Quantum simulations of SARS-CoV-2 main protease Mpro enable high-quality scoring of diverse ligands.

The COVID-19 pandemic has led to unprecedented efforts to identify drugs that can reduce its associated morbidity/mortality rate. Computational chemistry approaches hold the potential for triaging potential candidates far more quickly than their experimental counterparts. These methods have been widely used to search for small molecules that can inhibit critical proteins involved in the SARS-CoV-2 replication cycle. An important target is the SARS-CoV-2 main protease Mpro, an enzyme that cleaves the viral polyproteins into individual proteins required for viral replication and transcription. Unfortunately, standard computational screening methods face difficulties in ranking diverse ligands to a receptor due to disparate ligand scaffolds and varying charge states. Here, we describe full density functional quantum mechanical (DFT) simulations of Mpro in complex with various ligands to obtain absolute ligand binding energies. Our calculations are enabled by a new cloud-native parallel DFT implementation running on computational resources from Amazon Web Services (AWS). The results we obtain are promising: the approach is quite capable of scoring a very diverse set of existing drug compounds for their affinities to M pro and suggest the DFT approach is potentially more broadly applicable to repurpose screening against this target. In addition, each DFT simulation required only ~ 1 h (wall clock time) per ligand. The fast turnaround time raises the practical possibility of a broad application of large-scale quantum mechanics in the drug discovery pipeline at stages where ligand diversity is essential.

Long-term safety and efficacy studies of epinephrine HFA metered-dose inhaler (Primatene® Mist): a two-stage randomized controlled trial.

Objective: A new epinephrine hydrofluoroalkane (HFA) asthma metered-dose inhaler (MDI) was reformulated to replace the previously marketed epinephrine chlorofluorocarbon (CFC) MDI. In addition to the HFA propellant change, several enhanced modifications (i.e. changed from solution to suspension, 43% dose reduction, etc.) were made to the formulation of epinephrine HFA MDI. This study evaluates the 6-month long-term safety and efficacy profile of the new epinephrine HFA MDI.Method: The long-term safety study consists of two 3-month, multi-center, double- or evaluator-blinded, parallel-group, placebo, and active controlled stages. In each stage, subjects aged ≥12 years with intermittent or mild-to-moderate persistent asthma were randomized to receive epinephrine HFA (2 × 125 mcg/inhalation), placebo HFA, or epinephrine CFC (2 × 220 mcg/inhalation). Bronchodilator efficacy was assessed in Stage 1 and was determined primarily by the change in the forced expiratory volume in 1 s (ΔFEV1) at Week 12, relative to the same day baseline.Results: The primary efficacy endpoint (AUC0-6hrs of %ΔFEV1 at Week 12) for epinephrine HFA (47.3 ± 54.2) closely paralleled those for the active control, epinephrine CFC (41.0 ± 43.4). Both groups were found to be overall comparable in bronchodilator efficacy. Both also showed low incidence rates of AEs with tremor being most commonly reported for epinephrine HFA. All AEs found were non-serious and non-significant. The observed changes in vital signs, ECG, serum glucose, and potassium were minimal and not clinically relevant.Conclusion: This study demonstrated that the new epinephrine HFA is overall comparable, in both safety and efficacy, to the previous epinephrine CFC.

Rigorous Free Energy Simulations in Virtual Screening.

Virtual high throughput screening (vHTS) in drug discovery is a powerful approach to identify hits: when applied successfully, it can be much faster and cheaper than experimental high-throughput screening approaches. However, mainstream vHTS tools have significant limitations: ligand-based methods depend on knowledge of existing chemical matter, while structure-based tools such as docking involve significant approximations that limit their accuracy. Recent advances in scientific methods coupled with dramatic speedups in computational processing with GPUs make this an opportune time to consider the role of more rigorous methods that could improve the predictive power of vHTS workflows. In this Perspective, we assert that alchemical binding free energy methods using all-atom molecular dynamics simulations have matured to the point where they can be applied in virtual screening campaigns as a final scoring stage to prioritize the top molecules for experimental testing. Specifically, we propose that alchemical absolute binding free energy (ABFE) calculations offer the most direct and computationally efficient approach within a rigorous statistical thermodynamic framework for computing binding energies of diverse molecules, as is required for virtual screening. ABFE calculations are particularly attractive for drug discovery at this point in time, where the confluence of large-scale genomics data and insights from chemical biology have unveiled a large number of promising disease targets for which no small molecule binders are known, precluding ligand-based approaches, and where traditional docking approaches have foundered to find progressible chemical matter.

Alchemical Binding Free Energy Calculations in AMBER20: Advances and Best Practices for Drug Discovery.

Predicting protein-ligand binding affinities and the associated thermodynamics of biomolecular recognition is a primary objective of structure-based drug design. Alchemical free energy simulations offer a highly accurate and computationally efficient route to achieving this goal. While the AMBER molecular dynamics package has successfully been used for alchemical free energy simulations in academic research groups for decades, widespread impact in industrial drug discovery settings has been minimal because of the previous limitations within the AMBER alchemical code, coupled with challenges in system setup and postprocessing workflows. Through a close academia-industry collaboration we have addressed many of the previous limitations with an aim to improve accuracy, efficiency, and robustness of alchemical binding free energy simulations in industrial drug discovery applications. Here, we highlight some of the recent advances in AMBER20 with a focus on alchemical binding free energy (BFE) calculations, which are less computationally intensive than alternative binding free energy methods where full binding/unbinding paths are explored. In addition to scientific and technical advances in AMBER20, we also describe the essential practical aspects associated with running relative alchemical BFE calculations, along with recommendations for best practices, highlighting the importance not only of the alchemical simulation code but also the auxiliary functionalities and expertise required to obtain accurate and reliable results. This work is intended to provide a contemporary overview of the scientific, technical, and practical issues associated with running relative BFE simulations in AMBER20, with a focus on real-world drug discovery applications.

Fluticasone furoate/vilanterol versus fluticasone propionate in patients with asthma and exercise-induced bronchoconstriction.

Objective: To investigate whether once-daily (OD) fluticasone furoate (FF)/vilanterol (VI) provides greater long-term protection from postexercise fall in forced expiratory volume in 1 s (FEV1) than twice-daily (BD) fluticasone propionate (FP) in patients with asthma and exercise-induced bronchoconstriction. Methods: A randomized, double-blind, crossover study was conducted in patients (aged 12-50 years) on low-/mid-dose maintenance inhaled corticosteroid. Following a 4-week run-in period (FP 250 µg BD), patients with a ≥ 20% decrease in postexercise FEV1 received FF/VI 100/25 µg OD or FP 250 µg BD for 2 weeks. Exercise challenges were carried out 23 h after the first dose of study medication, and 12 and 23 h after evening clinic dose at the end of the 2-week treatment period. After a 2-week washout period (FP 250 µg), patients crossed over treatments, with procedures and tests repeated. The primary endpoint was mean maximal percentage decrease from pre-exercise FEV1 following exercise challenge 12-h postevening dose on Day 14. Results: The mean maximal percentage decrease from pre-exercise FEV1 after the 12-h exercise challenge (Day 14) was 15.02% with FF/VI, and 16.71% with FP (difference, -1.69; 95% confidence interval, -3.76 to 0.39; p = 0.109). After the 23-h exercise challenge (Day 14), respective mean maximal decreases were 11.90% and 14.05% (difference, -2.15; 95% confidence interval, -4.31 to 0.01). Conclusion: The study failed to show a difference between FF/VI and FP at providing long-term protection from exercise-induced bronchoconstriction.

Efficacy and safety of budesonide/formoterol pMDI vs budesonide pMDI in asthmatic children (6-<12 years).

BACKGROUND: The efficacy and safety of budesonide/formoterol pressurized metered-dose inhaler (pMDI) have been demonstrated in patients with asthma at least 12 years old. OBJECTIVE: To evaluate the efficacy of 2 formoterol doses added to budesonide as fixed combinations vs budesonide alone in children 6 to younger than 12 years with asthma. METHODS: This randomized, double-blinded, parallel-group, multicenter study (NCT02091986; CHASE 3) included children 6 to younger than 12 years with asthma previously receiving a medium-dose inhaled corticosteroid (ICS) or an ICS plus a long-acting ß2-agonist. Children symptomatic during a 7-28-day run-in on low-dose ICS, 1 inhalation of budesonide dry powder inhaler 90 µg twice daily (BID), were randomized to receive 2 inhalations of budesonide/formoterol pMDI 80/4.5 µg (160/9 µg) BID (n = 92), budesonide/formoterol pMDI 80/2.25 µg (160/4.5 µg) BID (n = 95), or budesonide pMDI 80 µg (160 µg) BID (n = 92) for 12 weeks. RESULTS: Change in forced expiratory volume in 1 second from baseline to 1 hour after dosing (primary end point), change in forced expiratory volume in 1 second 15 minutes after dosing, and peak expiratory flow 1 hour after dosing at week 12 were statistically significantly greater for budesonide/formoterol 160/9 µg vs budesonide (P ≤ .015 for all comparisons), but not for budesonide/formoterol 160/4.5 µg vs budesonide. Bronchodilator effects, evident 15 minutes after the dose on day 1, were maintained at week 12. Incidence of protocol-defined asthma exacerbations and improvements in asthma symptom-related and quality-of-life outcomes were similar across treatments. There were no notable safety differences among treatments. CONCLUSION: Budesonide/formoterol pMDI 160/9 µg showed statistically significant and clinically meaningful lung function improvements vs budesonide pMDI 160 µg, demonstrating appropriateness as a therapeutic option for children 6 to younger than 12 years with asthma symptomatic on ICS alone. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02091986.

Dupilumab in persistent asthma with elevated eosinophil levels.

BACKGROUND: Moderate-to-severe asthma remains poorly treated. We evaluated the efficacy and safety of dupilumab (SAR231893/REGN668), a fully human monoclonal antibody to the alpha subunit of the interleukin-4 receptor, in patients with persistent, moderate-to-severe asthma and elevated eosinophil levels. METHODS: We enrolled patients with persistent, moderate-to-severe asthma and a blood eosinophil count of at least 300 cells per microliter or a sputum eosinophil level of at least 3% who used medium-dose to high-dose inhaled glucocorticoids plus long-acting beta-agonists (LABAs). We administered dupilumab (300 mg) or placebo subcutaneously once weekly. Patients were instructed to discontinue LABAs at week 4 and to taper and discontinue inhaled glucocorticoids during weeks 6 through 9. Patients received the study drug for 12 weeks or until a protocol-defined asthma exacerbation occurred. The primary end point was the occurrence of an asthma exacerbation; secondary end points included a range of measures of asthma control. Effects on various type 2 helper T-cell (Th2)-associated biomarkers and safety and tolerability were also evaluated. RESULTS: A total of 52 patients were assigned to the dupilumab group, and 52 patients were assigned to the placebo group. Baseline characteristics were similar in the two groups. Three patients had an asthma exacerbation with dupilumab (6%) versus 23 with placebo (44%), corresponding to an 87% reduction with dupilumab (odds ratio, 0.08; 95% confidence interval, 0.02 to 0.28; P<0.001). Significant improvements were observed for most measures of lung function and asthma control. Dupilumab reduced biomarkers associated with Th2-driven inflammation. Injection-site reactions, nasopharyngitis, nausea, and headache occurred more frequently with dupilumab than with placebo. CONCLUSIONS: In patients with persistent, moderate-to-severe asthma and elevated eosinophil levels who used inhaled glucocorticoids and LABAs, dupilumab therapy, as compared with placebo, was associated with fewer asthma exacerbations when LABAs and inhaled glucocorticoids were withdrawn, with improved lung function and reduced levels of Th2-associated inflammatory markers. (Funded by Sanofi and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT01312961.).

Efficacy and safety of budesonide administered by pressurized metered-dose inhaler in children with asthma.

BACKGROUND: Budesonide is approved for delivery using a nebulized solution and dry-powder inhaler, but its use through a pressurized metered-dose inhaler (pMDI) in pediatric patients with asthma has not been determined. OBJECTIVE: To examine the efficacy and safety of 160 µg twice daily of budesonide through a pMDI vs placebo in children 6 to younger than 12 years with asthma and a demonstrated need for inhaled corticosteroids. METHODS: A 6-week, international, multicenter, double-blinded, parallel-group, phase 2 study randomized 304 pediatric patients (mean age, 9 years; 21.7% <8 years) 1:1 to 160 µg (80 µg × 2 inhalations) twice daily of budesonide through a pMDI or placebo after a 7- to 21-day run-in period. The primary efficacy end point was change from baseline in morning peak expiratory flow (PEF); safety end points included adverse events, vital signs, and discontinuations. RESULTS: Budesonide treatment significantly improved morning PEF vs placebo; mean treatment effect (budesonide vs placebo) was 13.6 L/min (P < .0001). Budesonide also showed significant improvements vs placebo for forced expiratory volume in 1 second, evening PEF, forced expiratory flow at 25% to 75% of pulmonary volume, reliever medication use, nighttime awakenings, awakenings with reliever use, and percentage of patients with at least 15- and at least 30-L/min increase in morning PEF from baseline. The numbers of patients experiencing adverse events and discontinuations were smaller in the budesonide than in the placebo group. No serious adverse events were reported. CONCLUSION: Budesonide at 160 µg twice daily through a pMDI was generally well tolerated and significantly improved lung function, symptoms, rescue medication use, and nighttime awakenings vs placebo in children 6 to younger than 12 years with asthma and a demonstrated need for inhaled corticosteroids.

Physics-based enzyme design: predicting binding affinity and catalytic activity.

Computational enzyme design is an emerging field that has yielded promising success stories, but where numerous challenges remain. Accurate methods to rapidly evaluate possible enzyme design variants could provide significant value when combined with experimental efforts by reducing the number of variants needed to be synthesized and speeding the time to reach the desired endpoint of the design. To that end, extending our computational methods to model the fundamental physical-chemical principles that regulate activity in a protocol that is automated and accessible to a broad population of enzyme design researchers is essential. Here, we apply a physics-based implicit solvent MM-GBSA scoring approach to enzyme design and benchmark the computational predictions against experimentally determined activities. Specifically, we evaluate the ability of MM-GBSA to predict changes in affinity for a steroid binder protein, catalytic turnover for a Kemp eliminase, and catalytic activity for α-Gliadin peptidase variants. Using the enzyme design framework developed here, we accurately rank the most experimentally active enzyme variants, suggesting that this approach could provide enrichment of active variants in real-world enzyme design applications.

Antibody structure determination using a combination of homology modeling, energy-based refinement, and loop prediction.

We present the blinded prediction results in the Second Antibody Modeling Assessment (AMA-II) using a fully automatic antibody structure prediction method implemented in the programs BioLuminate and Prime. We have developed a novel knowledge based approach to model the CDR loops, using a combination of sequence similarity, geometry matching, and the clustering of database structures. The homology models are further optimized with a physics-based energy function (VSGB2.0), which improves the model quality significantly. H3 loop modeling remains the most challenging task. Our ab initio loop prediction performs well for the H3 loop in the crystal structure context, and allows improved results when refining the H3 loops in the context of homology models. For the 10 human and mouse derived antibodies in this assessment, the average RMSDs for the homology model Fv and framework regions are 1.19 Å and 0.74 Å, respectively. The average RMSDs for five non-H3 CDR loops range from 0.61 Å to 1.05 Å, and the H3 loop average RMSD is 2.91 Å using our knowledge-based loop prediction approach. The ab initio H3 loop predictions yield an average RMSD of 1.28 Å when performed in the context of the crystal structure and 2.67 Å in the context of the homology modeled structure. Notably, our method for predicting the H3 loop in the crystal structure environment ranked first among the seven participating groups in AMA-II, and our method made the best prediction among all participants for seven of the ten targets.

The effect of GSK2190915, a 5-lipoxygenase-activating protein inhibitor, on exercise-induced bronchoconstriction.

Exercise-induced bronchoconstriction (EIB) describes the condition whereby exercise causes airflow obstruction that lasts for up to 90 minutes without treatment. This double-blind, placebo-controlled, five-way crossover study investigated the dose response and duration of action of a 5-lipoxygenase-activating protein inhibitor, GSK2190915, to inhibit EIB in subjects with asthma. Forty-seven subjects with EIB were enrolled. Exercise challenge testing was scheduled at 2, 9.5, and 24 hours after receiving a single dose of GSK2190915 (10, 50, 100, and 200 mg) or placebo in randomized order. GSK2190915 at 200 and 100 mg significantly attenuated the response to exercise at 2 and 9.5 hours postdose, respectively, compared with placebo. The adjusted mean maximum percentage change from baseline forced expiratory volume at 1 second within 60 minutes postexercise challenge (FEV1 (0-60)) treatment difference for GSK2190915 at 200 mg compared with placebo at 2 hours postdose was 6.30% (95% CI, 3.06, 9.54), corresponding to a 40% attenuation of the placebo response to exercise; the treatment difference between GSK2190915 at 100 mg and placebo at 9.5 hours postdose was 3.49% (95% CI, 1.02, 5.95), corresponding to a 41% attenuation of the placebo response to exercise. No significant effect was seen at 24 hours postdose with any dose; however, investigation of statistically significant treatment-related effects at this time point was limited because of the small fall in adjusted mean FEV1 (0-60) (-7.61%; 95% CI, -10.23, -4.99) after placebo. GSK2190915 may be of benefit in EIB prevention. GSK Clinical Study LPA112025; ClinicalTrials.gov identifier number: NCT00812929.

Safety profile and clinical activity of multiple subcutaneous doses of MEDI-528, a humanized anti-interleukin-9 monoclonal antibody, in two randomized phase 2a studies in subjects with asthma.

BACKGROUND: Interleukin-9 (IL-9)-targeted therapies may offer a novel approach for treating asthmatics. Two randomized placebo-controlled studies were conducted to assess the safety profile and potential efficacy of multiple subcutaneous doses of MEDI-528, a humanized anti-IL-9 monoclonal antibody, in asthmatics. METHODS: Study 1: adults (18-65 years) with mild asthma received MEDI-528 (0.3, 1, 3 mg/kg) or placebo subcutaneously twice weekly for 4 weeks. Study 2: adults (18-50 years) with stable, mild to moderate asthma and exercise-induced bronchoconstriction received 50 mg MEDI-528 or placebo subcutaneously twice weekly for 4 weeks. Adverse events (AEs), pharmacokinetics (PK), immunogenicity, asthma control (including asthma exacerbations), and exercise challenge test were evaluated in study 1, study 2, or both. RESULTS: In study 1 (N = 36), MEDI-528 showed linear serum PK; no anti-MEDI-528 antibodies were detected. Asthma control: 1/27 MEDI-528-treated subjects had 1 asthma exacerbation, and 2/9 placebo-treated subjects had a total of 4 asthma exacerbations (one considered a serious AE). In study 2, MEDI-528 (n = 7) elicited a trend in the reduction in mean maximum decrease in FEV1 post-exercise compared to placebo (n = 2) (-6.49% MEDI-528 vs -12.60% placebo; -1.40% vs -20.10%; -5.04% vs -15.20% at study days 28, 56, and 150, respectively). Study 2 was halted prematurely due to a serious AE in an asymptomatic MEDI-528-treated subject who had an abnormal brain magnetic resonance imaging that was found to be an artifact on further evaluation. CONCLUSIONS: In these studies, MEDI-528 showed an acceptable safety profile and findings suggestive of clinical activity that support continued study in subjects with mild to moderate asthma.

Reproducibility of the airway response to an exercise protocol standardized for intensity, duration, and inspired air conditions, in subjects with symptoms suggestive of asthma.

BACKGROUND: Exercise testing to aid diagnosis of exercise-induced bronchoconstriction (EIB) is commonly performed. Reproducibility of the airway response to a standardized exercise protocol has not been reported in subjects being evaluated with mild symptoms suggestive of asthma but without a definite diagnosis. This study examined reproducibility of % fall in FEV1 and area under the FEV1 time curve for 30 minutes in response to two exercise tests performed with the same intensity and duration of exercise, and inspired air conditions. METHODS: Subjects with mild symptoms of asthma exercised twice within approximately 4 days by running for 8 minutes on a motorized treadmill breathing dry air at an intensity to induce a heart rate between 80-90% predicted maximum; reproducibility of the airway response was expressed as the 95% probability interval. RESULTS: Of 373 subjects challenged twice 161 were positive (≥ 10% fall FEV1 on at least one challenge). The EIB was mild and 77% of subjects had <15% fall on both challenges. Agreement between results was 76.1% with 56.8% (212) negative (< 10% fall FEV1) and 19.3% (72) positive on both challenges. The remaining 23.9% of subjects had only one positive test. The 95% probability interval for reproducibility of the % fall in FEV1 and AUC0-30 min was ± 9.7% and ± 251% for all 278 adults and ± 13.4% and ± 279% for all 95 children. The 95% probability interval for reproducibility of % fall in FEV1 and AUC0-30 min for the 72 subjects with two tests ≥ 10% fall FEV1 was ± 14.6% and ± 373% and for the 34 subjects with two tests ≥ 15% fall FEV1 it was ± 12.2% and ± 411%. Heart rate and estimated ventilation achieved were not significantly different either on the two test days or when one test result was positive and one was negative. CONCLUSIONS: Under standardized, well controlled conditions for exercise challenge, the majority of subjects with mild symptoms of asthma demonstrated agreement in test results. Performing two tests may need to be considered when using exercise to exclude or diagnose EIB, when prescribing prophylactic treatment to prevent EIB and when designing protocols for clinical trials.

Baseline predictors of placebo response in exercise-induced bronchoconstriction (EIB): pooled regression analysis >from three studies of montelukast in EIB.

BACKGROUND: Exercise-induced bronchoconstriction (EIB) can be variable in its presentation and severity. Evaluating patterns of placebo response and patient-related factors driving placebo response could facilitate more efficient clinical trials for EIB. METHODS: Data were pooled from three randomized, double-blind, crossover trials evaluating single-dose montelukast 10 mg or placebo in patients (N = 160) 15-45 years of age with EIB, defined as maximum % fall in forced expiratory volume in one second (FEV1) ≥20% after two screening exercise challenges. Serial exercise challenges were performed at 2, 8.5-12, and 24 h postdose. The authors evaluated the distribution and variability of placebo response. They also evaluated possible drivers of response, analyzing all baseline patient demographic and prerandomization screening visit pulmonary function data as single covariates in a simple univariate regression model for maximum % fall in FEV1 while on placebo at 2 h postdose. All covariates with p values <.1 were entered into both stepwise forward and backward regression procedures to select the "best" model. RESULTS: Placebo response was variable, and showed a significant non-normal distribution (p < .001). Significant predictors of a greater response to placebo included: higher screening FEV1 % predicted (p <.001), smaller maximum % fall in FEV1 in screening (p < .001), shorter time to recovery in screening (p = .007), more asthma-related health care visits in the previous year (p = .004), older age (p = .001), less frequent asthma awakenings in the previous month (p = .003), and less frequent asthma symptoms in the past year (p = .011). CONCLUSION: Predictors of a larger placebo response were generally markers of less severe asthma and/or EIB. This may be related to EIB variability, spontaneous improvement, or the extent of placebo response relative to the outcomes in less severe patients.

Twenty-six-week efficacy and safety study of mometasone furoate/formoterol 200/10 microg combination treatment in patients with persistent asthma previously receiving medium-dose inhaled corticosteroids.

Asthma is a heterogeneous condition characterized by reduced lung function, chronic inflammation, and periodic asthma deteriorations. This study was performed to evaluate the effect of mometasone furoate (MF)/formoterol (F) combination, 200/10 microg, administered twice daily (b.i.d.) on asthma deteriorations and pulmonary function in patients with asthma uncontrolled on medium-dose inhaled corticosteroid (ICS). After 2- to 3-week open-label run-in with MF 200 microg b.i.d., patients (>or=12 years) were randomized to 26 weeks of treatment with MF/F 200/10 microg, MF 200 microg, F 10 microg, or placebo b.i.d. Coprimary end points were time to first asthma deterioration (MF/F versus F) and bronchodilation, assessed by the area under the curve of the change in forced expiratory volume in 1 second 0-12 hours (FEV(1) AUC(0-12h); MF/F versus MF). A total of 781 patients were randomized. Treatment with MF/F 200/10 microg reduced asthma deteriorations and clinically judged deteriorations (i.e., deterioration resulting in emergency treatment, hospitalization, or treatment with additional excluded asthma medication [i.e., systemic corticosteroids]). The proportion of patients experiencing asthma deteriorations was MF/F, 30.4%; MF, 33.9%; F, 54.0%; placebo, 55.6% (p < 0.001, MF/F versus F and placebo). There was a sixfold reduction in clinically judged deteriorations with MF/F versus F and placebo (p < 0.001). Lung function improved more rapidly with MF/F than MF and placebo. Mean change from baseline FEV(1) AUC(0-12h) at week 12 was MF/F, 11.7% versus MF, 5.7%; F, 8.5%; and placebo, 3.9% (p < 0.001). Treatment-related AEs were rare and similar across groups. Treatment with MF/F 200/10 microg was effective in reducing the risk of asthma deteriorations. MF/F was safe and provided rapid and sustained bronchodilation in patients with asthma.

Comparison of mannitol and methacholine to predict exercise-induced bronchoconstriction and a clinical diagnosis of asthma.

BACKGROUND: Asthma can be difficult to diagnose, but bronchial provocation with methacholine, exercise or mannitol is helpful when used to identify bronchial hyperresponsiveness (BHR), a key feature of the disease. The utility of these tests in subjects with signs and symptoms of asthma but without a clear diagnosis has not been investigated. We investigated the sensitivity and specificity of mannitol to identify exercise-induced bronchoconstriction (EIB) as a manifestation of BHR; compared this with methacholine; and compared the sensitivity and specificity of mannitol and methacholine for a clinician diagnosis of asthma. METHODS: 509 people (6-50 yr) were enrolled, 78% were atopic, median FEV1 92.5% predicted, and a low NAEPPII asthma score of 1.2. Subjects with symptoms of seasonal allergy were excluded. BHR to exercise was defined as a > or = 10% fall in FEV1 on at least one of two tests, to methacholine a PC20 < or = 16 mg/ml and to mannitol a 15% fall in FEV1 at < or = 635 mg or a 10% fall between doses. The clinician diagnosis of asthma was made on examination, history, skin tests, questionnaire and response to exercise but they were blind to the mannitol and methacholine results. RESULTS: Mannitol and methacholine were therapeutically equivalent to identify EIB, a clinician diagnosis of asthma, and prevalence of BHR. The sensitivity/specificity of mannitol to identify EIB was 59%/65% and for methacholine it was 56%/69%. The BHR was mild. Mean EIB % fall in FEV1 in subjects positive to exercise was 19%, (SD 9.2), mannitol PD15 158 (CI:129,193) mg, and methacholine PC20 2.1(CI:1.7, 2.6) mg/ml. The prevalence of BHR was the same: for exercise (43.5%), mannitol (44.8%), and methacholine (41.6%) with a test agreement between 62 & 69%. The sensitivity and specificity for a clinician diagnosis of asthma was 56%/73% for mannitol and 51%/75% for methacholine. The sensitivity increased to 73% and 72% for mannitol and methacholine when two exercise tests were positive. CONCLUSION: In this group with normal FEV1, mild symptoms, and mild BHR, the sensitivity and specificity for both mannitol and methacholine to identify EIB and a clinician diagnosis of asthma were equivalent, but lower than previously documented in well-defined populations. TRIAL REGISTRATION: This was a multi-center trial comprising 25 sites across the United States of America.

Mometasone furoate improves nasal and ocular symptoms of seasonal allergic rhinitis in adolescents.

Seasonal allergic rhinitis (SAR) is common in adolescents. However, few studies have investigated the effectiveness of intranasal corticosteroids (INSs) for nasal and ocular symptoms of SAR solely in adolescents. The purpose of this study was to determine the safety and efficacy of the INS mometasone furoate nasal spray (MFNS) in adolescents; a post hoc analysis was conducted of adolescents who had participated in a study with adults. Data were analyzed retrospectively for subjects aged 12-17 years with moderate or severe SAR randomized to mometasone furoate, 200 mcg once daily (n = 86), or placebo (n = 82) for 15 days in a multicenter, double-blind, placebo-controlled study. Symptom scores (0 = none to 3 = severe) were recorded in diaries twice daily. End points included changes from baseline in total nasal symptom score (TNSS), individual nasal symptom score (rhinorrhea, congestion, itching, and sneezing), and total ocular symptom score (TOSS). Over 15 days, a significantly greater decrease from baseline in mean TNSS was observed in subjects receiving mometasone furoate (-2.47; -28.8%) compared with those receiving placebo (-0.9; -9.6%; p < 0.001). Significant improvement versus placebo was seen for each full day of treatment. Mometasone furoate significantly improved individual nasal symptoms (p < or = 0.03) and TOSS (p = 0.011) versus placebo. The incidence of adverse events was similar for both treatment groups. MFNS, 200 mcg once daily, is an effective and well-tolerated treatment for symptoms of SAR in adolescents.

Predicting mutations deleterious to function in beta-lactamase TEM1 using MM-GBSA.

Missense mutations can have disastrous effects on the function of a protein. And as a result, they have been implicated in numerous diseases. However, the majority of missense variants only have a nominal impact on protein function. Thus, the ability to distinguish these two classes of missense mutations would greatly aid drug discovery efforts in target identification and validation as well as medical diagnosis. Monitoring the co-occurrence of a given missense mutation and a disease phenotype provides a pathway for classifying functionally disrupting missense mutations. But, the occurrence of a specific missense variant is often extremely rare making statistical links challenging to infer. In this study, we benchmark a physics-based approach for predicting changes in stability, MM-GBSA, and apply it to classifying mutations as functionally disrupting. A large and diverse dataset of 990 residue mutations in beta-lactamase TEM1 is used to assess performance as it is rich in both functionally disrupting mutations and functionally neutral/beneficial mutations. On this dataset, we compare the performance of MM-GBSA to alternative strategies for predicting functionally disrupting mutations. We observe that the MM-GBSA method obtains an area under the curve (AUC) of 0.75 on the entire dataset, outperforming all other predictors tested. More importantly, MM-GBSA's performance is robust to various divisions of the dataset, speaking to the generality of the approach. Though there is one notable exception: Mutations on the surface of the protein are the mutations that are the most difficult to classify as functionally disrupting for all methods tested. This is likely due to the many mechanisms available to surface mutations to disrupt function, and thus provides a direction of focus for future studies.

FURSMASA: a new approach to rapid scoring functions that uses a MD-averaged potential energy grid and a solvent-accessible surface area term with parameters GA fit to experimental data.

We demonstrate a new approach to the development of scoring functions through the formulation and parameterization of a new function, which can be used both for rapidly ranking the binding of ligands to proteins and for estimating relative aqueous molecular solubilities. The intent of this work is to introduce a new paradigm for creation of scoring functions, wherein we impose the following criteria upon the function: (1) simple; (2) intuitive; (3) requires no postparameterization tweaking; (4) can be applied (without reparameterization) to multiple target systems; and (5) can be rapidly evaluated for any potential ligand. Following these criteria, a new function, FURSMASA (function for rapid scoring using an MD-averaged grid and the accessible surface area) has been developed. Three novel features of the function include: (1) use of an MD-averaged potential energy grid for ligand-protein interactions, rather than a simple static grid; (2) inclusion of a term that depends on the change in the solvent-accessible surface area changes on an atomic (not molecular) basis; and (3) use of the recently derived predictive index (PI) target when optimizing the function, which focuses the function on its intended purpose of relative ranking. A genetic algorithm is used to optimize the function against test data sets that include ligands for the following proteins: IMPDH, p38, gyrase B, HIV-1, and TACE, as well as the Syracuse Research solubility database. We find that the function is predictive, and can simultaneously fit all the test data sets with cross-validated predictive indices ranging from 0.68 to 0.82. As a test of the ability of this function to predict binding for systems not in the training set, the resulting fitted FURSAMA function is then applied to 23 ligands of the COX-2 enzyme. Comparing the results for COX-2 against those obtained using a variety of well-known rapid scoring functions demonstrates that FURSMASA outperforms all of them in terms of the PI and correlation coefficient. We also find that the FURSAMA function is able to reliably predict the water solubility for 1032 compounds from the Syracuse Research solubility database with a cross-correlated PI of 0.84 and a correlation coefficient R(2) of 0.69. This prediction, which is based solely on a term derived from the atom-based solvent-accessible surface areas, compares favorably with the best prediction methods in the literature, most of which are more complex and/or require experimental data. Finally, as a rigorous test of the applicability to database screening, we apply FURSMASA to large active/decoy ligand databases for IMPDH (400 actives vs. 10,000 decoys), p38 (502 actives vs. 10,000 decoys), and HIV (787 actives vs. 10,000 decoys) used in earlier work to critically evaluate many popular scoring functions, and find that FURSMASA performs surprisingly well for IMPDH and HIV.

Deterioration in asthma control when subjects receiving fluticasone propionate/salmeterol 100/50 mcg Diskus are "stepped-down".

In this study, 647 subjects stable on fluticasone propionate/salmeterol Diskus 100/50 mcg BID (FSC) were randomized to continue FSC 100/50 mcg BID or "step down" to either fluticasone propionate (FP) 100 mcg BID, salmeterol (SAL) 50 mcg BID, or montelukast (MON) 10 mg once daily for 16 weeks. Overall asthma control significantly improved in the FSC group; whereas, "stepping down" to FP, SAL, or MON resulted in deterioration in asthma control, as determined by decreased measures of lung function and clinical features. This study provides support that treatment of both inflammation and smooth muscle dysfunction may be necessary to achieve and maintain asthma control in patients uncontrolled on ICS.