Stability enhancing N-Terminal PEGylation of oxytocin exploiting different polymer architectures and conjugation approaches

Oxytocin, a cyclic nine amino acid neurohypophyseal hormone therapeutic, is effectively used in the control of postpartum hemorrhaging (PPH) and is on the WHO List of Essential Medicines. However, oxytocin has limited shelf life stability in aqueous solutions, particularly at temperatures in excess of 25 °C and injectable aqueous oxytocin formulations require refrigeration (<8 °C). This is particularly problematic in the hot climates often found in many developing countries where daytime temperatures can exceed 40 °C and where reliable cold-chain storage is not always achievable. The purpose of this study was to develop N-terminal amine targeted PEGylation strategies utilizing both linear PEG and polyPEG “comb” polymers as an effective method for stabilizing solution formulations of this peptide for prolonged storage in the absence of efficient cold-chain storage. The conjugation chemistries investigated herein include irreversible amine targeted conjugation methods utilizing NHS ester and aldehyde reductive amination chemistry. Additionally, one reversible conjugation method using a Schiff base approach was explored to allow for the release of the native peptide, thus, ensuring that biological activity remains unaffected. The reversibility of this approach was investigated for the different polymer architectures, alongside a nonpolymer oxytocin analogue to monitor how pH can tune native peptide release. Elevated temperature degradation studies of the polymer conjugates were evaluated to assess the stability of the PEGylated analogues in comparison to the native peptide in aqueous formulations to mimic storage conditions in developing nations and regions where storage under appropriate conditions is challenging

What does it take to redesign a degree? A case study

BACKGROUND The Bachelor of Pharmaceutical Science course at Monash University is presently in the second year of a three-year redevelopment. Initiated to address the recommendations of a 2015 course review, the redevelopment has grown into a full course redesign with multiple aims including the deep embedding of skill development, authentic learning and lifelong learning skills into the curriculum; strengthening integrated and interdisciplinary learning; and improving the student experience. AIMS This case study maps the key stages in the full redesign of an applied science degree with the aim of identifying internal and external factors that have shaped its conception and implementation. METHODS Multiple data sources (institutional data, planning documents, personal notes and recollections) are used to describe the redesign process over a four-year period. The elements of the process are analysed with respect to literature examples and models of educational change and curriculum design. RESULTS AND CONCLUSIONS The present case has employed a mixed approach, addressing both institutional and individual practices, that is mostly consistent with an integration strategy of curriculum change

Inhibiting heat-shock protein 90 reverses sensory hypoalgesia in diabetic mice

Increasing the expression of Hsp70 (heat-shock protein 70) can inhibit sensory neuron degeneration after axotomy. Since the onset of DPN (diabetic peripheral neuropathy) is associated with the gradual decline of sensory neuron function, we evaluated whether increasing Hsp70 was sufficient to improve several indices of neuronal function. Hsp90 is the master regulator of the heat-shock response and its inhibition can up-regulate Hsp70. KU-32 (N-{7-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy]-8-methyl-2-oxo-2H-chromen-3-yl}acetamide) was developed as a novel, novobiocin-based, C-terminal inhibitor of Hsp90 whose ability to increase Hsp70 expression is linked to the presence of an acetamide substitution of the prenylated benzamide moiety of novobiocin. KU-32 protected against glucose-induced death of embryonic DRG (dorsal root ganglia) neurons cultured for 3 days in vitro. Similarly, KU-32 significantly decreased neuregulin 1-induced degeneration of myelinated Schwann cell DRG neuron co-cultures prepared from WT (wild-type) mice. This protection was lost if the co-cultures were prepared from Hsp70.1 and Hsp70.3 KO (knockout) mice. KU-32 is readily bioavailable and was administered once a week for 6 weeks at a dose of 20 mg/kg to WT and Hsp70 KO mice that had been rendered diabetic with streptozotocin for 12 weeks. After 12 weeks of diabetes, both WT and Hsp70 KO mice developed deficits in NCV (nerve conduction velocity) and a sensory hypoalgesia. Although KU-32 did not improve glucose levels, HbA1c (glycated haemoglobin) or insulin levels, it reversed the NCV and sensory deficits in WT but not Hsp70 KO mice. These studies provide the first evidence that targeting molecular chaperones reverses the sensory hypoalgesia associated with DPN.This work was supported by grants from the Juvenile Diabetes Research Foundation and the National Institutes of Health [NS054847 and DK073594] (to R.T.D.) and [CA120458 and CA109265] (to B.S.J.B.)

Global testing of a consensus solubility assessment to enhance robustness of the WHO biopharmaceutical classification system

The WHO Biopharmaceutical Classification System (BCS) is a practical tool to identify active pharmaceutical ingredients (APIs) that scientifically qualify for a waiver of in vivo bioequivalence studies. The focus of this study was to engage a global network of laboratories to experimentally quantify the pH-dependent solubility of the highest therapeutic dose of 16 APIs using a harmonized protocol. Intra-laboratory variability was ≤5 %, and no apparent association of inter-laboratory variability with API solubility was discovered. Final classification “low solubility” vs “high solubility” was consistent among laboratories. In comparison to the literature-based provisional 2006 WHO BCS classification, three compounds were re-classified from “high” to “low-solubility”. To estimate the consequences of these experimental solubility results on BCS classification, dose-adjusted in silico predictions of the fraction absorbed in humans were performed using GastroPlus®. Further expansion of these experimental efforts to qualified APIs from the WHO Essential Medicines List is anticipated to empower regulatory authorities across the globe to issue scientifically-supported guidance regarding the necessity of performing in vivo bioequivalence studies. Ultimately, this will improve access to affordable generic products, which is a critical prerequisite to reach Universal Health Coverage

Knowledge, perception and practice towards oxytocin stability and quality: A qualitative study of stakeholders in three resource-limited countries

Background: Oxytocin is the gold standard drug for the prevention of postpartum haemorrhage, but limitations in cold chain systems in resource-constrained settings can severely compromise the quality of oxytocin product available in these environments. This study investigated the perspectives and practices of stakeholders in low and lower-middle income countries towards oxytocin, its storage requirements and associated barriers, and the quality of product available. Methods: Qualitative inquiries were undertaken in Ethiopia, India and Myanmar, where data was collected through Focus Group Discussions (FGDs) and In-Depth Interviews (IDIs). A total of 12 FGDs and 106 IDIs were conducted with 158 healthcare providers (pharmacists, midwives, nurses, doctors and obstetricians) and 40 key informants (supply chain experts, program managers and policy-makers). Direct observations of oxytocin storage practices and cold chain resources were conducted at 51 healthcare facilities. Verbatim transcripts of FGDs and IDIs were translated to English and analysed according to a thematic content analysis framework. Findings: Stakeholder awareness of oxytocin heat sensitivity and the requirement for cold storage of the drug was widespread in Ethiopia but more limited in Myanmar and India. A consistent finding across all study regions was the significant barriers to maintaining a consistent cold chain, with the lack of refrigeration facilities and unreliability of electricity cited as major challenges. Perceptions of compromised oxytocin quality were expressed by some stakeholders in each country. Conclusion: Knowledge of the heat sensitivity of oxytocin and the potential impacts of inconsistent cold storage on product quality is not widespread amongst healthcare providers, policy makers and supply chain experts in Myanmar, Ethiopia and India. Targeted training and advocacy messages are warranted to emphasise the importance of cold storage to maintain oxytocin quality

Inhibiting heat-shock protein 90 reverses sensory hypoalgesia in diabetic mice

Increasing the expression of Hsp70 (heat-shock protein 70) can inhibit sensory neuron degeneration after axotomy. Since the onset of DPN (diabetic peripheral neuropathy) is associated with the gradual decline of sensory neuron function, we evaluated whether increasing Hsp70 was sufficient to improve several indices of neuronal function. Hsp90 is the master regulator of the heat-shock response and its inhibition can up-regulate Hsp70. KU-32 (N-{7-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy]-8-methyl-2-oxo-2H-chromen-3-yl}acetamide) was developed as a novel, novobiocin-based, C-terminal inhibitor of Hsp90 whose ability to increase Hsp70 expression is linked to the presence of an acetamide substitution of the prenylated benzamide moiety of novobiocin. KU-32 protected against glucose-induced death of embryonic DRG (dorsal root ganglia) neurons cultured for 3 days in vitro. Similarly, KU-32 significantly decreased neuregulin 1-induced degeneration of myelinated Schwann cell DRG neuron co-cultures prepared from WT (wild-type) mice. This protection was lost if the co-cultures were prepared from Hsp70.1 and Hsp70.3 KO (knockout) mice. KU-32 is readily bioavailable and was administered once a week for 6 weeks at a dose of 20 mg/kg to WT and Hsp70 KO mice that had been rendered diabetic with streptozotocin for 12 weeks. After 12 weeks of diabetes, both WT and Hsp70 KO mice developed deficits in NCV (nerve conduction velocity) and a sensory hypoalgesia. Although KU-32 did not improve glucose levels, HbA1c (glycated haemoglobin) or insulin levels, it reversed the NCV and sensory deficits in WT but not Hsp70 KO mice. These studies provide the first evidence that targeting molecular chaperones reverses the sensory hypoalgesia associated with DPN

Polygenic risk for coronary artery disease is associated with cognitive ability in older adults

Background: Coronary artery disease (CAD) is associated with cognitive decrements and risk of later dementia, but it is not known if shared genetic factors underlie this association. We tested whether polygenic risk for CAD was associated with cognitive ability in community-dwelling cohorts of middle-aged and older adults. Methods: Individuals from Generation Scotland: Scottish Family Health Study (GS:SFHS, N = 9865) and from the Lothian Birth Cohorts of 1921 (LBC1921, N  = 517) and 1936 (LBC1936, N  = 1005) provided cognitive data and genome-wide genotype data. Polygenic risk profile scores for CAD were calculated for all of the cohorts using the largest available genome-wide association studies (GWAS) data set, the CARDIoGRAM consortium (22 233 cases and 64 762 controls). Polygenic risk profile scores for CAD were then tested for their association with cognitive abilities in the presence and absence of manifest cardiovascular disease. Results: A meta-analysis of all three cohorts showed a negative association between CAD polygenic risk and fluid cognitive ability (β = −0.022, P  = 0.016), verbal intelligence (β = −0.024, P  = 0.011) and memory (β = −0.021, P  = 0.028). Conclusions: Increased polygenic risk for CAD is associated with lower cognitive ability in older adults. Common genetic variants may underlie some of the association between age-related cognitive decrements and the risk for CAD

In situ conjugation of dithiophenol maleimide polymers and oxytocin for stable and reversible polymer–peptide conjugates

The in situ one-pot synthesis of peptide–polymer bioconjugates is reported. Conjugation occurs efficiently without the need for purification of dithiophenol maleimide functionalized polymer as a disulfide bridging agent for the therapeutic oxytocin. Conjugation of polymers was demonstrated to be reversible and to significantly improve the solution stability of oxytocin

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Association analysis in over 329,000 individuals identifies 116 independent variants influencing neuroticism

Neuroticism is a relatively stable personality trait characterized by negative emotionality (for example, worry and guilt)1; heritability estimated from twin studies ranges from 30 to 50%2, and SNP-based heritability ranges from 6 to 15%3,4,5,6. Increased neuroticism is associated with poorer mental and physical health7,8, translating to high economic burden9. Genome-wide association studies (GWAS) of neuroticism have identified up to 11 associated genetic loci3,4. Here we report 116 significant independent loci from a GWAS of neuroticism in 329,821 UK Biobank participants; 15 of these loci replicated at P &lt; 0.00045 in an unrelated cohort (N = 122,867). Genetic signals were enriched in neuronal genesis and differentiation pathways, and substantial genetic correlations were found between neuroticism and depressive symptoms (rg = 0.82, standard error (s.e.) = 0.03), major depressive disorder (MDD; rg = 0.69, s.e. = 0.07) and subjective well-being (rg = –0.68, s.e. = 0.03) alongside other mental health traits. These discoveries significantly advance understanding of neuroticism and its association with MDD