A randomized, controlled, crossover pilot study of losartan for pediatric nonalcoholic fatty liver disease

A randomized, controlled, crossover pilot study of losartan for pediatric nonalcoholic fatty... Background: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in children, and currently, there are no FDA-approved therapies. Plasminogen activator inhibitor-1 (PAI-1) is elevated in children with NAFLD and associated with increased disease severity. Losartan potassium (losartan) is an angiotensin II receptor blocker (ARB) that reduces PAI-1 production and improves insulin sensitivity that has been proposed as a treatment for pediatric NAFLD but has not previously been tested. Methods: This was an 8-week randomized, double-blind, placebo-controlled, phase 2a, crossover study (with a 6- week washout between conditions) for safety and preliminary efficacy of losartan 50 mg a day taken orally in 12 normotensive children with biopsy proven nonalcoholic steatohepatitis (NASH). Results: Twelve children enrolled in the study, and nine completed all visits. No changes in blood pressure or serious adverse events occurred during the study. Trends in improvement in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and homeostatic model assessment insulin resistance (HOMA-IR) were seen with losartan treatment compared to the placebo time-period. More participants decreased ALT on losartan as compared to placebo (89% [8 out 9] vs. 56% [5 out of 9], respectively). Conclusions: This data provides preliminary evidence that losartan treatment is safe over 8 weeks in children with NAFLD and supports consideration of larger studies to test its efficacy. Trial registration: URL and trial identification number: https://clinicaltrials.gov/show/NCT01913470, NCT01913470. Date registered: August 1, 2013. Keywords: Children, Treatment, Pilot, Fatty liver disease, Plasminogen activator inhibitor-1, Insulin resistance Background (CVD) such as increased triglycerides, low high-density Nonalcoholic fatty liver disease (NAFLD) is the leading lipoprotein (HDL), and post-prandial lipemia [5, 6]. cause of liver disease in children [1]. The clinical import- Current treatment recommendations include lifestyle ance of NAFLD extends beyond liver injury to include changes, including attempting to improve diet and increased cardiovascular disease [2], type 2 diabetes [3], increase physical activity; however, lifestyle changes and increased overall mortality [4]. Our work and others typically do not achieve resolution of NAFLD [7]. have demonstrated that adolescents with NAFLD have Plasminogen activator inhibitor-1 (PAI-1) is an increased traditional markers of cardiovascular disease acute-phase protein which increases in states of insulin resistance, inflammation, and injury [8] and is elevated in both adults and children with hepatic steatosis [9–11]. * Correspondence: mvos@emory.edu Furthermore, PAI-1 correlates with fibrosis stage [12, 13]. Division of Gastroenterology, Hepatology, and Nutrition, Department of The renin-angiotensin system (RAS) has been suggested Pediatrics, School of Medicine, Emory University, Room W-450, 1760 Haygood Dr NE, Atlanta, GA 30322, USA to be involved in liver damage pathways and might play a Children’s Healthcare of Atlanta, Atlanta, GA, USA critical role in the pathogenesis of NAFLD [14]. Blockage Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Vos et al. Pilot and Feasibility Studies (2018) 4:109 Page 2 of 5 of the RAS significantly inhibits the expression of PAI-1 in Fasting blood samples were collected (typically between 7 the liver [15], and angiotensin receptor blockers (ARB), a and 9 am) at weeks 0, 8, 14, and 22. All participants con- class of medications that antagonize the angiotensin recep- tinued their usual healthy diet and exercise as recom- tor and suppress RAS, have been proposed as a novel treat- mended by their NAFLD physician and were asked not to ment for NAFLD in part not only because they decrease make major changes during the study. Patients started PAI-1 but also because they improve insulin resistance [16]. losartan or identical placebo pills at 25 mg per day for No data is available on RAS blocking medications in chil- 1 week and 50 mg for 7 weeks, and then the alternate dren with NAFLD, although losartan (an ARB) is approved therapy for 8 weeks (25 mg daily for 1 week and 50 mg for treatment of hypertension in children. daily for 7 weeks) after a 6-week washout. The primary Because no data existed for losartan in children with side effect associated with losartan is reduction of blood NAFLD, a phase 2a, proof of concept study was designed. pressure. To ensure safety, parents were provided and We completed an 8-week, randomized, double-blind, taught to use an automated blood pressure cuff for home placebo-controlled, pilot crossover trial aiming to test pre- monitoring of blood pressure. liminary efficacy and safety of losartan potassium (losar- tan, an ARB) on pediatric normotensive NAFLD patients. Laboratory measurements Concentrations of ALT and AST were measured at Methods Children’s Healthcare of Atlanta clinical laboratory. This was a randomized, double-blind, placebo-controlled, Freshly collected plasma samples were protected from crossover study (phase IIa clinical therapeutic development light and transported on ice pack for further processing trial) that aimed to establish the safety and efficacy of losar- using AU480 chemistry analyzer (Beckman Coulter, taninanewNAFLD population.Thisstudy was approved Inc.). Plasma glucose levels were measured by enzymatic by the Emory University Institutional Review Board and methods (Beckman Diagnostics, Fullerton, CA) and in- listed on Clinical Trials.gov (NCT01913470). Adolescents sulin was determined using immunoturbidometric with biopsy proven NASH, who failed to normalize liver methods (Sekisui Diagnostics, Exton, PA). HOMA-IR, enzymes with conventional therapy (attempted lifestyle the homeostatic model assessment for insulin resistance changes including healthier diet and physical activity), were index, was calculated as fasting glucose (mg/dl) × insulin consented and assessed for enrollment eligibility at a (mU/L)/405. Plasma PAI-1 concentrations were mea- screening visit. The liver biopsies were obtained during rou- sured in duplicate using a commercially available tine clinical care and assessed by one of several hospital pa- enzyme-linked immunosorbent assay kit (Catalog# thologists. The inclusion criteria were age 11–19 years at KHC3071) from Thermo Fisher Scientific (Waltham, enrollment; body weight ≥ 62.5 kg; BMI > 85th for age and MA), according to the manufacturer’s instruction. gender; history of definite or borderline NASH based upon histology using NASH clinical research network (CRN) cri- Statistical analysis teria [17]; ALT ≥ 3 times normal (69 U/L for girls, 78 U/L Because this was a feasibility study, the sample size was for boys) at enrollment; and at least 2 months of attempted modeled after other pilot pediatric NAFLD studies lifestyle changes after liver biopsy. Exclusion criteria were [18, 19]. A well-powered trial of ALT in pediatric NAFLD history of cirrhosis and liver synthetic dysfunction (inter- typically requires > 70 patients per arm, and given the lack national normalized ratio (INR) ≥ 1.5); history of of preliminary and safety data for losartan in children with hypotension; diagnosis of diabetes (or fasting glucose NAFLD, we selected to include 12 subjects and to exam- > 125 mg/dl); renal insufficiency (glomerular filtration rate ine safety, tolerability and preliminary efficacy. The pri- (GFR) < 30); any other chronic disease requiring daily medi- mary outcome was change in ALT level. cation (except medications for acid reflux, allergies or After the study was completed, we tested for carryover asthma); acute illness within past 2 weeks prior to enroll- and crossover effects for both outcomes ALT and AST. ment (fever > 100.4 °F); and any anti-oxidant therapy or Significance of the carryover effect was tested at the 0.1 supplements within past 4 weeks before enrollment. level, and analyses were conducted using SAS v.9.4 After a 4-week stabilization period, subjects were (Cary, NC). Critically, an unexpected, significant carry- randomized to losartan or placebo for 8 weeks followed by over effect of losartan was detected in ALT, the primary a 6-week washout and then the alternate therapy for outcome, meaning that participants did not return their 8 weeks using a randomized code prepared by the statisti- ALT concentrations to baseline levels. Because of the cian and placed in sealed envelopes. Participants, investiga- carryover of improvement into the second treatment tors, and coordinators were blinded to the intervention period, it was determined to be statistically incorrect to throughout the study, and the pills appeared identical. Par- compare combined treatment groups as previously ticipants’ study visits were at weeks 4, 8, 12, 14, 22, and 28 planned in the study design. Raw data at the individual for safety monitoring as well as intermediate efficacy. patient level is provided. Vos et al. Pilot and Feasibility Studies (2018) 4:109 Page 3 of 5 Table 1 Descriptive variables (N =9) points (Table 1). Enrollment continued from 2013 to 2015. Data from these participants was used if “pre” and Parameters Mean (SD) “post” values of a period treatment were available. Age (years) 13.8 (1.99) Sixty-seven percent of subjects were Hispanic with mean Male (n, %) 5 (55.6) age of 14 years and BMI z-score of 2.32. No significant Hispanics (n, %) 7 (77.8) weight or BMI change was observed in the losartan vs. BMI z-score 2.25 (0.43) placebo time periods during the study (p > 0.05). There SBP (mm Hg) 121 (10.7) were no serious adverse events reported and adverse DBP (mm Hg) 73.9 (5.90) events were balanced between treatment periods. Carryover effect analysis showed that participants did ALT (U/L) 90.0 (61.0) not return to baseline after the first 8-week period of AST (U/L) 55.3 (12.2) treatment for the primary measured outcome, ALT con- GGT (U/L) 37.7 (16.0) centration. Participants that were initially randomized to HOMA-IR 10.1 (23.5) losartan continued to decline even after they crossed Triglyceride (mg/dl) 83.0 (28.5) over to the placebo treatment, while the majority of par- HDL (mg/dl) 44.5 (4.39) ticipants initially randomized to placebo had higher ALT at the start of the second treatment (week 14, losartan) PAI-1 (ng/ml) 5.56 (2.01) than at baseline. No changes in blood pressure were ob- NASH (%) 6 (66.7%) served within and between losartan and placebo periods Values are median (IQR) for the duration of the study. The systolic blood pres- Results sures were 122.8 ± 11.6 mmHg and 117.0 ± 16.7 mmHg Twelve children were enrolled in the study; however, pre- and post-losartan period and 120.1 ± 8.7 mmHg data from ten participants was available for the analysis. and 121.6 ± 5.4 mmHg pre- and post-placebo period, Three participants withdrew from the study due to non- respectively. The diastolic blood pressures were 71.6 compliance (n = 1), self-withdrawal (n = 1), and loss to ± 7.8 mmHg and 71.4 ± 10.4 mmHg, pre- and follow-up (n = 1). Nine subjects completed all time post-losartan period, and 70.3 ± 6.4 and 71.1 ± 6.1 pre- Fig. 1 a–d Concentrations of liver enzymes, PAI-1 and HOMA-IR pre- and post-treatment periods Vos et al. Pilot and Feasibility Studies (2018) 4:109 Page 4 of 5 and post-placebo period. Individual changes in ALT, AST serious adverse events reported and stable blood pres- and HOMA-IR and PAI-1 over 8 weeks of losartan and sure throughout the study. We found preliminary trends placebo periods are shown in Fig. 1a–d. More participants of improved ALT and insulin resistance by losartan sup- decreased ALT while on losartan as compared to while on porting further testing of losartan for pediatric NAFLD placebo (89% [8 out 9] vs. 56% [5 out of 9], respectively). in a larger, randomized controlled trial with parallel For AST, 78% of participants (7 out of 9) had decreased groups. AST concentrations by week 8 while on losartan, while in Abbreviations the placebo period 44% (4 out of 9) had decreased AST by ALT: Alanine aminotransferase; ARB: Angiotensin receptor blockers; week 8 of treatment. At week 8 of the losartan period, AST: Aspartate aminotransferase; CRN: Clinical research network; most participants showed improvement in insulin resist- CVD: Cardiovascular disease; GFR: Glomerular filtration rate; HDL: High-density lipoprotein; HOMA-IR: Homeostatic model assessment insulin resistance; ance, as measured by HOMA-IR (78% [7 out of 9]) while INR: International normalized ratio; NAFLD: Nonalcoholic fatty liver disease; after the placebo period this number dropped to 44% NASH: Nonalcoholic steatohepatitis; PAI-1: Plasminogen activator inhibitor-1; (4 out of 9). No difference was seen in PAI-1 from baseline RAS: Renin-angiotensin system to end of each treatment (Fig. 1d). Acknowledgements We appreciate the patients and families who volunteered and participated in this study. Discussion This is a brief report of an early phase clinical trial test- Funding ing preliminary efficacy and safety and evaluating other The study was funded, in part, from grants from the National Institutes of biologic response variables. Losartan was found to be NIH R03 DK096157 (Vos) and NIH K23 DK080953-05 (Vos) and the National Center for Advancing Translational Sciences of the National Institutes of well tolerated by pediatric NAFLD participants, despite Health under Award Number UL1TR000454. The content is solely the respon- their normotensive state at baseline. We saw a promising sibility of the authors and does not necessarily represent the official views of trend in improvement of both insulin resistance and the National Institutes of Health. liver enzymes. Interestingly, there was no change in Availability of data and materials PAI-1 concentrations, despite this being an expected The datasets from the study are available from the corresponding author on effect of losartan. Our findings of improved insulin re- reasonable request. sistance in this pediatric cohort are in accordance with previous studies in adults in which treatment with losar- Authors’ contributions MBV contributed to the conception and design of study, interpretation of tan reduces insulin resistance.[20] Losartan treatment is the data of the work and drafting of the manuscript. RJ contributed to the thought to improve insulin resistance through direct data analysis, interpretation of data, drafting of the manuscript and critical effects on the RAS system [20] rather than suppression revision of the manuscript. JVK contributed to the data analysis, interpretation of data and critical revisions of the manuscript. RC contributed of PAI-1, which is consistent with our results. to the study design and data acquisition. JC contributed to the study design This was a phase 2a pilot study and therefore was and data acquisition. JF contributed to the interpretation of the data. SK intentionally small and short. We utilized a crossover de- contributed to the interpretation of the data and helped in critically revising the manuscript for important intellectual content. DSR contributed to the sign to minimize inter-subject variability and increase the study design and interpretation of the data. CM contributed to the critical study power. However, there was an unexpected carryover revisions of the manuscript, data analysis and interpretation of data. JW effect of losartan in those randomized to losartan first, des- contributed to the interpretation of the data and helped in critically revising the manuscript for important intellectual content. All authors read and pite the 6-week washout period. Previous pediatric pharma- approved the final manuscript. cokinetic studies of losartan did not show carryover effect past 2 weeks [21]. The carryover in ALT improvement from Ethics approval and consent to participate losartan over 6 weeks after going off treatment was unfor- The study was approved by Emory University Institutional Review Board and by Children’s Healthcare of Atlanta Review Board prior to initiation. tunate for the analysis of this phase 2a study, but it demon- strates strong justification for further evaluation of losartan, Consent for publication as sustained improvement in ALT and insulin resistance Is contained within the IRB approval. from a medication is a benefit to children with NAFLD. A major strength of this study is that extensive monitoring of Competing interests Dr. Vos receives grant funding and industry consulting in the area of blood potassium levels and blood pressures were carried pediatric NAFLD as follows: Dr. Miriam Vos receives research support from out throughout the study with no adverse events observed. Immuron, Shire, AMRA, Resonance Health, Target PharmaSolutions, NIH and Larger, later phase studies will be needed to determine Nutrition Science Initiative (NuSI) and consults for Intercept, Allergan, Aegerion, Shire, Immuron, Target PharmaSolutions. The other authors declare longer-term safety and efficacy of losartan. that they have no competing interests. Conclusions Publisher’sNote In summary, losartan treatment for 8 weeks was safe in Springer Nature remains neutral with regard to jurisdictional claims in a pediatric normotensive NAFLD cohort, with no published maps and institutional affiliations. Vos et al. Pilot and Feasibility Studies (2018) 4:109 Page 5 of 5 Author details 19. Dohil R, Schmeltzer S, Cabrera BL, Wang T, Durelle J, Duke KB, et al. Enteric- Division of Gastroenterology, Hepatology, and Nutrition, Department of coated cysteamine for the treatment of paediatric non-alcoholic fatty liver Pediatrics, School of Medicine, Emory University, Room W-450, 1760 disease. Aliment Pharmacol Ther. 2011;33:1036–44. Haygood Dr NE, Atlanta, GA 30322, USA. Children’s Healthcare of Atlanta, 20. Aksnes TA, Seljeflot I, Torjesen PA, Hoieggen A, Moan A, Kjeldsen SE. Atlanta, GA, USA. Department of Pediatrics, School of Medicine, Emory Improved insulin sensitivity by the angiotensin II-receptor blocker losartan is University, Atlanta, GA, USA. not explained by adipokines, inflammatory markers, or whole blood viscosity. Metab Clin Exp. 2007;56:1470–7. Received: 28 February 2018 Accepted: 25 May 2018 21. Shahinfar S, Cano F, Soffer BA, Ahmed T, Santoro EP, Zhang Z, et al. A double-blind, dose-response study of losartan in hypertensive children. Am J Hypertens. 2005;18:183–90. References 1. Welsh JA, Karpen S, Vos MB. Increasing prevalence of nonalcoholic fatty liver disease among United States adolescents, 1988–1994 to 2007–2010. J Peds. 2013;162:496–500. 2. Targher G, Byrne CD, Lonardo A, Zoppini G, Barbui C. Non-alcoholic fatty liver disease and risk of incident cardiovascular disease: a meta-analysis. J Hepatol. 2016;65:589–600. 3. Newton KP, Hou J, Crimmins NA, Lavine JE, Barlow SE, Xanthakos SA, et al. Prevalence of prediabetes and type 2 diabetes in children with nonalcoholic fatty liver disease. JAMA Pediatr. 2016;170:e161971. 4. Angulo P, Kleiner DE, Dam-Larsen S, Adams LA, Bjornsson ES, Charatcharoenwitthaya P, et al. Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease. Gastroenterology. 2015;149:389–97.e10. 5. Schwimmer JB, Pardee PE, Lavine JE, Blumkin AK, Cook S. Cardiovascular risk factors and the metabolic syndrome in pediatric nonalcoholic fatty liver disease. Circulation. 2008;118:277–83. 6. Fraser A, Longnecker MP, Lawlor DA. Prevalence of elevated alanine aminotransferase among US adolescents and associated factors: NHANES 1999-2004. Gastroenterology. 2007;133:1814–20. 7. Vos MB, Abrams SH, Barlow SE, Caprio S, Daniels SR, Kohli R, et al. NASPGHAN clinical practice guideline for the diagnosis and treatment of nonalcoholic fatty liver disease in children: recommendations from the expert committee on NAFLD (ECON) and the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN). J Pediatr Gastroenterol Nutr. 2017;64:319–34. 8. Cesari M, Pahor M, Incalzi RA. Plasminogen activator inhibitor-1 (PAI-1): a key factor linking fibrinolysis and age-related subclinical and clinical conditions. Cardiovasc Ther. 2010;28:e72–91. 9. Thuy S, Ladurner R, Volynets V, Wagner S, Strahl S, Konigsrainer A, et al. Nonalcoholic fatty liver disease in humans is associated with increased plasma endotoxin and plasminogen activator inhibitor 1 concentrations and with fructose intake. J Nutr. 2008;138:1452–5. 10. Alisi A, Manco M, Devito R, Piemonte F, Nobili V. Endotoxin and plasminogen activator inhibitor-1 serum levels associated with nonalcoholic steatohepatitis in children. J Pediatr Gastroenterol Nutr. 2010;50:645–9. 11. Holzberg JR, Jin R, Le NA, Ziegler TR, Brunt EM, McClain CJ, et al. PAI-1 predicts quantity of hepatic steatosis independent of insulin resistance and body weight. J Pediatr Gastroenterol Nutr. 2015;62(6):819–23. 12. Verrijken A, Francque S, Mertens I, Prawitt J, Caron S, Hubens G, Van Marck E, Staels B, Michielsen P, Van Gaal L. Prothrombotic factors in histologically proven nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Hepatology (Baltimore, MDMd). 2014;59:121–9. 13. Jin R, Krasinskas A, Le NA, Konomi JV, Holzberg J, Romero R, et al. Association between plasminogen activator inhibitor-1 and severity of liver injury and cardiovascular risk in children with non-alcoholic fatty liver disease. Pediatr Obes. 2016;13(1):23–9. 14. Matthew Morris E, Fletcher JA, Thyfault JP, Rector RS. The role of angiotensin II in nonalcoholic steatohepatitis. Mol Cell Endocrinol. 2013; 378:29–40. 15. Rosselli MS, Burgueno AL, Carabelli J, Schuman M, Pirola CJ, Sookoian S. Losartan reduces liver expression of plasminogen activator inhibitor-1 (PAI- 1) in a high fat-induced rat nonalcoholic fatty liver disease model. Atherosclerosis. 2009;206:119–26. 16. Lee MH, Song HK, Ko GJ, Kang YS, Han SY, Han KH, et al. Angiotensin receptor blockers improve insulin resistance in type 2 diabetic rats by modulating adipose tissue. Kidney Int. 2008;74:890–900. 17. Kleiner DE, Brunt EM. Nonalcoholic fatty liver disease: pathologic patterns and biopsy evaluation in clinical research. Semin Liver Dis. 2012;32:3–13. 18. Lavine JE. Vitamin E treatment of nonalcoholic steatohepatitis in children: a pilot study. J Pediatr. 2000;136:734–8. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Pilot and Feasibility Studies Springer Journals

A randomized, controlled, crossover pilot study of losartan for pediatric nonalcoholic fatty liver disease

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Medicine & Public Health; Medicine/Public Health, general; Biomedicine, general; Statistics for Life Sciences, Medicine, Health Sciences
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Abstract

Background: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in children, and currently, there are no FDA-approved therapies. Plasminogen activator inhibitor-1 (PAI-1) is elevated in children with NAFLD and associated with increased disease severity. Losartan potassium (losartan) is an angiotensin II receptor blocker (ARB) that reduces PAI-1 production and improves insulin sensitivity that has been proposed as a treatment for pediatric NAFLD but has not previously been tested. Methods: This was an 8-week randomized, double-blind, placebo-controlled, phase 2a, crossover study (with a 6- week washout between conditions) for safety and preliminary efficacy of losartan 50 mg a day taken orally in 12 normotensive children with biopsy proven nonalcoholic steatohepatitis (NASH). Results: Twelve children enrolled in the study, and nine completed all visits. No changes in blood pressure or serious adverse events occurred during the study. Trends in improvement in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and homeostatic model assessment insulin resistance (HOMA-IR) were seen with losartan treatment compared to the placebo time-period. More participants decreased ALT on losartan as compared to placebo (89% [8 out 9] vs. 56% [5 out of 9], respectively). Conclusions: This data provides preliminary evidence that losartan treatment is safe over 8 weeks in children with NAFLD and supports consideration of larger studies to test its efficacy. Trial registration: URL and trial identification number: https://clinicaltrials.gov/show/NCT01913470, NCT01913470. Date registered: August 1, 2013. Keywords: Children, Treatment, Pilot, Fatty liver disease, Plasminogen activator inhibitor-1, Insulin resistance Background (CVD) such as increased triglycerides, low high-density Nonalcoholic fatty liver disease (NAFLD) is the leading lipoprotein (HDL), and post-prandial lipemia [5, 6]. cause of liver disease in children [1]. The clinical import- Current treatment recommendations include lifestyle ance of NAFLD extends beyond liver injury to include changes, including attempting to improve diet and increased cardiovascular disease [2], type 2 diabetes [3], increase physical activity; however, lifestyle changes and increased overall mortality [4]. Our work and others typically do not achieve resolution of NAFLD [7]. have demonstrated that adolescents with NAFLD have Plasminogen activator inhibitor-1 (PAI-1) is an increased traditional markers of cardiovascular disease acute-phase protein which increases in states of insulin resistance, inflammation, and injury [8] and is elevated in both adults and children with hepatic steatosis [9–11]. * Correspondence: mvos@emory.edu Furthermore, PAI-1 correlates with fibrosis stage [12, 13]. Division of Gastroenterology, Hepatology, and Nutrition, Department of The renin-angiotensin system (RAS) has been suggested Pediatrics, School of Medicine, Emory University, Room W-450, 1760 Haygood Dr NE, Atlanta, GA 30322, USA to be involved in liver damage pathways and might play a Children’s Healthcare of Atlanta, Atlanta, GA, USA critical role in the pathogenesis of NAFLD [14]. Blockage Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Vos et al. Pilot and Feasibility Studies (2018) 4:109 Page 2 of 5 of the RAS significantly inhibits the expression of PAI-1 in Fasting blood samples were collected (typically between 7 the liver [15], and angiotensin receptor blockers (ARB), a and 9 am) at weeks 0, 8, 14, and 22. All participants con- class of medications that antagonize the angiotensin recep- tinued their usual healthy diet and exercise as recom- tor and suppress RAS, have been proposed as a novel treat- mended by their NAFLD physician and were asked not to ment for NAFLD in part not only because they decrease make major changes during the study. Patients started PAI-1 but also because they improve insulin resistance [16]. losartan or identical placebo pills at 25 mg per day for No data is available on RAS blocking medications in chil- 1 week and 50 mg for 7 weeks, and then the alternate dren with NAFLD, although losartan (an ARB) is approved therapy for 8 weeks (25 mg daily for 1 week and 50 mg for treatment of hypertension in children. daily for 7 weeks) after a 6-week washout. The primary Because no data existed for losartan in children with side effect associated with losartan is reduction of blood NAFLD, a phase 2a, proof of concept study was designed. pressure. To ensure safety, parents were provided and We completed an 8-week, randomized, double-blind, taught to use an automated blood pressure cuff for home placebo-controlled, pilot crossover trial aiming to test pre- monitoring of blood pressure. liminary efficacy and safety of losartan potassium (losar- tan, an ARB) on pediatric normotensive NAFLD patients. Laboratory measurements Concentrations of ALT and AST were measured at Methods Children’s Healthcare of Atlanta clinical laboratory. This was a randomized, double-blind, placebo-controlled, Freshly collected plasma samples were protected from crossover study (phase IIa clinical therapeutic development light and transported on ice pack for further processing trial) that aimed to establish the safety and efficacy of losar- using AU480 chemistry analyzer (Beckman Coulter, taninanewNAFLD population.Thisstudy was approved Inc.). Plasma glucose levels were measured by enzymatic by the Emory University Institutional Review Board and methods (Beckman Diagnostics, Fullerton, CA) and in- listed on Clinical Trials.gov (NCT01913470). Adolescents sulin was determined using immunoturbidometric with biopsy proven NASH, who failed to normalize liver methods (Sekisui Diagnostics, Exton, PA). HOMA-IR, enzymes with conventional therapy (attempted lifestyle the homeostatic model assessment for insulin resistance changes including healthier diet and physical activity), were index, was calculated as fasting glucose (mg/dl) × insulin consented and assessed for enrollment eligibility at a (mU/L)/405. Plasma PAI-1 concentrations were mea- screening visit. The liver biopsies were obtained during rou- sured in duplicate using a commercially available tine clinical care and assessed by one of several hospital pa- enzyme-linked immunosorbent assay kit (Catalog# thologists. The inclusion criteria were age 11–19 years at KHC3071) from Thermo Fisher Scientific (Waltham, enrollment; body weight ≥ 62.5 kg; BMI > 85th for age and MA), according to the manufacturer’s instruction. gender; history of definite or borderline NASH based upon histology using NASH clinical research network (CRN) cri- Statistical analysis teria [17]; ALT ≥ 3 times normal (69 U/L for girls, 78 U/L Because this was a feasibility study, the sample size was for boys) at enrollment; and at least 2 months of attempted modeled after other pilot pediatric NAFLD studies lifestyle changes after liver biopsy. Exclusion criteria were [18, 19]. A well-powered trial of ALT in pediatric NAFLD history of cirrhosis and liver synthetic dysfunction (inter- typically requires > 70 patients per arm, and given the lack national normalized ratio (INR) ≥ 1.5); history of of preliminary and safety data for losartan in children with hypotension; diagnosis of diabetes (or fasting glucose NAFLD, we selected to include 12 subjects and to exam- > 125 mg/dl); renal insufficiency (glomerular filtration rate ine safety, tolerability and preliminary efficacy. The pri- (GFR) < 30); any other chronic disease requiring daily medi- mary outcome was change in ALT level. cation (except medications for acid reflux, allergies or After the study was completed, we tested for carryover asthma); acute illness within past 2 weeks prior to enroll- and crossover effects for both outcomes ALT and AST. ment (fever > 100.4 °F); and any anti-oxidant therapy or Significance of the carryover effect was tested at the 0.1 supplements within past 4 weeks before enrollment. level, and analyses were conducted using SAS v.9.4 After a 4-week stabilization period, subjects were (Cary, NC). Critically, an unexpected, significant carry- randomized to losartan or placebo for 8 weeks followed by over effect of losartan was detected in ALT, the primary a 6-week washout and then the alternate therapy for outcome, meaning that participants did not return their 8 weeks using a randomized code prepared by the statisti- ALT concentrations to baseline levels. Because of the cian and placed in sealed envelopes. Participants, investiga- carryover of improvement into the second treatment tors, and coordinators were blinded to the intervention period, it was determined to be statistically incorrect to throughout the study, and the pills appeared identical. Par- compare combined treatment groups as previously ticipants’ study visits were at weeks 4, 8, 12, 14, 22, and 28 planned in the study design. Raw data at the individual for safety monitoring as well as intermediate efficacy. patient level is provided. Vos et al. Pilot and Feasibility Studies (2018) 4:109 Page 3 of 5 Table 1 Descriptive variables (N =9) points (Table 1). Enrollment continued from 2013 to 2015. Data from these participants was used if “pre” and Parameters Mean (SD) “post” values of a period treatment were available. Age (years) 13.8 (1.99) Sixty-seven percent of subjects were Hispanic with mean Male (n, %) 5 (55.6) age of 14 years and BMI z-score of 2.32. No significant Hispanics (n, %) 7 (77.8) weight or BMI change was observed in the losartan vs. BMI z-score 2.25 (0.43) placebo time periods during the study (p > 0.05). There SBP (mm Hg) 121 (10.7) were no serious adverse events reported and adverse DBP (mm Hg) 73.9 (5.90) events were balanced between treatment periods. Carryover effect analysis showed that participants did ALT (U/L) 90.0 (61.0) not return to baseline after the first 8-week period of AST (U/L) 55.3 (12.2) treatment for the primary measured outcome, ALT con- GGT (U/L) 37.7 (16.0) centration. Participants that were initially randomized to HOMA-IR 10.1 (23.5) losartan continued to decline even after they crossed Triglyceride (mg/dl) 83.0 (28.5) over to the placebo treatment, while the majority of par- HDL (mg/dl) 44.5 (4.39) ticipants initially randomized to placebo had higher ALT at the start of the second treatment (week 14, losartan) PAI-1 (ng/ml) 5.56 (2.01) than at baseline. No changes in blood pressure were ob- NASH (%) 6 (66.7%) served within and between losartan and placebo periods Values are median (IQR) for the duration of the study. The systolic blood pres- Results sures were 122.8 ± 11.6 mmHg and 117.0 ± 16.7 mmHg Twelve children were enrolled in the study; however, pre- and post-losartan period and 120.1 ± 8.7 mmHg data from ten participants was available for the analysis. and 121.6 ± 5.4 mmHg pre- and post-placebo period, Three participants withdrew from the study due to non- respectively. The diastolic blood pressures were 71.6 compliance (n = 1), self-withdrawal (n = 1), and loss to ± 7.8 mmHg and 71.4 ± 10.4 mmHg, pre- and follow-up (n = 1). Nine subjects completed all time post-losartan period, and 70.3 ± 6.4 and 71.1 ± 6.1 pre- Fig. 1 a–d Concentrations of liver enzymes, PAI-1 and HOMA-IR pre- and post-treatment periods Vos et al. Pilot and Feasibility Studies (2018) 4:109 Page 4 of 5 and post-placebo period. Individual changes in ALT, AST serious adverse events reported and stable blood pres- and HOMA-IR and PAI-1 over 8 weeks of losartan and sure throughout the study. We found preliminary trends placebo periods are shown in Fig. 1a–d. More participants of improved ALT and insulin resistance by losartan sup- decreased ALT while on losartan as compared to while on porting further testing of losartan for pediatric NAFLD placebo (89% [8 out 9] vs. 56% [5 out of 9], respectively). in a larger, randomized controlled trial with parallel For AST, 78% of participants (7 out of 9) had decreased groups. AST concentrations by week 8 while on losartan, while in Abbreviations the placebo period 44% (4 out of 9) had decreased AST by ALT: Alanine aminotransferase; ARB: Angiotensin receptor blockers; week 8 of treatment. At week 8 of the losartan period, AST: Aspartate aminotransferase; CRN: Clinical research network; most participants showed improvement in insulin resist- CVD: Cardiovascular disease; GFR: Glomerular filtration rate; HDL: High-density lipoprotein; HOMA-IR: Homeostatic model assessment insulin resistance; ance, as measured by HOMA-IR (78% [7 out of 9]) while INR: International normalized ratio; NAFLD: Nonalcoholic fatty liver disease; after the placebo period this number dropped to 44% NASH: Nonalcoholic steatohepatitis; PAI-1: Plasminogen activator inhibitor-1; (4 out of 9). No difference was seen in PAI-1 from baseline RAS: Renin-angiotensin system to end of each treatment (Fig. 1d). Acknowledgements We appreciate the patients and families who volunteered and participated in this study. Discussion This is a brief report of an early phase clinical trial test- Funding ing preliminary efficacy and safety and evaluating other The study was funded, in part, from grants from the National Institutes of biologic response variables. Losartan was found to be NIH R03 DK096157 (Vos) and NIH K23 DK080953-05 (Vos) and the National Center for Advancing Translational Sciences of the National Institutes of well tolerated by pediatric NAFLD participants, despite Health under Award Number UL1TR000454. The content is solely the respon- their normotensive state at baseline. We saw a promising sibility of the authors and does not necessarily represent the official views of trend in improvement of both insulin resistance and the National Institutes of Health. liver enzymes. Interestingly, there was no change in Availability of data and materials PAI-1 concentrations, despite this being an expected The datasets from the study are available from the corresponding author on effect of losartan. Our findings of improved insulin re- reasonable request. sistance in this pediatric cohort are in accordance with previous studies in adults in which treatment with losar- Authors’ contributions MBV contributed to the conception and design of study, interpretation of tan reduces insulin resistance.[20] Losartan treatment is the data of the work and drafting of the manuscript. RJ contributed to the thought to improve insulin resistance through direct data analysis, interpretation of data, drafting of the manuscript and critical effects on the RAS system [20] rather than suppression revision of the manuscript. JVK contributed to the data analysis, interpretation of data and critical revisions of the manuscript. RC contributed of PAI-1, which is consistent with our results. to the study design and data acquisition. JC contributed to the study design This was a phase 2a pilot study and therefore was and data acquisition. JF contributed to the interpretation of the data. SK intentionally small and short. We utilized a crossover de- contributed to the interpretation of the data and helped in critically revising the manuscript for important intellectual content. DSR contributed to the sign to minimize inter-subject variability and increase the study design and interpretation of the data. CM contributed to the critical study power. However, there was an unexpected carryover revisions of the manuscript, data analysis and interpretation of data. JW effect of losartan in those randomized to losartan first, des- contributed to the interpretation of the data and helped in critically revising the manuscript for important intellectual content. All authors read and pite the 6-week washout period. Previous pediatric pharma- approved the final manuscript. cokinetic studies of losartan did not show carryover effect past 2 weeks [21]. The carryover in ALT improvement from Ethics approval and consent to participate losartan over 6 weeks after going off treatment was unfor- The study was approved by Emory University Institutional Review Board and by Children’s Healthcare of Atlanta Review Board prior to initiation. tunate for the analysis of this phase 2a study, but it demon- strates strong justification for further evaluation of losartan, Consent for publication as sustained improvement in ALT and insulin resistance Is contained within the IRB approval. from a medication is a benefit to children with NAFLD. A major strength of this study is that extensive monitoring of Competing interests Dr. Vos receives grant funding and industry consulting in the area of blood potassium levels and blood pressures were carried pediatric NAFLD as follows: Dr. Miriam Vos receives research support from out throughout the study with no adverse events observed. Immuron, Shire, AMRA, Resonance Health, Target PharmaSolutions, NIH and Larger, later phase studies will be needed to determine Nutrition Science Initiative (NuSI) and consults for Intercept, Allergan, Aegerion, Shire, Immuron, Target PharmaSolutions. The other authors declare longer-term safety and efficacy of losartan. that they have no competing interests. Conclusions Publisher’sNote In summary, losartan treatment for 8 weeks was safe in Springer Nature remains neutral with regard to jurisdictional claims in a pediatric normotensive NAFLD cohort, with no published maps and institutional affiliations. Vos et al. Pilot and Feasibility Studies (2018) 4:109 Page 5 of 5 Author details 19. Dohil R, Schmeltzer S, Cabrera BL, Wang T, Durelle J, Duke KB, et al. Enteric- Division of Gastroenterology, Hepatology, and Nutrition, Department of coated cysteamine for the treatment of paediatric non-alcoholic fatty liver Pediatrics, School of Medicine, Emory University, Room W-450, 1760 disease. Aliment Pharmacol Ther. 2011;33:1036–44. Haygood Dr NE, Atlanta, GA 30322, USA. Children’s Healthcare of Atlanta, 20. Aksnes TA, Seljeflot I, Torjesen PA, Hoieggen A, Moan A, Kjeldsen SE. Atlanta, GA, USA. Department of Pediatrics, School of Medicine, Emory Improved insulin sensitivity by the angiotensin II-receptor blocker losartan is University, Atlanta, GA, USA. not explained by adipokines, inflammatory markers, or whole blood viscosity. Metab Clin Exp. 2007;56:1470–7. Received: 28 February 2018 Accepted: 25 May 2018 21. Shahinfar S, Cano F, Soffer BA, Ahmed T, Santoro EP, Zhang Z, et al. A double-blind, dose-response study of losartan in hypertensive children. Am J Hypertens. 2005;18:183–90. References 1. Welsh JA, Karpen S, Vos MB. Increasing prevalence of nonalcoholic fatty liver disease among United States adolescents, 1988–1994 to 2007–2010. J Peds. 2013;162:496–500. 2. Targher G, Byrne CD, Lonardo A, Zoppini G, Barbui C. Non-alcoholic fatty liver disease and risk of incident cardiovascular disease: a meta-analysis. J Hepatol. 2016;65:589–600. 3. Newton KP, Hou J, Crimmins NA, Lavine JE, Barlow SE, Xanthakos SA, et al. Prevalence of prediabetes and type 2 diabetes in children with nonalcoholic fatty liver disease. JAMA Pediatr. 2016;170:e161971. 4. Angulo P, Kleiner DE, Dam-Larsen S, Adams LA, Bjornsson ES, Charatcharoenwitthaya P, et al. Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease. Gastroenterology. 2015;149:389–97.e10. 5. Schwimmer JB, Pardee PE, Lavine JE, Blumkin AK, Cook S. Cardiovascular risk factors and the metabolic syndrome in pediatric nonalcoholic fatty liver disease. Circulation. 2008;118:277–83. 6. Fraser A, Longnecker MP, Lawlor DA. Prevalence of elevated alanine aminotransferase among US adolescents and associated factors: NHANES 1999-2004. Gastroenterology. 2007;133:1814–20. 7. Vos MB, Abrams SH, Barlow SE, Caprio S, Daniels SR, Kohli R, et al. NASPGHAN clinical practice guideline for the diagnosis and treatment of nonalcoholic fatty liver disease in children: recommendations from the expert committee on NAFLD (ECON) and the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN). J Pediatr Gastroenterol Nutr. 2017;64:319–34. 8. Cesari M, Pahor M, Incalzi RA. Plasminogen activator inhibitor-1 (PAI-1): a key factor linking fibrinolysis and age-related subclinical and clinical conditions. Cardiovasc Ther. 2010;28:e72–91. 9. Thuy S, Ladurner R, Volynets V, Wagner S, Strahl S, Konigsrainer A, et al. Nonalcoholic fatty liver disease in humans is associated with increased plasma endotoxin and plasminogen activator inhibitor 1 concentrations and with fructose intake. J Nutr. 2008;138:1452–5. 10. Alisi A, Manco M, Devito R, Piemonte F, Nobili V. Endotoxin and plasminogen activator inhibitor-1 serum levels associated with nonalcoholic steatohepatitis in children. J Pediatr Gastroenterol Nutr. 2010;50:645–9. 11. Holzberg JR, Jin R, Le NA, Ziegler TR, Brunt EM, McClain CJ, et al. PAI-1 predicts quantity of hepatic steatosis independent of insulin resistance and body weight. J Pediatr Gastroenterol Nutr. 2015;62(6):819–23. 12. Verrijken A, Francque S, Mertens I, Prawitt J, Caron S, Hubens G, Van Marck E, Staels B, Michielsen P, Van Gaal L. Prothrombotic factors in histologically proven nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Hepatology (Baltimore, MDMd). 2014;59:121–9. 13. Jin R, Krasinskas A, Le NA, Konomi JV, Holzberg J, Romero R, et al. Association between plasminogen activator inhibitor-1 and severity of liver injury and cardiovascular risk in children with non-alcoholic fatty liver disease. Pediatr Obes. 2016;13(1):23–9. 14. Matthew Morris E, Fletcher JA, Thyfault JP, Rector RS. The role of angiotensin II in nonalcoholic steatohepatitis. Mol Cell Endocrinol. 2013; 378:29–40. 15. Rosselli MS, Burgueno AL, Carabelli J, Schuman M, Pirola CJ, Sookoian S. Losartan reduces liver expression of plasminogen activator inhibitor-1 (PAI- 1) in a high fat-induced rat nonalcoholic fatty liver disease model. Atherosclerosis. 2009;206:119–26. 16. Lee MH, Song HK, Ko GJ, Kang YS, Han SY, Han KH, et al. Angiotensin receptor blockers improve insulin resistance in type 2 diabetic rats by modulating adipose tissue. Kidney Int. 2008;74:890–900. 17. Kleiner DE, Brunt EM. Nonalcoholic fatty liver disease: pathologic patterns and biopsy evaluation in clinical research. 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Pilot and Feasibility StudiesSpringer Journals

Published: Jun 5, 2018

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