In vivo mRNA expression of a multi-mechanistic mAb combination protects against Staphylococcus aureus infection.

Single monoclonal antibodies (mAbs) can be expressed in vivo through gene delivery of their mRNA formulated with lipid nanoparticles (LNPs). However, delivery of a mAb combination could be challenging due to the risk of heavy and light variable chain mispairing. We evaluated the pharmacokinetics of a three mAb combination against Staphylococcus aureus first in single chain variable fragment scFv-Fc and then in immunoglobulin G 1 (IgG1) format in mice. Intravenous delivery of each mRNA/LNP or the trio (1 mg/kg each) induced functional antibody expression after 24 h (10-100 µg/mL) with 64%-78% cognate-chain paired IgG expression after 3 days, and an absence of non-cognate chain pairing for scFv-Fc. We did not observe reduced neutralizing activity for each mAb compared with the level of expression of chain-paired mAbs. Delivery of the trio mRNA protected mice in an S. aureus-induced dermonecrosis model. Intravenous administration of the three mRNA in non-human primates achieved peak serum IgG levels ranging between 2.9 and 13.7 µg/mL with a half-life of 11.8-15.4 days. These results suggest nucleic acid delivery of mAb combinations holds promise and may be a viable option to streamline the development of therapeutic antibodies.

Physical activity, inactivity and sleep during the Diabetes Remission Clinical Trial (DiRECT).

AIMS: As sustained weight loss is vital for achieving remission of type 2 diabetes, we explored whether randomisation to weight loss plus maintenance in the DiRECT trial was associated with physical activity, inactivity or sleep. METHODS: Participants were randomised to either a dietary weight management programme or best-practice care. The weight management group were encouraged to increase daily physical activity to their sustainable maximum. Objective measurement was achieved using a wrist-worn GENEActiv accelerometer for 7 days at baseline, 12 and 24 months in both groups. RESULTS: Despite average weight loss of 10 kg at 12 months in the intervention (n = 66) group, there were no differences in total physical activity or inactivity compared with the control (n = 104) at any time point. However, in our exploratory analysis, those who lost more than 10% of their baseline body weight performed on average 11 mins/day more light activity than the <10% group at 24 months (p = 0.033) and had significantly lower bouts of Inactivity30min (interaction, p = 0.005) across 12 and 24 months. At 24 months, the ≥10% group had higher daily acceleration (38.5 ± 12.1 vs. 33.2 ± 11.1 mg, p = 0.020), and higher accelerations in the most active 5-hour period (59.4 ± 21.8 vs. 50.6 ± 18.3 mg, p = 0.023). Wakefulness after sleep onset decreased in the intervention group compared with the control group and also in the ≥10% weight loss group at 12 and 24 months. CONCLUSIONS: Randomisation to a successful intensive weight loss intervention, including regular physical activity encouragement, was not associated with increased physical activity although sleep parameters improved. Physical activity was greater, and night-time waking reduced in those who maintained >10% weight loss at 12 and 24 months. TRIAL REGISTRATION ISRCTN03267836.

Delivering the Diabetes Remission Clinical Trial (DiRECT) in primary care: Experiences of healthcare professionals.

OBJECTIVE: The Diabetes Remission Clinical Trial (DiRECT) used a formula total diet replacement programme followed by structured weight loss maintenance to induce and sustain weight loss and remission of type 2 diabetes (T2D) in 36% of participants after 2 years. Nurses and dietitians delivering DiRECT in 22 primary care practices in Tyneside and Scotland provided behavioural support to participants. Participant experiences with DiRECT highlighted the key role of support by healthcare professionals (HCPs). We evaluated HCPs' experiences with DiRECT. RESEARCH DESIGN AND METHODS: Healthcare professionals delivering DiRECT were interviewed at 12 months, while general practices (GPs) were sent an implementation questionnaire. The interviews were analysed thematically. The questionnaires were analysed using frequencies and a narrative synthesis. RESULTS: Healthcare professionals representing 11 of 22 intervention practices were interviewed and 10 of 22 GPs completed questionnaires. HCPs' initial concerns over perceived potential negative intervention effects, particularly withdrawing anti-diabetes and anti-hypertensive medications, were barriers to engagement. Trust of HCPs towards the research team and perceived credibility of the study facilitated engagement and adoption. Ongoing support by research dietitians was key to the management of participants. Involvement in DiRECT inspired more focus on behaviour modification in the treatment of other people living with T2D in routine practice. CONCLUSIONS: Diabetes Remission Clinical Trial was considered highly appropriate for the management of T2D in primary care when supported by trained dietitians. Addressing limitations, including varying training needs of HCPs may improve intervention scale-up and tailoring to clinical contexts.

Participant experiences in the Diabetes REmission Clinical Trial (DiRECT).

INTRODUCTION: The Diabetes REmission Clinical Trial (DiRECT) has shown that sustained remission of type 2 diabetes in primary care is achievable through weight loss using total diet replacement (TDR) with continued behavioural support. Understanding participants' experiences can help optimise the intervention, support implementation into healthcare, and understand the process of behaviour change. METHODS: Thirty-four DiRECT participants were recruited into this embedded qualitative evaluation study. In-person and telephone interviews were conducted before the TDR; at week 6-8 of the TDR; 2 weeks into food reintroduction (FR); and at 1 year, to learn about participant experiences with the programme. Transcribed narratives were analysed thematically, and we used interpretation to develop overarching themes. RESULTS: Initiation of the TDR and transition to FR were challenging and required increased behavioural support. In general, adhering to TDR proved easier than the participants had anticipated. Some participants chose the optional extension of TDR. Rapid weight loss and changes in diabetes markers provided ongoing motivation. Further weight loss, behavioural support and occasional use of TDR facilitated weight loss maintenance (WLM). A process of behaviour adaptation to change following regime disruption was identified in three stages: (1) expectations of the new, (2) overcoming difficulties with adherence, and (3) acceptance of continuous effort and establishment of routines. CONCLUSIONS: The DiRECT intervention was acceptable and regularity, continuity, and tailoring of behavioural support was instrumental in its implementation in primary care. The adaptation process accounts for some of the individual variability of experiences with the intervention and highlights the need for programme flexibility.

Antihypertensive medication needs and blood pressure control with weight loss in the Diabetes Remission Clinical Trial (DiRECT).

AIMS/HYPOTHESIS: Our aim was to evaluate the safety and efficacy of a planned therapeutic withdrawal of all antihypertensive and diuretic medications, on commencing a formula low-energy diet replacement, targeting remission of type 2 diabetes. METHODS: Post hoc analysis of changes in BP, antihypertensive medication prescriptions and symptoms during the initial total diet replacement phase was performed in the intervention arm of the Diabetes Remission Clinical Trial (n = 143) and in the subset (n = 69) who discontinued antihypertensive medications at the start of total diet replacement. The Counterweight-Plus total diet replacement provided about 3470 kJ/day (830 kcal) with automatic reductions in all nutrients, including sodium, to achieve marked negative energy balance and rapid weight loss over 12-20 weeks, with regular BP monitoring and an antihypertensive reintroduction protocol based on current clinical guidelines. RESULTS: Of 143 intervention group participants who commenced total diet replacement, 78 (55%) were on treatment for hypertension at baseline. The overall mean BP fell significantly from the start of total diet replacement (week 1) and was significantly lower at week 20, after total diet replacement finished, and also at 12 and 24 months. Of the 78 participants previously on treatment for hypertension, 65 (83%) stopped all antihypertensive and diuretic medications as per protocol, and four (5%) stopped some drugs. These 69 participants experienced no immediate (within the first week) change in BP, but their mean BP fell significantly from 9 weeks. No excessive rises in BP were recorded in individuals, but antihypertensive medications were reintroduced during total diet replacement to manage raised BP for 19/69 (27.5%) participants, mostly within the first 3-7 weeks, despite some weight loss. Reintroduction of antihypertensive medications was necessary for 5/19 participants previously on one drug, and for 14/19 previously on two or more drugs. Of the 69 who stopped antihypertensives, 19 (28%) remained off medications at 24 months. Among the 53 participants who achieved sustained remissions of diabetes at 24 months (with a mean weight loss of 11.4 kg), 31 had been previously treated for hypertension. Twenty-seven stopped medication at baseline, and 15/27 required reintroduction of antihypertensive medications. Mild to moderate dizziness, suggesting some postural hypotension, was reported during total diet replacement by 51 participants, 15 of whom had recorded dizziness at baseline prior to starting total diet replacement, with nine of these on antihypertensive or diuretic medications. CONCLUSIONS/INTERPRETATION: Replacing antihypertensive medications with a 3470 kJ/day (830 kcal) diet to induce weight loss reduces BP substantially and may increase mild dizziness. It is safe to stop antihypertensives, but BP should be monitored regularly, particularly for those taking two or more antihypertensives, as over two-thirds will require reintroduction of some medications. Long-term support to maintain weight loss is vital. TRIAL REGISTRATION: ISRCTN registry, number 03267836.

Brief formula low-energy-diet for relapse management during weight loss maintenance in the Diabetes Remission Clinical Trial (DiRECT).

BACKGROUND: Weight loss maintenance (WLM) is critical for sustaining type 2 diabetes (T2D) remission, but poorly evidenced. We evaluated brief return to formula low-energy-diet (LED) as relapse treatments (RTs) during the WLM phase of the Diabetes Remission Clinical Trial (DiRECT). METHODS: This post-hoc evaluation included all participants commencing the WLM phase of DiRECT. The protocol offered RT when regain of >2 kg occurred. RESULTS: In total, 123/149 (83%) DiRECT intervention participants commenced the WLM phase after 26 (17%) had withdrawn prior to the WLM phase. Most participants [99/123 (80%)] regained >2 kg during the WLM phase, among whom 60/99 (61%) were recorded as using RT and 39/99 (39%) not using any RT. At baseline, RT users had a higher mean (SD) body mass index [35.8 (4.9) kg m-2 vs. 33.8 (3.9) kg m-2 , p = 0.0231] and had greater social deprivation (P = 0.0003) than non-users, although otherwise the groups were similar. Weight loss ≥ 2k g was achieved in 30/93 (32%) of RT attempts. At 2 years, those regaining >2 kg and using RT (n = 60) had mean (SD) weight losses of 7.4 (6.1) kg, with 25 (42%) remissions and 7 (12%) programme withdrawals. Those regaining >2 kg but not using RT (n = 39) had weight losses of 8.8 (6.0) kg, with 21 (54%) remissions and 4 (10%) programme withdrawals (all not significant). Twelve participants were never recorded as having regained >2 kg or using RTs and, at 2 years, their weight losses were 12.9 (9.2) kg, with 4 (33%) remissions and 8 (67%) programme withdrawals. CONCLUSIONS: Most people with T2D experience weight regain >2 kg during the 2 years after substantial weight loss with a LED. Only one-third of RTs corrected their 2-kg regain, resulting in similar weight losses, remissions and programme withdrawals at 2 years compared to those not using RTs; however, both groups had weight losses below those not recorded as regaining >2 kg during WLM.

Weight loss-induced increase in fasting ghrelin concentration is a predictor of weight regain: Evidence from the Diabetes Remission Clinical Trial (DiRECT).

AIM: To investigate whether appetite-related hormones were predictors of weight regain in the Diabetes Remission Clinical Trial (DiRECT). MATERIALS AND METHODS: DiRECT is a cluster-randomized clinical trial, designed to assess the effect of weight loss on type 2 diabetes remission. For this post hoc analysis, data were available for 253 (147 interventions, 106 controls) individuals with type 2 diabetes (age 53.6 ± 7.5 years, body mass index 34.7 ± 4.4 kg/m2 , 59% men). Intervention participants received a 24-month weight management programme, and controls remained on usual diabetes care. Fasting plasma concentrations of leptin, ghrelin, glucagon-like peptide-1 and peptide YY were measured at baseline, 12 months and 24 months in all participants, and at 5 months in a subset of participants in the intervention (n = 56) and control groups (n = 22). Potential predictors were examined using multivariable linear regression models. RESULTS: The intervention group lost 14.3 ± 6.0% body weight at 5 months but regained weight over time, with weight losses of 10.0 ± 7.5% at 12 months and 7.6 ± 6.3% at 24 months. Weight loss in controls was 1.1 ± 3.7% and 2.1 ± 5.0% at 12 and 24 months, respectively. Body weight increased by 2.3% (95% confidence interval [CI] 0.4, 4.1; P = 0.019) between 12 and 24 months for every 1-ng/mL increase in ghrelin between baseline and 12 months, and weight regain between 12 and 24 months was increased by 1.1% (95% CI 0.2, 2.0; P = 0.023) body weight for every 1-ng/mL increase in ghrelin at 12 months. CONCLUSION: The rise in ghrelin (but not any other measured hormone) during diet-induced weight loss was a predictor of weight regain during follow-up, and concentrations remained elevated over time, suggesting a small but significant compensatory drive to regain weight. Attenuating the effects of ghrelin may improve weight-loss maintenance.

Weight loss induced increase in fasting ghrelin concentration is a predictor of weight regain: evidence from the Diabetes Remission Clinical Trial.

AIM: To investigate whether appetite-related hormones were predictors of weight regain in the Diabetes Remission Clinical Trial (DiRECT). MATERIALS AND METHODS: DiRECT is a cluster-randomised clinical trial designed to assess the effect of weight-loss on type 2 diabetes remission. For this post hoc analysis, data were available for 253 (147 interventions, 106 controls) individuals with type 2 diabetes (aged 53.6±7.5 years, BMI 34.7±4.4 kg/m2, 59% males). Intervention participants received a 24-month weight-management programme and controls remained on usual diabetes care. Fasting plasma concentrations of leptin, ghrelin, GLP-1, and PYY were measured at baseline, 12 and 24-months in all participants, and at 5-months in a subset of interventions (n=56) and controls (n=22). Potential predictors were examined using multivariable linear regression models. RESULTS: The intervention group lost 14.3±6.0% body-weight at 5-months but regained over time, with weight-losses of 10.0±7.5% at 12-months and 7.6±6.3% at 24-months. Weight-loss in controls was 1.1±3.7% and 2.1±5.0% at 12 and 24-months, respectively. Body-weight increased by 2.3% [95% CI: 0.4,4.1]; p=0.019) between 12 and 24-months for every 1 ng/ml increase in ghrelin between baseline and 12-months, and weight regain between 12 and 24-months was increased by 1.1% (95% CI: 0.2,2.0; p=0.023) body-weight for every 1 ng/ml increase in ghrelin at 12-months. CONCLUSION: The rise in ghrelin (but not any other measured hormone) during diet-induced weight-loss was a predictor of weight regain during follow-up, and concentrations remained elevated over time, suggesting a small but significant compensatory drive to regain weight. Attenuating the effects of ghrelin may improve WLM. This article is protected by copyright. All rights reserved.

Primary care-led weight management for remission of type 2 diabetes (DiRECT): an open-label, cluster-randomised trial.

BACKGROUND: Type 2 diabetes is a chronic disorder that requires lifelong treatment. We aimed to assess whether intensive weight management within routine primary care would achieve remission of type 2 diabetes. METHODS: We did this open-label, cluster-randomised trial (DiRECT) at 49 primary care practices in Scotland and the Tyneside region of England. Practices were randomly assigned (1:1), via a computer-generated list, to provide either a weight management programme (intervention) or best-practice care by guidelines (control), with stratification for study site (Tyneside or Scotland) and practice list size (>5700 or ≤5700). Participants, carers, and research assistants who collected outcome data were aware of group allocation; however, allocation was concealed from the study statistician. We recruited individuals aged 20-65 years who had been diagnosed with type 2 diabetes within the past 6 years, had a body-mass index of 27-45 kg/m2, and were not receiving insulin. The intervention comprised withdrawal of antidiabetic and antihypertensive drugs, total diet replacement (825-853 kcal/day formula diet for 3-5 months), stepped food reintroduction (2-8 weeks), and structured support for long-term weight loss maintenance. Co-primary outcomes were weight loss of 15 kg or more, and remission of diabetes, defined as glycated haemoglobin (HbA1c) of less than 6·5% (<48 mmol/mol) after at least 2 months off all antidiabetic medications, from baseline to 12 months. These outcomes were analysed hierarchically. This trial is registered with the ISRCTN registry, number 03267836. FINDINGS: Between July 25, 2014, and Aug 5, 2017, we recruited 306 individuals from 49 intervention (n=23) and control (n=26) general practices; 149 participants per group comprised the intention-to-treat population. At 12 months, we recorded weight loss of 15 kg or more in 36 (24%) participants in the intervention group and no participants in the control group (p<0·0001). Diabetes remission was achieved in 68 (46%) participants in the intervention group and six (4%) participants in the control group (odds ratio 19·7, 95% CI 7·8-49·8; p<0·0001). Remission varied with weight loss in the whole study population, with achievement in none of 76 participants who gained weight, six (7%) of 89 participants who maintained 0-5 kg weight loss, 19 (34%) of 56 participants with 5-10 kg loss, 16 (57%) of 28 participants with 10-15 kg loss, and 31 (86%) of 36 participants who lost 15 kg or more. Mean bodyweight fell by 10·0 kg (SD 8·0) in the intervention group and 1·0 kg (3·7) in the control group (adjusted difference -8·8 kg, 95% CI -10·3 to -7·3; p<0·0001). Quality of life, as measured by the EuroQol 5 Dimensions visual analogue scale, improved by 7·2 points (SD 21·3) in the intervention group, and decreased by 2·9 points (15·5) in the control group (adjusted difference 6·4 points, 95% CI 2·5-10·3; p=0·0012). Nine serious adverse events were reported by seven (4%) of 157 participants in the intervention group and two were reported by two (1%) participants in the control group. Two serious adverse events (biliary colic and abdominal pain), occurring in the same participant, were deemed potentially related to the intervention. No serious adverse events led to withdrawal from the study. INTERPRETATION: Our findings show that, at 12 months, almost half of participants achieved remission to a non-diabetic state and off antidiabetic drugs. Remission of type 2 diabetes is a practical target for primary care. FUNDING: Diabetes UK.

Clinical and metabolic features of the randomised controlled Diabetes Remission Clinical Trial (DiRECT) cohort.

AIMS/HYPOTHESIS: Substantial weight loss in type 2 diabetes can achieve a return to non-diabetic biochemical status, without the need for medication. The Diabetes Remission Clinical Trial (DiRECT), a cluster-randomised controlled trial, is testing a structured intervention designed to achieve and sustain this over 2 years in a primary care setting to determine practicability for routine clinical practice. This paper reports the characteristics of the baseline cohort. METHODS: People with type 2 diabetes for <6 years with a BMI of 27-45 kg/m2 were recruited in 49 UK primary care practices, randomised to either best-practice diabetes care alone or with an additional evidence-based weight management programme (Counterweight-Plus). The co-primary outcomes, at 12 months, are weight loss ≥15 kg and diabetes remission (HbA1c <48 mmol/mol [6.5%]) without glucose-lowering therapy for at least 2 months. Outcome assessors are blinded to group assignment. RESULTS: Of 1510 people invited, 423 (28%) accepted; of whom, 306 (72%) were eligible at screening and gave informed consent. Seven participants were later found to have been randomised in error and one withdrew consent, leaving 298 (176 men, 122 women) who will form the intention to treat (ITT) population for analysis. Mean (SD) age was 54.4 (7.6) years, duration of diabetes 3.0 (1.7) years, BMI 34.6 (4.4) kg/m2 for all participants (34.2 (4.2) kg/m2 in men and 35.3 (4.6) kg/m2 in women) and baseline HbA1c (on treatment) 59.3 (12.7) mmol/mol (7.6% [1.2%]). The recruitment rate in the intervention and control groups, and comparisons between the subgroups recruited in Scotland and England, showed few differences. CONCLUSIONS/INTERPRETATION: DiRECT has recruited a cohort of people with type 2 diabetes with characteristics similar to those seen in routine practice, indicating potential widespread applicability. Over 25% of the eligible population wished to participate in the study, including a high proportion of men, in line with the prevalence distribution of type 2 diabetes. TRIAL REGISTRATION: www.controlled-trials.com/ISRCTN03267836 ; date of registration 20 December 2013.

Intracellular sorting and transcytosis of the rat transferrin receptor antibody OX26 across the blood-brain barrier in vitro is dependent on its binding affinity.

The blood-brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re-directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter-study comparisons. In this study, the efficiency of transcytosis in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (Kd from 5 to174 nM) of the rat TfR-binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics-selected reaction monitoring and fluorescent imaging with markers of early- and late endosomes. The OX26 variants with affinities of 76 and 108 nM showed improved trancytosis (Papp values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co-localized with late-endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co-localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14193.

Is There an Optimal Diet for Weight Management and Metabolic Health?

Individuals can lose body weight and improve health status on a wide range of energy (calorie)-restricted dietary interventions. In this paper, we have reviewed the effectiveness of the most commonly utilized diets, including low-fat, low-carbohydrate, and Mediterranean approaches, in addition to commercial slimming programs, meal replacements, and newly popularized intermittent fasting diets. We also consider the role of artificial sweeteners in weight management. Low-fat diets tend to improve low-density lipoprotein cholesterol the most, while lower-carbohydrate diets may preferentially improve triglycerides and high-density lipoprotein cholesterol. However, differences between diets are marginal. Weight loss improves almost all obesity-related co-morbidities and metabolic markers, regardless of the macronutrient composition of the diet, but individuals do vary in preferences and ability to adhere to different diets. Optimizing adherence is the most important factor for weight loss success, and this is enhanced by regular professional contact and supportive behavioral change programs. Maintaining weight losses in the long term remains the biggest challenge, and is undermined by an "obesogenic" environment and biological adaptations that accompany weight loss.

Enhanced Delivery of Galanin Conjugates to the Brain through Bioengineering of the Anti-Transferrin Receptor Antibody OX26.

The blood-brain barrier (BBB) is a formidable obstacle for brain delivery of therapeutic antibodies. However, antibodies against the transferrin receptor (TfR), enriched in brain endothelial cells, have been developed as delivery carriers of therapeutic cargoes into the brain via a receptor-mediated transcytosis pathway. In vitro and in vivo studies demonstrated that either a low-affinity or monovalent binding of these antibodies to the TfR improves their release on the abluminal side of the BBB and target engagement in brain parenchyma. However, these studies have been performed with mouse-selective TfR antibodies that recognize different TfR epitopes and have varied binding characteristics. In this study, we evaluated serum pharmacokinetics and brain and CSF exposure of the rat TfR-binding antibody OX26 affinity variants, having KDs of 5 nM, 76 nM, 108 nM, and 174 nM, all binding the same epitope in bivalent format. Pharmacodynamic responses were tested in the Hargreaves chronic pain model after conjugation of OX26 affinity variants with the analgesic and antiepileptic peptide, galanin. OX26 variants with affinities of 76 nM and 108 nM showed enhanced brain and cerebrospinal fluid (CSF) exposure and higher potency in the Hargreaves model, compared to a 5 nM affinity variant; lowering affinity to 174 nM resulted in prolonged serum pharmacokinetics, but reduced brain and CSF exposure. The study demonstrates that binding affinity optimization of TfR-binding antibodies could improve their brain and CSF exposure even in the absence of monovalent TfR engagement.

Brain penetration, target engagement, and disposition of the blood-brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1.

Receptor mediated transcytosis harnessing the cellular uptake and transport of natural ligands across the blood-brain barrier (BBB) has been identified as a means for antibody delivery to the CNS. In this study, we characterized bispecific antibodies in which a BBB-crossing antibody fragment FC5 was used as a BBB carrier. Cargo antibodies were either a high-affinity, selective antibody antagonist of the metabotropic glutamate receptor-1 (BBB-mGluR1), a widely abundant CNS target, or an IgG that does not bind the CNS target (BBB-NiP). Both BBB-NiP and BBB-mGluR1 demonstrated a similar 20-fold enhanced rate of transcytosis across an in vitro BBB model compared with mGluR1 IgG fused to a control antibody fragment. All 3 bispecific antibodies exhibited identical pharmacokinetics in vivo Comparative assessment of BBB-NiP and BBB-mGluR1 revealed that, whereas their serum pharmacokinetics and BBB penetration were identical, their central disposition (brain levels) and elimination (cerebrospinal fluid levels) were widely different, due to central target-mediated removal of the mGluR1-engaging antibody. Central mGluR1 target engagement after systemic administration was demonstrated by a dose-dependent inhibition of mGluR-1-mediated thermal hyperalgesia and by colocalization of the antibody with thalamic neurons involved in mGluR1-mediated pain processing. We demonstrate the feasibility of targeting central G-protein-coupled receptors using a BBB-crossing bispecific antibody approach and emerging principles that govern brain distribution and disposition of these antibodies. These data will be important for designing safe and selective CNS antibody therapeutics.-Webster, C. I., Caram-Salas, N., Haqqani, A. S., Thom, G., Brown, L., Rennie, K., Yogi, A., Costain, W., Brunette, E., Stanimirovic, D. B. Brain penetration, target engagement, and disposition of the blood-brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1.

The Diabetes Remission Clinical Trial (DiRECT): protocol for a cluster randomised trial.

BACKGROUND: Despite improving evidence-based practice following clinical guidelines to optimise drug therapy, Type 2 diabetes (T2DM) still exerts a devastating toll from vascular complications and premature death. Biochemical remission of T2DM has been demonstrated with weight loss around 15kg following bariatric surgery and in several small studies of non-surgical energy-restriction treatments. The non-surgical Counterweight-Plus programme, running in Primary Care where obesity and T2DM are routinely managed, produces >15 kg weight loss in 33% of all enrolled patients. The Diabetes UK-funded Counterpoint study suggested that this should be sufficient to reverse T2DM by removing ectopic fat in liver and pancreas, restoring first-phase insulin secretion. The Diabetes Remission Clinical Trial (DiRECT) was designed to determine whether a structured, intensive, weight management programme, delivered in a routine Primary Care setting, is a viable treatment for achieving durable normoglycaemia. Other aims are to understand the mechanistic basis of remission and to identify psychological predictors of response. METHODS/DESIGN: Cluster-randomised design with GP practice as the unit of randomisation: 280 participants from around 30 practices in Scotland and England will be allocated either to continue usual guideline-based care or to add the Counterweight-Plus weight management programme, which includes primary care nurse or dietitian delivery of 12-20weeks low calorie diet replacement, food reintroduction, and long-term weight loss maintenance. Main inclusion criteria: men and women aged 20-65 years, all ethnicities, T2DM 0-6years duration, BMI 27-45 kg/m(2). Tyneside participants will undergo Magnetic Resonance (MR) studies of pancreatic and hepatic fat, and metabolic studies to determine mechanisms underlying T2DM remission. Co-primary endpoints: weight reduction ≥ 15 kg and HbA1c <48 mmol/mol at one year. Further follow-up at 2 years. DISCUSSION: This study will establish whether a structured weight management programme, delivered in Primary Care by practice nurses or dietitians, is a viable treatment to achieve T2DM remission. Results, available from 2018 onwards, will inform future service strategy. TRIAL REGISTRATION: Current Controlled Trials ISRCTN03267836 . Date of Registration 20/12/2013.

5-year follow-up of the randomised Diabetes Remission Clinical Trial (DiRECT) of continued support for weight loss maintenance in the UK: an extension study.

BACKGROUND: In DiRECT, a randomised controlled effectiveness trial, weight management intervention after 2 years resulted in mean weight loss of 7·6 kg, with 36% of participants in remission of type 2 diabetes. Of 36 in the intervention group who maintained over 10 kg weight loss at 2 years, 29 (81%) were in remission. Continued low-intensity dietary support was then offered up to 5 years from baseline to intervention participants, aiming to maintain weight loss and gain clinical benefits. This extension study was designed to provide observed outcomes at 5 years. METHODS: The DiRECT trial took place in primary care practices in the UK. Participants were individuals aged 20-65 years who had less than 6 years' duration of type 2 diabetes, a BMI greater than 27 kg/m2, and were not on insulin. The intervention consisted of withdrawal of antidiabetic and antihypertensive drugs, total diet replacement (825-853 kcal per day formula diet for 12-20 weeks), stepped food reintroduction (2-8 weeks), and then structured support for weight-loss maintenance. After sharing the 2-year results with all participants, UK National Health Service data were collected annually until year 5 from remaining intervention participants who received low-intensity dietary support, intervention withdrawals, and the original randomly allocated groups. The primary outcome was remission of type 2 diabetes; having established in the DiRECT trial that sustained weight loss was the dominant driver of remission, this was assumed for the Extension study. The trial is registered with the ISRCTN registry, number 03267836. FINDINGS: Between July 25, 2014, and Aug 5, 2016, 149 participants were randomly assigned to the intervention group and 149 were assigned to the control group in the original DiRECT study. After 2 years, all intervention participants still in the trial (101 [68%] of 149) were approached to receive low-intensity support for a further 3 years. 95 (94%) of 101 were able to continue and consented and were allocated to the DiRECT extension group. 54 participants were allocated to the non-extension group, where intervention was withdrawn. At 5 years, DiRECT extension participants (n=85) lost an average of 6·1 kg, with 11 (13%) of 85 in remission. Compared with the non-extension group, DiRECT extension participants had more visits with HbA1c <48 mmol/mol (<6·5%; 36% vs 17%, p=0·0004), without glucose-lowering medication (62% vs 30%, p<0·0001), and in remission (34% vs 12%, p<0·0001). Original control participants (n=149) had mean weight loss 4·6 kg (n=82), and 5 (5%) of 93 were in remission. Compared with control participants, original intervention participants had more visits with weight more than 5% below baseline (61% vs 29%, p<0·0001), HbA1c below 48 mmol/mol (29% vs 15%, p=0·0002), without antidiabetic medication (51% vs 16%, p<0·0001), and in remission (27% vs 4%, p<0·0001). Of those in remission at year 2, 26% remained in remission at 5 years. Serious adverse events in the original intervention group (4·8 events per 100 patient-years) were under half those in the control group (10·2 per 100 patient-years, p=0·0080). INTERPRETATION: The extended DiRECT intervention was associated with greater aggregated and absolute weight loss, and suggested improved health status over 5 years. FUNDING: Diabetes UK.

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier.

BACKGROUND: Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. METHODS: Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. RESULTS: Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS endothelial cell types can be obtained, and these can be passaged onto large numbers of cell culture inserts for in vitro permeability studies. The passaged brain and spinal cord endothelial cells are pure and express endothelial markers, tight junction proteins and intracellular transport machinery. Further, both models exhibit tight, functional barrier characteristics that are discriminating against large and small molecules in permeability assays and show functional expression of the pharmaceutically important P-gp efflux transporter. CONCLUSIONS: Our techniques allow the provision of high yields of robust sister cultures of endothelial cells that accurately model the blood-CNS barriers in vitro. These models are ideally suited for use in studying the biology of the blood-brain barrier and blood-spinal cord barrier in vitro and for pre-clinical drug discovery.

Dietary weight-management for type 2 diabetes remissions in South Asians: the South Asian diabetes remission randomised trial for proof-of-concept and feasibility (STANDby).

Background: We aimed to assess whether a structured weight management programme incorporating a total diet replacement (TDR) (3-5 months ∼850 kcal/day formula diet) weight loss phase is acceptable to people of South Asian ethnicity and can achieve type 2 diabetes (T2D) remissions similarly to other populations. Methods: Adults of South Asian ethnicity, aged 18-65 years, with T2D for ≤4 years, and BMI 25-45 kg/m2 were recruited from primary care and social media, and randomised to commence TDR either immediately (iTDR), or delayed (dTDR) for 3-5 months as a usual care control arm during this period. Intervention effects were tested in randomised comparisons powered to detect significant weight loss, and in an expanded observational analysis to determine remission effect size, including both iTDR and dTDR groups. Acceptability in those recruited was explored by questionnaire and weight change. Trial registration: Current Controlled Trials, ISRCTN10720065. Date of Registration 27/09/2017. Findings: Twenty-five eligible individuals were recruited. Mean baseline (SD) age was 45.8 (11.1) years, weight 88.2 (13.7) kg, BMI 32.1 (3.8) kg/m2, HbA1c 60.4 (11.3) mmol/mol, liver fat by MRI 15.6 (9.4)%. In the RCT, mean(SD) weight change after TDR was -7.7 (7.2)% in the intervention group (n = 13), and -1.2 (1.4)% in the usual-care control group (n = 12) (p = 0.005), with T2D remission achieved by 5/13, compared to 0/12 respectively (p = 0.039). In the observational study, 23/25 started TDR and 19/23 participants completed the TDR phase. Median time spent in TDR was 105 days (IQR 77-134 days). T2D remission was achieved in 10/23 (43%), and weight changes were concordant with the RCT. Overall, 8/23 (35%) lost over 10% bodyweight. Absolute liver fat proportion near halved from 15.3% at the start of TDR to 8.6% (p < 0.001). Interpretation: In UK-based South Asians, TDR-led weight loss and T2D remission rates are comparable to those observed in white cohorts, and the intervention was acceptable in most of those recruited. There is potential to further improve outcomes, but one-third lost >10% body weight, and the mechanism underpinning T2D remission appears similar, driven by weight change with loss of excess ectopic body-fat. Funding: We gratefully acknowledge funding for the MRI scans from the, Miss MJM Smith Trust (registered charity: SC040586). No other external funds were provided for this trial. NS is supported by the British Heart Foundation Research Excellence Award (RE/18/6/34217).

Total Diet Replacement Within an Integrated Intensive Lifestyle Intervention for Remission of Type 2 Diabetes: Lessons From DiRECT.

The prognosis for people with type 2 diabetes (T2D) remains concerning, yet its seriousness is often underestimated. T2D is a manifestation, in susceptible individuals, of the disease-process of obesity, and at diagnosis, 10-year survival rates for T2D are around 50%. Here, we will examine: (a) the role of weight loss in T2D, (b) use of total diet replacements (TDRs) to induce weight loss, (c) the Diabetes Remission Clinical Trial (DiRECT) protocol and key results, (d) other dietary interventions related to T2D remission, (e) remission in real life, and (f) future directions. Remission of short-duration T2D will usually require 10-15% body weight loss, and results from the DiRECT trial demonstrated that this can be achieved within routine care in nearly half of all people undertaking a supported, TDR-led behavioural weight management programme. In light of these findings, which have since been replicated in the Diabetes Intervention Accentuating Diet and Enhancing Metabolism (DIADEM-I) trial conducted in the Middle East and North Africa, it is now time to prioritize weight loss programmes for T2D remission from diagnosis, and with increasing acceptance and availability of digital healthcare, there is an opportunity to scale up delivery of remission programmes in a cost effective manner.

Antibody-oligonucleotide conjugate achieves CNS delivery in animal models for spinal muscular atrophy.

Antisense oligonucleotides (ASOs) have emerged as one of the most innovative new genetic drug modalities. However, their high molecular weight limits their bioavailability for otherwise-treatable neurological disorders. We investigated conjugation of ASOs to an antibody against the murine transferrin receptor, 8D3130, and evaluated it via systemic administration in mouse models of the neurodegenerative disease spinal muscular atrophy (SMA). SMA, like several other neurological and neuromuscular diseases, is treatable with single-stranded ASOs that modulate splicing of the survival motor neuron 2 (SMN2) gene. Administration of 8D3130-ASO conjugate resulted in elevated levels of bioavailability to the brain. Additionally, 8D3130-ASO yielded therapeutic levels of SMN2 splicing in the central nervous system of adult human SMN2-transgenic (hSMN2-transgenic) mice, which resulted in extended survival of a severely affected SMA mouse model. Systemic delivery of nucleic acid therapies with brain-targeting antibodies offers powerful translational potential for future treatments of neuromuscular and neurodegenerative diseases.