Clinical use of HIV integrase inhibitors : a systematic review and meta-analysis

Background: Optimal regimen choice of antiretroviral therapy is essential to achieve long-term clinical success. Integrase inhibitors have swiftly been adopted as part of current antiretroviral regimens. The purpose of this study was to review the evidence for integrase inhibitor use in clinical settings. Methods: MEDLINE and Web-of-Science were screened from April 2006 until November 2012, as were hand-searched scientific meeting proceedings. Multiple reviewers independently screened 1323 citations in duplicate to identify randomized controlled trials, nonrandomized controlled trials and cohort studies on integrase inhibitor use in clinical practice. Independent, duplicate data extraction and quality assessment were conducted. Results: 48 unique studies were included on the use of integrase inhibitors in antiretroviral therapy-naive patients and treatment-experienced patients with either virological failure or switching to integrase inhibitors while virologically suppressed. On the selected studies with comparable outcome measures and indication (n = 16), a meta-analysis was performed based on modified intention-to-treat (mITT), on-treatment (OT) and as-treated (AT) virological outcome data. In therapy-naive patients, favorable odds ratios (OR) for integrase inhibitor-based regimens were observed, (mITT OR 0.71, 95% CI 0.59-0.86). However, integrase inhibitors combined with protease inhibitors only did not result in a significant better virological outcome. Evidence further supported integrase inhibitor use following virological failure (mITT OR 0.27; 95% CI 0.11-0.66), but switching to integrase inhibitors from a high genetic barrier drug during successful treatment was not supported (mITT OR 1.43; 95% CI 0.89-2.31). Integrase inhibitor-based regimens result in similar immunological responses compared to other regimens. A low genetic barrier to drug-resistance development was observed for raltegravir and elvitegravir, but not for dolutegravir. Conclusion: In first-line therapy, integrase inhibitors are superior to other regimens. Integrase inhibitor use after virological failure is supported as well by the meta-analysis. Careful use is however warranted when replacing a high genetic barrier drug in treatment-experienced patients switching successful treatment

Fixed-dose combination bictegravir, emtricitabine, and tenofovir alafenamide versus dolutegravir-containing regimens for initial treatment of HIV-1 infection: week 144 results from two randomised, double-blind, multicentre, phase 3, non-inferiority trials

Background: In the primary week-48 analyses of two phase 3 studies, coformulated bictegravir, emtricitabine, and tenofovir alafenamide was non-inferior to a dolutegravir-containing regimen in treatment-naive people with HIV. We report week-144 efficacy and safety results from these studies. Methods: We did two double-blind, active-controlled studies (now in open-label extension phase). Study 1 randomly assigned (1:1) HLA-B*5701-negative adults without hepatitis B virus co-infection to receive coformulated bictegravir 50 mg, emtricitabine 200 mg, and tenofovir alafenamide 25 mg, or coformulated dolutegravir 50 mg, abacavir 600 mg, and lamivudine 300 mg once daily. Study 2 randomly assigned (1:1) adults to bictegravir, emtricitabine, and tenofovir alafenamide, or dolutegravir 50 mg given with coformulated emtricitabine 200 mg and tenofovir alafenamide 25 mg. We previously reported non-inferiority at the primary endpoint. Here, we report the week-144 secondary outcome of proportion of participants with plasma HIV-1 RNA less than 50 copies per mL at week 144, by US Food and Drug Administration Snapshot algorithm, analysed in the same manner. These studies were registered with ClinicalTrials.gov, NCT02607930 and NCT02607956. Findings: 629 participants were randomly assigned and treated in study 1 (314 to bictegravir, emtricitabine, and tenofovir alafenamide, and 315 to dolutegravir, abacavir, and lamivudine) and 645 in study 2 (327 to bictegravir, emtricitabine, and tenofovir alafenamide, 325 to dolutegravir, emtricitabine, tenofovir alafenamide). At week 144, bictegravir, emtricitabine, and tenofovir alafenamide was non-inferior to both dolutegravir-containing regimens for efficacy. In study 1, 256 (82%) of 314 participants had plasma HIV-1 RNA less than 50 copies per mL in the bictegravir, emtricitabine, and tenofovir alafenamide group and 265 (84%) of 315 in the dolutegravir, abacavir, and lamivudine group (difference −2·6%, 95% CI −8·5 to 3·4). In study 2, 262 (82%) of 320 participants had plasma HIV-1 RNA less than 50 copies per mL in the bictegravir, emtricitabine, and tenofovir alafenamide group and 273 (84%) of 325 in the dolutegravir, emtricitabine, and tenofovir alafenamide group (difference −1·9%, −7·8 to 3·9). In both studies, no participant had treatment-emergent resistance to study drugs up to week 144. All treatment regimens were well tolerated with additional exposure. Adverse events that led to study drug discontinuation were reported for no participants in the bictegravir, emtricitabine, and tenofovir alafenamide group versus five (2%) of 315 in the dolutegravir, abacavir, and lamivudine group (study 1), and six (2%) of 320 in the bictegravir, emtricitabine, and tenofovir alafenamide versus six (2%) of 325 in the dolutegravir, emtricitabine, and tenofovir alafenamide group (study 2). In study 1, statistically significant differences were observed in median changes from baseline in fasting total cholesterol (14 mg/dL vs 10 mg/dL; p=0·034), direct LDL (21 mg/dL vs 14 mg/dL; p=0·004), and total cholesterol to HDL ratio (−0·1 vs −0·3; p=0·007) at week 144; no differences were observed between groups in study 2. Weight gain was seen across all treatment groups in both studies, with no differences in median changes from baseline in weight at week 144 for either study. Interpretation: These long-term data support the use of bictegravir, emtricitabine, and tenofovir alafenamide as a safe, well tolerated, and durable treatment for people with HIV, with no emergent resistance. Funding: Gilead Sciences. © 2020 Elsevier Lt

CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases

GacA is essential for Group A <i>Streptococcus </i>and defines a new class of monomeric dTDP-4-dehydrorhamnose reductases (RmlD)

The sugar nucleotide dTDP-L-rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A Streptococcus (GAS). The final step of the four-step dTDP-L-rhamnose biosynthesis pathway is catalyzed by dTDP-4-dehydrorhamnose reductases (RmlD). RmlD from the Gram-negative bacterium Salmonella is the only structurally characterized family member and requires metal-dependent homo-dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram-negative and Gram-positive RmlD homologues predicts that enzymes from all Gram-positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gacA in a S. mutans rmlD knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn-sequencing and generation of a conditional-expression mutant identified gacA as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram-positive bacteria and a subset of Gram-negative bacteria. These results will help future screens for novel inhibitors of dTDP-L-rhamnose biosynthesis

Serum miRNAs miR-206, 143-3p and 374b-5p as potential biomarkers for amyotrophic lateral sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative condition characteris loss of motor neurones and progressive muscle wasting. There is no diagnostic test fo therefore robust biomarkers would not only be valuable for diagnosis, but also the classification of disease subtypes, monitoring responses to drugs and tracking diseas progression. As regulators of gene expression, microRNAs (miRNAs) are increasingly for diagnostic and prognostic purposes in various disease states with increasing explo in neurodegenerative disorders. We hypothesise that circulating blood based miRNAs serve as biomarkers and use miRNA profiling to determine miRNA signatures from th serum of sporadic (sALS) patients compared to healthy controls and patients with dise that mimic ALS. A number of differentially expressed miRNAs were identified in each patient comparisons. Validation in an additional patient cohort showed that miR-206 a miR-143-3p were increased and miR-374b-5p was decreased compared to controls. A continued change in miRNA expression persisted during disease progression indicatin potential use of these particular miRNAs as longitudinal biomarkers in ALS

The use of the SAEM algorithm in MONOLIX software for estimation of population pharmacokinetic-pharmacodynamic-viral dynamics parameters of maraviroc in asymptomatic HIV subjects

Using simulated viral load data for a given maraviroc monotherapy study design, the feasibility of different algorithms to perform parameter estimation for a pharmacokinetic-pharmacodynamic-viral dynamics (PKPD-VD) model was assessed. The assessed algorithms are the first-order conditional estimation method with interaction (FOCEI) implemented in NONMEM VI and the SAEM algorithm implemented in MONOLIX version 2.4. Simulated data were also used to test if an effect compartment and/or a lag time could be distinguished to describe an observed delay in onset of viral inhibition using SAEM. The preferred model was then used to describe the observed maraviroc monotherapy plasma concentration and viral load data using SAEM. In this last step, three modelling approaches were compared; (i) sequential PKPD-VD with fixed individual Empirical Bayesian Estimates (EBE) for PK, (ii) sequential PKPD-VD with fixed population PK parameters and including concentrations, and (iii) simultaneous PKPD-VD. Using FOCEI, many convergence problems (56%) were experienced with fitting the sequential PKPD-VD model to the simulated data. For the sequential modelling approach, SAEM (with default settings) took less time to generate population and individual estimates including diagnostics than with FOCEI without diagnostics. For the given maraviroc monotherapy sampling design, it was difficult to separate the viral dynamics system delay from a pharmacokinetic distributional delay or delay due to receptor binding and subsequent cellular signalling. The preferred model included a viral load lag time without inter-individual variability. Parameter estimates from the SAEM analysis of observed data were comparable among the three modelling approaches. For the sequential methods, computation time is approximately 25% less when fixing individual EBE of PK parameters with omission of the concentration data compared with fixed population PK parameters and retention of concentration data in the PD-VD estimation step. Computation times were similar for the sequential method with fixed population PK parameters and the simultaneous PKPD-VD modelling approach. The current analysis demonstrated that the SAEM algorithm in MONOLIX is useful for fitting complex mechanistic models requiring multiple differential equations. The SAEM algorithm allowed simultaneous estimation of PKPD and viral dynamics parameters, as well as investigation of different model sub-components during the model building process. This was not possible with the FOCEI method (NONMEM version VI or below). SAEM provides a more feasible alternative to FOCEI when facing lengthy computation times and convergence problems with complex models

Adherence to Antiretroviral Treatment and Correlation with Risk of Hospitalization among Commercially Insured HIV Patients in the United States

Purpose: A lower daily pill burden may improve adherence to antiretroviral treatment (ART) and clinical outcomes in patients with human immunodeficiency virus (HIV). This study assessed differences in adherence using the number of pills taken per day, and evaluated how adherence correlated with hospitalization. Methodology: Commercially insured patients in the LifeLink database with an HIV diagnosis (International Classification of Diseases, 9th Revision, Clinical Modification code 042.xx) between 6/1/2006 and 12/31/2008 and receipt of a complete ART regimen were selected for inclusion. Patients were grouped according to their daily pill count and remained on ART for at least 60 days. Outcomes included adherence and rates of hospitalization. Adherence was measured as the proportion of days between the start and end of the regimen in which the patient maintained supply of all initiated ART components. Logistic regressions assessed the relationship between pills per day, adherence, and hospitalization, controlling for demographics, comorbidities, and ART-naïve (vs. experienced) status. Results: 7,073 patients met the study inclusion criteria, and 33.4%, 5.8%, and 60.8 % received an ART regimen comprising one, two, or three or more pills per day, respectively. Regression analysis showed patients receiving a single pill per day were significantly more likely to reach a 95 % adherence threshold versus patients receiving three or more pills per day (odds ratio [OR] = 1.59; P,0.001). Regardless of the number of pills received per day, patients were over 40 % less likely to have

Trehalose-6-phosphate-mediated toxicity determines essentiality of OtsB2 in Mycobacterium tuberculosis in vitro and in mice

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors

A High-Density Genome-Wide Association Screen of Sporadic ALS in US Veterans

Following reports of an increased incidence of amyotrophic lateral sclerosis (ALS) in U.S. veterans, we have conducted a high-density genome-wide association study (GWAS) of ALS outcome and survival time in a sample of U.S. veterans. We tested ∼1.3 million single nucleotide polymorphisms (SNPs) for association with ALS outcome in 442 incident Caucasian veteran cases diagnosed with definite or probable ALS and 348 Caucasian veteran controls. To increase power, we also included genotypes from 5909 publicly-available non-veteran controls in the analysis. In the survival analysis, we tested for association between SNPs and post-diagnosis survival time in 639 Caucasian veteran cases with definite or probable ALS. After this discovery phase, we performed follow-up genotyping of 299 SNPs in an independent replication sample of Caucasian veterans and non-veterans (ALS outcome: 183 cases and 961 controls; survival: 118 cases). Although no SNPs reached genome-wide significance in the discovery phase for either phenotype, three SNPs were statistically significant in the replication analysis of ALS outcome: rs6080539 (177 kb from PCSK2), rs7000234 (4 kb from ZNF704), and rs3113494 (13 kb from LOC100506746). Two SNPs located in genes that were implicated by previous GWA studies of ALS were marginally significant in the pooled analysis of discovery and replication samples: rs17174381 in DPP6 (p = 4.4×10−4) and rs6985069 near ELP3 (p = 4.8×10−4). Our results underscore the difficulty of identifying and convincingly replicating genetic associations with a rare and genetically heterogeneous disorder such as ALS, and suggest that common SNPs are unlikely to account for a substantial proportion of patients affected by this devastating disorder

Inherent Interfacial Mechanical Gradients in 3D Hydrogels Influence Tumor Cell Behaviors

Cells sense and respond to the rigidity of their microenvironment by altering their morphology and migration behavior. To examine this response, hydrogels with a range of moduli or mechanical gradients have been developed. Here, we show that edge effects inherent in hydrogels supported on rigid substrates also influence cell behavior. A Matrigel hydrogel was supported on a rigid glass substrate, an interface which computational techniques revealed to yield relative stiffening close to the rigid substrate support. To explore the influence of these gradients in 3D, hydrogels of varying Matrigel content were synthesized and the morphology, spreading, actin organization, and migration of glioblastoma multiforme (GBM) tumor cells were examined at the lowest (<50 µm) and highest (>500 µm) gel positions. GBMs adopted bipolar morphologies, displayed actin stress fiber formation, and evidenced fast, mesenchymal migration close to the substrate, whereas away from the interface, they adopted more rounded or ellipsoid morphologies, displayed poor actin architecture, and evidenced slow migration with some amoeboid characteristics. Mechanical gradients produced via edge effects could be observed with other hydrogels and substrates and permit observation of responses to multiple mechanical environments in a single hydrogel. Thus, hydrogel-support edge effects could be used to explore mechanosensitivity in a single 3D hydrogel system and should be considered in 3D hydrogel cell culture systems