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The FLAIR study demonstrated noninferiority of monthly long-acting cabotegravir + rilpivirine versus daily oral dolutegravir/abacavir/lamivudine for maintaining virologic suppression. Three participants who received long-acting therapy had confirmed virologic failure (CVF) at Week 48, and all had HIV-1 that was originally classified as subtype A1 and contained the baseline integrase polymorphism L74I; updated classification algorithms reclassified all 3 as HIV-1 subtype A6. Retrospectively, the impact of L74I on in vitro sensitivity and durability of response to cabotegravir in HIV-1 subtype B and A6 backgrounds was studied. Site-directed L74I and mutations observed in participants with CVF were generated in HIV-1 subtype B and a consensus integrase derived from 3 subtype A6 CVF baseline sequences. Rilpivirine susceptibility was assessed in HIV-1 subtype B and A1 containing reverse transcriptase mutations observed in participants with CVF. HIV-1 subtype B L74I and L74I/G140R mutants and HIV-1 subtype A6 I74L and I74/G140R mutants remained susceptible to cabotegravir; L74I/Q148R double mutants exhibited reduced susceptibility in HIV-1 subtypes B and A6 (half maximal effective capacity fold change, 4.4 and 4.1, respectively). Reduced rilpivirine susceptibility was observed across HIV-1 subtypes B and A1 with resistance-associated mutations K101E or E138K (half maximal effective capacity fold change, 2.21 to 3.09). In cabotegravir breakthrough experiments, time to breakthrough was similar between L74 and I74 viruses across HIV-1 subtypes B and A6; Q148R was selected at low cabotegravir concentrations. Therefore, the L74I integrase polymorphism did not differentially impact in vitro sensitivity to cabotegravir across HIV-1 subtype B and A6 integrase genes (ClinicalTrials.gov identifier: NCT02938520).
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Fármacos Anti-HIV , Infecções por HIV , Integrase de HIV , HIV-1 , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Dicetopiperazinas , Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , Integrase de HIV/genética , HIV-1/genética , Humanos , Integrases , Piridonas/farmacologia , Piridonas/uso terapêutico , Estudos Retrospectivos , Rilpivirina/farmacologia , Rilpivirina/uso terapêuticoRESUMO
Non-invasive bioluminescent imaging (NIBLI) of HIV-1 infection dynamics allows for real-time monitoring of viral spread and the localization of infected cell populations in living animals. In this report, we describe full-length replication-competent GFP and Nanoluciferase (Nluc) expressing HIV-1 reporter viruses from two clinical transmitted / founder (T/F) strains: TRJO.c and Q23.BG505. By infecting humanized mice with these HIV-1 T/F reporter viruses, we were able to directly monitor longitudinal viral spread at whole-animal resolution via NIBLI at a sensitivity of as few as 30-50 infected cells. Bioluminescent signal strongly correlated with HIV-1 infection and responded proportionally to virus suppression in vivo in animals treated daily with a combination antiretroviral therapy (cART) regimen. Longitudinal NIBLI following cART withdrawal visualized tissue-sites that harbored virus during infection recrudescence. Notably, we observed rebounding infection in the same lymphoid tissues where infection was first observed prior to ART treatment. Our work demonstrates the utility of our system for studying in vivo viral infection dynamics and identifying infected tissue regions for subsequent analyses.
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Fármacos Anti-HIV/farmacologia , Modelos Animais de Doenças , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Medições Luminescentes/métodos , Animais , Infecções por HIV/tratamento farmacológico , Humanos , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/virologia , Camundongos , Replicação Viral/efeitos dos fármacosRESUMO
The HIV-1 pandemic affecting over 37 million people worldwide continues, with nearly one-half of the infected population on highly active antiretroviral therapy (HAART). Major therapeutic challenges remain because of the emergence of drug-resistant HIV-1 strains, limitations because of safety and toxicity with current HIV-1 drugs, and patient compliance for lifelong, daily treatment regimens. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) that target the viral polymerase have been a key component of the current HIV-1 combination drug regimens; however, these issues hamper them. Thus, the development of novel more effective NNRTIs as anti-HIV-1 agents with fewer long-term liabilities, efficacy on new drug-resistant HIV-1 strains, and less frequent dosing is crucial. Using a computational and structure-based design strategy to guide lead optimization, a 5 µM virtual screening hit was transformed to a series of very potent nanomolar to picomolar catechol diethers. One representative, compound I, was shown to have nanomolar activity in HIV-1-infected T cells, potency on clinically relevant HIV-1 drug-resistant strains, lack of cytotoxicity and off-target effects, and excellent in vivo pharmacokinetic behavior. In this report, we show the feasibility of compound I as a late-stage preclinical candidate by establishing synergistic antiviral activity with existing HIV-1 drugs and clinical candidates and efficacy in HIV-1-infected humanized [human peripheral blood lymphocyte (Hu-PBL)] mice by completely suppressing viral loads and preventing human CD4+ T-cell loss. Moreover, a long-acting nanoformulation of compound I [compound I nanoparticle (compound I-NP)] in poly(lactide-coglycolide) (PLGA) was developed that shows sustained maintenance of plasma drug concentrations and drug efficacy for almost 3 weeks after a single dose.
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Fármacos Anti-HIV/administração & dosagem , Sistemas de Liberação de Medicamentos , Infecções por HIV/tratamento farmacológico , HIV-1 , Animais , Fármacos Anti-HIV/farmacocinética , Simulação por Computador , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Camundongos , Camundongos Endogâmicos BALB C , NanopartículasRESUMO
The clinical benefits of HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are hindered by their unsatisfactory pharmacokinetic (PK) properties along with the rapid development of drug-resistant variants. However, the clinical efficacy of these inhibitors can be improved by developing compounds with enhanced pharmacological profiles and heightened antiviral activity. We used computational and structure-guided design to develop two next-generation NNRTI drug candidates, compounds I and II, which are members of a class of catechol diethers. We evaluated the preclinical potential of these compounds in BALB/c mice because of their high solubility (510 µg/ml for compound I and 82.9 µg/ml for compound II), low cytotoxicity, and enhanced antiviral activity against wild-type (WT) HIV-1 RT and resistant variants. Additionally, crystal structures of compounds I and II with WT RT suggested an optimal binding to the NNRTI binding pocket favoring the high anti-viral potency. A single intraperitoneal dose of compounds I and II exhibited a prolonged serum residence time of 48 hours and concentration maximum (Cmax) of 4000- to 15,000-fold higher than their therapeutic/effective concentrations. These Cmax values were 4- to 15-fold lower than their cytotoxic concentrations observed in MT-2 cells. Compound II showed an enhanced area under the curve (0-last) and decreased plasma clearance over compound I and efavirenz, the standard of care NNRTI. Hence, the overall (PK) profile of compound II was excellent compared with that of compound I and efavirenz. Furthermore, both compounds were very well tolerated in BALB/c mice without any detectable acute toxicity. Taken together, these data suggest that compounds I and II possess improved anti-HIV-1 potency, remarkable in vivo safety, and prolonged in vivo circulation time, suggesting strong potential for further development as new NNRTIs for the potential treatment of HIV infection.
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Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Infecções por HIV/tratamento farmacológico , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/uso terapêutico , Alcinos , Animais , Benzoxazinas/química , Benzoxazinas/farmacologia , Cristalografia por Raios X , Ciclopropanos , Feminino , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Camundongos Endogâmicos BALB C , Inibidores da Transcriptase Reversa/farmacocinética , Inibidores da Transcriptase Reversa/toxicidade , SolubilidadeRESUMO
RNA-guided endonucleases (RGENs) derived from the CRISPR/Cas system represent an efficient tool for genome editing. RGENs consist of two components: Cas9 protein and guide RNA. Plasmid-mediated delivery of these components into cells can result in uncontrolled integration of the plasmid sequence into the host genome, and unwanted immune responses and potential safety problems that can be caused by the bacterial sequences. Furthermore, this delivery method requires transfection tools. Here we show that simple treatment with cell-penetrating peptide (CPP)-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs leads to endogenous gene disruptions in human cell lines. The Cas9 protein was conjugated to CPP via a thioether bond, whereas the guide RNA was complexed with CPP, forming condensed, positively charged nanoparticles. Simultaneous and sequential treatment of human cells, including embryonic stem cells, dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinoma cells, with the modified Cas9 and guide RNA, leads to efficient gene disruptions with reduced off-target mutations relative to plasmid transfections, resulting in the generation of clones containing RGEN-induced mutations. Our CPP-mediated RGEN delivery process provides a plasmid-free and additional transfection reagent-free method to use this tool with reduced off-target effects. We envision that our method will facilitate RGEN-directed genome editing.
Assuntos
Sistemas CRISPR-Cas , Peptídeos Penetradores de Células/química , RNA Guia de Cinetoplastídeos/genética , Transfecção/métodos , Linhagem Celular Tumoral , Genoma Humano , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida/métodos , Nanopartículas/química , Edição de RNA , RNA Guia de Cinetoplastídeos/químicaAssuntos
Infecções por HIV , HIV-1 , Animais , Fármacos Anti-HIV , Modelos Animais de Doenças , CamundongosRESUMO
Stem cells are poorly permissive to non-viral gene transfection reagents. In this study, we explored the possibility of improving gene delivery into human embryonic (hESC) and mesenchymal (hMSC) stem cells by synergizing the activity of a cell-binding ligand with a polymer that releases nucleic acids in a cytoplasm-responsive manner. A 29 amino acid long peptide, RVG, targeting the nicotinic acetylcholine receptor (nAchR) was identified to bind both hMSC and H9-derived hESC. Conjugating RVG to a redox-sensitive biodegradable dendrimer-type arginine-grafted polymer (PAM-ABP) enabled nanoparticle formation with plasmid DNA without altering the environment-sensitive DNA release property and favorable toxicity profile of the parent polymer. Importantly, RVG-PAM-ABP quantitatively enhanced transfection into both hMSC and hESC compared to commercial transfection reagents like Lipofectamine 2000 and Fugene. â¼60% and 50% of hMSC and hESC were respectively transfected, and at increased levels on a per cell basis, without affecting pluripotency marker expression. RVG-PAM-ABP is thus a novel bioreducible, biocompatible, non-toxic, synthetic gene delivery system for nAchR-expressing stem cells. Our data also demonstrates that a cell-binding ligand like RVG can cooperate with a gene delivery system like PAM-ABP to enable transfection of poorly-permissive cells.
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Materiais Biocompatíveis/química , Técnicas de Transferência de Genes , Vetores Genéticos/química , Peptídeos/química , Células-Tronco/citologia , Animais , Arginina/química , Citoplasma/metabolismo , DNA/química , Células-Tronco Embrionárias/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Proteínas de Fluorescência Verde/química , Humanos , Ligantes , Lipídeos/química , Células-Tronco Mesenquimais/citologia , Camundongos , Oxirredução , Fenótipo , Plasmídeos/metabolismo , Polímeros/química , Receptores Nicotínicos/metabolismo , TransfecçãoRESUMO
Previous data suggest a lack of cross-resistance between the gp120-directed attachment inhibitor temsavir (active moiety of fostemsavir) and the CD4-directed post-attachment inhibitor ibalizumab. Recently, analysis of HIV-1 envelopes with reduced sensitivity to both inhibitors was undertaken to determine whether they shared genotypic correlates of resistance. Sequences from 2 envelopes with reduced susceptibility to both agents were mapped onto a temsavir-bound gp120 structure. Residues within 5.0 Å of the temsavir binding site were evaluated using reverse genetics. Broader applicability and contextual determinants of key substitutions were further assessed using envelopes from participants in the phase 3 BRIGHTE study. Temsavir sensitivity was measured by half-maximal inhibitory concentration (IC50) and ibalizumab sensitivity by IC50 and maximum percent inhibition (MPI). One envelope required substitutions of E113D and T434M for full restoration of temsavir susceptibility. Neither substitution nor their combination affected ibalizumab sensitivity. However, in the second envelope, an E202 substitution (HXB2, T202) was sufficient for observed loss of susceptibility to both inhibitors. One BRIGHTE participant with no ibalizumab exposure had an emergent K202E substitution at protocol-defined virologic failure, with reduced sensitivity to both inhibitors. Introducing T202E into previously susceptible clinical isolates reduced temsavir potency by ≥ 40-fold and ibalizumab MPI from >99% to â¼80%. Interestingly, introduction of the gp120 V5 region from a highly ibalizumab-susceptible envelope mitigated the E202 effect on ibalizumab but not temsavir. A rare HIV-1 gp120 E202 mutation reduced temsavir susceptibility, and depending on sequence context, could result in reduced susceptibility to ibalizumab.
Assuntos
Fármacos Anti-HIV , Farmacorresistência Viral , Proteína gp120 do Envelope de HIV , Infecções por HIV , HIV-1 , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Fármacos Anti-HIV/farmacologia , Organofosfatos/farmacologia , Sítios de Ligação , Concentração Inibidora 50 , Anticorpos Monoclonais , PiperazinasRESUMO
Temsavir binds directly to the HIV-1 envelope glycoprotein gp120 and selectively inhibits interactions between HIV-1 and CD4 receptors. Previous studies identified gp120 amino acid positions where substitutions are associated with reduced susceptibility to temsavir. The mechanism by which temsavir susceptibility is altered in these envelope glycoproteins was evaluated. Pseudoviruses encoding gp120 substitutions alone (S375H/I/M/N, M426L, M434I, M475I) or in combination (S375H + M475I) were engineered on a wild-type JRFL background. Temsavir-gp120 and CD4-gp120 binding kinetics and ability of temsavir to block CD4-gp120 binding were evaluated using the purified polymorphic gp120 proteins and a Creoptix® WAVE Delta grating-coupled interferometry system. Fold-change in half-maximal inhibitory concentration (IC50) in JRFL-based pseudoviruses containing the aforementioned polymorphisms relative to that of wild-type ranged from 4-fold to 29,726-fold, while temsavir binding affinity for the polymorphic gp120 proteins varied from 0.7-fold to 73.7-fold relative to wild-type gp120. Strong correlations between temsavir IC50 and temsavir binding affinity (r = 0.7332; P = 0.0246) as well as temsavir binding on-rate (r = -0.8940; P = 0.0011) were observed. Binding affinity of gp120 proteins for CD4 varied between 0.4-fold and 3.1-fold compared with wild-type gp120; no correlations between temsavir IC50 and CD4 binding kinetic parameters were observed. For all polymorphic gp120 proteins, temsavir was able to fully block CD4 binding; 3 polymorphs required higher temsavir concentrations. Loss of susceptibility to temsavir observed for gp120 polymorphisms strongly correlated with reductions in temsavir binding on-rate. Nonetheless, temsavir retained the ability to fully block CD4-gp120 engagement given sufficiently high concentrations.
Assuntos
Fármacos Anti-HIV , Antígenos CD4 , Proteína gp120 do Envelope de HIV , HIV-1 , Ligação Proteica , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/química , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Fármacos Anti-HIV/farmacologia , Antígenos CD4/metabolismo , Antígenos CD4/genética , Substituição de Aminoácidos , Polimorfismo Genético , Farmacorresistência Viral , Concentração Inibidora 50 , CinéticaRESUMO
Amyloid-ß (Aß) peptide aggregation in the brain is a key factor in Alzheimer's disease. However, direct inhibition of ß-secretase or γ-secretase proves ineffective in reducing Aß accumulation and improving cognition in Alzheimer's. Recent findings suggest that inhibiting gamma-secretase activating protein (GSAP) can decrease Aß generation without affecting crucial γ-secretase substrates. Dimerization of Lep9R3LC (diLep9R3LC) was confirmed by Ellman's test. The peptide-small interfering RNA (siRNA) complex ratio, particle size, and surface charge were analyzed using electrophoretic mobility shift assay, and dynamic light scattering, respectively. In a 3xTg mice model of Alzheimer's disease, diLep9R3LC:siRNA complexes were intravenously administered twice a week for 8 weeks. Assessments included gene silencing, protein expression, and behavioral improvement using reverse transcription polymerase chain reaction, quantitative polymerase chain reaction, western blotting, Y-maze, and object recognition tests. The efficacy of Lep9R3LC dimerization was ~80% after a 3-d reaction by Ellman's test. In N2a cells, diLep9R3LC:siGSAP complexes achieved ~70% silencing at 48 h posttransfection. In 7-month-old male 3xTg mice, GSAP knockdown was ~30% in the cortex and ~50% in the hippocampus. The behavior improved in mice treated with diLep9R3LC:siGSAP complexes, showing a 60% increase in entries and an 80% increase object recognition. A novel dipeptide, diLep9R3LC, complexed with siRNA targeting GSAP (siGSAP), efficiently delivers siRNA to the mouse brain, targeting the hippocampus. The treatment inhibits Aß accumulation, reduces GSK-3ß-associated with tau hyperphosphorylation, and improves Alzheimer's behavior. Our findings highlight diLep9R3LC:siGSAP's potential for Alzheimer's and as a siRNA carrier for central nervous system-related diseases.
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Therapeutic anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (MAbs) provide immunosuppressed and vulnerable populations with prophylactic and treatment interventions against coronavirus disease 2019 (COVID-19). AZD7442 (tixagevimab-cilgavimab) is a combination of extended-half-life neutralizing MAbs that bind to distinct epitopes on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The Omicron variant of concern carries mutations at >35 positions in the spike protein and has undergone further genetic diversification since its emergence in November 2021. Here, we characterize the in vitro neutralization activity of AZD7442 toward major viral subvariants circulating worldwide during the first 9 months of the Omicron wave. BA.2 and its derived subvariants showed the highest susceptibility to AZD7442, while BA.1 and BA.1.1 showed a lower susceptibility. BA.4/BA.5 had a susceptibility level intermediate between BA.1 and BA.2. Mutagenesis of parental Omicron subvariant spike proteins was performed to establish a molecular model to describe the underlying determinants of neutralization by AZD7442 and its component MAbs. The concurrent mutation of residues at positions 446 and 493, located in the tixagevimab and cilgavimab binding sites, was sufficient to enhance in vitro susceptibility of BA.1 to AZD7442 and its component MAbs to levels similar to the Wuhan-Hu-1+D614G virus. AZD7442 maintained neutralization activity against all Omicron subvariants tested up to and including BA.5. The evolving nature of the SARS-CoV-2 pandemic warrants continuing real-time molecular surveillance and assessment of in vitro activity of MAbs used in prophylaxis against and the treatment of COVID-19. IMPORTANCE MAbs are key therapeutic options for COVID-19 prophylaxis and treatment in immunosuppressed and vulnerable populations. Due to the emergence of SARS-CoV-2 variants, including Omicron, it is vital to ensure that neutralization is maintained for MAb-based interventions. We studied the in vitro neutralization of AZD7442 (tixagevimab-cilgavimab), a cocktail of two long-acting MAbs targeting the SARS-CoV-2 spike protein, toward Omicron subvariants circulating from November 2021 to July 2022. AZD7442 neutralized major Omicron subvariants up to and including BA.5. The mechanism of action responsible for the lower in vitro susceptibility of BA.1 to AZD7442 was investigated using in vitro mutagenesis and molecular modeling. A combination of mutations at two spike protein positions, namely, 446 and 493, was sufficient to enhance BA.1 susceptibility to AZD7442 to levels similar to the Wuhan-Hu-1+D614G ancestral virus. The evolving nature of the SARS-CoV-2 pandemic warrants continuing real-time global molecular surveillance and mechanistic studies of therapeutic MAbs for COVID-19.
RESUMO
INTRODUCTION: AZD7442 (tixagevimab/cilgavimab) comprises neutralising monoclonal antibodies (mAbs) that bind to distinct non-overlapping epitopes on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Viral evolution during mAb therapy can select for variants with reduced neutralisation susceptibility. We examined treatment-emergent SARS-CoV-2 variants during TACKLE (NCT04723394), a phase 3 study of AZD7442 for early outpatient treatment of coronavirus disease 2019 (COVID-19). METHODS: Non-hospitalised adults with mild-to-moderate COVID-19 were randomised and dosed ≤ 7 days from symptom onset with AZD7442 (n = 452) or placebo (n = 451). Next-generation sequencing of the spike gene was performed on SARS-CoV-2 reverse-transcription polymerase chain reaction-positive nasopharyngeal swabs at baseline and study days 3, 6, and 15 post dosing. SARS-CoV-2 lineages were assigned using spike nucleotide sequences. Amino acid substitutions were analysed at allele fractions (AF; % of sequence reads represented by substitution) ≥ 25% and 3% to 25%. In vitro susceptibility to tixagevimab, cilgavimab, and AZD7442 was evaluated for all identified treatment-emergent variants using a pseudotyped microneutralisation assay. RESULTS: Longitudinal spike sequences were available for 461 participants (AZD7442, n = 235; placebo, n = 226) and showed that treatment-emergent variants at any time were rare, with 5 (2.1%) AZD7442 participants presenting ≥ 1 substitution in tixagevimab/cilgavimab binding sites at AF ≥ 25%. At AF 3% to 25%, treatment-emergent variants were observed in 15 (6.4%) AZD7442 and 12 (5.3%) placebo participants. All treatment-emergent variants showed in vitro susceptibility to AZD7442. CONCLUSION: These data indicate that AZD7442 creates a high genetic barrier for resistance and is a feasible option for COVID-19 treatment.
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BACKGROUND: Temsavir (TMR), the active agent of the gp120-directed attachment inhibitor fostemsavir (FTR), the CD4-directed attachment inhibitor ibalizumab (IBA), and the CCR5 antagonist maraviroc (MVC) are antiretroviral agents that target steps in HIV-1 viral entry. Although mechanisms of inhibition of the three agents are different, it is important to understand whether there is potential for cross-resistance between these agents, as all involve interactions with gp120. METHODS: Envelopes derived from plasma samples from participants in the BRIGHTE study who experienced protocol-derived virologic failure (PDVF) and were co-dosed with FTR and either IBA or MVC were analyzed for susceptibility to the agents. Also, CCR5-tropic MVC-resistant envelopes from the MOTIVATE trials were regenerated and studies were performed to understand whether susceptibility to multiple agents were linked. RESULTS: The cloned envelopes exhibited reduced susceptibility to TMR and resistance to the co-dosed agent. At PDVF, emergent or preexisting amino acid substitutions were present at TMR positions of interest. When amino acid substitutions at these positions were reverted to the consensus sequence, full susceptibility to TMR was restored without effecting resistance to the co-dosed agent. In addition, five envelopes from MOTIVATE were regenerated and exhibited R5-tropic-MVC-resistance. Only one exhibited reduced susceptibility to TMR and it contained an M426L polymorphism. When reverted to 426M, full sensitivity for TMR was restored, but it remained MVC resistant. CONCLUSION: The data confirm that decreased susceptibility to TMR and resistance to IBA or MVC are not linked and that there is no cross-resistance between either of these two agents and FTR.
Assuntos
Fármacos Anti-HIV , Infecções por HIV , Fármacos Anti-HIV/uso terapêutico , Anticorpos Monoclonais/farmacologia , Antagonistas dos Receptores CCR5/farmacologia , Antagonistas dos Receptores CCR5/uso terapêutico , Cicloexanos/farmacologia , Cicloexanos/uso terapêutico , Farmacorresistência Viral , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/tratamento farmacológico , Humanos , MaravirocRESUMO
The HIV pandemic has affected over 38 million people worldwide with close to 26 million currently accessing antiretroviral therapy (ART). A major challenge in the long-term treatment of HIV-1 infection is nonadherence to ART. Long-acting antiretroviral (LA-ARV) formulations, that reduce dosing frequency to less than once a day, are an urgent need that could tackle the adherence issue. Here, we have developed two LA-ART interventions, one an injectable nanoformulation, and the other, a removable implant, for the delivery of a synergistic two-drug ARV combination comprising a pre-clinical nonnucleoside reverse transcriptase inhibitor (NNRTI), Compound I, and the nucleoside reverse transcriptase inhibitor (NRTI), 4'-ethynyl-2-fluoro-2'-deoxyadenosine. The nanoformulation is poly(lactide-co-glycolide)-based and the implant is a copolymer of ω-pentadecalactone and p-dioxanone, poly(PDL-co-DO), a novel class of biocompatible, biodegradable materials. Both the interventions, packaged independently with each ARV, released sustained levels of the drugs, maintaining plasma therapeutic indices for over a month, and suppressed viremia in HIV-1-infected humanized mice for up to 42 days with maintenance of CD4+ T cells. These data suggest promise in the use of these new drugs as LA-ART formulations in subdermal implant and injectable mode.
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Small CD4-mimetic compounds (CD4mc) sensitize HIV-1-infected cells to antibody-dependent cellular cytotoxicity (ADCC) by facilitating antibody recognition of epitopes that are otherwise occluded on the unliganded viral envelope (Env). Combining CD4mc with two families of CD4-induced (CD4i) antibodies, which are frequently found in plasma of HIV-1-infected individuals, stabilizes Env in a conformation that is vulnerable to ADCC. We employed new-generation SRG-15 humanized mice, supporting natural killer (NK) cell and Fc-effector functions to demonstrate that brief treatment with CD4mc and CD4i-Abs significantly decreases HIV-1 replication, the virus reservoir and viral rebound after ART interruption. These effects required Fc-effector functions and NK cells, highlighting the importance of ADCC. Viral rebound was also suppressed in HIV-1+-donor cell-derived humanized mice supplemented with autologous HIV-1+-donor-derived plasma and CD4mc. These results indicate that CD4mc could have therapeutic utility in infected individuals for decreasing the size of the HIV-1 reservoir and/or achieving a functional cure.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Antivirais/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD4/química , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Epitopos/imunologia , Feminino , Glicoproteínas/química , Glicoproteínas/imunologia , Células HEK293 , Infecções por HIV/virologia , HIV-1/química , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Camundongos SCID , Modelos Animais , Conformação Proteica , Replicação Viral/efeitos dos fármacos , Produtos do Gene env do Vírus da Imunodeficiência Humana/químicaRESUMO
Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 µL of solution in 1-2 µL droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.
Assuntos
Sistema Nervoso Central/metabolismo , Sistemas de Liberação de Medicamentos , RNA Interferente Pequeno/administração & dosagem , Administração Intranasal , Animais , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos/instrumentação , Sistemas de Liberação de Medicamentos/métodos , Inativação Gênica , Glicoproteínas/administração & dosagem , Glicoproteínas/genética , Humanos , Camundongos , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , RNA Interferente Pequeno/genética , Proteínas Virais/administração & dosagem , Proteínas Virais/genéticaRESUMO
Combination antiretroviral therapy (cART) has been proven effective in inhibiting human immunodeficiency virus type 1 (HIV-1) infection and has significantly improved the health outcomes in acquired immune deficiency syndrome (AIDS) patients. The therapeutic benefits of cART have been challenged because of the toxicity and emergence of drug-resistant HIV-1 strains along with lifelong patient compliance resulting in non-adherence. These issues also hinder the clinical benefits of non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are one of the vital components of cART for the treatment of HIV-1 infection. In this study, using a computational and structural based drug design approach, we have discovered an effective HIV -1 NNRTI, compound I (Cmpd I) that is very potent in biochemical assays and which targets key residues in the allosteric binding pocket of wild-type (WT)-RT as revealed by structural studies. Furthermore, Cmpd I exhibited very potent antiviral activity in HIV-1 infected T cells, lacked cytotoxicity (therapeutic index >100,000), and no significant off-target effects were noted in pharmacological assays. To address the issue of non-adherence, we developed a long-acting nanoformulation of Cmpd I (Cmpd I-NP) using poly (lactide-coglycolide) (PLGA) particles. The pharmacokinetic studies of free and nanoformulated Cmpd I were carried out in BALB/c mice. Intraperitoneal administration of Cmpd I and Cmpd I-NP in BALB/c mice revealed prolonged serum residence time of 48â¯h and 30 days, respectively. The observed serum concentrations of Cmpd I in both cases were sufficient to provide >97% inhibition in HIV-1 infected T-cells. The significant antiviral activity along with favorable pharmacological and pharmacokinetic profile of Cmpd I, provide compelling and critical support for its further development as an anti-HIV therapeutic agent.
Assuntos
Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacocinética , Fármacos Anti-HIV/farmacologia , Cristalografia por Raios X , Sistemas de Liberação de Medicamentos/métodos , Desenho de Fármacos , Infecções por HIV/virologia , Transcriptase Reversa do HIV/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/uso terapêutico , Nanopartículas/virologia , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacocinética , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
No vaccines or therapeutics are licensed for West Nile virus (WNV), a mosquito-transmitted neuroencephalitic flavivirus. The small interfering RNA siFvEJW targets a conserved sequence within the WNV E protein and limits virus infection. Using a rabies virus-derived neuron-targeting peptide (RVG9R) and an intranasal route for delivering siFvEJW to the CNS, we demonstrate that treatment of WNV-infected mice at late stages of neuroinvasive disease results in recovery. Selectively targeting virus in the CNS lowers viral burdens in the brain, reduces neuropathology, and results in a 90% survival rate at 5-6 days post-infection (when viral titers peak in the CNS), while placebo-treated mice succumb by days 9-10. Importantly, CNS virus clearance is achieved by humoral and cell-mediated immune responses to WNV infection in peripheral tissues, which also engender sterilizing immunity against subsequent WNV infection. These results indicate that intranasal RVG9R-siRNA treatment offers efficient late-stage therapy and facilitates natural long-term immunity against neuroinvasive flaviviruses.
Assuntos
Encéfalo/virologia , Portadores de Fármacos , RNA Interferente Pequeno/metabolismo , Vírus da Raiva/genética , Replicação Viral/efeitos dos fármacos , Febre do Nilo Ocidental/prevenção & controle , Vírus do Nilo Ocidental/fisiologia , Animais , Encéfalo/imunologia , Modelos Animais de Doenças , Camundongos , Placebos/administração & dosagem , Análise de Sobrevida , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/imunologiaRESUMO
Ischemic stroke-induced neuronal cell death results in the permanent disabling of brain function. Apoptotic mechanisms are thought to play a prominent role in neuronal injury and ample evidence implicates Fas signaling in mediating cell death. In this study, we describe the neuroprotective effects of a Fas-blocking peptide (FBP) that by obstructing Fas signaling in cerebral ischemia inhibits apoptosis. Using an intranasal administration route in a rat model of focal cerebral ischemia, we demonstrate that nose-to-brain delivery of FBP after middle cerebral artery occlusion (MCAO) surgery results in the delivery and retention of FBP in Fas-expressing ischemic areas of the brain. A single intranasal administration of 2 mg/kg FBP resulted in significantly reduced neuronal cell death by inhibiting Fas-mediated apoptosis leading to decreased infarct volumes, reduced neurologic deficit scores and recovery from cerebral ischemia. Intranasally delivered FBP might be a promising strategy for the treatment of cerebral ischemic stroke.
Assuntos
Apoptose/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Peptídeos/farmacologia , Receptor fas/antagonistas & inibidores , Animais , Biomarcadores , Encéfalo/irrigação sanguínea , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/etiologia , Linhagem Celular , Humanos , Imuno-Histoquímica , Camundongos , Peptídeos/administração & dosagem , RatosRESUMO
siRNA entrapment within endosomes is a significant problem encountered with siRNA delivery platforms that co-opt receptor-mediated entry pathways. Attachment of a cell-penetrating peptide (CPP), such as nona-arginine (9R) to a cell receptor-binding ligand like the Rabies virus glycoprotein, RVG, allows effective siRNA delivery to the cytoplasm by non-endocytic pathways, but a significant amount of siRNA complexes also enters the cell by ligand-induced receptor endocytosis and remain localized in endosomes. Here, we report that the incorporation of trileucine (3 Leu) residues as an endo-osmolytic moiety in the peptide improves endosomal escape and intracellular delivery of siRNA. The trileucine motif did not affect early non-endosomal mechanism of cytoplasmic siRNA delivery but enhanced target gene silencing by >20% only beyond 24 h of transfection when siRNA delivery is mostly through the endocytic route and siRNA trapped in the endosomes at later stages were subject to release into cytoplasm. The mechanism may involve endosomal membrane disruption as trileucine residues lysed RBCs selectively under endosomal pH conditions. Interestingly <3 Leu or >3 Leu residues were not as effective, suggesting that 3 Leu residues are useful for enhancing cytoplasmic delivery of siRNA routed through endosomes.