Diversity of US participants in AstraZeneca-sponsored clinical trials.

BACKGROUND: To develop medicines that are safe and efficacious to all patients, clinical trials must enroll appropriate target populations, but imbalances related to race, ethnicity and sex have been reported. A comprehensive analysis and improvement in understanding representativeness of patient enrollment in industry-sponsored trials are key public health needs. METHODS: We assessed race/ethnicity and sex representation in AstraZeneca (AZ)-sponsored clinical trials in the United States (US) from 2010 to 2022, compared with the 2019 US Census. RESULTS: In total, 246 trials representing 95,372 patients with complete race/ethnicity and sex records were analyzed. The proportions of different race/ethnicity subgroups in AZ-sponsored clinical trials and the US Census were similar (White: 69.5% vs 60.1%, Black or African American: 13.3% vs 12.5%, Asian: 1.8% vs 5.8%, Hispanic: 14.4% vs 18.5%). We also observed parity in the proportions of males and females between AZ clinical trials and US Census (males: 52.4% vs 49.2%, females: 47.6% vs 50.8%). Comparisons of four distinct therapy areas within AZ (Respiratory and Immunology [R&I]; Cardiovascular, Renal, and Metabolism [CVRM]; Solid Tumors; and Hematological Malignancies), including by trial phases, revealed greater variability, with proportions observed above and below US Census levels. CONCLUSION: This analysis provides the first detailed insights into the representativeness of AZ trials. Overall, the proportions of different race/ethnicity and sex subgroups in AZ-sponsored clinical trials were broadly aligned with the US Census. We outline some of AZ's planned health equity initiatives that are intended to continue to improve equitable patient enrollment.

Immunogenicity and safety of AZD2816, a beta (B.1.351) variant COVID-19 vaccine, and AZD1222 (ChAdOx1 nCoV-19) as third-dose boosters for previously vaccinated adults: a multicentre, randomised, partly double-blinded, phase 2/3 non-inferiority immunobridging study in the UK and Poland.

BACKGROUND: This study aimed to evaluate AZD2816, a variant-updated COVID-19 vaccine expressing the full-length SARS-CoV-2 beta (B.1.351) variant spike protein that is otherwise similar to AZD1222 (ChAdOx1 nCoV-19), and AZD1222 as third-dose boosters. METHODS: This phase 2/3, partly double-blinded, randomised, active-controlled study was done at 19 sites in the UK and four in Poland. Adult participants who had received a two-dose AZD1222 or mRNA vaccine primary series were randomly assigned by means of an Interactive Response Technology-Randomisation and Trial Supply Management system (1:1 within each primary-series cohort, stratified by age, sex, and comorbidities) to receive AZD1222 or AZD2816 (intramuscular injection; 5 × 1010 viral particles). Participants, investigators, and all sponsor staff members involved in study conduct were masked to randomisation. AZD1222 and AZD2816 doses were prepared by unmasked study staff members. The primary objectives were to evaluate safety and humoral immunogenicity (non-inferiority of day-29 pseudovirus neutralising antibody geometric mean titre [GMT] against ancestral SARS-CoV-2: AZD1222 booster vs AZD1222 primary series [historical controls]; margin 0·67; SARS-CoV-2-seronegative participants). This study is registered with ClinicalTrials.gov, NCT04973449, and is completed. FINDINGS: Between June 27 and Sept 30, 2021, 1394 participants of the 1741 screened were randomly assigned to AZD1222 or AZD2816 following an AZD1222 (n=373, n=377) or mRNA vaccine (n=322, n=322) primary series. In SARS-CoV-2-seronegative participants receiving AZD1222 or AZD2816, 78% and 80% (AZD1222 primary series) and 90% and 93%, respectively (mRNA vaccine primary series) reported solicited adverse events to the end of day 8; 2%, 2%, 1%, and 1% had serious adverse events and 12%, 12%, 10%, and 11% had adverse events of special interest, respectively, to the end of day 180. The primary immunogenicity non-inferiority endpoint was met: day-29 neutralising antibody GMT ratios (ancestral SARS-CoV-2) were 1·02 (95% CI 0·90-1·14) and 3·47 (3·09-3·89) with AZD1222 booster versus historical controls (AZD1222 and mRNA vaccine primary series, respectively). Responses against beta were greater with AZD2816 versus AZD1222 (GMT ratios, AZD1222, mRNA vaccine primary series 1·84 [1·63-2·08], 2·22 [1·99-2·47]). INTERPRETATION: Both boosters were well tolerated, with immunogenicity against ancestral SARS-CoV-2 similar to AZD1222 primary-series vaccination. AZD2816 gave greater immune responses against beta versus AZD1222. FUNDING: AstraZeneca.

Rare variant associations with plasma protein levels in the UK Biobank.

Integrating human genomics and proteomics can help elucidate disease mechanisms, identify clinical biomarkers and discover drug targets1-4. Because previous proteogenomic studies have focused on common variation via genome-wide association studies, the contribution of rare variants to the plasma proteome remains largely unknown. Here we identify associations between rare protein-coding variants and 2,923 plasma protein abundances measured in 49,736 UK Biobank individuals. Our variant-level exome-wide association study identified 5,433 rare genotype-protein associations, of which 81% were undetected in a previous genome-wide association study of the same cohort5. We then looked at aggregate signals using gene-level collapsing analysis, which revealed 1,962 gene-protein associations. Of the 691 gene-level signals from protein-truncating variants, 99.4% were associated with decreased protein levels. STAB1 and STAB2, encoding scavenger receptors involved in plasma protein clearance, emerged as pleiotropic loci, with 77 and 41 protein associations, respectively. We demonstrate the utility of our publicly accessible resource through several applications. These include detailing an allelic series in NLRC4, identifying potential biomarkers for a fatty liver disease-associated variant in HSD17B13 and bolstering phenome-wide association studies by integrating protein quantitative trait loci with protein-truncating variants in collapsing analyses. Finally, we uncover distinct proteomic consequences of clonal haematopoiesis (CH), including an association between TET2-CH and increased FLT3 levels. Our results highlight a considerable role for rare variation in plasma protein abundance and the value of proteogenomics in therapeutic discovery.

Direct intramyocardial injection of VEGF mRNA in patients undergoing coronary artery bypass grafting.

Vascular endothelial growth factor A (VEGF-A) has therapeutic cardiovascular effects, but delivery challenges have impeded clinical development. We report the first clinical study of naked mRNA encoding VEGF-A (AZD8601) injected into the human heart. EPICCURE (ClinicalTrials.gov: NCT03370887) was a randomized, double-blind study of AZD8601 in patients with left ventricular ejection fraction (LVEF) 30%-50% who were undergoing elective coronary artery bypass surgery. Thirty epicardial injections of AZD8601 (total 3 mg) or placebo in citrate-buffered saline were targeted to ischemic but viable myocardial regions mapped using quantitative [15O]-water positron emission tomography. Seven patients received AZD8601 and four received placebo and were followed for 6 months. There were no deaths or treatment-related serious adverse events and no AZD8601-associated infections, immune reactions, or arrhythmias. Exploratory outcomes indicated potential improvement in LVEF, Kansas City Cardiomyopathy Questionnaire scores, and N-terminal pro-B-type natriuretic peptide levels, but the study is limited in size, and significant efficacy conclusions are not possible from the dataset. Naked mRNA without lipid encapsulation may provide a safe delivery platform for introducing genetic material to cardiac muscle, but further studies are needed to confirm efficacy and safety in a larger patient pool.

AZD7442 (Tixagevimab/Cilgavimab) for Post-Exposure Prophylaxis of Symptomatic Coronavirus Disease 2019.

BACKGROUND: This phase 3 trial assessed AZD7442 (tixagevimab/cilgavimab) for post-exposure prophylaxis against symptomatic coronavirus disease 2019 (COVID-19). METHODS: Adults without prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or COVID-19 vaccination were enrolled within 8 days of exposure to a SARS-CoV-2-infected individual and randomized 2:1 to a single 300-mg AZD7442 dose (one 1.5-mL intramuscular injection each of tixagevimab and cilgavimab) or placebo. Primary end points were safety and first post-dose SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR)-positive symptomatic COVID-19 event before day 183. RESULTS: A total of 1121 participants were randomized and dosed (AZD7442, n = 749; placebo, n = 372). Median (range) follow-up was 49 (5-115) and 48 (20-113) days for AZD7442 and placebo, respectively. Adverse events occurred in 162 of 749 (21.6%) and 111 of 372 (29.8%) participants with AZD7442 and placebo, respectively, mostly mild/moderate. RT-PCR-positive symptomatic COVID-19 occurred in 23 of 749 (3.1%) and 17 of 372 (4.6%) AZD7442- and placebo-treated participants, respectively (relative risk reduction, 33.3%; 95% confidence interval [CI], -25.9 to 64.7; P = .21). In predefined subgroup analyses of 1073 (96%) participants who were SARS-CoV-2 RT-PCR-negative (n = 974, 87%) or missing an RT-PCR result (n = 99, 9%) at baseline, AZD7442 reduced RT-PCR-positive symptomatic COVID-19 by 73.2% (95% CI, 27.1 to 90.1) vs placebo. CONCLUSIONS: This study did not meet the primary efficacy end point of post-exposure prevention of symptomatic COVID-19. However, analysis of participants who were SARS-CoV-2 RT-PCR-negative or missing an RT-PCR result at baseline support a role for AZD7442 in preventing symptomatic COVID-19. Clinical Trials Registration. NCT04625972.

Human genetics uncovers MAP3K15 as an obesity-independent therapeutic target for diabetes.

We performed collapsing analyses on 454,796 UK Biobank (UKB) exomes to detect gene-level associations with diabetes. Recessive carriers of nonsynonymous variants in MAP3K15 were 30% less likely to develop diabetes (P = 5.7 × 10-10) and had lower glycosylated hemoglobin (ß = -0.14 SD units, P = 1.1 × 10-24). These associations were independent of body mass index, suggesting protection against insulin resistance even in the setting of obesity. We replicated these findings in 96,811 Admixed Americans in the Mexico City Prospective Study (P < 0.05)Moreover, the protective effect of MAP3K15 variants was stronger in individuals who did not carry the Latino-enriched SLC16A11 risk haplotype (P = 6.0 × 10-4). Separately, we identified a Finnish-enriched MAP3K15 protein-truncating variant associated with decreased odds of both type 1 and type 2 diabetes (P < 0.05) in FinnGen. No adverse phenotypes were associated with protein-truncating MAP3K15 variants in the UKB, supporting this gene as a therapeutic target for diabetes.

Efficacy and safety of intramuscular administration of tixagevimab-cilgavimab for early outpatient treatment of COVID-19 (TACKLE): a phase 3, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Early intramuscular administration of SARS-CoV-2-neutralising monoclonal antibody combination, tixagevimab-cilgavimab, to non-hospitalised adults with mild to moderate COVID-19 has potential to prevent disease progression. We aimed to evaluate the safety and efficacy of tixagevimab-cilgavimab in preventing progression to severe COVID-19 or death. METHODS: TACKLE is an ongoing, phase 3, randomised, double-blind, placebo-controlled study conducted at 95 sites in the USA, Latin America, Europe, and Japan. Eligible participants were non-hospitalised adults aged 18 years or older with a laboratory-confirmed SARS-CoV-2 infection (determined by RT-PCR or an antigen test) from any respiratory tract specimen collected 3 days or less before enrolment and who had not received a COVID-19 vaccination. A WHO Clinical Progression Scale score from more than 1 to less than 4 was required for inclusion and participants had to receive the study drug 7 days or less from self-reported onset of mild to moderate COVID-19 symptoms or measured fever. Participants were randomly assigned (1:1) to receive either a single tixagevimab-cilgavimab 600 mg dose (two consecutive 3 mL intramuscular injections, one each of 300 mg tixagevimab and 300 mg cilgavimab) or placebo. Randomisation was stratified (using central blocked randomisation with randomly varying block sizes) by time from symptom onset, and high-risk versus low-risk of progression to severe COVID-19. Participants, investigators, and sponsor staff involved in the treatment or clinical evaluation and monitoring of the participants were masked to treatment-group assignments. The primary endpoints were severe COVID-19 or death from any cause through to day 29, and safety. This study is registered with ClinicalTrials.gov, NCT04723394. FINDINGS: Between Jan 28, 2021, and July 22, 2021, 1014 participants were enrolled, of whom 910 were randomly assigned to a treatment group (456 to receive tixagevimab-cilgavimab and 454 to receive placebo). The mean age of participants was 46·1 years (SD 15·2). Severe COVID-19 or death occurred in 18 (4%) of 407 participants in the tixagevimab-cilgavimab group versus 37 (9%) of 415 participants in the placebo group (relative risk reduction 50·5% [95% CI 14·6-71·3]; p=0·0096). The absolute risk reduction was 4·5% (95% CI 1·1-8·0; p<0·0001). Adverse events occurred in 132 (29%) of 452 participants in the tixagevimab-cilgavimab group and 163 (36%) of 451 participants in the placebo group, and were mostly of mild or moderate severity. There were three COVID-19-reported deaths in the tixagevimab-cilgavimab group and six in the placebo group. INTERPRETATION: A single intramuscular tixagevimab-cilgavimab dose provided statistically and clinically significant protection against progression to severe COVID-19 or death versus placebo in unvaccinated individuals and safety was favourable. Treating mild to moderate COVID-19 earlier in the disease course with tixagevimab-cilgavimab might lead to more favourable outcomes. FUNDING: AstraZeneca.

Intramuscular AZD7442 (Tixagevimab-Cilgavimab) for Prevention of Covid-19.

BACKGROUND: The monoclonal-antibody combination AZD7442 is composed of tixagevimab and cilgavimab, two neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that have an extended half-life and have been shown to have prophylactic and therapeutic effects in animal models. Pharmacokinetic data in humans indicate that AZD7442 has an extended half-life of approximately 90 days. METHODS: In an ongoing phase 3 trial, we enrolled adults (≥18 years of age) who had an increased risk of an inadequate response to vaccination against coronavirus disease 2019 (Covid-19), an increased risk of exposure to SARS-CoV-2, or both. Participants were randomly assigned in a 2:1 ratio to receive a single dose (two consecutive intramuscular injections, one containing tixagevimab and the other containing cilgavimab) of either 300 mg of AZD7442 or saline placebo, and they were followed for up to 183 days in the primary analysis. The primary safety end point was the incidence of adverse events after a single dose of AZD7442. The primary efficacy end point was symptomatic Covid-19 (SARS-CoV-2 infection confirmed by means of reverse-transcriptase-polymerase-chain-reaction assay) occurring after administration of AZD7442 or placebo and on or before day 183. RESULTS: A total of 5197 participants underwent randomization and received one dose of AZD7442 or placebo (3460 in the AZD7442 group and 1737 in the placebo group). The primary analysis was conducted after 30% of the participants had become aware of their randomized assignment. In total, 1221 of 3461 participants (35.3%) in the AZD7442 group and 593 of 1736 participants (34.2%) in the placebo group reported having at least one adverse event, most of which were mild or moderate in severity. Symptomatic Covid-19 occurred in 8 of 3441 participants (0.2%) in the AZD7442 group and in 17 of 1731 participants (1.0%) in the placebo group (relative risk reduction, 76.7%; 95% confidence interval [CI], 46.0 to 90.0; P<0.001); extended follow-up at a median of 6 months showed a relative risk reduction of 82.8% (95% CI, 65.8 to 91.4). Five cases of severe or critical Covid-19 and two Covid-19-related deaths occurred, all in the placebo group. CONCLUSIONS: A single dose of AZD7442 had efficacy for the prevention of Covid-19, without evident safety concerns. (Funded by AstraZeneca and the U.S. government; PROVENT ClinicalTrials.gov number, NCT04625725.).

Improving the efficiency and effectiveness of an industrial SARS-CoV-2 diagnostic facility.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.

The SARS-CoV-2 monoclonal antibody combination, AZD7442, is protective in nonhuman primates and has an extended half-life in humans.

Despite the success of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there remains a need for more prevention and treatment options for individuals remaining at risk of coronavirus disease 2019 (COVID-19). Monoclonal antibodies (mAbs) against the viral spike protein have potential to both prevent and treat COVID-19 and reduce the risk of severe disease and death. Here, we describe AZD7442, a combination of two mAbs, AZD8895 (tixagevimab) and AZD1061 (cilgavimab), that simultaneously bind to distinct, nonoverlapping epitopes on the spike protein receptor binding domain to neutralize SARS-CoV-2. Initially isolated from individuals with prior SARS-CoV-2 infection, the two mAbs were designed to extend their half-lives and reduce effector functions. The AZD7442 mAbs individually prevent the spike protein from binding to angiotensin-converting enzyme 2 receptor, blocking virus cell entry, and neutralize all tested SARS-CoV-2 variants of concern. In a nonhuman primate model of SARS-CoV-2 infection, prophylactic AZD7442 administration prevented infection, whereas therapeutic administration accelerated virus clearance from the lung. In an ongoing phase 1 study in healthy participants (NCT04507256), a 300-mg intramuscular injection of AZD7442 provided SARS-CoV-2 serum geometric mean neutralizing titers greater than 10-fold above those of convalescent serum for at least 3 months, which remained threefold above those of convalescent serum at 9 months after AZD7442 administration. About 1 to 2% of serum AZD7442 was detected in nasal mucosa, a site of SARS-CoV-2 infection. Extrapolation of the time course of serum AZD7442 concentration suggests AZD7442 may provide up to 12 months of protection and benefit individuals at high-risk of COVID-19.

Phase 3 Safety and Efficacy of AZD1222 (ChAdOx1 nCoV-19) Covid-19 Vaccine.

BACKGROUND: The safety and efficacy of the AZD1222 (ChAdOx1 nCoV-19) vaccine in a large, diverse population at increased risk for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the United States, Chile, and Peru has not been known. METHODS: In this ongoing, double-blind, randomized, placebo-controlled, phase 3 clinical trial, we investigated the safety, vaccine efficacy, and immunogenicity of two doses of AZD1222 as compared with placebo in preventing the onset of symptomatic and severe coronavirus disease 2019 (Covid-19) 15 days or more after the second dose in adults, including older adults, in the United States, Chile, and Peru. RESULTS: A total of 32,451 participants underwent randomization, in a 2:1 ratio, to receive AZD1222 (21,635 participants) or placebo (10,816 participants). AZD1222 was safe, with low incidences of serious and medically attended adverse events and adverse events of special interest; the incidences were similar to those observed in the placebo group. Solicited local and systemic reactions were generally mild or moderate in both groups. Overall estimated vaccine efficacy was 74.0% (95% confidence interval [CI], 65.3 to 80.5; P<0.001) and estimated vaccine efficacy was 83.5% (95% CI, 54.2 to 94.1) in participants 65 years of age or older. High vaccine efficacy was consistent across a range of demographic subgroups. In the fully vaccinated analysis subgroup, no severe or critical symptomatic Covid-19 cases were observed among the 17,662 participants in the AZD1222 group; 8 cases were noted among the 8550 participants in the placebo group (<0.1%). The estimated vaccine efficacy for preventing SARS-CoV-2 infection (nucleocapsid antibody seroconversion) was 64.3% (95% CI, 56.1 to 71.0; P<0.001). SARS-CoV-2 spike protein binding and neutralizing antibodies increased after the first dose and increased further when measured 28 days after the second dose. CONCLUSIONS: AZD1222 was safe and efficacious in preventing symptomatic and severe Covid-19 across diverse populations that included older adults. (Funded by AstraZeneca and others; ClinicalTrials.gov number, NCT04516746.).

Direct Interaction of PP2A Phosphatase with GABAB Receptors Alters Functional Signaling.

Addictive drugs usurp the brain's intrinsic mechanism for reward, leading to compulsive and destructive behaviors. In the ventral tegmental area (VTA), the center of the brain's reward circuit, GABAergic neurons control the excitability of dopamine (DA) projection neurons and are the site of initial psychostimulant-dependent changes in signaling. Previous work established that cocaine/methamphetamine exposure increases protein phosphatase 2A (PP2A) activity, which dephosphorylates the GABABR2 subunit, promotes internalization of the GABAB receptor (GABABR) and leads to smaller GABABR-activated G-protein-gated inwardly rectifying potassium (GIRK) currents in VTA GABA neurons. How the actions of PP2A become selective for a particular signaling pathway is poorly understood. Here, we demonstrate that PP2A can associate directly with a short peptide sequence in the C terminal domain of the GABABR1 subunit, and that GABABRs and PP2A are in close proximity in rodent neurons (mouse/rat; mixed sexes). We show that this PP2A-GABABR interaction can be regulated by intracellular Ca2+ Finally, a peptide that potentially reduces recruitment of PP2A to GABABRs and thereby limits receptor dephosphorylation increases the magnitude of baclofen-induced GIRK currents. Thus, limiting PP2A-dependent dephosphorylation of GABABRs may be a useful strategy to increase receptor signaling for treating diseases.SIGNIFICANCE STATEMENT Dysregulation of GABAB receptors (GABABRs) underlies altered neurotransmission in many neurological disorders. Protein phosphatase 2A (PP2A) is involved in dephosphorylating and subsequent internalization of GABABRs in models of addiction and depression. Here, we provide new evidence that PP2A B55 regulatory subunit interacts directly with a small region of the C-terminal domain of the GABABR1 subunit, and that this interaction is sensitive to intracellular Ca2+ We demonstrate that a short peptide corresponding to the PP2A interaction site on GABABR1 competes for PP2A binding, enhances phosphorylation GABABR2 S783, and affects functional signaling through GIRK channels. Our study highlights how targeting PP2A dependent dephosphorylation of GABABRs may provide a specific strategy to modulate GABABR signaling in disease conditions.

Phosphorylation of Glutamine Synthetase on Threonine 301 Contributes to Its Inactivation During Epilepsy.

The astrocyte-specific enzyme glutamine synthetase (GS), which catalyzes the amidation of glutamate to glutamine, plays an essential role in supporting neurotransmission and in limiting NH4 + toxicity. Accordingly, deficits in GS activity contribute to epilepsy and neurodegeneration. Despite its central role in brain physiology, the mechanisms that regulate GS activity are poorly defined. Here, we demonstrate that GS is directly phosphorylated on threonine residue 301 (T301) within the enzyme's active site by cAMP-dependent protein kinase (PKA). Phosphorylation of T301 leads to a dramatic decrease in glutamine synthesis. Enhanced T301 phosphorylation was evident in a mouse model of epilepsy, which may contribute to the decreased GS activity seen during this trauma. Thus, our results highlight a novel molecular mechanism that determines GS activity under both normal and pathological conditions.

Clusterin Is Required for ß-Amyloid Toxicity in Human iPSC-Derived Neurons.

Our understanding of the molecular processes underlying Alzheimer's disease (AD) is still limited, hindering the development of effective treatments, and highlighting the need for human-specific models. Advances in identifying components of the amyloid cascade are progressing, including the role of the protein clusterin in mediating ß-amyloid (Aß) toxicity. Mutations in the clusterin gene (CLU), a major genetic AD risk factor, are known to have important roles in Aß processing. Here we investigate how CLU mediates Aß-driven neurodegeneration in human induced pluripotent stem cell (iPSC)-derived neurons. We generated a novel CLU-knockout iPSC line by CRISPR/Cas9-mediated gene editing to investigate Aß-mediated neurodegeneration in cortical neurons differentiated from wild type and CLU knockout iPSCs. We measured response to Aß using an imaging assay and measured changes in gene expression using qPCR and RNA sequencing. In wild type neurons imaging indicated that neuronal processes degenerate following treatment with Aß25-35 peptides and Aß1-42 oligomers, in a dose dependent manner, and that intracellular levels of clusterin are increased following Aß treatment. However, in CLU knockout neurons Aß exposure did not affect neurite length, suggesting that clusterin is an important component of the amyloid cascade. Transcriptomic data were analyzed to elucidate the pathways responsible for the altered response to Aß in neurons with the CLU deletion. Four of the five genes previously identified as downstream to Aß and Dickkopf-1 (DKK1) proteins in an Aß-driven neurotoxic pathway in rodent cells were also dysregulated in human neurons with the CLU deletion. AD and lysosome pathways were the most significantly dysregulated pathways in the CLU knockout neurons, and pathways relating to cytoskeletal processes were most dysregulated in Aß treated neurons. The absence of neurodegeneration in the CLU knockout neurons in response to Aß compared to the wild type neurons supports the role of clusterin in Aß-mediated AD pathogenesis.

Impact of a five-dimensional framework on R&D productivity at AstraZeneca.

In 2011, AstraZeneca embarked on a major revision of its research and development (R&D) strategy with the aim of improving R&D productivity, which was below industry averages in 2005-2010. A cornerstone of the revised strategy was to focus decision-making on five technical determinants (the right target, right tissue, right safety, right patient and right commercial potential). In this article, we describe the progress made using this '5R framework' in the hope that our experience could be useful to other companies tackling R&D productivity issues. We focus on the evolution of our approach to target validation, hit and lead optimization, pharmacokinetic/pharmacodynamic modelling and drug safety testing, which have helped improve the quality of candidate drug nomination, as well as the development of the right culture, where 'truth seeking' is encouraged by more rigorous and quantitative decision-making. We also discuss where the approach has failed and the lessons learned. Overall, the continued evolution and application of the 5R framework are beginning to have an impact, with success rates from candidate drug nomination to phase III completion improving from 4% in 2005-2010 to 19% in 2012-2016.

Estradiol modulates the efficacy of synaptic inhibition by decreasing the dwell time of GABAA receptors at inhibitory synapses.

Estrogen plays a critical role in many physiological processes and exerts profound effects on behavior by regulating neuronal excitability. While estrogen has been established to exert effects on dendritic morphology and excitatory neurotransmission its role in regulating neuronal inhibition is poorly understood. Fast synaptic inhibition in the adult brain is mediated by specialized populations of γ-c aA receptors (GABAARs) that are selectively enriched at synapses, a process dependent upon their interaction with the inhibitory scaffold protein gephyrin. Here we have assessed the role that estradiol (E2) plays in regulating the dynamics of GABAARs and stability of inhibitory synapses. Treatment of cultured cortical neurons with E2 reduced the accumulation of GABAARs and gephyrin at inhibitory synapses. However, E2 exposure did not modify the expression of either the total or the plasma membrane GABAARs or gephyrin. Mechanistically, single-particle tracking revealed that E2 treatment selectively reduced the dwell time and thereby decreased the confinement of GABAARs at inhibitory synapses. Consistent with our cell biology measurements, we observed a significant reduction in amplitude of inhibitory synaptic currents in both cultured neurons and hippocampal slices exposed to E2, while their frequency was unaffected. Collectively, our results suggest that acute exposure of neurons to E2 leads to destabilization of GABAARs and gephyrin at inhibitory synapses, leading to reductions in the efficacy of GABAergic inhibition via a postsynaptic mechanism.

Deficits in the activity of presynaptic γ-aminobutyric acid type B receptors contribute to altered neuronal excitability in fragile X syndrome.

The behavioral and anatomical deficits seen in fragile X syndrome (FXS) are widely believed to result from imbalances in the relative strengths of excitatory and inhibitory neurotransmission. Although modified neuronal excitability is thought to be of significance, the contribution that alterations in GABAergic inhibition play in the pathophysiology of FXS are ill defined. Slow sustained neuronal inhibition is mediated by γ-aminobutyric acid type B (GABAB) receptors, which are heterodimeric G-protein-coupled receptors constructed from R1a and R2 or R1b and R2 subunits. Via the activation of Gi/o, they limit cAMP accumulation, diminish neurotransmitter release, and induce neuronal hyperpolarization. Here we reveal that selective deficits in R1a subunit expression are seen in Fmr1 knock-out mice (KO) mice, a widely used animal model of FXS, but the levels of the respective mRNAs were unaffected. Similar trends of R1a expression were seen in a subset of FXS patients. GABAB receptors (GABABRs) exert powerful pre- and postsynaptic inhibitory effects on neurotransmission. R1a-containing GABABRs are believed to mediate presynaptic inhibition in principal neurons. In accordance with this result, deficits in the ability of GABABRs to suppress glutamate release were seen in Fmr1-KO mice. In contrast, the ability of GABABRs to suppress GABA release and induce postsynaptic hyperpolarization was unaffected. Significantly, this deficit contributes to the pathophysiology of FXS as the GABABR agonist (R)-baclofen rescued the imbalances between excitatory and inhibitory neurotransmission evident in Fmr1-KO mice. Collectively, our results provided evidence that selective deficits in the activity of presynaptic GABABRs contribute to the pathophysiology of FXS.

Pioneering government-sponsored drug repositioning collaborations: progress and learning.

A new model for translational research and drug repositioning has recently been established based on three-way partnerships between public funders, the pharmaceutical industry and academic investigators. Through two pioneering initiatives - one involving the Medical Research Council in the United Kingdom and one involving the National Center for Advancing Translational Sciences of the National Institutes of Health in the United States - new investigations of highly characterized investigational compounds have been funded and are leading to the exploration of known mechanisms in new disease areas. This model has been extended beyond these first two initiatives. Here, we discuss the progress to date and the unique requirements and challenges for this model.

Purinergic receptor activation facilitates astrocytic GABAB receptor calcium signalling.

Gamma-aminobutyric acid B receptors (GABABRs) are heterodimeric G-protein coupled receptors, which mediate slow synaptic inhibition in the brain. Emerging evidence suggests astrocytes also express GABABRs, although their physiological significance remains unknown. To begin addressing this issue, we have used imaging and biochemical analysis to examine the role GABABRs play in regulating astrocytic Ca(2+) signalling. Using live imaging of cultured cortical astrocytes loaded with calcium indicator Fluo-4/AM, we found that astrocytic GABABRs are able to induce astrocytic calcium transients only if they are pre-activated by P2 purinoceptors (P2YRs). The GABABR-mediated calcium transients were attenuated by the removal of extracellular calcium. Furthermore, P2YRs enhance the phosphorylation of astrocytic GABABR R2 subunits on both serine 783 (S783) and serine 892 (S892), two phosphorylation sites that are well known to regulate the activity and the cell surface stability of GABABRs. Collectively these results suggest that P2YR mediated signalling is an important determinant of GABABR activity and phosphorylation in astrocytes.