ABSTRACT
Broadening our understanding of the abundance and phenotype of B cell subsets that are induced or perturbed by exogenous Ags will improve the vaccine evaluation process. Mass cytometry (CyTOF) is being used to increase the number of markers that can be investigated in single cells, and therefore characterize cell phenotype at an unprecedented level. We designed a panel of CyTOF Abs to compare the B cell response in cynomolgus macaques at baseline, and 8 and 28 d after the second homologous immunization with modified vaccinia virus Ankara. The spanning-tree progression analysis of density-normalized events (SPADE) algorithm was used to identify clusters of CD20(+) B cells. Our data revealed the phenotypic complexity and diversity of circulating B cells at steady-state and significant vaccine-induced changes in the proportions of some B cell clusters. All SPADE clusters, including those altered quantitatively by vaccination, were characterized phenotypically and compared using double hierarchical clustering. Vaccine-altered clusters composed of previously described subsets including CD27(hi)CD21(lo) activated memory and CD27(+)CD21(+) resting memory B cells, and subphenotypes with novel patterns of marker coexpression. The expansion, followed by the contraction, of a single memory B cell SPADE cluster was positively correlated with serum anti-vaccine Ab titers. Similar results were generated by a different algorithm, automatic classification of cellular expression by nonlinear stochastic embedding. In conclusion, we present an in-depth characterization of B cell subphenotypes and proportions, before and after vaccination, using a two-step clustering analysis of CyTOF data, which is suitable for longitudinal studies and B cell subsets and biomarkers discovery.
Subject(s)
B-Lymphocytes/immunology , Flow Cytometry , Vaccines/immunology , Animals , Cluster Analysis , Macaca fascicularis , Male , PhenotypeABSTRACT
RNA is a major drug target, but the design of small molecules that modulate RNA function remains a great challenge. In this context, a series of structurally homologous 'polyamide amino acids' (PAA) was studied as HIV-1 trans-activating response (TAR) RNA ligands. An extensive thermodynamic study revealed the occurence of an enthalpy-entropy compensation phenomenon resulting in very close TAR affinities for all PAA. However, their binding modes and their ability to compete with the Tat fragment strongly differ according to their structure. Surprisingly, PAA that form loose complexes with TAR were shown to be stronger Tat competitors than those forming tight ones, and thermal denaturation studies demonstrated that loose complexes are more stable than tight ones. This could be correlated to the fact that loose and tight ligands induce distinct RNA conformational changes as revealed by circular dichroism experiments, although nuclear magnetic resonance (NMR) experiments showed that the TAR binding site is the same in all cases. Finally, some loose PAA also display promising inhibitory activities on HIV-infected cells. Altogether, these results lead to a better understanding of RNA interaction modes that could be very useful for devising new ligands of relevant RNA targets.
Subject(s)
Anti-HIV Agents/chemistry , HIV Long Terminal Repeat/drug effects , RNA, Viral/drug effects , Thermodynamics , tat Gene Products, Human Immunodeficiency Virus/metabolism , Amino Acids/chemistry , Anti-HIV Agents/metabolism , Anti-HIV Agents/pharmacology , Binding, Competitive , Circular Dichroism , Fluorescence Resonance Energy Transfer , Ligands , Nuclear Magnetic Resonance, Biomolecular , Nylons/chemistry , Nylons/metabolism , Nylons/pharmacology , RNA, Viral/chemistry , RNA, Viral/metabolismABSTRACT
In the domain of health, one important challenge is the efficient delivery of drugs in the body using non-toxic nanocarriers. Most of the existing carrier materials show poor drug loading (usually less than 5 wt% of the transported drug versus the carrier material) and/or rapid release of the proportion of the drug that is simply adsorbed (or anchored) at the external surface of the nanocarrier. In this context, porous hybrid solids, with the ability to tune their structures and porosities for better drug interactions and high loadings, are well suited to serve as nanocarriers for delivery and imaging applications. Here we show that specific non-toxic porous iron(III)-based metal-organic frameworks with engineered cores and surfaces, as well as imaging properties, function as superior nanocarriers for efficient controlled delivery of challenging antitumoural and retroviral drugs (that is, busulfan, azidothymidine triphosphate, doxorubicin or cidofovir) against cancer and AIDS. In addition to their high loadings, they also potentially associate therapeutics and diagnostics, thus opening the way for theranostics, or personalized patient treatments.
Subject(s)
Diagnostic Imaging , Drug Carriers/chemistry , Nanostructures/chemistry , Organometallic Compounds/chemistry , Animals , Cell Line, Tumor , Contrast Media/chemistry , Contrast Media/metabolism , Contrast Media/toxicity , Drug Carriers/metabolism , Drug Carriers/toxicity , Female , Humans , Macrophages/drug effects , Magnetic Resonance Imaging , Mice , Organometallic Compounds/metabolism , Organometallic Compounds/toxicity , Particle Size , Porosity , RatsABSTRACT
The HIV-1 envelope, gp120, which features the binding determinants for both CD4 and coreceptor recognition, is key for virus entry and represents an attractive pharmacological target. However, critical domains for entry (coreceptor and CD4 binding sites) are either cryptic or located in partially occluded cavities. Here we developed a chemical approach to synthesize a CD4-mimetic peptide linked to a heparan sulfate dodecasaccharide. This molecule binds to gp120, induces the exposure of the coreceptor binding domain and renders it available for interaction with the oligosaccharide. The linkage between the CD4 mimetic and the heparan sulfate derivative provides strong cooperative effects, resulting in low-nanomolar antiviral activity toward both CCR5- and CXCR4-tropic HIV-1 strains. This compound, which has the unique ability to simultaneously target two critical and highly conserved regions of gp120, establishes a new type of inhibitor and suggests a general concept for the inhibition of numerous other biological systems.
Subject(s)
Anti-HIV Agents/pharmacology , CD4 Antigens/pharmacology , Glycoconjugates/pharmacology , HIV-1/drug effects , Heparitin Sulfate/pharmacology , Receptors, CCR5/metabolism , Receptors, CXCR4/metabolism , Virus Attachment/drug effects , Virus Internalization/drug effects , Anti-HIV Agents/chemistry , Binding Sites , CD4 Antigens/chemistry , Glycoconjugates/chemistry , HIV Envelope Protein gp120/metabolism , HIV-1/physiology , Heparitin Sulfate/chemistry , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/virology , Models, MolecularABSTRACT
Enfuvirtide (also known as Fuzeon, T-20, or DP-178) is an antiretroviral fusion inhibitor which prevents human immunodeficiency virus type 1 (HIV-1) from entering host cells. This linear 36-mer synthetic peptide is indicated, in combination with other antiretroviral agents, for the treatment of HIV-1-infected individuals and AIDS patients with multidrug-resistant HIV infections. Although enfuvirtide is an efficient anti-HIV-1 drug, its clinical use is limited by a short plasma half-life, i.e., approximately 2 h, which requires twice-daily subcutaneous injections, often resulting in skin sensitivity reaction side effects at the injection sites. Ultimately, 80% of patients stop enfuvirtide treatment within 6 months because of these side effects. We report on the development of long-lasting enfuvirtide conjugates by the use of the site-specific conjugation of enfuvirtide to an antithrombin-binding carrier pentasaccharide (CP) through polyethylene glycol (PEG) linkers of various lengths. These conjugates showed consistent and broad anti-HIV-1 activity in the nanomolar range. The coupling of the CP to enfuvirtide only moderately affected the in vitro anti-HIV-1 activity in the presence of antithrombin. Most importantly, one of these conjugates, enfuvirtide-PEG(12)-CP (EP40111), exhibited a prolonged elimination half-life of more than 10 h in rat plasma compared to the half-life of native enfuvirtide, which was 2.8 h. On the basis of the pharmacokinetic properties of antithrombin-binding pentasaccharides, the anticipated half-life of EP40111 in humans would putatively be about 120 h, which would allow subcutaneous injection once a week instead of twice daily. In conclusion, EP40111 is a promising compound with strong potency as a novel long-lasting anti-HIV-1 drug.
Subject(s)
Anti-HIV Agents/administration & dosage , Anti-HIV Agents/pharmacology , HIV Envelope Protein gp41/administration & dosage , HIV Envelope Protein gp41/pharmacology , HIV-1/drug effects , Peptide Fragments/administration & dosage , Peptide Fragments/pharmacology , Amino Acid Sequence , Animals , Anti-HIV Agents/chemical synthesis , Antithrombins/metabolism , Cell Line , Cell Survival , Chromatography, High Pressure Liquid , Delayed-Action Preparations , Drug Carriers , Enfuvirtide , Factor Xa Inhibitors , Female , HIV Envelope Protein gp41/chemical synthesis , Half-Life , Humans , Molecular Sequence Data , Neutrophils/drug effects , Neutrophils/virology , Peptide Fragments/chemical synthesis , Polyethylene Glycols/chemistry , Polysaccharides/chemistry , Rats , Rats, Wistar , Tandem Mass SpectrometryABSTRACT
4-(N)-1,1',2-trisnor-squalenoyldideoxycytidine monophosphate (SQddC-MP) and 4-(N)-1,1',2-trisnor-squalenoylgemcitabine monophosphate (SQdFdC-MP) were synthesized using phosphoramidite chemistry. These amphiphilic molecules self-assembled to about hundred nanometers size nanoassemblies in aqueous medium. Nanoassemblies of SQddC-MP displayed significant anti-HIV activity whereas SQdFdC-MP nanoassemblies displayed promising anticancer activity on leukemia cells. These results suggested that squalene conjugate of negatively charged nucleotide analogues efficiently penetrated within cells. Thus, we propose a new prodrug strategy for improved delivery of nucleoside analogues to ameliorate their biological efficacy.
Subject(s)
Anti-HIV Agents/chemistry , Antineoplastic Agents/chemistry , Nanoparticles/chemistry , Nucleosides/chemistry , Prodrugs/chemistry , Squalene/chemistry , Animals , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Mice , Nanoparticles/ultrastructure , Nucleosides/chemical synthesis , Nucleosides/pharmacology , Particle Size , Prodrugs/chemical synthesis , Prodrugs/pharmacologyABSTRACT
Based on a split-and-mix strategy, a library of trimeric Polyamide Amino Acids (PAA) incorporating four different amino acids (Lys, Ala, Arg, and Phe) has been prepared. Screening of the batches for HIV TAR RNA binding in a fluorescent assay allowed the identification of several components that interact with TAR RNA at a micromolar concentration, with a good TAR versus tRNA specificity. Some of these compounds compete efficiently with the association of TAR and Tat protein. In cell cultures, these compounds display a moderate antiviral activity, associated nevertheless with some toxicity. Overall, these results confirm that this new family can be a basis for the design of novel RNA targeting drugs.
Subject(s)
Amino Acids/chemistry , Anti-HIV Agents/chemistry , HIV Long Terminal Repeat , Nylons/chemistry , RNA, Viral/chemistry , Amino Acid Sequence , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Fluorescence Resonance Energy Transfer , Ligands , Nylons/chemical synthesis , Nylons/pharmacology , RNA, Viral/metabolism , tat Gene Products, Human Immunodeficiency Virus/chemistry , tat Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
Telomerase reverse transcriptase (TERT) is highly expressed in more than 90% of canine cancer cells and low to absent in normal cells. Given that immune tolerance to telomerase is easily broken both naturally and experimentally, telomerase is an attractive tumor associated antigen for cancer immunotherapy. Indeed, therapeutic trials using human telomerase peptides have been performed. We have developed an immunogenic yet catalytically inactive human telomerase DNA construct that is in clinical trials with patients presenting solid tumors. Paralleling this human construct, we have developed a canine telomerase DNA vaccine, called pDUV5. When administered intradermally to mice combined with electrogene transfer, pDUV5 induced canine TERT specific cytotoxic T-cells as measured by IFN-γ ELISpot assay. Intradermal vaccination of healthy dogs with 400 µg of pDUV5 generated strong, broad and long lasting TERT specific cellular immune responses. In vitro immunization with cTERT peptides revealed the maintenance of cTERT specific T-cells in PBMCs from tumor bearing dogs showing that this repertoire was not depleted. This study highlights the potential of pDUV5 as a cancer vaccine and supports its evaluation for the treatment of spontaneous canine tumors.
ABSTRACT
Four novel N-isobutyryl-L-cysteine/2-mercaptoethylamine (MEA, cysteamine) conjugates have been designed and synthesized. The antioxidant activities of these new series were evaluated by three different free radical scavenging methods (DPPH test, ABTS test, and deoxyribose assay) and their metal binding capacity was evaluated by the ethidium bromide fluorescence binding assay. These results were compared with those obtained with their pro-GSH acetyl analogues recently developed in our laboratory. We observed that most of these compounds exhibit free radical-scavenging activities similar to those of Trolox, but always superior than NAC. While none of these new derivatives had pro-GSH activities, they displayed anti-HIV properties in human monocyte-derived macrophages infected in vitro. The present study demonstrates that these new N-isobutyryl derivatives, which are expected to have a greater bioavailability than their acetyl analogues, may have useful applications in HIV infection in respect to their antioxidant and anti-HIV activities.
Subject(s)
Anti-HIV Agents/chemistry , Cysteine/analogs & derivatives , Free Radical Scavengers/chemistry , Macrophages/virology , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Antioxidants/chemistry , Cysteamine/analogs & derivatives , Cysteamine/therapeutic use , Cysteine/therapeutic use , Humans , Structure-Activity RelationshipABSTRACT
Type I interferons (IFNs) are widely used to treat viral diseases. Depressive symptoms and suicide attempts are common neuropsychiatric side-effects during treatment with type I IFNs. Activation of indoleamine-2,3-dioxygenase (IDO), the first and rate-limiting enzyme of the kynurenine pathway by IFNs, leads to an increase in tryptophan (Trp) catabolism. Low levels of Trp lead to decrease of serotonin synthesis, which is likely to be related to the depressive symptoms. Ovine type I interferon-tau (IFN-tau) has a more potent antiretroviral effect and is less toxic than human type I IFN-alpha. Effects of IFN-tau and IFN-alpha on IDO expression and activity in primary cultures of human macrophages were compared in parallel to those of IFN-gamma, considered as one of the most potent IDO inducer. We found that both IFN-alpha and IFN-tau were poor inducers of IDO compared with IFN-gamma. However, IDO activation was slightly and significantly lower with ovine IFN-tau than human IFN-alpha, suggesting that ovine IFN-tau might have a lower impact on serotoninergic pathway compared with human IFN-alpha.
Subject(s)
Antiviral Agents/pharmacology , Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Interferon Type I/pharmacology , Interferon-alpha/pharmacology , Macrophages/drug effects , Pregnancy Proteins/pharmacology , Animals , Cells, Cultured , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Macrophages/enzymology , RNA, Messenger/metabolism , SheepABSTRACT
Medical countermeasures to treat biothreat agent infections require broad-spectrum therapeutics that do not induce agent resistance. A cell-based high-throughput screen (HTS) against ricin toxin combined with hit optimization allowed selection of a family of compounds that meet these requirements. The hit compound Retro-2 and its derivatives have been demonstrated to be safe in vivo in mice even at high doses. Moreover, Retro-2 is an inhibitor of retrograde transport that affects syntaxin-5-dependent toxins and pathogens. As a consequence, it has a broad-spectrum activity that has been demonstrated both in vitro and in vivo against ricin, Shiga toxin-producing O104:H4 entero-hemorrhagic E. coli and Leishmania sp. and in vitro against Ebola, Marburg and poxviruses and Chlamydiales. An effect is anticipated on other toxins or pathogens that use retrograde trafficking and syntaxin-5. Since Retro-2 targets cell components of the host and not directly the pathogen, no selection of resistant pathogens is expected. These lead compounds need now to be developed as drugs for human use.
Subject(s)
Benzamides/pharmacology , Chlamydiales/metabolism , Ebolavirus/metabolism , Leishmania/metabolism , Ricin/metabolism , Shiga Toxins/metabolism , Thiophenes/pharmacology , Animals , Benzamides/chemistry , Body Weight/drug effects , Chlamydiales/drug effects , Ebolavirus/drug effects , Escherichia coli/metabolism , HEK293 Cells , HeLa Cells , Humans , Injections, Intraperitoneal , Leishmania/drug effects , Mice , Mice, Inbred BALB C , Mitomycin/pharmacology , Models, Animal , RAW 264.7 Cells , Ricin/antagonists & inhibitors , Shiga Toxins/antagonists & inhibitors , Thiophenes/chemistryABSTRACT
Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.
Subject(s)
Adamantane/analogs & derivatives , Benzyl Compounds/pharmacology , Endosomes/drug effects , Ricin/antagonists & inhibitors , Toxins, Biological/antagonists & inhibitors , Adamantane/chemistry , Adamantane/pharmacology , Animals , Benzyl Compounds/chemistry , Benzylamines , Cell Compartmentation/drug effects , Endoplasmic Reticulum/drug effects , Golgi Apparatus/drug effects , HeLa Cells , Humans , Lysosomes/drug effects , Mice , Ricin/drug effects , Ricin/toxicity , Toxins, Biological/chemistry , Toxins, Biological/toxicityABSTRACT
Amongst the many strategies aiming at inhibiting HIV-1 infection, blocking viral entry has been recently recognized as a very promising approach. Using diverse in vitro models and a broad range of HIV-1 primary patient isolates, we report here that IND02, a type A procyanidin polyphenol extracted from cinnamon, that features trimeric and pentameric forms displays an anti-HIV-1 activity against CXCR4 and CCR5 viruses with 1-7 µM ED50 for the trimer. Competition experiments, using a surface plasmon resonance-based binding assay, revealed that IND02 inhibited envelope binding to CD4 and heparan sulphate (HS) as well as to an antibody (mAb 17b) directed against the gp120 co-receptor binding site with an IC50 in the low µM range. IND02 has thus the remarkable property of simultaneously blocking gp120 binding to its major host cell surface counterparts. Additionally, the IND02-trimer impeded up-regulation of the inhibitory receptors Tim-3 and PD-1 on CD4+ and CD8+ cells, thereby demonstrating its beneficial effect by limiting T cell exhaustion. Among naturally derived products significantly inhibiting HIV-1, the IND02-trimer is the first component demonstrating an entry inhibition property through binding to the viral envelope glycoprotein. These data suggest that cinnamon, a widely consumed spice, could represent a novel and promising candidate for a cost-effective, natural entry inhibitor for HIV-1 which can also down-modulate T cell exhaustion markers Tim-3 and PD-1.
Subject(s)
Biflavonoids/pharmacology , Catechin/pharmacology , Cinnamomum zeylanicum/chemistry , HIV Envelope Protein gp120/metabolism , Heparitin Sulfate/metabolism , Hepatitis A Virus Cellular Receptor 2/metabolism , Proanthocyanidins/pharmacology , Programmed Cell Death 1 Receptor/metabolism , T-Lymphocytes/drug effects , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Biflavonoids/chemistry , Binding Sites , CD4 Antigens/metabolism , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Catechin/chemistry , Humans , Proanthocyanidins/chemistry , Proanthocyanidins/metabolism , T-Lymphocytes/immunology , Up-Regulation/drug effectsABSTRACT
Interferon-tau (IFN-tau) is a type I IFN responsible for maternal recognition of the fetus in ruminants. In addition to its physiologic role, IFN-tau also inhibits HIV replication in human lymphocytes and macrophages and displays immunomodulatory effects but lacks the toxicity associated with other type I IFNs. Human IFN-alpha promotes a Th1 response, whereas IFN-tau has anti-inflammatory properties, inducing the production of Th2 cytokines in murine models of experimental autoimmune encephalitis (EAE) or fetal loss. We compared the effects of ovine IFN-tau (OvIFN-tau) and human IFN-alpha (HuIFN-alpha) on cytokine mRNA and protein production in human peripheral blood mononuclear cells (PBMCs) activated with a recall antigen, such as purified protein derivative (PPD) of tuberculin or with a proinflammatory stimulus, such as lipopolysaccharide (LPS). In both cases, IFN-alpha increased IFN-gamma production, whereas IFN-tau did not and thereby promoted Th2 cytokine production. This original property renders IFN-tau a potential candidate for therapeutic applications in immune disorders, such as multiple sclerosis (MS), but its therapeutic use in the treatment of HIV infection should be considered with caution.
Subject(s)
Interferon Type I/pharmacology , Interferon-gamma/biosynthesis , Lymphocytes/drug effects , Lymphocytes/metabolism , Pregnancy Proteins/pharmacology , Cell Proliferation/drug effects , Cells, Cultured , Cytokines/genetics , Humans , Interferon Type I/immunology , Interferon-alpha/pharmacology , Interferon-gamma/immunology , Lipopolysaccharides/pharmacology , Lymphocytes/cytology , Lymphocytes/immunology , Pregnancy Proteins/immunology , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
We evaluated the immunogenicity of a prime/boost vaccine strategy combining 5 lipopeptides (HIV-Lipo-5) and a recombinant modified vaccinia virus Ankara (rMVA-HIV) in cynomolgus macaques. Both of these vaccine components deliver HIV LAI Gag, Pol, and Nef antigens. Systemic and local safety was excellent in all groups. Immunization with HIV-Lipo-5 alone induced significant serum anti-HIV antibody titers which were not modified by rMVA-HIV immunization. However, induction of T-cell responses, as measured by IFNγ and IL-2 producing cells upon short-term stimulation with HIV peptide pools, required combined immunization with rMVA-HIV. Responses were preferentially observed against Gag antigen. Interestingly, HIV-Lipo-5 efficiently primed HIV induced T-cell responses upon the injection of rMVA-HIV, which may help to reduce the required number of vector injections. Our results provide a rationale for the use of a strategy involving HIV-Lipo-5 priming followed by rMVA-HIV booster immunization as a prophylactic or therapeutic vaccine approach against HIV infection and AIDS.
Subject(s)
AIDS Vaccines/administration & dosage , AIDS Vaccines/immunology , HIV Antibodies/blood , HIV Antigens/immunology , Lipopeptides/immunology , T-Lymphocytes/immunology , Vaccinia virus/genetics , AIDS Vaccines/genetics , Animals , CD8-Positive T-Lymphocytes/immunology , Cytokines/blood , Cytokines/immunology , Enzyme-Linked Immunospot Assay , HIV Antibodies/immunology , HIV Antigens/administration & dosage , Immunization, Secondary , Interferon-gamma/immunology , Interleukin-2/immunology , Macaca fascicularis , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunologyABSTRACT
Adenosine is a pleiotropic endogenous nucleoside with potential neuroprotective pharmacological activity. However, clinical use of adenosine is hampered by its extremely fast metabolization. To overcome this limitation, we recently developed a new squalenoyl nanomedicine of adenosine [Squalenoyl-Adenosine (SQAd)] by covalent linkage of this nucleoside to the squalene, a natural lipid. The resulting nanoassemblies (NAs) displayed a dramatic pharmacological activity both in cerebral ischemia and spinal cord injury pre-clinical models. The aim of the present study was to investigate the plasma profile and tissue distribution of SQAd NAs using both Squalenoyl-[(3)H]-Adenosine NAs and [(14)C]-Squalenoyl-Adenosine NAs as respective tracers of adenosine and squalene moieties of the SQAd bioconjugate. This study was completed by radio-HPLC analysis allowing to determine the metabolization profile of SQAd. We report here that SQAd NAs allowed a sustained circulation of adenosine under its prodrug form (SQAd) for at least 1h after intravenous administration, when free adenosine was metabolized within seconds after injection. Moreover, the squalenoylation of adenosine and its formulation as NAs also significantly modified biodistribution, as SQAd NAs were mainly captured by the liver and spleen, allowing a significant release of adenosine in the liver parenchyma. Altogether, these results suggest that SQAd NAs provided a reservoir of adenosine into the bloodstream which may explain the previously observed neuroprotective efficacy of SQAd NAs against cerebral ischemia and spinal cord injury.
Subject(s)
Adenosine , Nanoparticles , Prodrugs , Squalene , Adenosine/administration & dosage , Adenosine/chemistry , Adenosine/pharmacokinetics , Animals , Carbon Radioisotopes , Chromatography, High Pressure Liquid , Male , Mice , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Prodrugs/administration & dosage , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Squalene/administration & dosage , Squalene/chemistry , Squalene/pharmacokinetics , Tissue Distribution , TritiumABSTRACT
It is now widely accepted that neuronal damage in HIV infection results mainly from microglial activation and involves apoptosis, oxidative stress and glutamate-mediated neurotoxicity. Glutamate toxicity acts via 2 distinct pathways: an excitotoxic one in which glutamate receptors are hyperactivated, and an oxidative one in which cystine uptake is inhibited, resulting in glutathione depletion and oxidative stress. A number of studies show that astrocytes normally take up glutamate, keeping extracellular glutamate concentration low in the brain and preventing excitotoxicity. This action is inhibited in HIV infection, probably due to the effects of inflammatory mediators and viral proteins. Other in vitro studies as well as in vivo experiments in rodents following mechanical stimulation, show that activated microglia and brain macrophages express high affinity glutamate transporters. These data have been confirmed in chronic inflammation of the brain, particularly in SIV infection, where activated microglia and brain macrophages also express glutamine synthetase. Recent studies in humans with HIV infection show that activated microglia and brain macrophages express the glutamate transporter EAAT-1 and that expression varies according to the disease stage. This suggests that, besides their recognized neurotoxic properties in HIV infection, these cells also have a neuroprotective function, and may partly make up for the inhibited astrocytic function, at least temporarily. This hypothesis might explain the discrepancy between microglial activation which occurs early in the disease, and neuronal apoptosis and neuronal loss which is a late event. In this review article, we discuss the possible neuroprotective and neurotrophic roles of activated microglia and macrophages that may be generated by the expression of high affinity glutamate transporters and glutamine synthetase, 2 major effectors of glial glutamate metabolism, and the implications for HIV-induced neuronal dysfunction, the underlying cause of HIV dementia.
Subject(s)
Amino Acid Transport System X-AG/genetics , Glutamate-Ammonia Ligase/genetics , HIV Infections/metabolism , Macrophages/metabolism , Microglia/metabolism , Symporters/genetics , AIDS Dementia Complex/physiopathology , Animals , Brain/metabolism , Brain/pathology , Gene Expression Regulation , Glutamate Plasma Membrane Transport Proteins , HIV Infections/immunology , HIV Infections/pathology , Humans , Macrophages/immunology , Macrophages/pathology , Mice , Microglia/immunology , Microglia/pathology , Neuroprotective Agents/metabolism , RatsABSTRACT
OBJECTIVES: To investigate whether P-glycoprotein (P-gp) and multidrug resistance proteins (MRPs), which limit the bioavailability of HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs), modulate the anti-HIV activity of NRTIs, non-NRTIs and PIs in vitro. DESIGN: We used primary cultures of major HIV target cells: human monocyte-derived macrophages (MDMs) and lymphocytes. METHODS: P-gp and MRP expression in response to long-term zidovudine (3'-azido-3'-deoxythymidine; AZT) or indinavir treatment was quantified by RT-PCR. MDM and lymphocytes were infected in vitro with HIV-1/Ba-L and HIV-1-LAI, respectively, and treated with antiretroviral drugs. We evaluated the activity of these drugs in combination with PSC833, a P-gp inhibitor, and/or probenecid, an MRP1 inhibitor. Intracellular AZT triphosphate derivative (AZT-TP) was quantified by HPLC-MSMS. P-gp ATPase activity was measured with inside-out native membrane vesicles enriched in P-gp. RESULTS: Levels of MDR1, mrp4 and mrp5 mRNA were high following AZT treatment. In infected MDM, PSC833 and probenecid increased the anti-HIV activity of AZT and indinavir. AZT (5 nM) decreased HIV replication by 34% alone and by 72% in combination with P-gp/MRP inhibitors. Indinavir (10 nM) gave 14% inhibition alone and 81% in combination. The increase in anti-HIV activity of AZT was correlated with an increase in intracellular AZT-TP concentration. However, unlike PIs, neither AZT nor its metabolites interacted with P-gp. CONCLUSION: AZT increases the expression of multidrug transporters, thereby decreasing its pharmacological activity. The cellular efflux of AZT probably involves MRP4 or MRP5. In contrast, increases in indinavir anti-HIV activity require the inhibition of both P-gp and MRP1.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B/metabolism , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , Indinavir/pharmacology , Leukocytes, Mononuclear/drug effects , Macrophages/drug effects , Reverse Transcriptase Inhibitors/pharmacology , Zidovudine/pharmacology , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Cells, Cultured , Cyclosporins/pharmacology , HIV-1/metabolism , Humans , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/virology , Macrophages/metabolism , Macrophages/virology , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Probenecid/pharmacology , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
New formamidine-3TC (3TC = 2',3'-dideoxy-3'-thiacytidine) analogues have been synthesized through various methods, and their antiviral activities (HIV, HBV) have been evaluated in vitro. Anti-HIV-1 in acutely infected MT-4 cells and peripheral blood monocellular cells (PBMCs) showed that compounds substituted by N,N-diarylformamidine side chains at the 4-N nucleic base position (compounds 3 and 8-11) had at least equivalent anti-HIV activity as 3TC (EC50 = 0.5 and 11.6 microM, respectively). Moreover, the newly synthesized compounds demonstrated higher anti-HBV activity (EC50 ranging from 0.01 to 0.05 microM) compared to the parent nucleoside 3TC (EC50 = 0.2 microM). It should be underlined that these new promising derivatives inhibited HIV in cells of a macrophage lineage, which are known to be cellular reservoir for HIV. These results were particularly of interest, since the antiviral activities appeared not to be mediated through the formamidine bond hydrolysis and consequently the release of free 3TC. These new analogue series were found to be highly stable to hydrolysis even after prolonged incubation in different biological media (t(1/2) ranged from 48 to 120 h). This enzymatic stability, coupled to the fact that no delay in the antiviral response was observed compared to the free 3TC antiviral response, suggest that this new N,N-diarylformamidine nucleoside series should not be considered as classical prodrugs.
Subject(s)
Amidines/chemical synthesis , Antiviral Agents/chemical synthesis , Lamivudine/analogs & derivatives , Lamivudine/chemical synthesis , Amidines/pharmacology , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Antiviral Agents/pharmacology , Cell Extracts , Cell Line , Culture Media , Drug Stability , HIV-1/drug effects , Hepatitis B virus/drug effects , Humans , Hydrolysis , In Vitro Techniques , Lamivudine/pharmacology , Monocytes/drug effects , Monocytes/virology , Structure-Activity RelationshipABSTRACT
We synthesized a series of N-(N-acetyl-L-cysteinyl)-S-acetylcysteamine (10) analogues bearing various acyl groups on thiol cysteine or cysteamine residues, to investigate the structure-activity relationship for pro-GSH and anti-HIV properties in human macrophages. The S-substituents were ranked in the following order of efficacy: H > or = acetyl > isobutyryl > pivaloyl > benzoyl. We found that none of these derivatives had pro-GSH or antiviral activities in vitro higher than that of 10, but several displayed similar levels of anti-HIV activity, making them possible candidates for use as adjuvant therapies in conjunction with HAART, for treating neurological aspects of HIV infection.