Efficacy and safety of Treamid in the rehabilitation of patients after COVID-19 pneumonia: a phase 2, randomized, double-blind, placebo-controlled trial.

BACKGROUND: Many patients who recovered from COVID are still suffering from pulmonary dysfunction that can be persistent even for months after infection. Therefore, treatment to prevent irreversible impairment of lung function is needed. Treamid (bisamide derivative of dicarboxylic acid, BDDA) was shown to have anti-inflammatory and antifibrotic effects in animal models of pulmonary fibrosis. This study was designed to assess the safety, tolerability, and efficacy of Treamid in the rehabilitation of patients after COVID pneumonia. The aim was to establish whether Treamid could be effective in ameliorating post-COVID sequelae. METHODS: The phase 2, randomized, double-blind, placebo-controlled clinical trial was done at 8 medical centers in Russia. Patients with a diagnosis of COVID in the past medical history (with the first symptoms of COVID appear no earlier than 2 months before screening) and having fibrotic changes in the lungs, decreased lung function (percentage of predicted FVC and/or DLCO < 80%), and moderate or severe dyspnea according to mMRC scale were enrolled and randomly assigned in a 1:1 ratio (stratified by the initial degree of lung damage, age, and concomitant chronic diseases) by use of interactive responsive technology to peroral administration of Treamid 50 mg or placebo once a day for 4 weeks. The primary outcome was the proportion of patients who achieved clinically significant improvement in FVC and/or DLCO (defined as a relative increase in FVC of ≥ 10% or a relative increase in FVC in the range of ≥ 5 to < 10% plus a relative increase in DLCO of ≥ 15%) at week 4 compared with baseline. Secondary endpoints included changes from baseline in dyspnea scoring evaluated by the modified Borg and mMRC scales, pulmonary function (FEV1, FVC, FEV1/FVC ratio, DLCO, TLC, FRC), 6-min walk distance, the overall score of the KBILD questionnaire, and the proportion of patients with a reduction in the degree of lung damage assessed by CT scores. This trial was registered on ClinicalTrials.gov (Identifier: NCT04527354). The study was fully funded by PHARMENTERPRISES LLC. RESULTS: 12 out of 29 patients (41%) in Treamid group achieved clinically significant improvement in FVC and/or DLCO compared to 5 out of 30 patients (17%) in placebo group (p = 0.036). There was a significant decrease of dyspnea according to modified Borg scale observed in the Treamid group (- 0.9 ± 0.7 vs. - 0.4 ± 0.8, p = 0.018). No significant differences in the adverse events were noted. Exploratory analysis of the female population indicated superiority of Treamid over placebo by decreasing dyspnea and the extent of lung damage as well as increasing TLC. CONCLUSIONS: 4 weeks oral administration of 50 mg Treamid was associated with clinically significant improvement in the post-COVID patients, evident by an increase in FVC and/or DLCO as well as decreasing dyspnea. Treamid was well tolerated and can be safely administered to patients discharged after COVID. Treamid was more effective in women visible by superior improvement of COVID sequalae after 4 weeks treatment. Considering that female gender is a risk factor associated with the development of post-COVID symptoms, Treamid might offer a pharmacological treatment for long-term sequalae after COVID and supports further investigation in future clinical trials in post-COVID patients.

Imidazole-4-N-acetamide Derivatives as a Novel Scaffold for Selective Targeting of Cyclin Dependent Kinases.

The rational design of cyclin-dependent protein kinase (CDK) inhibitors presumes the development of approaches for accurate prediction of selectivity and the activity of small molecular weight anticancer drug candidates. Aiming at attenuation of general toxicity of low selectivity compounds, we herein explored the new chemotype of imidazole-4-N-acetamide substituted derivatives of the pan-CDK inhibitor PHA-793887. Newly synthesized compounds 1-4 containing an aliphatic methyl group or aromatic radicals at the periphery of the scaffold were analyzed for the prediction of relative free energies of binding to CDK1, -2, -5, and -9 using a protocol based on non-equilibrium (NEQ) thermodynamics. This methodology allows for the demonstration of a good correlation between the calculated parameters of interaction of 1-4 with individual targets and the values of inhibitory potencies in in vitro kinase assays. We provide evidence in support of NEQ thermodynamics as a time sparing, precise, and productive approach for generating chemical inhibitors of clinically relevant anticancer targets.

Thrifty energy phenotype predicts weight regain in postmenopausal women with overweight or obesity and is related to FGFR1 signaling.

BACKGROUND&AIMS: Long term improvement of body weight and metabolism is highly requested in obesity. The specific impact of weight loss associated temporary negative energy balance or modified body composition on metabolism and weight regain is unclear. METHODS: We randomly assigned 80 post-menopausal women (BMI 33.9 (32.2-36.8)kg/m2) to an intervention (IG) or control group (CG). IG underwent a dietary three month-weight loss intervention followed by a four week-weight maintenance period without negative energy balance. The CG was instructed to keep their weight stable. Phenotyping was performed at baseline (M0), after weight loss (M3), the maintenance period (M4) and 24-month follow-up (M24). Co-primary outcomes were changes of insulin sensitivity (ISIClamp) and lean body mass (LBM). Energy metabolism and adipose gene expression were secondary endpoints. RESULTS: Between March 2012 and July 2015, 479 subjects were screened for eligibility. 80 subjects were randomly assigned to IG (n = 40) or CG (n = 40). The total number of dropouts was 18 (IG: n = 13, CG: n = 5). LBM and ISIClamp were stable in the CG between M0 and M3, but were changed in the IG at M3 (LBM: -1.4 (95%CI -2.2-(-0.6)) kg and ISIClamp: +0.020 (95%CI 0.012-0.028) mg·kg-1·min-1/(mU·l-1)) (p < 0.01 and p < 0.05 for IG vs. CG, respectively). Effects on LBM, ISIClamp, FM and BMI were preserved until M4. Lower resting energy expenditure per LBM (REELBM) at M3 and stronger difference of REELBM between M3 and M4 (ΔREELBM-M3M4), which indicates a thrifty phenotype, were positively associated with FM regain at M24 (p = 0.022 and p = 0.044, respectively). Gene set enrichment analysis revealed a relationship of this phenotype to weight loss-induced adaption of adipose FGFR1 signaling. CONCLUSION: Negative energy balance had no additional effect on insulin sensitivity. FGFR1 signaling might be involved in the adaption of energy expenditure to temporary negative energy balance, which indicates a thrifty phenotype susceptible to weight regain. TRIAL REGISTRATION: ClinicalTrials.gov number: NCT01105143, https://clinicaltrials.gov/ct2/show/NCT01105143, date of registration: April 16th, 2010.

Fetuin-B, a potential link of liver-adipose tissue cross talk during diet-induced weight loss-weight maintenance.

BACKGROUND/OBJECTIVES: Numerous hepatokines are involved in inter-organ cross talk regulating tissue-specific insulin sensitivity. Adipose tissue lipolysis represents a crucial element of adipose insulin sensitivity and is substantially involved in long-term body weight regulation after dietary weight loss. Thus, we aimed to analyze the impact of the hepatokine Fetuin-B in the context of weight loss induced short- and long-term modulation of adipose insulin sensitivity. SUBJECTS/METHODS: 143 subjects (age > 18; BMI ≥ 27 kg/m2) were analyzed before (T-3) and after (T0) a standardized 12-week dietary weight reduction program. Afterward, subjects were randomized to a 12-month lifestyle intervention or a control group. After 12 months (T12) no further intervention was performed until 6 months later (T18) (Maintain-Adults trial). Tissue-specific insulin sensitivity was estimated by HOMA-IR (predominantly liver), ISIClamp (predominantly skeletal muscle), and free fatty acid suppression during hyperinsulinemic-euglycemic clamp (FFASupp) (predominantly adipose tissue). Fetuin-B was measured at all concomitant time points. RESULTS: Circulating Fetuin-B levels correlated significantly with estimates of obesity, hepatic steatosis as well as HOMA-IR, ISIClamp, FFASupp at baseline. Fetuin-B decreased during dietary weight loss (4.2 (3.5-4.9) vs. 3.8 (3.2-4.6) µg/ml; p = 2.1 × 10-5). This change was associated with concomitant improvement of HOMA-IR (r = 0.222; p = 0.008) and FFASupp (r = -0.210; p = 0.013), suggesting a particular relationship to hepatic and adipose tissue insulin sensitivity. Weight loss induced improvements of insulin resistance were almost completely preserved until months 12 and 18 and most interestingly, the short and long-term improvement of FFASupp was partially predicted by baseline level of Fetuin-B. CONCLUSIONS: Our data suggest that Fetuin-B might be a potential mediator of liver-adipose cross talk involved in short- and long-term regulation of adipose insulin sensitivity, especially in the context of diet-induced weight changes. TRIAL REGISTRATION: ClinicalTrials.gov number: NCT00850629, https://clinicaltrials.gov/ct2/show/NCT00850629 , date of registration: February 25, 2009.

Metabolic impact of weight loss induced reduction of adipose ACE-2 - Potential implication in COVID-19 infections?

BACKGROUND & AIMS: Angiotensin converting enzyme (ACE)-2 is a modulator of adipose tissue metabolism. However, human data of adipose ACE-2 is rarely available. Considering that, ACE-2 is believed to be the receptor responsible for cell entry of SARS-CoV-2, a better understanding of its regulation is desirable. We therefore characterized the modulation of subcutaneous adipose ACE-2 mRNA expression during weight loss and the impact of ACE-2 expression on weight loss induced short- and long-term improvements of glucose metabolism. METHODS: 143 subjects (age > 18; BMI ≥ 27 kg/m2) were analyzed before and after a standardized 12-week dietary weight reduction program. Afterwards subjects were randomized to a 12-month lifestyle intervention or a control group (Maintain-Adults trial). Insulin sensitivity (IS) was estimated by HOMA-IR (as an estimate of liver IS) and ISIClamp (as an estimate of skeletal muscle IS). ACE-2 mRNA expression (ACE-2AT) was measured in subcutaneous adipose tissue before and after weight loss. RESULTS: ACE-2AT was not affected by obesity, but was reduced in insulin resistant subjects. Weight loss resulted in a decline of ACE-2AT (29.0 (20.0-47.9) vs. 21.0 (13.0-31.0); p = 1.6 ∗ 10-7). A smaller reduction of ACE-2 AT (ΔACE-2AT) was associated with a larger improvement of ISIClamp (p = 0.013) during weight reduction over 3 months, but not with the extend of weight loss. The degree of changes in insulin resistance were preserved until month 12 and was also predicted by the weight loss induced degree of ΔACE-2AT (p = 0.011). CONCLUSIONS: Our data indicate that subcutaneous adipose ACE-2 expression correlates with insulin sensitivity. Weight loss induced decline of subcutaneous adipose ACE-2 expression might affect short- and long-term improvement of myocellular insulin sensitivity, which might be also relevant in the context of ACE-2 downregulation by SARS-CoV-2. TRIAL REGISTRATION: ClinicalTrials.gov number: NCT00850629, https://clinicaltrials.gov/ct2/show/NCT00850629, date of registration: February 25, 2009.

Genetic Nicotinamide N-Methyltransferase (Nnmt) Deficiency in Male Mice Improves Insulin Sensitivity in Diet-Induced Obesity but Does Not Affect Glucose Tolerance.

Antisense oligonucleotide knockdown (ASO-KD) of nicotinamide N-methyltransferase (NNMT) in high-fat diet (HFD)-fed mice has been reported to reduce weight gain and plasma insulin levels and to improve glucose tolerance. Using NNMT-ASO-KD or NNMT knockout mice (NNMT-/-), we tested the hypothesis that Nnmt deletion protects against diet-induced obesity and its metabolic consequences in males and females on obesity-inducing diets. We also examined samples from a human weight reduction (WR) study for adipose NNMT (aNNMT) expression and plasma 1-methylnicotinamide (MNAM) levels. In Western diet (WD)-fed female mice, NNMT-ASO-KD reduced body weight, fat mass, and insulin level and improved glucose tolerance. Although NNMT-/- mice fed a standard diet had no obvious phenotype, NNMT-/- males fed an HFD showed strongly improved insulin sensitivity (IS). Furthermore, NNMT-/- females fed a WD showed reduced weight gain, less fat, and lower insulin levels. However, no improved glucose tolerance was observed in NNMT-/- mice. Although NNMT expression in human fat biopsy samples increased during WR, corresponding plasma MNAM levels significantly declined, suggesting that other mechanisms besides aNNMT expression modulate circulating MNAM levels during WR. In summary, upon NNMT deletion or knockdown in males and females fed different obesity-inducing diets, we observed sex- and diet-specific differences in body composition, weight, and glucose tolerance and estimates of IS.

An Integrated Understanding of the Molecular Mechanisms of How Adipose Tissue Metabolism Affects Long-term Body Weight Maintenance.

Lifestyle-based weight loss interventions frequently demonstrate long-term inefficiency and weight regain. Identification of underlying mechanisms and predictors to identify subjects who will benefit from lifestyle-based weight loss strategies is urgently required. We analyzed 143 adults of the randomized Maintain trial (Maintain-Adults) after intended weight loss to identify mechanisms contributing to the regulation of body weight maintenance. Unbiased RNA sequencing of adipose and skeletal muscle biopsies revealed fatty acid metabolism as a key pathway modified by weight loss. Variability of key enzymes of this pathway, estimates of substrate oxidation, and specific serum acylcarnitine (AC) species, representing a systemic snapshot of in vivo substrate flux, predicted body weight maintenance (defined as continuous or dichotomized [< or ≥3% weight regain] variable) 18 months after intended weight loss in the entire cohort. Key results were confirmed in a similar randomized controlled trial in 137 children and adolescents (Maintain-Children), which investigated the same paradigm in a pediatric cohort. These data suggest that adaption of lipid utilization in response to negative energy balance contributes to subsequent weight maintenance. Particularly a functional role for circulating ACs, which have been suggested to reflect intracellular substrate utilization, as mediators between peripheral energy stores and control of long-term energy homeostasis was indicated.

Effects of a combined dietary, exercise and behavioral intervention and sympathetic system on body weight maintenance after intended weight loss: Results of a randomized controlled trial.

BACKGROUND: Lifestyle based weight loss interventions are hampered by long-term inefficacy. Prediction of individuals successfully reducing body weight would be highly desirable. Although sympathetic activity is known to contribute to energy homeostasis, its predictive role in body weight maintenance has not yet been addressed. OBJECTIVES: We investigated, whether weight regain could be modified by a weight maintenance intervention and analyzed the predictive role of weight loss-induced changes of the sympathetic system on long-term weight regain. DESIGN: 156 subjects (age > 18; BMI ≥ 27 kg/m2) participated in a 12-week weight reduction program. After weight loss (T0), 143 subjects (weight loss > 8%) were randomized to a 12-month lifestyle intervention or a control group. After 12 months (T12) no further intervention was performed until month 18 (T18). Weight regain at T18 (regainBMI) was the primary outcome. Evaluation of systemic and tissue specific estimates of sympathetic system was a pre-defined secondary outcome. RESULTS: BMI was reduced by 4.67 ±â€¯1.47 kg/m2 during the initial weight loss period. BMI maintained low in subjects of the intervention group until T12 (+0.07 ±â€¯2.98 kg/m2; p = 0.58 compared to T0), while control subjects regained +0.98 ±â€¯1.93 kg/m2 (p < 0.001 compared to T0). The intervention group regained more weight than controls after ceasing the intervention (1.17 ±â€¯1.34 vs. 0.57 ±â€¯0.93 kg/m2) until T18. Consequently, BMI was not different at T18 (33.49 (32.64; 34.33) vs. 34.18 (33.61; 34.75) kg/m2; p=0.17). Weight loss-induced modification of urinary metanephrine excretion independently predicted regainBMI (R2 = 0.138; p < 0.05). The lifestyle intervention did not modify the course of urinary metanephrines after initial weight loss. CONCLUSIONS: Our lifestyle intervention successfully maintained body weight during the intervention period. However, no long-term effect could be observed beyond the intervention period. Predictive sympathetic activity was not persistently modified by the intervention, which may partially explain the lack of long-term success of such interventions.

Correction: Chronic Activation of Hepatic Nrf2 Has No Major Effect on Fatty Acid and Glucose Metabolism in Adult Mice.

[This corrects the article DOI: 10.1371/journal.pone.0166110.].

Weight Loss Partially Restores Glucose-Driven Betatrophin Response in Humans.

CONTEXT: Recently a potential role of betatrophin was shown in the postprandial switch from lipid to glucose metabolism. OBJECTIVE: The objective of the study was to analyze whether obesity is associated with altered postprandial betatrophin response and whether this could be restored by weight loss. Design, Setting, Participants, and Intervention: Oral glucose load was performed in 12 lean individuals at baseline as well as in 20 obese subjects before and after a 12-week structured weight-loss program at an endocrinology research center. Euglycemic hyperinsulinemic clamps were performed in the obese cohort. The effect of insulin and different glucose concentrations on betatrophin expression were analyzed in 3T3-L1 adipocytes. MAIN OUTCOME MEASURE: Circulating betatrophin levels during a glucose challenge were measured. RESULTS: The betatrophin level decreases after an oral glucose intake (P < .001). This correlates with the increase of glucose levels (r = -0.396; P < .05). Hyperinsulinemia results in an increase of betatrophin. In vitro experiments in 3T3-L1 adipocytes confirmed that insulin and low glucose concentration increases betatrophin expression, whereas a further elevation of glucose levels blunts this effect. Obese subjects are characterized by lower fasting betatrophin (600.6 ± 364.4 vs 759.5 ± 197.9 pg/mL; P < .05) and a more pronounced betatrophin suppression during the glucose challenge. The impaired betatrophin response in obese subjects is restored after weight loss and is comparable with lean individuals. CONCLUSIONS: Obesity is associated with increased betatrophin suppression after an oral glucose load, which is driven by increased hyperglycemia. Given the metabolic properties of betatrophin, this may indicate that betatrophin is tightly linked to obesity-associated metabolic disturbances. In line with such an assumption, weight loss almost completely eliminated this phenomenon.

Chronic Activation of Hepatic Nrf2 Has No Major Effect on Fatty Acid and Glucose Metabolism in Adult Mice.

The transcription factor NF-E2-related factor 2 (Nrf2) induces cytoprotective genes, but has also been linked to the regulation of hepatic energy metabolism. In order to assess the pharmacological potential of hepatic Nrf2 activation in metabolic disease, Nrf2 was activated over 7 weeks in mice on Western diet using two different siRNAs against kelch-like ECH-associated protein 1 (Keap1), the inhibitory protein of Nrf2. Whole genome expression analysis followed by pathway analysis demonstrated successful knock-down of Keap1 expression and induction of Nrf2-dependent genes involved in anti-oxidative stress defense and biotransformation, proving the activation of Nrf2 by the siRNAs against Keap1. Neither the expression of fatty acid- nor carbohydrate-handling proteins was regulated by Keap1 knock-down. Metabolic profiling of the animals did also not show effects on plasma and hepatic lipids, energy expenditure or glucose tolerance. The data indicate that hepatic Keap1/Nrf2 is not a major regulator of glucose or lipid metabolism in mice.

ANP system activity predicts variability of fat mass reduction and insulin sensitivity during weight loss.

INTRODUCTION: In weight loss trials, a considerable inter-individual variability in reduction of fat mass and changes of insulin resistance is observed, even under standardized study conditions. The underlying mechanisms are not well understood. Given the metabolic properties of the atrial natriuretic peptide (ANP) system, we hypothesized that ANP signaling might be involved in this phenomenon by changes of ANP secretion or receptor balance. Therefore, we investigated the impact of systemic, adipose and myocellular ANP system on metabolic and anthropometric improvements during weight loss. METHODS: We comprehensively investigated 143 subjects (31 male, 112 female) before and after a 3 month-standardized weight loss program. The time course of BMI, fat mass, insulin sensitivity, circulating mid-regional proANP (MR-proANP) levels as well as adipose and myocellular natriuretic receptor A (NPR-A) and C (NPR-C) mRNA expression were investigated. RESULTS: BMI decreased by -12.6±3.7%. This was accompanied by a remarkable decrease of adipose NPR-C expression (1005.0±488.4 vs. 556.7±465.6; p<0.001) as well as a tendency towards increased adipose NPR-A expression (4644.7±946.8 vs. 4877.6±869.8; p=0.051). Weight loss induced changes in NPR-C (ΔNPR-C) was linked to relative reduction of total fat mass (ΔFM) (r=0.281; p<0.05), reduction of BMI (r=0.277; p<0.01), and increase of free fatty acids (ΔFFA) (r=-0.258; p<0.05). Basal NPR-C expression and weight loss induced ΔNPR-C independently explained 22.7% of ΔFM. In addition, ΔMR-proANP was independently associated with improvement of insulin sensitivity (standardized ß=0.246, p<0.01). DISCUSSION: ANP receptor expression predicted the degree of weight loss induced fat mass reduction. Our comprehensive human data support that peripheral ANP signalling is involved in control of adipose tissue plasticity and function during weight loss. (Funded by the Deutsche Forschungsgemeinschaft (KFO281/2), the Berlin Institute of Health (BIH) and the German Centre for Cardiovascular Research (DZHK/BMBF); ClinicalTrials.gov number: NCT00850629).