Nordic Bioscience Research Articles

#900 Endotrophin is associated with long-term risk of kidney function decline in normoalbuminuric persons with type 2 diabetes in the Joslin Kidney Study

Abstract Background and Aims Endotrophin is a pro-fibrotic and pro-inflammatory molecule derived from the post-translational processing of type VI collagen. Evidence for an association of increased circulating levels of endotrophin with increased risk of kidney and cardiovascular-related outcome and all-cause mortality in persons with type 2 diabetes have recently emerged. Here we want to investigate endotrophin as an early marker of kidney-related outcome risk in persons with type 2 diabetes with normal levels of albuminuria. Method Endotrophin was measured using the nordicPRO-C6TM assay (Nordic Bioscience, Herlev, Denmark) in serum of 1088 individuals with type 2 diabetes from the Joslin Diabetes Study. 635 individuals had normal to mildly increased albuminuria (<30 mg/g), 379 had moderately increased albuminuria (30-300 mg/g) and 74 had severely increased albuminuria (>300 mg/g). All patients had eGFR >60 and where hence classified as CKD stage 1 and 2. Risk of kidney disease progression was defined as a 40% decline in eGFR within 10 years. Results In the cohort (n = 1088), a total of 128 patients progressed to 40% eGFR decline. High levels of circulating endotrophin were associated with an increased risk of kidney disease progression in unadjusted logistic regression analysis (OR for T3 vs T1+T2 (PRO-C6): 2.1 [95% CI: 1.4-3.0], p = 0.0001) and after adjustment for eGFR, HbA1c, age and sex (OR for T3 vs T1+T2 (PRO-C6): 1.8 [95% CI: 1.1-2.6], p = 0.005). When looking at patients classified in albuminuria stages, endotrophin was significantly associated with kidney disease progression in the individuals with normal to mildly increased albuminuria (39 events) (OR for T3 vs T1+T2 (PRO-C6): 2.6 [95% CI: 1.3-4.9], p = 0.005 in unadjusted analysis and 2.3 [95% CI: 1.1-4.5], p = 0.02 after adjustment for eGFR, HbA1c, age and sex, but it was not significantly associated to kidney progression in the groups with moderately (49 events) and severely (40 events) increased albuminuria. Conclusion This study sheds further light on the potential of endotrophin as an early marker of long-term adverse kidney outcome in persons with type 2 diabetes. The association of circulating endotrophin with increased risk of kidney disease progression had already been reported both in CKD and DKD patients (for shorter-term outcome), and this study further expands on the applicability of endotrophin as a biomarker in persons with type 2 diabetes without an overt kidney disease, who will nevertheless progress to develop CKD in a 10-year time horizon.

426-P: Semaglutide, Empagliflozin, and Cagrilintide Reduce Levels of Serum Endotrophin in Type 2 Diabetic Rats

Background: Glucagon-like peptide 1 receptor agonists (GLP-1 RAs) and sodium-glucose co-transporter-2 inhibitors (SGLT2i) demonstrated cardiovascular (CV) and kidney outcomes benefit in persons with type 2 diabetes (T2D). The long-acting amylin analogue, cagrilintide, is under investigation for the treatment of obesity and T2D. Endotrophin, a pro-fibrotic fragment derived from collagen type VI, is an independent risk marker of mortality, CV and kidney outcomes, and dulaglutide treatment decreased levels of this biomarker compared with insulin glargine in persons with T2D. We investigated for the first time the effect of semaglutide, empagliflozin, and cagrilintide on endotrophin in type 2 diabetic rats. Method: We measured endotrophin (by the rodent PRO-C6 ELISA) in serum collected at baseline and study end from diabetic ZDF rats treated with either vehicle (n = 10), semaglutide (n = 7), empagliflozin (n = 4), or cagrilintide (n = 10). The dose chosen for semaglutide (50 nmol/kg), empagliflozin (30 mg/kg), and cagrilintide (10 nmol/kg) was based on previous research. Results: Levels of endotrophin increased significantly during the study in vehicle-treated rats, reflecting the disease progression (median [95% CI] for increase: 182.5 [55.87-298.0], P < 0.01). Interestingly, treatment with semaglutide, empagliflozin, and cagrilintide each attenuated this increase, resulting in significantly lower levels of endotrophin compared to vehicle at study end (P < 0.01), (P < 0.05), and (P < 0.01), respectively. Conclusion: We developed a novel, robust assay to detect the clinically relevant endotrophin biomarker in rodents. Levels of serum endotrophin increased over time in vehicle-treated rats, which is in line with endotrophin predicting disease progression in persons with T2D. In addition to the beneficial effects on metabolic factors, semaglutide, empagliflozin, and cagrilintide each reduced levels of endotrophin, suggesting a potential effect to reduce fibrosis. Disclosure A.Møller: None. S.A.Melander: None. A.T.Larsen: Employee; Nordic Bioscience A/S. C.F.G.Laursen: None. F.Genovese: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S. M.A.Karsdal: Speaker's Bureau; Pfizer Inc. D.Rasmussen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S. K.Henriksen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S.

861-P: The Effect of Dual Amylin and Calcitonin Receptor Agonist Treatment on Spatial Memory in Rat Models of Metabolic Syndrome

Obesity and metabolic-related complications, especially insulin resistance, have been linked to cognitive dysfunction. Dual amylin and calcitonin receptor agonists (DACRAs) elicit beneficial effects on body weight, glucose control, and insulin action, making them novel candidates for treating obesity and related comorbidities. Whether DACRAs affect cognition has yet to be addressed. With these studies, we evaluated the effect of DACRA treatment on spatial learning and memory in rat models of metabolic syndrome. Spatial learning and spatial memory retention were evaluated by Morris Water Maze in ZDSD rats treated with KBP-336 (4.5 nmol/kg Q3D) for 7 months and ZSF1 rats treated with KBP-336 (4.5 nmol/kg Q3D) for 2 months. In the ZDSD study, rats performed 8 training trials with a hidden platform in a constant position and a 60 s probe without the platform. At the study end, rats performed a 60 s probe followed by 4 re-training trials and another probe. At study end of the ZSF1 study, rats performed 6 training trials with a hidden platform in a constant position followed by a 60 s probe without the platform. In both studies, KBP-336 treatment improved the overall metabolic status, including glucose control and insulin action. In ZDSD rats, KBP-336-treated rats spent more time and traveled longer distances in Whishaw's corridor than vehicle-treated rats during the probes at the study end. Together indicating a possible treatment improvement in spatial memory on KBP-336. In ZSF1 rats, KBP-336 treatment resulted in increased time spent in the platform quadrant and Whishaw's corridor, as well as increased distance, traveled in Whishaw's corridor during the probe. In both studies, the treatment did not affect the spatial learning evaluated as latency to reach the hidden platform during the training trials. Altogether, we found that the improved metabolic status obtained with KBP-336 treatment might have a beneficial effect on spatial memory in diabetic and obese rat models. Disclosure K.Mohamed: Employee; Nordic Bioscience. A.T.Larsen: Employee; Nordic Bioscience A/S. S.A.Melander: None. M.A.Karsdal: Speaker's Bureau; Pfizer Inc. K.Henriksen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S. Funding Danish Research Foundation; Innovation Fund Denmark; European Union’s Horizon 2020 Research and Innovation Program (814244)

840-P: Dual Amylin and Calcitonin Receptor Agonist Treatment Improves Blood Pressure and Glucose Control in ZSF1 Rats

Long-acting dual amylin and calcitonin receptor agonists (DACRAs) are novel candidates for the treatment of type 2 diabetes and obesity due to their beneficial effects on both body weight, glucose control and insulin action. However, whether DACRAs affect hypertension, and diabetes-related cardiovascular complications remain unknown. In this study, the cardiovascular protective effect of the DACRA KBP-336 was evaluated in obese Zucker diabetic fatty-Spontaneously hypertensive heart failure F1 hybrid (ZSF1) rats. Obese ZSF1 rats were treated for 60 days with the KBP-336 (4.5 nmol/kg Q3D) and treatment efficacy on glucose control and blood pressure measured by tail cuff was evaluated. Furthermore, serum biomarkers of cardiovascular complications were evaluated. KBP-336 treatment significantly lowered fasting blood glucose levels compared to vehicle (P<0.001). This was also illustrated in the HbA1c levels at study end, where KBP-336 resulted in significantly (P<0.001) lower levels than vehicle. Importantly, KBP-336 significantly reduced both systolic (AUC levels reduced by 13.3%, P<0.001) and diastolic (AUC levels reduced by 17.0%, P<0.001) blood pressure compared to vehicle without affecting the heart rate. Notably, the beneficial effects on both glucose control and blood pressure were obtained independent of weight loss as KBP-336 only resulted in a small and transient weight reduction. Furthermore, serum levels of the marker of cardiovascular disease, rPRO-C6 (collagen type VI formation marker) were 6.4% lower in KBP-336 treated rats compared to vehicle, supporting improvement in cardiovascular parameters. In summary, we show for the first time that KBP-336 benefits blood pressure in a metabolic rat model suffering from hypertension. In addition, KBP-336 improved glucose control independent of weight loss. These data highlight KBP-336 as a promising candidate for treating obesity and related comorbidities including type 2 diabetes and hypertension. Disclosure S.A.Melander: None. A.T.Larsen: Employee; Nordic Bioscience A/S. K.Mohamed: Employee; Nordic Bioscience. M.A.Karsdal: Speaker's Bureau; Pfizer Inc. K.Henriksen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S. Funding Danish Research Foundation; Innovation Fund Denmark; European Union’s Horizon 2020 Research and Innovation Program (814244)

425-P: Renin-Angiotensin System Inhibition in Combination with Glucagon-Like Peptide 1 Receptor Agonist Treatment Improve Levels of a Kidney Fibrosis Biomarker in Persons with Type 2 Diabetes

Background: The drug combinations for rewriting trajectories of renal pathologies in type 2 diabetes (DC-ren) study focuses on DKD, a complication of diabetes often accompanied by cardiovascular disease. Novel biomarkers for identifying persons at increased risk of disease progression and predicting treatment response are currently needed. Levels of urinary C3M, a marker of type III collagen degradation, have been shown to correlate with CKD progression, and dulaglutide treatment improved levels of this biomarker compared with insulin glargine in persons with type 2 diabetes (T2D). We investigated the effect of combination therapy on urinary C3M in persons with T2D. Method: We measured urinary C3M in urine collected at baseline and after years 1, 2, and 3 from 229 individuals with T2D enrolled in the DC-ren study. All persons were on renin-angiotensin system inhibitor (RASi) treatment at baseline and during follow-up. While some persons remained on RASi treatment (n = 184), others received glucagon-like peptide-1 receptor agonist (GLP-1 RA) on top of RASi (n = 45). Results: The cohort consisted of 48% males (age of 66 ± 8 years and eGFR was 65 ± 17 ml/min/1.73 m2). Lower baseline levels of urinary C3M were associated with lower eGFR, and this relation remained consistent over time. Levels of urinary C3M were higher in persons after treatment with GLP-1 RA in combination with RASi compared to RASi alone (P = 0.034). In adjusted linear models (for the RASi group), an association of current urinary C3M with future eGFR was found (P < 0.01), indicating that urinary C3M is a prognostic risk marker for kidney function decline. Conclusion: RASi in combination with GLP-1 RA treatment was associated with increased type III collagen degradation, suggesting a potential effect to reduce kidney fibrosis. This anti-fibrotic effect could be a mechanism for the beneficial effects observed with GLP-1 RA treatment on CKD in T2D. Disclosure A.Møller: None. S.Thöni: None. C.F.G.Laursen: None. M.A.Karsdal: Speaker's Bureau; Pfizer Inc. F.Genovese: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S. D.Rasmussen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S.

211-OR: Dual Amylin and Calcitonin Receptor Agonists Increase Mitochondrial Respiratory Capacity in Adipose Tissues of Obese Rat

Dual amylin and calcitonin receptor agonists (DACRAs) are potential therapeutic candidates for treatment of obesity. Preclinical studies have shown favorable effects of DACRAs on appetite regulation contributing to a reduction in body weight. Interestingly, the DACRA-induced effects on weight loss are superior to the effects of a suppressed food intake, suggesting an effect on energy expenditure potentially by targeting the mitochondria. We investigated the effects of long-term treatment with a class of DACRAs, namely KBPs, on body weight, food intake and mitochondrial respiratory capacity (MRC) in Sprague-Dawley rats fed a high-fat diet and treated s.c. with KBPs for 8 weeks. Moreover, a pair-fed (PF) group was used to examine food intake-independent effects of KBPs on MRC. At study end, MRC was analyzed in perirenal (pAT) and inguinal (iAT) adipose tissue using high resolution respirometry. Expectedly, KBP treatment significantly reduced body weight compared to PF and vehicle. PF was successfully KBP-matched in terms of accumulated food intake and the food intake was significantly lower than that of vehicle-treated rats. Coupled and uncoupled MRC supported by complex I+II substrates and fatty acids were significantly greater in iAT and pAT after KBP treatment indicating effects on both carbohydrate and lipid metabolism. Additionally, an increased oligomycin-induced leak-respiration was found to be independent of food intake in iAT. Further analysis will shed light on whether these changes in MRC are a result of an increased mitochondrial content or of the respiratory capacity of each mitochondrion. In conclusion, treatment with KBPs in obese rats is associated with an increased MRC in pAT and iAT and elicits effects additional to those obtained by diet-induced weight loss. This highlights DACRAs’ potential as anti-obesity agents with possible benefits on energy expenditure as a contributing cause to the DACRA-induced weight loss. Disclosure E.A.Petersen: Employee; Nordic Bioscience A/S. I.Blom: None. S.A.Melander: None. M.A.Karsdal: Speaker's Bureau; Pfizer Inc. S.Larsen: None. K.Henriksen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S. A.T.Larsen: Employee; Nordic Bioscience A/S. Funding Innovation Fund Denmark

Type III collagen formation is significantly associated with risk of outcome in HFpEF patients but loses its significant association with underlying AF

Abstract Introduction Heart failure with preserved ejection fraction (HFpEF) is the most common type of heart failure, associated with high morbidity and mortality. Atrial fibrillation (AF) has been associated with structural remodelling and fibrosis and can coexist with HFpEF. Type III collagen is the second most abundant collagen in the heart wall and has been associated with cardiac fibrosis. It has been previously shown that type III collagen formation, measured by PRO-C3, is prognostic for all-cause mortality in HFpEF. Purpose In this study, we aimed at investigating if the prognostic power of PRO-C3 in relation to all-cause mortality would be affected by underlying AF in HFpEF patients. Methods The analysis included 166 individuals with hypertensive HFpEF. The patients were classified in three groups according to NYHA classification, 43.2% in NYHA Class II, 52.5% NYHA Class III and 2.5% NYHA Class IV. A previous history of AF was present in 53.4% of the patients. Cardiac function was assessed by echocardiography and standard clinical measures. Type III collagen formation was evaluated by means of ELISA with the biomarker PRO-C3, which targets the released N-terminal pro-peptide of type III collagen. Results Levels of PRO-C3 were significantly elevated in HFpEF patients with AF (p=0.0063). PRO-C3 was significantly predictive of all-cause mortality in HFpEF patients (AUC=0.643, p=0.0053), but lost its significant association when factoring in AF (AUC=0.581, p=0.235). There was an increased risk of all-cause mortality (p=0.0089) but not statistically significant differences in HFpEF patients with AF (p=0.178) with increasing tertiles of PRO-C3. Conclusions Type III collagen formation as measured by PRO-C3, was increased in patients with HFpEF and previously diagnosed AF. However, while PRO-C3 can be predictive of all-cause mortality in HFpEF patients, it lacks predicting ability when AF is considered. The presented data suggest a potential role of increased type III collagen formation in HFpEF patients with adverse outcomes, which is consistent with the presence of increased fibrosis, and can potentially be used as a tool of risk stratification. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Nordic Bioscience A/S

1153-P: Association of Extracellular Matrix Remodeling Biomarkers with Complications of Type 2 Diabetes

Background: Persons with diabetes have a high risk of complications related to the micro- and macrovascular circulation. One of the pathological processes involved in these complications is the onset of abnormal extracellular matrix (ECM) remodeling in different organs, leading to fibrosis. The quantification of ECM remodeling may identify patients that are at higher risk for adverse outcomes. Method: We measured biomarkers of collagen type III (PRO-C3) and VI (PRO-C6) formation and MMP-mediated degradation of type I (C1M) , III (C3M) , and IV (C4M and Tumstatin; TUM) in serum from 267 persons with T2DM from the CMR in T2DM study (NCT02684331) . Myocardial fibrosis (LGE) and ECV were determined by gadolinium-contrast CMR. Serum samples from 79 healthy controls were measured for comparison. Results: Levels of all biomarkers were significantly elevated in persons with T2DM compared to healthy controls (all P<0.0001) . Some of the markers were associated with complications, PRO-C6 levels were: significantly elevated in patients with hypertension compared to persons without known CVD (P<0.0001) ; could discriminate patients with CKD - eGFR < 60 ml/min/1.73m2 with an AUC of 0.83 (P<0.001) ; were significantly increased in patients with ischemic LGE (P<0.05) , suggesting an association of PRO-C6 with fibrosis; and in a multiple linear regression analysis, higher PRO-C6 levels were significantly associated with higher HbA1c (r=0.15, P=0.0397) , lower eGFR (r=−0.41, P<0.0001) , higher proANP (r=0.24, P=0.0005) , and higher ECV (r=0.24, P=0.0006) . PRO-C3 levels were significantly elevated in patients with CKD compared to non-CKD, and levels of C3M and C4M increased significantly with presence of albuminuria. Conclusion: Biomarkers of ECM remodeling, particularly PRO-C6, may identify persons with active pro-fibrotic processes at risk for complications related to T2DM. The potential of the biomarkers to predict adverse outcomes will be investigated once follow-up data have been collected. Disclosure A.Møller: None. A.S.Bojer: None. F.Genovese: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S. M.H.Sørensen: None. P.Gæde: Advisory Panel; AstraZeneca, Research Support; Novo Nordisk. M.A.Karsdal: Employee; Nordic Bioscience A/S, Nordic Bioscience A/S, Nordic Bioscience A/S. P.L.Madsen: None. D.G.Rasmussen: Employee; Nordic Bioscience A/S.

823-P: The Long-Acting Dual Amylin and Calcitonin Receptor Agonist KBP Has Increased Efficacy on Weight Loss and Glucose Control Compared with Cagrilintide in Obese and Diabetic Rats

Long-acting dual amylin and calcitonin receptor agonists (DACRAs) are novel candidates for treatment of type 2 diabetes (T2D) and obesity due to their beneficial effects on both body weight, glucose control and insulin action. Cagrilintide, which is currently in clinical trials, has shown promising effects on weight loss. In this study we compared a new long-acting DACRA (KBP) to cagrilintide in pre-clinical models of obesity and T2D. In vitro potencies were assessed using receptor assays. In vivo efficacies were investigated head-to-head in high fat diet (HFD) fed obese and T2D (ZDF) rat models. In vitro data showed that both peptides active both the amylin and the calcitonin receptor, with KBP being more potent on both receptors. This was further confirmed in vivo by assessment of acute effects on food intake and CTX suppression. KBP (1.5, 4.5 and 13.5 nmol/kg) and cagrilintide (10, 30 and 100 nmol/kg) induced a potent and dose-dependent weight loss in HFD rats, with the highest dose of KBP being superior to cagrilintide. In diabetic ZDF rats, DACRA treatment improved glucose control and preserved plasma insulin compared to vehicle. Interestingly, despite similar levels of plasma insulin, KBP treatment was superior to cagrilintide in improving glucose control. This was further reflected in the HbA1c levels at study end, where KBP treatment resulted in significantly lower levels compared to cagrilintide. In summary, both DACRAs induced weight loss and improved glucose tolerance, insulin action as well as glucose control. However, KBP treatment results in superior efficacy on both weight loss and glucose control. These findings highlight KBP as a promising once-weekly agent for treatment of obesity and T2D. Disclosure A.T.Larsen: Employee; Nordic Bioscience. N.Sonne: None. K.Mohamed: None. E.Bredtoft: None. F.Andersen: None. M.A.Karsdal: Employee; Nordic Bioscience A/S, Nordic Bioscience A/S, Nordic Bioscience A/S. K.Henriksen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S.

824-P: Dual Amylin and Calcitonin Receptor Agonists Improve Insulin Sensitivity and Increase Tissue-Specific Glucose Uptake Independent of Weight Loss

Dual amylin and calcitonin receptor agonists (DACRAs) are known to induce a significant weight loss and improve glucose tolerance and glucose control in rats. However, it is unknown if DACRAs has an insulin sensitizing effect beyond that induced by weight loss and if DACRAs affect tissue specific glucose uptake. Additionally, clamp studies with DACRAs have not previously been performed in diabetic rats. Pre-diabetic ZDSD and diabetic ZDF rats were treated with the DACRA KBP-088 (1.5 nmol//kg, s.c.) for 12 days, and a hyperinsulinemic clamp was conducted on day 13, 24 h post-dosing. In ZDSD rats a hyperinsulinemic euglycemic clamp was carried out, while a hyperinsulinemic isoglycemic clamp was carried out in ZDF rats. In addition, specific glucose uptake was assessed during a hyperinsulinemic isoglycemic clamp in ZDF rats. In ZDSD rats, KBP-088 treatment resulted in a significant reduction in body weight and fasting blood glucose, and improved insulin sensitivity by increasing the glucose infusion rate (GIR) (vehicle: 8.6, KBP-088: 12 mg glucose/kg/min) . In ZDF rats, KBP-088 significantly reduced fasting blood glucose and improved insulin sensitivity (GIR, vehicle: 1.5, KBP-088: 16.5 mg glucose/kg/min) , independent on weight loss. In both studies, KBP-088 increased the rate of glucose clearance, likely be increasing glucose storage, but without altering the endogenous glucose production. Direct assessment of tissue specific glucose uptake showed, that KBP-088 significantly increased glucose uptake in both muscles and adipose tissues when compared to vehicle. In summary, KBP-088 significantly improved insulin sensitivity in both pre-diabetic and diabetic rats and markedly increased tissue specific glucose uptake. Importantly, KBP-088 has an insulin sensitizing effect independent of weight loss, highlighting DACRAs as promising agents for treatment of diabetes. Disclosure A.T.Larsen: Employee; Nordic Bioscience. N.Sonne: None. E.Bredtoft: None. M.A.Karsdal: Employee; Nordic Bioscience A/S, Nordic Bioscience A/S, Nordic Bioscience A/S. K.Henriksen: Employee; Nordic Bioscience A/S, Stock/Shareholder; Nordic Bioscience A/S.

MO747: Endotrophin Levels are Extremely Elevated in Dialysis Patients with Heart Failure with Preserved Ejection Fraction (HFPEF) and are Influenced by Background Treatment with Diuretics and ARBS/ACEI

Abstract BACKGROUND AND AIMS Endotrophin, a bioactive molecule derived from the cleavage of the collagen type VI pro-peptide, released during collagen maturation, has been associated with multiple pathogenic processes, including inflammation and fibrosis. PRO-C6, a biomarker reflecting circulating endotrophin levels, has been associated with an adverse prognosis in multiple disease settings, including chronic kidney disease and heart failure with preserved ejection fraction (HFpEF). Here, we assessed levels of PRO-C6 in a population of dialysis patients with and without heart failure, and we observed an association of PRO-C6 with imaging and soluble biomarkers of heart disease, presence of co-morbidities and medications. METHOD PRO-C6 was measured with the enzyme-linked immunosorbent assay developed by Nordic Bioscience (Denmark) in serum of 46 patients undergoing dialysis (mean age 70 years, 37% female) and with HFpEF (61%), HFmrEF (7%), HFrEF (2%) or no HF (30%) collected at the Clínica Universidad de Navarra (Spain). The samples were collected pre-dialysis. PRO-C6 was also measured in serum of 50 healthy individuals (mean age 61 years, 50% female) collected at the same site. Differences between groups were estimated with the Kruskal–Wallis test, correlations were estimated with Spearman’s rank correlation coefficient. RESULTS Levels of PRO-C6 were extremely elevated in the serum of dialysis patients compared with healthy individuals (81 ng/mL versus 6.3 ng/mL; P < 0.0001), and also compared with previously seen levels in HFpEF patients (patients with levels above 16 ng/mL were at increased risk of worse outcome in the TOPCAT cohort). Among the dialysis patients, patients with HFpEF had significantly increased PRO-C6 levels compared with patients with no HF (90.2 ng/mL versus 61.8 ng/mL; P = 0.03). Due to the low number of events, the numerical increase in PRO-C6 levels in patients that died, had myocardial infarction and pulmonary hypertension did not reach statistical significance. PRO-C6 showed modest direct correlations with age, NT-proBNP, hsTNT and collagen type I C-terminal telopeptide (CITP), and a modest inverse correlation with eGFR. When looking at echo parameters, PRO-C6 was modestly directly correlated with left ventricular posterior wall thickness and relative wall thickness and modestly inversely correlated with left ventricular end diastolic volume. Interestingly, patients treated with ARB/ACEi and diuretics presented significantly lower levels of PRO-C6 compared with those that were not receiving these drugs (64.4 ng/mL versus 90.2 ng/mL; P = 0.01 and 59.0 ng/mL versus 87.1 ng/mL; P = 0.008, respectively). Patients treated with antiplatelets on the contrary had higher levels of PRO-C6 (88.9 ng/mL versus 67.7 ng/mL; P = 0.04). CONCLUSION Even in a population of kidney failure patients, presenting very high levels of circulating endotrophin, the presence of HFpEF was associated with further increased levels of endotrophin. Moreover, endotrophin levels were associated with echo parameters and biomarkers reflecting diastolic disfunction and left ventricular stress and damage. We have shown here for the first time that patients treated with ARB/ACEi and diuretics present lower levels of circulating endotrophin. PRO-C6 measuring endotrophin confirms being a biomarker of interest in HFpEF patients, even in the presence of kidney failure.

MO063: A New Tool for Preclinical Research and Drug Discovery: Extracellular Matrix Remodeling Quantification in Human Precision-Cut Kidney Slices

Abstract BACKGROUND AND AIMS Accumulation of extracellular matrix (ECM) proteins is a hallmark of renal fibrosis, leading to altered tissue homeostasis, kidney failure and ultimately death. Current treatment strategies are ineffective in halting renal fibrogenesis, and drug discovery is hampered by the lack of clinically relevant biomarkers. Thus, there is an urgent need for early and pharmacodynamic biomarkers of renal fibrosis to support the development of new therapeutic treatments. In this study, we explored the use of non-invasive biomarkers of ECM turnover in human precision-cut kidney slices (PCKS): we observed the release of these markers over time in the slice supernatants and measured the changes following treatment with a prostaglandin E2 (PGE2) EP1 receptor antagonist. METHOD PCKS were prepared from healthy and fibrotic human kidney tissue obtained from patients treated at Aarhus University Hospital, Skejby. The slices were cultured in 48-well plates in medium (300 µl/well) and incubated for up to 72 hours to stimulate the development of renal fibrosis. The medium was collected at hours 0, 24, 48 and 72. Biomarkers of collagen type I (PRO-C1), III (PRO-C3), fibronectin (FN; FBN-C) and α-SMA formation and MMP-mediated degradation of collagen type I (C1M), III (C3M) and IV (Tumstatin; TUM) were measured in the medium by ELISA assays developed at Nordic Bioscience. mRNA expression levels of COL1A1, FN and α-SMA were measured by qPCR at all time points. Viability was assessed by measuring the ATP content of the slices at all time points. The anti-fibrotic effect of SC-19 220—a PGE2 EP1 receptor antagonist—was studied in fibrotic PCKS at hours 24 and 48 after treatment. RESULTS Fibrosis developed spontaneously in human kidney slices, as shown by increasing levels of PRO-C1 (P < 0.0001), PRO-C3 (P < 0.0001), FBN-C (P < 0.0001) and α-SMA (P < 0.0001) over time. Levels of C1M (not significant) and C3M (P < 0.01) did not increase to the same extent, and levels of TUM decreased over time (P < 0.001), indicating that this model reflects the process of fibrogenesis over fibrolysis. In addition, protein levels of PRO-C1 and FBN-C correlated significantly with mRNA levels of COL1A1 (r = 0.92, P = 0.0088) and FN (r = 0.86, P = 0.0277), respectively. Treatment with SC-19 220 reduced the gene expression of COL1A1, FN and α-SMA, and at a protein level it decreased levels of PRO-C1, PRO-C3 and FBN-C. PCKS retained constant ATP levels up to 72 hours. CONCLUSION In conclusion, we demonstrated that quantification of ECM remodeling in human PCKS via noninvasive biomarkers can be a valuable preclinical tool to study renal fibrogenesis and the effect of anti-fibrotic treatments. This study highlights that the EP1 receptor is a promising target for halting the production of ECM proteins. Since this is a human model, results may be translated to clinical care with higher confidence.

Prophylactic Treatment with Rurioctocog Alfa Pegol Results in a Dose-Dependent Normalization of Biomarkers of Joint Health in Severe Hemophilia a: An Exploratory Analysis from the Propel Study

Prophylactic Treatment with Rurioctocog Alfa Pegol Results in a Dose-Dependent Normalization of Biomarkers of Joint Health in Severe Hemophilia a: An Exploratory Analysis from the Propel Study

COL4A3 is degraded in allergic asthma and degradation predicts response to anti-IgE therapy.

Asthma is a heterogeneous syndrome substantiating the urgent requirement for endotype-specific biomarkers. Dysbalance of fibrosis and fibrolysis in asthmatic lung tissue leads to reduced levels of the inflammation-protective collagen 4 (COL4A3). To delineate the degradation of COL4A3 in allergic airway inflammation and evaluate the resultant product as a biomarker for anti-IgE therapy response. The serological COL4A3 degradation marker C4Ma3 (Nordic Bioscience, Denmark) and serum cytokines were measured in the ALLIANCE cohort (paediatric cases/controls: n=134/n=35; adult cases/controls: n=149/n=31). Exacerbation of allergic airway disease in mice was induced by sensitising to ovalbumin (OVA), challenge with OVA aerosol and instillation of poly(cytidylic-inosinic). Fulacimstat (chymase inhibitor; Bayer) was used to determine the role of mast cell chymase in COL4A3 degradation. Patients with cystic fibrosis (n=14) and cystic fibrosis with allergic bronchopulmonary aspergillosis (ABPA; n=9) as well as patients with severe allergic uncontrolled asthma (n=19) were tested for COL4A3 degradation. Omalizumab (anti-IgE) treatment was assessed using the Asthma Control Test. Serum levels of C4Ma3 were increased in asthma in adults and children alike and linked to a more severe, exacerbating allergic asthma phenotype. In an experimental asthma mouse model, C4Ma3 was dependent on mast cell chymase. Serum C4Ma3 was significantly elevated in cystic fibrosis plus ABPA and at baseline predicted the success of the anti-IgE therapy in allergic, uncontrolled asthmatics (diagnostic OR 31.5). C4Ma3 levels depend on lung mast cell chymase and are increased in a severe, exacerbating allergic asthma phenotype. C4Ma3 may serve as a novel biomarker to predict anti-IgE therapy response.

736-P: Proposed Biosimilar Gan & Lee Insulin Lispro (GL-LIS) Shows Pharmacokinetic (PK) and Pharmacodynamic (PD) Bioequivalence (BE) vs. U.S.- and EU-Licensed Insulin Lispro (LIS)

This randomized, double-blind, three-way crossover trial (NCT04235439) compared the PK and PD properties of GL-LIS, a proposed biosimilar LIS by Gan & Lee, US- and EU-licensed Humalog® (US-LIS and EU-LIS) in 38 healthy male subjects (mean±SD age 41±15 years, BMI 24.4±2.8 kg/m²). Each subject received a single 0.2 U/kg dose of either GL-LIS, US-LIS or EU-LIS in each of the 3 euglycemic glucose clamp periods (ClampArt, clamp level 81 mg/dl, clamp duration 12h post-dose). GL-LIS and comparator insulins showed superimposable serum LIS concentration- and glucose infusion rate (GIR) profiles (Figure). PK BE was demonstrated between GL-LIS and the two comparator insulins as serum LIS concentration point estimates (using log-transformed data) were close to 100% and the 90% CIs were well contained within the pre-defined similarity margins of 80-125% (Figure). Likewise, the primary PD endpoints met BE-criteria as did multiple secondary PK/PD-endpoints, including AUCLIS.0-2h, AUCLIS.0-4h, AUCLIS.0-6h, AUCGIR.0-2h, AUCGIR.0-4h, AUCGIR.0-6h, and AUCGIR.6-12h. Several sensitivity analyses confirmed BE. All insulins had a low incidence of adverse events (5-11 events per insulin). No injection site reactions were observed. In conclusion, GL-LIS demonstrated both PK- and PD-bioequivalence to US- and EU-licensed LIS formulations. Disclosure E. Zijlstra: Speaker’s Bureau; Self; Novo Nordisk A/S. T. Heise: Advisory Panel; Self; Novo Nordisk A/S, Valbiotis, Research Support; Self; ADOCIA, Afon Technology Ltd., AstraZeneca, Biocon, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Gan & Lee Pharmaceuticals, Johnson & Johnson, Julphar, Mylan N. V., Nestle, Neuraly, Nordic Bioscience, Novo Nordisk, Sanofi, Zealand Pharma A/S, Speaker’s Bureau; Self; Novo Nordisk A/S. M. Ermer: None. J. Lu: Employee; Self; Gan & Lee Pharmaceuticals. M. G. Wilson: Consultant; Self; Gan & Lee Pharmaceuticals, Employee; Spouse/Partner; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company, Stock/Shareholder; Spouse/Partner; Eli Lilly and Company. L. Plum-moerschel: None. Funding Gan & Lee Pharmaceuticals

197-OR: ADO09, a Coformulation of Insulin A21G and Pramlintide (Pram), Improves Blood Glucose Control and Reduces Body Weight in Subjects with T1D

ADO09 is a co-formulation of Pram and insulin A21G developed to deliver the positive effects of Pram without additional injections. This double-blind randomized cross-over trial investigated the effects of pre-meal ADO09 vs. insulin aspart (ASP) on mixed meal tolerance tests (MMTT) and CGM over 24 days in 16 T1D subjects (BMI 30.5 ± 3.1 kg/m²) using daily prandial insulin doses of 45-75 U. ADO09 reduced incremental AUC glucose 0-4h during MMTT by 69% (p=0.006) and deltaPG 1h by 82 mg/dL (p<0.001) vs. ASP (figure). Post-meal glucagon levels were lowered by up to 85% and speed of gastric emptying (acetaminophen absorption) reduced by >50% with ADO09. CGM over the final 3 weeks showed higher time in range (+58 min), lower time >180 mg/dL (-71 min) and slightly higher time <70 mg/dL (+13mins) with ADO09. Body weight decreased by 1.6 kg with ADO09 (p=0.007) as did daily prandial insulin doses (-12 U vs. ASP, median 35.0 U vs. 47.3; p=0.0004). Slightly more hypoglycemic episodes were noted with ADO09 (n=96 vs. 79) with no difference in nocturnal hypoglycemia. While more adverse events (AEs) occurred with ADO09 (22 vs. 10), most AEs were mild and of gastro-intestinal nature (nausea, decreased appetite).In conclusion, ADO09 improved blood glucose control and reduced both body weight and prandial insulin doses vs. insulin aspart in T1D subjects with high prandial insulin needs.View largeDownload slideView largeDownload slide DisclosureG. Meiffren: Employee; Self; ADOCIA, Stock/Shareholder; Self; ADOCIA. J. Devries: Advisory Panel; Self; ADOCIA, Novo Nordisk Inc., Zealand Pharma A/S, Speaker’s Bureau; Self; Novo Nordisk Inc. T. Heise: Advisory Panel; Self; Novo Nordisk A/S, Valbiotis, Research Support; Self; ADOCIA, Afon Technology Ltd., AstraZeneca, Biocon, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Gan & Lee Pharmaceuticals, Johnson & Johnson, Julphar, Mylan N. V., Nestlé, Neuraly, Nordic Bioscience, Novo Nordisk, Sanofi, Zealand Pharma A/S, Speaker’s Bureau; Self; Novo Nordisk A/S. G. Andersen: None. R. Eloy: Employee; Self; ADOCIA, Stock/Shareholder; Self; ADOCIA. C. Mégret: Employee; Self; ADOCIA, Stock/Shareholder; Self; ADOCIA. S. Famulla: None. C. Seroussi: Employee; Self; ADOCIA, Stock/Shareholder; Self; ADOCIA. Y. Chan: Employee; Self; ADOCIA. M. Gaudier: Employee; Self; ADOCIA, Stock/Shareholder; Self; ADOCIA. O. Soula: Employee; Self; ADOCIA, Employee; Spouse/Partner; ADOCIA, Stock/Shareholder; Self; ADOCIA, Stock/Shareholder; Spouse/Partner; ADOCIA.

1055-P: The Machine Learning Prediction Model NASHmap Identifies Higher Insulin Resistance in Type 2 Diabetes Mellitus (T2DM) Patients at Risk for Nonalcoholic Steatohepatitis (NASH)

Background: Phenotypic methods such as elevated liver tests and/or the presence of obesity or hypertension are often used to identify patients with T2DM at risk for NASH. Machine learning (ML) models allow the prediction of outcomes with many interacting features and may be superior to phenotype in the context of NASH and T2DM. Here we present an intergroup comparison between patients with T2DM predicted as positive or negative for NASH by NASHmap, a ML model showing high prediction performance in real-word datasets. Methods: We evaluated a subgroup of patients from a large T2DM clinical trial database (2002-2012) who had data for 14 NASHmap parameters. These include clinical observations and routine laboratory parameters but not HOMA-IR. We compared the profile of patients predicted by NASHmap (NASH positive or negative) using 17 parameters (14 NASHmap and 3 additional parameters). We compared HOMA-IR values between NASHmap positive and negative patients in the subgroup where the values were available. Results: The clinical database had 23038 metabolically compensated T2DM patients without chronic liver disease of other etiologies. Data for all 14 NASHmap parameters were available in 4336 patients. NASHmap positive patients were younger, had higher HbA1c, liver enzymes, triglycerides, WBC, body mass index, platelets, and a higher rate for hypertension. Data for HOMA-IR were available in 1208 patients. Median HOMA-IR value was 4.2 in NASHmap positive group vs. 2.6 in NASHmap negative group. Patients in both groups had a similar median duration of diabetes. Conclusion: Overall, the patients identified as at risk for NASH by NASHmap showed a metabolically unfavorable phenotype indicated by the higher HOMA-IR values in this group that the ML algorithm is also able to detect. ML models may be an important tool in effectively identifying patients at risk for NASH in electronic health records and clinical care. Disclosure A. Tietz: Employee; Self; Novartis AG, Stock/Shareholder; Self; Novartis AG. G. Bader: Employee; Self; Novartis AG. M. Docherty: Employee; Self; ZS Associates. B. Reinhart: Employee; Self; ZS Associates. M. Balp: Employee; Self; Novartis AG. M. Pedrosa: Employee; Self; Novartis AG. S. K. Acharya: Employee; Self; Novartis Healthcare Pvt. Ltd., Employee; Spouse/Partner; Novartis Healthcare Pvt. Ltd. J. Loeffler: Employee; Self; Novartis AG, Stock/Shareholder; Self; Novartis AG. J. M. Schattenberg: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Consultant; Self; Boehringer Ingelheim, BMS, Echosens, Genfit, Gilead Sciences, Intercept Pharmaceuticals, Madrigal, Novartis, Novo Nordisk, Nordic Bioscience, Pfizer, Roche, Sanofi, Siemens Healthcare GmbH, Zydus, Bristol-Myers Squibb Company.

738-P: Proposed Biosimilar Gan &amp; Lee Insulin Aspart (GL-ASP) Shows Pharmacokinetic (PK) and Pharmacodynamic (PD) Bioequivalence (BE) to U.S.- and EU-Licensed Insulin Aspart (ASP)

This randomized, double-blind, three-way crossover trial (NCT04237129) investigated the PK and PD properties of GL-ASP, a proposed biosimilar ASP by Gan & Lee, US licensed Novolog® and EU authorized NovoRapid® (US-ASP and EU-ASP) in 36 healthy male subjects (mean±SD age 36±12 years, BMI 24.3±2.7 kg/m²). Each subject received a single 0.2 U/kg dose of either GL-ASP, US-ASP or EU-ASP in each of the 3 euglycemic glucose clamp periods (ClampArt, clamp level 81 mg/dl, clamp duration 12h post-dose). GL-ASP and comparator insulins showed superimposable ASP concentration- and glucose infusion rate (GIR) profiles (Figure). PK BE was demonstrated between GL-ASP and the two comparator insulins as serum ASP concentration point estimates (using log-transformed data) were close to 100% and 90% CIs were well contained within the pre-defined similarity margins of 80-125% (Figure). Likewise, the primary PD endpoints met BE-criteria as did the secondary PK/PD-endpoints AUCASP.0-2h and AUCGIR.0-2h. Sensitivity analyses with untransformed data or exclusion of profiles with C-peptide increases confirmed PD BE. Adverse events were rare with all insulins (7-14 events per insulin, only 1 injection site reaction [mild erythema] with GL-ASP). In conclusion, GL-ASP demonstrated both PK- and PD-bioequivalence to US- and EU-licensed ASP formulations. Disclosure L. Plum-moerschel: None. E. H. Uhrmacher: None. E. Zijlstra: Speaker’s Bureau; Self; Novo Nordisk A/S. J. Lu: Employee; Self; Gan & Lee Pharmaceuticals. M. G. Wilson: Consultant; Self; Gan & Lee Pharmaceuticals, Employee; Spouse/Partner; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company, Stock/Shareholder; Spouse/Partner; Eli Lilly and Company. M. E. Barton: Employee; Self; Gan & Lee Pharmaceuticals. T. Heise: Advisory Panel; Self; Novo Nordisk A/S, Valbiotis, Research Support; Self; ADOCIA, Afon Technology Ltd., AstraZeneca, Biocon, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Gan & Lee Pharmaceuticals, Johnson & Johnson, Julphar, Mylan N. V., Nestle, Neuraly, Nordic Bioscience, Novo Nordisk, Sanofi, Zealand Pharma A/S, Speaker’s Bureau; Self; Novo Nordisk A/S. Funding Gan & Lee Pharmaceuticals

740-P: Proposed Biosimilar Gan &amp; Lee Insulin Glargine (GL-GLA) Shows Pharmacokinetic (PK) and Pharmacodynamic (PD) Bioequivalence (BE) to U.S.- and EU-Licensed Insulin Glargine (IG)

This randomized, double-blind, three-way crossover trial (NCT04236895) compared the PK and PD properties of GL-GLA, a proposed biosimilar IG by Gan & Lee, US- and EU-licensed Lantus® (US-IG and EU-IG) in 114 male subjects with type 1 diabetes (mean±SD age 42±8 years, BMI 25.8±2.0 kg/m²). Each subject received a single 0.5 U/kg dose of either GL-GLA, EU-IG, or US-IG in each of the 3 euglycemic glucose clamp periods (ClampArt, clamp level 100 mg/dl, clamp duration 30h post-dose). PK is presented as concentrations of the mainly absorbed IG metabolite M1. GL-GLA and comparator insulins showed superimposable profiles for PK and glucose infusion rates (GIR, Figure). PK BE was demonstrated between GL-GLA and the comparator insulins as the point estimates for measured metabolite M1 were close to 100% and 90% CIs were well contained within the pre-defined similarity margins of 80-125% (Figure). Likewise, the primary PD endpoints met BE-criteria as did secondary PK/PD-endpoints including AUCM1.0-12h, AUCM1.12-24h, AUCGIR.0-12h, and AUCGIR.12‑24h. All insulins were well tolerated with similar adverse event rates (19% [GL-GLA]; 26% [US-IG]; 21% [EU-IG]) and only few events of injection site reactions per insulin. In conclusion, GL-GLA demonstrated both PK- and PD-bioequivalence to US- and EU-licensed insulin glargine formulations. Disclosure T. Heise: Advisory Panel; Self; Novo Nordisk A/S, Valbiotis, Research Support; Self; ADOCIA, Afon Technology Ltd., AstraZeneca, Biocon, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Gan & Lee Pharmaceuticals, Johnson & Johnson, Julphar, Mylan N. V., Nestle, Neuraly, Nordic Bioscience, Novo Nordisk, Sanofi, Zealand Pharma A/S, Speaker’s Bureau; Self; Novo Nordisk A/S. L. Plum-moerschel: None. G. Andersen: None. J. Lu: Employee; Self; Gan & Lee Pharmaceuticals. M. G. Wilson: Consultant; Self; Gan & Lee Pharmaceuticals, Employee; Spouse/Partner; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company, Stock/Shareholder; Spouse/Partner; Eli Lilly and Company. E. Zijlstra: Speaker’s Bureau; Self; Novo Nordisk A/S. Funding Gan & Lee Pharmaceuticals

MO010COLLAGEN TYPE VI PRODUCTION BY KIDNEY FIBROBLASTS IS DEPENDENT ON THE RIGHT STIMULI AND MAY SELF-PERPETUATE FIBROSIS

Abstract Background and Aims Accumulation of extracellular matrix (ECM) proteins is a hallmark of kidney fibrosis, which can lead to altered tissue homeostasis, kidney failure, and ultimately death. Many different cell types are involved in this process, but fibroblasts are the main source of ECM proteins such as collagen type I (COL I), III (COL III), and VI (COL VI). Recently, it was suggested that a fragment of COL VI, released during collagen maturation, is a bioactive molecule (endotrophin; ETP) with signaling potential, indicating that collagens are not just passive structural proteins. In this study, we investigated the effect of different pro-fibrotic stimulants on COL VI production and the effect of ETP itself on human kidney fibroblasts in the scar-in-a-jar (SiaJ) cell model. Method Cells were seeded in 48-well plates at 30.000 cells/well and incubated for 24h in DMEM + 10% FBS for adherence. Cells were then starved by incubating them for further 24h in DMEM + 0.4% FBS. To induce fibrogenesis, fresh medium was added at day 0 with 225/150 mg/mL Ficoll 70/400 and 1% ascorbic acid, containing either 7-, 0.7-, or 0.07 nM PDGF-AA, 8-, 0.8-, or 0.08 nM PDGF-BB, 4-, 0.4-, or 0.04 nM PDGF-CC, 7-, 0.7-, or 0.07 nM PDGF-DD, 0.9-, or 0.09 nM CTGF, 0.02 nM TGF-β or 30 nM ETP. Medium was changed and collected on days 3, 6, 10, and 13. Biomarkers of COL I (PRO-C1), III (PRO-C3), and VI (PRO-C6) formation were assessed in the medium by enzyme-linked immunosorbent assays developed at Nordic Bioscience. Results The stimulation of kidney fibroblasts with PDGF-AA, -BB, -CC, and -DD caused an increase in PRO-C6 compared to the unstimulated cells at every time point (P&amp;lt;0.0001). The increase in formation peaked at day 10, and a dose-dependent increase in COL VI levels was observed with PDGF-DD treatment. Interestingly, CTGF treatment did not enhance the synthesis of COL VI at any time point, and TGF-β treatment suppressed PRO-C6 levels compared to the untreated cells (not significant). The stimulation with 30 nM ETP caused an increase in PRO-C1 (P&amp;lt;0.0001) and PRO-C3 (P&amp;lt;0.0001) compared to the unstimulated cells on days 6, 10, and 13. The increase in collagen formation peaked at day 10 for both markers, with a 7.28-fold increase for COL I and a 4.13-fold increase for COL III. Conclusion The production of COL VI, an important mediator of fibrosis and inflammation through its bioactive fragment endotrophin, shows a differential expression after stimulation of kidney fibroblasts with different pro-fibrotic growth factors. Interestingly, members of the PDGF family induced COL VI production, whereas CTGF and TGF-β did not. Moreover, we confirmed that ETP itself could stimulate kidney fibroblasts to produce more ECM proteins; hence COL VI may self-perpetuate fibrosis. This SiaJ model, combined with ECM formation biomarkers, could be used to elucidate the mechanisms behind acute and sustained matrix production profiles in vivo.