm that long-term administration of RE enriched in CA moderates weight gain and improves the plasma lipids profile, primarily in the lean animals. Our data also suggest that these effects may be caused, at least in part, by a significant inhibition of gastric lipase and subsequent reduction in fat absorption.

Abundant Non-canonical dUTP Found in Primary Human Macrophages Drives Its Frequent Incorporation by HIV-1 Reverse Transcriptase

Terminally differentiated/non-dividing macrophages contain extremely low cellular dNTP concentrations (20–40 nm), compared with activated CD4+ T cells (2–5 μm). However, our LC-MS/MS study revealed that the non-canonical dUTP concentration (2.9 μm) is ∼60 times higher than TTP in macrophages, whereas the concentrations of dUTP and TTP in dividing human primary lymphocytes are very similar. Specifically, we evaluated the contribution of HIV-1 reverse transcriptase to proviral DNA uracilation under the physiological conditions found in HIV-1 target cells. Indeed, biochemical simulation of HIV-1 reverse transcription demonstrates that HIV-1 RT efficiently incorporates dUTP in the macrophage nucleotide pools but not in the T cell nucleotide pools. Measurement of both pre-steady state and steady state kinetic parameters of dUTP incorporation reveals minimal selectivity of HIV-1 RT for TTP over dUTP, implying that the cellular dUTP/TTP ratio determines the frequency of HIV-1 RT-mediated dUTP incorporation. The RT of another lentivirus, simian immunodeficiency virus, also displays efficient dUTP incorporation in the dNTP/dUTP pools found in macrophages but not in T cells. Finally, 2′,3′-dideoxyuridine was inhibitory to HIV-1 proviral DNA synthesis in macrophages but not in T cells. The data presented demonstrates that the non-canonical dUTP was abundant relative to TTP, and efficiently incorporated during HIV-1 reverse transcription, particularly in non-dividing macrophages.

Viral decay kinetics in the highly active antiretroviral therapy-treated rhesus macaque model of AIDS.

To prevent progression to AIDS, persons infected with human immunodeficiency virus type 1 (HIV-1) must remain on highly active antiretroviral therapy (HAART) indefinitely since this modality does not eradicate the virus. The mechanisms involved in viral persistence during HAART are poorly understood, but an animal model of HAART could help elucidate these mechanisms and enable studies of HIV-1 eradication strategies. Due to the specificity of non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for HIV-1, we have used RT-SHIV, a chimeric virus of simian immunodeficiency virus with RT from HIV-1. This virus is susceptible to NNRTIs and causes an AIDS-like disease in rhesus macaques. In this study, two groups of HAART-treated, RT-SHIV-infected macaques were analyzed to determine viral decay kinetics. In the first group, viral loads were monitored with a standard TaqMan RT-PCR assay with a limit of detection of 50 viral RNA copies per mL. Upon initiation of HAART, viremia decayed in a bi-phasic manner with half-lives of 1.7 and 8.5 days, respectively. A third phase was observed with little further decay. In the second group, the macaques were followed longitudinally with a more sensitive assay utilizing ultracentrifugation to concentrate virus from plasma. Bi-phasic decay of viral RNA was also observed in these animals with half-lives of 1.8 and 5.8 days. Viral loads in these animals during a third phase ranged from 2–58 RNA copies/mL, with little decay over time. The viral decay kinetics observed in these macaques are similar to those reported for HIV-1 infected humans. These results demonstrate that low-level viremia persists in RT-SHIV-infected macaques despite a HAART regimen commonly used in humans.

Astragalus Root and Elderberry Fruit Extracts Enhance the IFN-β Stimulatory Effects of Lactobacillus acidophilus in Murine-Derived Dendritic Cells

Many foods and food components boost the immune system, but little data are available regarding the mechanisms by which they do. Bacterial strains have disparate effects in stimulating the immune system. Indendritic cells, the gram-negative bacteria Escherichia coli upregulates proinflammatory cytokines, whereas gram-positive Lactobacillus acidophilus induces a robust interferon (IFN)-β response. The immune-modulating effects of astragalus root and elderberry fruit extracts were examined in bone marrow-derived murine dendritic cells that were stimulated with L. acidophilus or E. coli. IFN-β and other cytokines were measured by ELISA and RT-PCR. Endocytosis of fluorescence-labeled dextran and L. acidophilus in the presence of elderberry fruit or astragalus root extract was evaluated in dendritic cells. Our results show that both extracts enhanced L. acidophilus-induced IFN-β production and slightly decreased the proinflammatory response to E. coli. The enhanced IFN-β production was associated with upregulation of toll-like receptor 3 and to a varying degree, the cytokines IL-12, IL-6, IL-1β and TNF-α. Both extracts increased endocytosis in immature dendritic cells, and only slightly influenced the viability of the cells. In conclusion, astragalus root and elderberry fruit extracts increase the IFN-β inducing activity of L. acidophilus in dendritic cells, suggesting that they may exert antiviral and immune-enhancing activity.

Cellular Pharmacology of 9-(β-D-1,3-dioxolan-4-yl) Guanine and its Lack of Drug Interactions with Zidovudine in Primary Human Lymphocytes

Amdoxovir, currently in Phase II clinical trials, is rapidly converted to 9-(β-D-1,3-dioxolan-4-yl)guanine (DXG) by adenosine deaminase in vitro and in humans. The cellular pharmacology of DXG in primary human lymphocytes, including dose-response relationships, intracellular half-life of DXG triphosphate (DXG-TP), and combination studies were determined. DXG produced high levels of DXG-TP with a long half-life (16 h) in activated human peripheral blood mononuclear cells. Since zidovudine (ZDV) and DXG select for different resistance mutations, co-formulation of the these two drugs is an attractive proposition. A combination study between DXG and ZDV showed no reduction of DXG-TP or ZDV-TP. Taken together, these results suggest that an appropriately designed DXG prodrug could be given once a day and that co-formulation with ZDV might be a possibility.

Lack of Pharmacokinetic Interaction between Amdoxovir and Reduced- and Standard-Dose Zidovudine in HIV-1-Infected Individuals

ABSTRACT Amdoxovir (AMDX) inhibits HIV-1 containing the M184V/I mutation and is rapidly absorbed and deaminated to its active metabolite, β-d-dioxolane guanosine (DXG). DXG is synergistic with zidovudine (ZDV) in HIV-1-infected primary human lymphocytes. A recent in silico pharmacokinetic (PK)/enzyme kinetic study suggested that ZDV at 200 mg twice a day (b.i.d.) may reduce toxicity without compromising efficacy relative to the standard 300-mg b.i.d. dose. Therefore, an intense PK clinical study was conducted using AMDX/placebo, with or without ZDV, in 24 subjects randomized to receive oral AMDX at 500 mg b.i.d., AMDX at 500 mg plus ZDV at 200 or 300 mg b.i.d., or ZDV at 200 or 300 mg b.i.d. for 10 days. Full plasma PK profiles were collected on days 1 and 10, and complete urine sampling was performed on day 9. Plasma and urine concentrations of AMDX, DXG, ZDV, and ZDV-5′-O-glucuronide (GZDV) were measured using a validated liquid chromatography-tandem mass spectrometry method. Data were analyzed using noncompartmental methods, and multiple comparisons were performed on the log-transformed parameters, at steady state. Coadministration of AMDX with ZDV did not significantly change either of the plasma PK parameters or percent recovery in the urine of AMDX, DXG, or ZDV/GZDV. Larger studies with AMDX/ZDV, with a longer duration, are warranted.

Specific phenolic compounds and sensory properties of a new dealcoholized red wine with pomegranate (Punica granatum L.) extract

The pomegranate (Punica granatum L.) fruit has a long history of human consumption and possesses notable antioxidant and cardiovascular properties. This work evaluated the feasibility to provide a new functional beverage based on a dealcoholized red wine matrix supplemented by a pomegranate extract. The potential bioactive compounds in the pomegranate extract, punicalagin A and B and ellagic acid, were analyzed during the downstream process in order to evaluate the functional dose in the final beverage. The addition of pomegranate extract to the dealcoholized red wine resulted in a product with more intense yeast odor, acidity, yeast flavor, and astringency and with a less intense berry flavor. Consumer acceptance of the product was also investigated and the results revealed the existence of a niche of consumers willing to consume dealcoholized wine enriched with pomegranate extract. After tasting, 50% and 40% of those consumers initially interested by this product concept declared to be interested to purchase the control sample and the functional beverage, respectively. The daily consumption of two servings of 250 mL of this new pomegranate-enriched dealcoholized wine provides 82 mg of total ellagitannins, corresponding to the sum of punicalagin A and B and ellagic acid.