STUDIES ON THE CONFORMATIONAL STABILITY, DYNAMICS AND VISCOSITY OF IMMUNOGLOBULINS

Proteins, such as immunoglobulins, are inherently dynamic molecules with unique biological functions. The intra- and intermolecular interactions that govern the dynamic nature and function of immunoglobulins may also influence their stability. A molecular understanding of interrelationship between dynamics, function and conformational stability of immunoglobulins, both in solution and in presence of co-solutes, can be important for their pharmaceutical development. In addition, it is important to understand the differences in any such correlations between closely related immunoglobulins. Furthermore, molecular interactions in immunoglobulins can be unique at low and high concentrations, which necessitate newer complementary approaches to investigate such complex systems. Therefore, a better understanding of interactions that govern protein structure, dynamics and conformational stability at low and high concentrations and in presence of excipients should aid in designing, optimizing and developing rational formulation conditions for protein based therapeutics. A variety of experimental methods sensitive to structure, dynamics and conformational stability of proteins in solution were employed in these studies. External perturbations such a change in pH, ionic strength and temperature were used to probe the response of proteins, both at low and high concentrations. Different monoclonal antibodies within IgG1 isotype were used to investigate interrelationships between protein dynamics and conformational stability in absence and presence of co-solutes. In addition, an ultraviolet spectroscopy based approach was developed to understand interactions modulating solution viscosity in high concentration immunoglobulin solutions. These studies provide evidence that immunoglobulins belonging to the same IgG1 subtype can have notable differences in their conformational stability, dynamics, aggregation propensity, hydration properties and their response to co-solutes. In addition, alterations in protein dynamics (at a global protein level and in local regions with differences in solvent exposures) by stabilizing or destabilizing excipients were found to modulate the conformational stability of a monoclonal antibody. Furthermore, it was determined that potential interactions and factors modulating solution viscosity at high protein concentrations may also result in changes in their extinction coefficients. The work presented in this dissertation provides evidence that factors modulating protein dynamics and those governing intra- and intermolecular interactions can influence the conformational stability, aggregation and viscosity of proteins in solution

Comparison of the Structural Stability and Dynamic Properties of Recombinant Anthrax Protective Antigen and its 2- Fluorohistidine Labeled Analogue

Protective antigen (PA) is the primary protein antigenic component of both the currently used anthrax vaccine and related recombinant vaccines under development. An analogue of recombinant PA (2-FHis rPA) has been recently shown to block the key steps of pore formation in the process of inducing cytotoxicity in cells, and thus can potentially be used as an antitoxin or a vaccine. This rPA analogue was produced by fermentation to incorporate the unnatural amino acid 2-fluorohistidine (2-FHis). In this study, the effects of 2-FHis labeling on rPA antigen’s conformational stability and dynamic properties were investigated by various biophysical techniques. Temperature/pH stability profiles of rPA and 2-FHis rPA were analyzed by the empirical phase diagram (EPD) approach, and physical stability differences between them were identified. Results showed that rPA and 2-FHis rPA had similar stability at pH 7–8. With decreasing solution pH, however, 2-FHis rPA was found to be more stable. Dynamic sensitive measurements of the two proteins at pH 5 found that 2-FHis rPA was more dynamic and/or differentially hydrated under acidic pH conditions. The biophysical characterization and stability data provide information useful for the potential development of 2-FHis rPA as a more stable rPA vaccine candidate

Interventions for female drug-using offenders (Review)

Kumar KM,

Interventions for female drug-using offenders

Background This review represents one in a family of three reviews focusing on the effectiveness of interventions in reducing drug use and criminal activity for offenders. Objectives To assess the effectiveness of interventions for female drug‐using offenders in reducing criminal activity, or drug use, or both. Search methods We searched 12 electronic bibliographic databases up to February 2019. Selection criteria We included randomised controlled trials (RCTs). Data collection and analysis We used standard methodological procedures expected by Cochrane. Main results We included 13 trials with 2560 participants. Interventions were delivered in prison (7/13 studies, 53%) and community (6/13 studies, 47%) settings. The rating of bias was affected by the lack of clear reporting by authors, and we rated many items as 'unclear'. In two studies (190 participants) collaborative case management in comparison to treatment as usual did not reduce drug use (risk ratio (RR) 0.65, 95% confidence interval (CI) 0.20 to 2.12; 1 study, 77 participants; low‐certainty evidence), reincarceration at nine months (RR 0.71, 95% CI 0.32 to 1.57; 1 study, 77 participants; low‐certainty evidence), and number of subsequent arrests at 12 months (RR 1.11, 95% CI 0.83 to 1.49; 1 study, 113 participants; low‐certainty evidence). One study (36 participants) comparing buprenorphine to placebo showed no significant reduction in self‐reported drug use at end of treatment (RR 0.57, 95% CI 0.27 to 1.20) and three months (RR 0.58, 95% CI 0.25 to 1.35); very low‐certainty evidence. No adverse events were reported. One study (38 participants) comparing interpersonal psychotherapy to a psychoeducational intervention did not find reduction in drug use at three months (RR 0.67, 95% CI 0.30 to 1.50; low‐certainty evidence). One study (31 participants) comparing acceptance and commitment therapy (ACT) to a waiting list showed no significant reduction in self‐reported drug use using the Addiction Severity Index (mean difference (MD) ‐0.04, 95% CI ‐0.37 to 0.29) and abstinence from drug use at six months (RR 2.89, 95% CI 0.73 to 11.43); low‐certainty evidence. One study (314 participants) comparing cognitive behavioural skills to a therapeutic community programme and aftercare showed no significant reduction in self‐reported drug use (RR 0.86, 95% CI 0.58 to 1.27), re‐arrest for any type of crime (RR 0.73, 95% CI 0.52 to 1.03); criminal activity (RR 0.80, 95% CI 0.63 to 1.03), or drug‐related crime (RR 0.95, 95% CI 0.68 to 1.32). A significant reduction for arrested (not for parole) violations at six months follow‐up was significantly in favour of cognitive behavioural skills (RR 0.43, 95% CI 0.25 to 0.77; very low‐certainty evidence). A second study with 115 participants comparing cognitive behavioural skills to an alternative substance abuse treatment showed no significant reduction in reincarceration at 12 months (RR 0.70, 95% CI 0.43 to 1.12; low certainty‐evidence. One study (44 participants) comparing cognitive behavioural skills and standard therapy versus treatment as usual showed no significant reduction in Addiction Severity Index (ASI) drug score at three months (MD 0.02, 95% CI ‐0.05 to 0.09) and six months (MD ‐0.02, 95% CI ‐0.09 to 0.05), and incarceration at three months (RR 0.46, 95% CI 0.04 to 4.68) and six months (RR 0.51, 95% CI 0.20 to 1.27); very low‐certainty evidence. One study (171 participants) comparing a single computerised intervention versus case management showed no significant reduction in the number of days not using drugs at three months (MD ‐0.89, 95% CI ‐4.83 to 3.05; low certainty‐evidence). One study (116 participants) comparing dialectic behavioural therapy and case management (DBT‐CM) versus a health promotion intervention showed no significant reduction at six months follow‐up in positive drug testing (RR 0.67, 95% CI 0.43 to 1.03), number of people not using marijuana (RR 1.23, 95% CI 0.95 to 1.59), crack (RR 1.00, 95% CI 0.87 to 1.14), cocaine (RR 1.02, 95% CI 0.93 to 1.12), heroin (RR 1.05, 95% CI 0.98 to 1.13), methamphetamine (RR 1.02, 95% CI 0.87 to 1.20), and self‐reported drug use for any drug (RR 1.20, 95% CI 0.92 to 1.56); very low‐certainty evidence. One study (211 participants) comparing a therapeutic community programme versus work release showed no significant reduction in marijuana use at six months (RR 1.03, 95% CI 0.19 to 5.65), nor 18 months (RR 1.00, 95% CI 0.07 to 14.45), heroin use at six months (RR 1.59, 95% CI 0.49 to 5.14), nor 18 months (RR 1.92, 95% CI 0.24 to 15.37), crack use at six months (RR 2.07, 95% CI 0.41 to 10.41), nor 18 months (RR 1.64, 95% CI 0.19 to 14.06), cocaine use at six months (RR 1.09, 95% CI 0.79 to 1.50), nor 18 months (RR 0.93, 95% CI 0.64 to 1.35). It also showed no significant reduction in incarceration for drug offences at 18 months (RR 1.45, 95% CI 0.87 to 2.42); with overall very low‐ to low‐certainty evidence. One study (511 participants) comparing intensive discharge planning and case management versus prison only showed no significant reduction in use of marijuana (RR 0.79, 95% CI 0.53 to 1.16), hard drugs (RR 1.12, 95% CI 0.88 to 1.43), crack cocaine (RR 1.08, 95% CI 0.75 to 1.54), nor positive hair testing for marijuana (RR 0.75, 95% CI 0.55 to 1.03); it found a significant reduction in arrests (RR 0.19, 95% CI 0.04 to 0.87), but no significant reduction in drug charges (RR 1.07, 95% CI 0.75 to 1.53) nor incarceration (RR 1.09, 95% CI 0.86 to 1.39); moderate‐certainty evidence. One narrative study summary (211 participants) comparing buprenorphine pre‐ and post‐release from prison showed no significant reduction in drug use at 12 months post‐release; low certainty‐evidence. No adverse effects were reported. Authors' conclusions The studies showed a high degree of heterogeneity for types of comparisons, outcome measures and small samples. Descriptions of treatment modalities are required. On one outcome of arrest (no parole violations), we identified a significant reduction when cognitive behavioural therapy (CBT) was compared to a therapeutic community programme. But for all other outcomes, none of the interventions were effective. Larger trials are required to increase the precision of confidence about the certainty of evidence

Pharmacological interventions for drug-using offenders : an update to a systematic review and meta-analysis

This updated systematic review assesses the effects of pharmacological interventions for drug-using offenders. Methods Systematic review protocols and conventions of the Cochrane Collaboration were followed to identify eligible studies. Studies were pooled in a meta-analysis to assess the impact of pharmacological interventions on drug use and criminal activity. An economic appraisal was conducted. Results The search strategies identified 22 studies containing 4372 participants. Meta-analyses revealed a small statistically significant mean difference favouring pharmacological interventions relative to psychological interventions in reducing drug use and criminal activity. When comparing the drugs to one another there were no significant differences between those included (methadone versus buprenorphine, naltrexone and cyclazocine). Conclusion Overall, the findings of this review suggest that methadone and naltrexone may have some impact on reducing drug use and reincarceration. Individual pharmacological drugs had differing (generally non-significant) effects. One study identified serious adverse events. Three studies reported cost and consequences information sufficient to conduct a full economic analysis but this was not comprehensive enough to be able to make judgements across all treatment options. Full economic analyses should be encouraged. The study findings were limited mainly to male adult offenders

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Evaluation of Antibacterial Efficacy of Vitex negundo Linn. extract as Root Canal Irrigant against Enterococcus faecalis and its penetration into Root Dentin: An in-vitro study

Introduction: The aim of this study was to evaluate the antibacterial efficacy of Vitex negundo Linn. extract as root canal irrigant against Enterococcus faecalis and its penetration into root dentin. Methods and Materials: Forty single rooted premolars were randomly divided into 4 groups: 3% Sodium hypochlorite (NaOCl), 2% Chlorhexidine (CHX) , 100mg/ml Vitex negundo Linn. and saline as control all mixed with Rhodamine B dye. Test samples were analysed for bacterial count before and after irrigation using absorbent paper points and the colony forming units were recorded and measured. Sectioning of the samples was performed at three levels 3mm,6mm,9mm from apex and then these samples were analysed using confocal laser scanning microscopy for penetration depth of the irrigant within the dentinal tubules. Paired t-test and ANOVA test were used to perform statistical analysis with level of significance set at 0.05 Results: The mean CFU/ml count of Enterococcus facealis reduced significantly in all the groups post irrigation. All the irrigants showed maximum penetration depth at coronal third level compared to middle and apical third level respectively. The penetration depth of NaOCl group was better when compared to CHX group and Vitex negundo Linn. group but the difference was statistically not significant. Conclusion: Although 3% NaOCl was the most effective irrigant, all agents exerted acceptable antimicrobial activity against Enterococcus faecalis and penetration depth within tubules of dentin