Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability.

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.

Displacement of Submacular Hemorrhage Using Subretinal Cocktail Injection versus Pneumatic Displacement: A Real-World Comparative Study.

INTRODUCTION: The objective of this study was to compare the outcome of submacular hemorrhage (SMH) displacement using pneumatic displacement with intravitreal expansile gas versus pars plana vitrectomy (PPV) with subretinal injection of tissue plasminogen activator (tPA), anti-vascular endothelial growth factor (VEGF) agent, and air as primary surgery. METHODS: Retrospective interventional case series of 63 patients who underwent surgical displacement of SMH secondary to neovascular age-related macular degeneration (nAMD) or polypoidal choroidal vasculopathy (PCV) from May 1, 2015, to October 31, 2022. Medical records were reviewed for diagnosis, logMAR visual acuity (VA), central subfield thickness (CST), and postoperative displacement rates and complications up to 12 months after operation. RESULTS: The diagnosis was nAMD in 24 (38.1%) and PCV in 39 (61.9%) eyes. There were 40 (63.5%) eyes in the pneumatic displacement group (38 received C3F8, 2 received SF6) and 23 (36.5%) eyes in the subretinal cocktail injection. Mean baseline VA was 1.46 and 1.62, respectively (p = 0.404). The subretinal injection group had more extensive SMH (p = 0.005), thicker CST (1,006.6 µm vs. 780.2 µm, p = 0.012), and longer interval between symptom and operation (10.65 vs. 5.53 days, p < 0.001). The mean postoperative VA at 6 months was 0.67 and 0.91 (p = 0.180) for pneumatic displacement and subretinal injection groups, respectively, though VA was significantly better in the pneumatic group at 12-month visit (0.64 vs. 1.03, p = 0.040). At least 10 mean change in VA were >10 letters gain in both groups up to 12 months. Postoperative CST reduction was greater (625.1 µm vs. 326.5 µm, p = 0.008) and complete foveal displacement (87.0% vs. 37.5%), p < 0.001, odds ratio [OR] = 11.1) and displacement to arcade or beyond (52.5% vs. 17.5%, p = 0.009, OR = 5.15) were more frequent in the subretinal injection group. Two patients with failed pneumatic displacement were successfully treated with subretinal cocktail injection as a second operation. CONCLUSION: Surgical displacement of SMH leads to clinically meaningful improvement in VA. PPV with subretinal cocktail injection is more effective than pneumatic displacement in displacing SMH with similar safety profile despite longer interval before operation, higher CST, and more extensive SMH at baseline. Retinal surgeons could consider this novel technique in cases with thick and extensive SMH or as a rescue secondary operation in selected cases.

Reaction Time in Fibromyalgia Patients.

BACKGROUND: Fibromyalgia has unknown aetiology and is associated with reduced information processing speed and therefore prolonged reaction time. However, the processes underlying this are unknown. OBJECTIVES: First, to compare the reaction time in a cohort of fibromyalgia patients and a matched group of normal controls. Second, to assess whether detailed symptoms of pain and autonomic function, as well as measures of tinnitus, fatigue, daytime sleepiness and Mycoplasma pneumoniae infection are predictors of reaction time in fibromyalgia. METHODS: The between-groups mean serial five-choice reaction time difference was assessed in a cohort of fibromyalgia patients and in a matched group of normal controls in an analytical casecontrolled study. With the mean serial five-choice reaction time as the dependent variable for the fibromyalgia group, a mixed stepwise multiple linear regression was performed with inputs relating to pain, dysautonomia, tinnitus, fatigue, daytime sleepiness and Mycoplasma pneumoniae infection. RESULTS: The mean (standard error) serial five-choice reaction time for the fibromyalgia group was 448.4 (23.0) ms, compared with 386.3 (8.3) ms for the control group (p = 0.007). The final multiple linear regression model (p < 0.001; adjusted R2 = 0.772) contained 13 predictors: eight sensory pain and three affective pain parameters, and Mycoplasma pneumoniae IgG and IgA assay results. CONCLUSION: Certain sensory and affective pain parameters, as well as Mycoplasma pneumoniae infection, appear to be predictors of reaction time in fibromyalgia. Further research into the pathophysiological mechanisms by which they affect information processing is warranted and may shed light on the aetiology of fibromyalgia.

T Lymphocyte Interferon-gamma Response to Anaplasmataceae-related Major Surface Proteins and Ankyrin A in Fibromyalgia.

BACKGROUND: The aetiology of fibromyalgia is unknown; its symptoms may be related to a T-lymphocyte-mediated response to infectious organisms. OBJECTIVES: First, to test the hypothesis that fibromyalgia is associated with increased interferon (IFN)-γ-secreting T-lymphocytes after stimulation with Anaplasmataceae-related major surface proteins (MSFs) and the macromolecular translocation type IV secretion system effector ankyrin repeat domain-containing protein A (AnkA). Second, to ascertain the relationship in fibromyalgia between (i) the IFN-γ-secreting T-lymphocyte response to stimulation with Anaplasmataceae-related MSFs and AnkA, and (ii) co-infection by Borrelia and Yersinia spp., and antinuclear antibodies. METHODS: Using a case-control design, patients fulfilling the American College of Rheumatology revised criteria for fibromyalgia, and controls, underwent the following blinded assessments: (i) enzyme- linked immune absorbent spot (ELISpot) IFN-γ release assay of T-lymphocyte reactivity to Anaplasmataceae-related MSFs and AnkA; (ii) ELISpot IFN-γ release assays of T-lymphocyte reactivity to three Borrelia antigens, namely Borrelia burgdorferi full antigen (B31); peptide mix (from Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii); and Borrelia burgdorferi lymphocyte function-associated antigen-1; (iii) immunoglobulin (Ig) A assay by enzyme-linked immunosorbent assay (ELISA) of antibodies to Yersinia spp.; (iv) IgG (ELISA) antibodies to Yersinia spp.; (v) serum antinuclear antibodies (immunofluorescence). RESULTS: The groups were age- and sex-matched. The mean (standard error) value of IFN-γ release for the fibromyalgia group was 1.52 (0.26), compared with 1.00 (0.22) for the controls. Generalised linear modelling (p<0.001) of IFN-γ release in the fibromyalgia patients showed significant main effects of all three indices of Borrelia infection and of antinuclear antibodies. CONCLUSION: Anaplasmataceae may play an aetiological role in fibromyalgia.

Comparative Analysis of 24-2C, 24-2, and 10-2 Visual field Tests for Detecting Mild-Stage Glaucoma with Central Visual field Defects.

PURPOSE: This study sought to identify the most effective testing program for detecting visual-field defects in mild-stage glaucoma with central visual-field defects. DESIGN: A multicenter, retrospective diagnostic testing evaluation. PARTICIPANTS: The study involved 93 eyes (83 patients) with mild-stage glaucoma (median mean deviation [interquartile range]: -1.79 [2.16] dB) with central visual-field defects and 69 eyes (63 patients; median mean deviation, -1.38 [2.31] dB) with mild-stage glaucoma without central visual-field defects, from Jikei University School of Medicine and Tajimi Iwase Eye Clinic. METHODS: Patients underwent 10-2 Swedish Interactive Thresholding Algorithm (SITA) Standard, 24-2 SITA Standard, and 24-2C SITA Faster tests. Central visual-field defects were defined using 10-2 SITA Standard and optical coherence tomography (OCT). A detection power of 4 points in the 24-2 that coincided with 10-2 (Center4), 12 points that lie within 10° (24-2-12), and 22 points that lie within 10° of 24-2C (24-2C-22) were analyzed using receiver operating characteristic (ROC) curves based on logistic regression analysis, using total deviation (TD) and pattern deviation (PD) probability plots. MAIN OUTCOME MEASURES: Area under the receiver operating characteristic curve (AUC) of the Center4, 24-2-12, and 24-2C-22 tests. RESULTS: In the upper-central visual field, AUCs of the TD plot were 0.50 (0.40-0.58) for the Center4, 0.75 (0.67-0.83) for 24-2-12, and 0.85 (0.78-0.91) for 24-2C-22, with 24-2C-22 AUC significantly exceeding 24-2-12 AUC. For the PD plot, AUCs were 0.53 (0.44-0.63), 0.81 (0.74-0.89), and 0.84 (0.77-0.90), respectively. In the lower-central visual field, using a total plot, AUCs were 0.27 (0.18-0.36), 0.57 (0.47-0.69), and 0.57 (0.46-0.68) for the Center4, 24-2-12, and 24-2C-22, respectively. Using the PD plot in the upper field, AUCs were 0.27 (0.19-0.36), 0.64 (0.53-0.75), and 0.81 (0.72-0.90), respectively, with the AUC of the 24-2C-22 significantly exceeding that of 24-2-12. The 24-2C test was significantly faster than both the 24-2 and 10-2 tests, reducing testing duration by 46% and 52%, respectively. CONCLUSIONS: The 24-2C SITA Faster test is highly effective and efficient for detecting mild-stage glaucoma with central visual-field defects. This, and its reduced duration, makes it a valuable tool in clinical settings.

Identification and neuroprotective properties of NA-184, a calpain-2 inhibitor.

Our laboratory has shown that calpain-2 activation in the brain following acute injury is directly related to neuronal damage and the long-term functional consequences of the injury, while calpain-1 activation is generally neuroprotective and calpain-1 deletion exacerbates neuronal injury. We have also shown that a relatively selective calpain-2 inhibitor, referred to as C2I, enhanced long-term potentiation and learning and memory, and provided neuroprotection in the controlled cortical impact (CCI) model of traumatic brain injury (TBI) in mice. Using molecular dynamic simulation and Site Identification by Ligand Competitive Saturation (SILCS) software, we generated about 130 analogs of C2I and tested them in a number of in vitro and in vivo assays. These led to the identification of two interesting compounds, NA-112 and NA-184. Further analyses indicated that NA-184, (S)-2-(3-benzylureido)-N-((R,S)-1-((3-chloro-2-methoxybenzyl)amino)-1,2-dioxopentan-3-yl)-4-methylpentanamide, selectively and dose-dependent inhibited calpain-2 activity without evident inhibition of calpain-1 at the tested concentrations in mouse brain tissues and human cell lines. Like NA-112, NA-184 inhibited TBI-induced calpain-2 activation and cell death in mice and rats, both male and females. Pharmacokinetic and pharmacodynamic analyses indicated that NA-184 exhibited properties, including stability in plasma and liver and blood-brain barrier permeability, that make it a good clinical candidate for the treatment of TBI.

Exercise microdosing for skeletal muscle health applications to spaceflight.

Findings from a recent 70-day bedrest investigation suggested intermittent exercise testing in the control group may have served as a partial countermeasure for skeletal muscle size, function, and fiber-type shifts. The purpose of the current study was to investigate the metabolic and skeletal muscle molecular responses to the testing protocols. Eight males (29 ± 2 yr) completed muscle power (6 × 4 s; peak muscle power: 1,369 ± 86 W) and V̇o2max (13 ± 1 min; 3.2 ± 0.2 L/min) tests on specially designed supine cycle ergometers during two separate trials. Blood catecholamines and lactate were measured pre-, immediately post-, and 4-h postexercise. Muscle homogenate and muscle fiber-type-specific [myosin heavy chain (MHC) I and MHC IIa] mRNA levels of exercise markers (myostatin, IκBα, myogenin, MuRF-1, ABRA, RRAD, Fn14, PDK4) and MHC I, IIa, and IIx were measured from vastus lateralis muscle biopsies obtained pre- and 4-h postexercise. The muscle power test altered (P ≤ 0.05) norepinephrine (+124%), epinephrine (+145%), lactate (+300%), and muscle homogenate mRNA (IκBα, myogenin, MuRF-1, RRAD, Fn14). The V̇o2max test altered (P ≤ 0.05) norepinephrine (+1,394%), epinephrine (+1,412%), lactate (+736%), and muscle homogenate mRNA (myostatin, IκBα, myogenin, MuRF-1, ABRA, RRAD, Fn14, PDK4). In general, both tests influenced MHC IIa muscle fibers more than MHC I with respect to the number of genes that responded and the magnitude of response. Both tests also influenced MHC mRNA expression in a muscle fiber-type-specific manner. These findings provide unique insights into the adaptive response of skeletal muscle to small doses of exercise and could help shape exercise dosing for astronauts and Earth-based individuals.NEW & NOTEWORTHY Declines in skeletal muscle health are a concern for astronauts on long-duration spaceflights. The current findings add to the growing body of exercise countermeasures data, suggesting that small doses of specific exercise can be beneficial for certain aspects of skeletal muscle health. This information can be used in conjunction with other components of existing exercise programs for astronauts and might translate to other areas focused on skeletal muscle health (e.g., sports medicine, rehabilitation, aging).

NASA SPRINT exercise program efficacy for vastus lateralis and soleus skeletal muscle health during 70 days of simulated microgravity.

The efficacy of the NASA SPRINT exercise countermeasures program for quadriceps (vastus lateralis) and triceps surae (soleus) skeletal muscle health was investigated during 70 days of simulated microgravity. Individuals completed 6° head-down-tilt bedrest (BR, n = 9), bedrest with resistance and aerobic exercise (BRE, n = 9), or bedrest with resistance and aerobic exercise and low-dose testosterone (BRE + T, n = 8). All groups were periodically tested for muscle (n = 9 times) and aerobic (n = 4 times) power during bedrest. In BR, surprisingly, the typical bedrest-induced decrements in vastus lateralis myofiber size and power were either blunted (myosin heavy chain, MHC I) or eliminated (MHC IIa), along with no change (P > 0.05) in %MHC distribution and blunted quadriceps atrophy. In BRE, MHC I (vastus lateralis and soleus) and IIa (vastus lateralis) contractile performance was maintained (P > 0.05) or increased (P < 0.05). Vastus lateralis hybrid fiber percentage was reduced (P < 0.05) and energy metabolism enzymes and capillarization were generally maintained (P > 0.05), while not all of these positive responses were observed in the soleus. Exercise offsets 100% of quadriceps and approximately two-thirds of soleus whole muscle mass loss. Testosterone (BRE + T) did not provide any benefit over exercise alone for either muscle and for some myocellular parameters appeared detrimental. In summary, the periodic testing likely provided a partial exercise countermeasure for the quadriceps in the bedrest group, which is a novel finding given the extremely low exercise dose. The SPRINT exercise program appears to be viable for the quadriceps; however, refinement is needed to completely protect triceps surae myocellular and whole muscle health for astronauts on long-duration spaceflights.NEW & NOTEWORTHY This study provides unique exercise countermeasures development information for astronauts on long-duration spaceflights. The NASA SPRINT program was protective for quadriceps myocellular and whole muscle health, whereas the triceps surae (soleus) was only partially protected as has been shown with other programs. The bedrest control group data may provide beneficial information for overall exercise dose and targeting fast-twitch muscle fibers. Other unique approaches for the triceps surae are needed to supplement existing exercise programs.

Discovery of GS-7682, a Novel 4'-Cyano-Modified C-Nucleoside Prodrug with Broad Activity against Pneumo- and Picornaviruses and Efficacy in RSV-Infected African Green Monkeys.

Acute respiratory viral infections, such as pneumovirus and respiratory picornavirus infections, exacerbate disease in COPD and asthma patients. A research program targeting respiratory syncytial virus (RSV) led to the discovery of GS-7682 (1), a novel phosphoramidate prodrug of a 4'-CN-4-aza-7,9-dideazaadenosine C-nucleoside GS-646089 (2) with broad antiviral activity against RSV (EC50 = 3-46 nM), human metapneumovirus (EC50 = 210 nM), human rhinovirus (EC50 = 54-61 nM), and enterovirus (EC50 = 83-90 nM). Prodrug optimization for cellular potency and lung cell metabolism identified 5'-methyl [(S)-hydroxy(phenoxy)phosphoryl]-l-alaninate in combination with 2',3'-diisobutyrate promoieties as being optimal for high levels of intracellular triphosphate formation in vitro and in vivo. 1 demonstrated significant reductions of viral loads in the lower respiratory tract of RSV-infected African green monkeys when administered once daily via intratracheal nebulized aerosol. Together, these findings support additional evaluation of 1 and its analogues as potential therapeutics for pneumo- and picornaviruses.

The Role of Chlamydia pneumoniae in the Aetiology of Autoimmune Diseases.

Introduction The most prevalent chronic human autoimmune disorder worldwide is rheumatoid arthritis. Synovial samples from acute-phase patients are polymerase chain reaction-positive for Chlamydia pneumoniae (C. pneumoniae) DNA and express chlamydial hsp60. Furthermore, anti-cyclic citrullinated peptide (anti-CCP) antibodies promote apoptosis of mature human Saos-2 osteoblasts via cell surface binding to citrullinated heat shock protein 60 (HSP60). Hence, we hypothesised that C. pneumoniae infection is associated with anti-CCP antibodies. Methods C. pneumoniae IgA and anti-CCP antibody levels were determined in 26 healthy subjects in this cross-sectional study. Serum C. pneumoniae IgA antibody levels were assessed using an enzyme-linked immunosorbent assay. Serum anti-CCP antibody levels were assessed using fluoroenzymeimmunoassay. Results There was a highly significant positive correlation between the two sets of antibodies (rs = 0.621; P = 0.0007). Linear regression analysis showed that this correlation was not the result of age or sex. Discussion A biologically plausible mechanism is put forward for these results, involving HSP60 acting as an endogenous ligand for toll-like receptor 4 (TLR4) and the interaction of TLR4 with lipopolysaccharides, which occur in the outer membrane of the C. pneumoniae elementary body. Pronounced pro-inflammatory mediator secretion then takes place. The release of Ca2+ ions may then activate local peptidylarginine deiminases, leading to the formation of CCPs and thus the reported finding. Confirmation of these results may have potential clinical implications in terms of diagnosis, including pre-symptomatic diagnosis, and treatment.

Human enteroviral infection in fibromyalgia: a case-control blinded study

SUMMARY OBJECTIVE: This study aimed to test the hypothesis that fibromyalgia is associated with a human enteroviral infection. METHODS: Venous peripheral blood samples from 27 patients fulfilling the American College of Rheumatology revised diagnostic criteria for fibromyalgia and from 26 age- and sex-matched controls, who underwent immunofluorescence assays for coxsackievirus A7 IgG, coxsackievirus B1 IgG, coxsackievirus A7 IgA, coxsackievirus B1 IgA, echovirus IgG, and echovirus IgA. These immunological tests were performed blind to group status. RESULTS: There were no significant differences between the patient and control groups in respect of positive results for coxsackievirus A7 IgG (p=0.467), coxsackievirus B1 IgG (p=0.491), coxsackievirus A7 IgA (p=0.586), coxsackievirus B1 IgA (p=0.467), echovirus IgG (p=0.236), and echovirus IgA (p=1). CONCLUSIONS: The results of this systematic study do not support the hypothesis that fibromyalgia is associated with infection by a human enterovirus.