Hyperinsulinemic hypoglycemia, a major cause of severe hypoglycemia

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1 brief report Sirolimus Therapy in Infants with Severe Hyperinsulinemic Hypoglycemia Senthil Senniappan, M.D., Sanda Alexandrescu, M.D., Nina Tatevian, M.D., Pratik Shah, M.D., Ved Arya, M.D., Sarah Flanagan, Ph.D., Sian Ellard, Ph.D., Dyanne Rampling, F.I.B.M.S., Michael Ashworth, M.D., Robert E. Brown, M.D., and Khalid Hussain, M.D. SUMMARY Hyperinsulinemic hypoglycemia is the most common cause of severe, persistent neonatal hypoglycemia. The treatment of hyperinsulinemic hypoglycemia that is unresponsive to diazoxide is subtotal pancreatectomy. We examined the effectiveness of the mammalian target of rapamycin (mtor) inhibitor sirolimus in four infants with severe hyperinsulinemic hypoglycemia that had been unresponsive to maximal doses of diazoxide (20 mg per kilogram of body weight per day) and octreotide (35 μg per kilogram per day). All the patients had a clear glycemic response to sirolimus, although one patient required a small dose of octreotide to maintain normoglycemia. There were no major adverse events during 1 year of follow-up. Hyperinsulinemic hypoglycemia, a major cause of severe hypoglycemia during the neonatal period, is characterized by inappropriate insulin secretion from pancreatic beta cells in the presence of low blood glucose levels. 1 The condition may result from defects in key genes involved in the regulation of insulin secretion from beta cells, including ABCC8, KCNJ11, GLUD1, GCK, HADH, SLC16A1, HNF4A, HNF1A, and UCP2. 1,2 Two major histologic subtypes have been described: diffuse and focal. 3 Mutations in ABCC8 and KCNJ11 are associated with severe hyperinsulinemic hypoglycemia that is unresponsive to medical treatment with diazoxide and octreotide. 1 The only treatment option currently available for patients with medically unresponsive forms of diffuse hyperinsulinemic hypoglycemia is a subtotal pancreatectomy, in which 95 to 98% of insulin-secreting cells are physically removed to alleviate the severe hypoglycemia. However, some patients who have undergone surgery continue to have recurrent hyperinsulinemic hypoglycemia, whereas diabetes mellitus and exocrine pancreatic insufficiency develop in others. In a recent study of 105 affected children who underwent pancreatectomy, 59% had persistent hyperinsulinemic hypoglycemia up to 5 years after surgery, and diabetes mellitus had developed in all the children by the time they reached early adolescence. 4 Hence, there is a need for a medical therapy that can be used as an alternative to subtotal pancreatectomy. A possible mechanism of hyperinsulinism and beta-cell hyperplasia in diffuse hyperinsulinemic hypoglycemia involves the constitutive activation of the mtor pathway. 5 The serine threonine protein kinase mtor has been implicated in the cellular response to nutrients and growth factor signaling. 6 The mtor pathway is From the Developmental Endocrinology Research Group, Clinical and Molecular Genetics Unit, Institute of Child Health, University College London (S.S., P.S., V.A., K.H.), and the Departments of Paediatric Endocrinology (S.S., P.S., V.A., K.H.) and Histopathology (D.P., M.A.), Great Ormond Street Hospital for Children, London, and the Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter (S.F., S.E.) all in the United Kingdom; the Department of Pathology, University of California, San Francisco, San Francisco (S.A.); and the Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston (N.T., R.E.B.). Address reprint requests to Dr. Hussain at the Developmental Endocrinology Research Group, Clinical and Molecular Genetics Unit, Institute of Child Health, University College London, 30 Guilford St., London WC1N 1EH, United Kingdom, or at khalid.hussain@ucl.ac.uk. N Engl J Med 2014;370: DOI: /NEJMoa Copyright 2014 Massachusetts Medical Society. n engl j med 370;12 nejm.org march 20,

2 abnormally activated in several neoplasms, including insulinoma, 7 and inhibitors of mtor have been increasingly recognized as a treatment option in patients with cancer. 8 The use of the mtor inhibitor sirolimus (formerly called rapamycin) in a patient with an insulinoma suggested a potential role for mtor inhibitors in both the reduction of beta-cell proliferation and the inhibition of insulin production. 9 In addition, therapy with everolimus, an mtor kinase inhibitor, was reported to result in a clinically significant glycemic response and regression in tumor size in four patients with metastatic insulinoma. 7 Given the potential role of the mtor pathway in the pathogenesis of hyperinsulinemic hypoglycemia and the promising use of mtor inhibitors in insulinoma, we studied the glycemic response to sirolimus in four consecutive patients with diffuse hyperinsulinemic hypoglycemia that had been unresponsive to diazoxide and octreotide. Methods Patients Four consecutive patients with severe hyperinsulinemic hypoglycemia that was unresponsive to maximal doses of diazoxide (20 mg per kilogram per day) and octreotide (35 μg per kilogram per day) were recruited to participate in the study. The characteristics of the patients are shown in Table 1. All the patients had severe symptomatic hypoglycemia (associated with seizures, poor feeding, and irritability) on day 1 of life in a local hospital, necessitating transfer to our center for further care. A central venous catheter was inserted, and high doses of dextrose and intravenous glucagon were administered to stabilize blood glucose levels at more than 63 mg per deci liter (3.5 mmol per liter). The diagnosis of hyperinsulinemic hypoglycemia was confirmed by means of standard testing (Table 2). 10 Since there had not been a response to maximal doses of diazoxide and octreotide in any of the four patients, our standard protocol would have called for subtotal pancreatectomy for all of them. We therefore recruited these infants for our study. The study of the genetic mechanisms of hypoglycemia was approved by the research ethics committee at Great Ormond Street Hospital in London. Approval for the study was also obtained from the drugs and therapeutics committee at the hospital. Detailed discussions were Table 1. Patient Characteristics and Assessment of Response to Treatment with Sirolimus.* Patient No. Sex Birth Weight Gestation Findings on 18F-DOPA PET Genetic Characteristics Age at Start of Sirolimus Treatment Treatment at Study Entry kg wk wk 1 Female Diffuse No mutation in ABCC8, KCNJ11, or HNF4A 2 Female Diffuse Maternally inherited heterozygous ABCC8 mutation p.r74w (c.220c T) 3 Male Diffuse Maternally inherited heterozygous ABCC8 mutation p.p1563t (c.4687c A) 4 Male Not performed Homozygous ABCC8 mutation (c g A) 11 Octreotide, 35 μg/kg/day 8 Octreotide, 35 μg/kg/day 7 Octreotide, 35 μg/kg/day 16 Octreotide, 35 μg/kg/day * All intravenous fluids contained glucose to maintain normoglycemia. 18F-DOPA denotes fluorine-18-l-dihydroxyphenylalanine, and PET positron-emission tomography n engl j med 370;12 nejm.org march 20, 2014

3 brief report held with the families, and the benefits and risks of the study protocol were explained, after which parents provided written informed consent. The protocol is available with the full text of this article at NEJM.org. Testing Genomic DNA was extracted with the use of standard methods. The coding regions and conserved splice sites of ABCC8, KCNJ11, and HNF4A were amplified by means of a polymerase-chainreaction assay, and the products were sequenced on an ABI 3730 capillary sequencer (Applied Biosystems). If no mutation was identified, testing for a partial or whole gene deletion was performed by means of multiplex ligation-dependent probe amplification. 11 We also tested for a recently identified deep intronic cryptic founder mutation in ABCC8. 12 To confirm diffuse disease, scanning with fluorine-18-l-dihydroxyphenylalanine (18F-DOPA) positron-emission tomography (PET) was performed in three of the four patients. The fourth patient had a homozygous ABCC8 mutation, which is strongly suggestive of diffuse disease; consequently, 18F-DOPA PET scanning was deemed unnecessary in this patient. Treatment All the patients received sirolimus at an initial dose of 0.5 mg per square meter of body-surface area per day (in one or two doses). The dose was gradually increased with the goal of reaching a serum trough level of 5 to 15 ng per milliliter. The serum trough level of sirolimus was measured every 5 days. Once the desired serum drug level had been reached and blood glucose levels were stable, intravenous glucose and glucagon infusions were gradually tapered. Regular monitoring was performed, including a complete blood count, measurement of serum lipid levels, and analysis of renal and liver function. After discharge, patients were followed up regularly for assessment of glycemic control and measurement of serum sirolimus levels. Results Molecular Genetic Testing Two infants had heterozygous maternally inherited ABCC8 mutations, p.r74w and p.p1563t. The p.r74w missense mutation has been reported in multiple patients. 13 The p.p1563t mutation is novel and affects a conserved amino acid within the nucleotide binding domain 1 (NBD1) of the Dose of Intravenous Glucose at Study Entry Response to Treatment with Sirolimus Side Effects Final Outcome mg/kg/min 10 Day 15: Intravenous fluids discontinued Day 17: Glucagon discontinued Day 20: Octreotide discontinued Mild elevation of triglyceride levels Discharged home to have oral feedings every 4 hours 9 Day 10: Intravenous fluids discontinued Day 14: Glucagon discontinued Day 20: Octreotide discontinued Transient elevation of liver aminotransferase levels Discharged home to have oral feedings every 4 hours 9 Day 10: Intravenous fluids discontinued Day 12: Glucagon discontinued Day 19: Octreotide discontinued Mild, transient elevation of cholesterol and triglyceride levels Discharged home to have ondemand oral feeding 12 Day 16: Intravenous fluids discontinued Day 19: Glucagon discontinued and octreotide subcutaneous injections continued at 10 μg/kg/day None Discharged home to have gastrostomy feedings every 4 hours n engl j med 370;12 nejm.org march 20,

4 Table 2. Clinical and Biochemical Data at Diagnosis, at Discharge, and at 24, 36, and 52 Weeks of Age. Assessment Age Duration of Treatment with Sirolimus Dose of Sirolimus Duration of Fasting Blood Glucose Level* Insulin Level NEFA Level 3β-Hydroxybutyrate Level wk mg/day hr mg/dl mu/liter mmol/liter Patient 1 Hypoglycemia screening at diagnosis 3 0 < Fasting results at discharge First follow-up assessment Second follow-up assessment < Third follow-up assessment Patient 2 Hypoglycemia screening at diagnosis 4 0 < <0.05 Fasting results at discharge < First follow-up assessment < Second follow-up assessment Third follow-up assessment < Patient 3 Hypoglycemia screening at diagnosis 2 0 < <0.05 Fasting results at discharge First follow-up assessment < Second follow-up assessment Third follow-up assessment Patient 4 Hypoglycemia screening at diagnosis 2 0 < < Fasting results at discharge First follow-up assessment < Second follow-up assessment < Third follow-up assessment * The normal range for blood glucose level is 63 to 99 mg per deciliter. To convert the values for glucose to millimoles per liter, multiply by Insulin levels during hypoglycemia should be less than 2 mu per liter. The normal range for the level of nonesterified fatty acids (NEFA) is 0.84 to 2.74 mu per liter. 10 The normal range for the level of 3β-hydroxybutyrate is 0.22 to 2.34 mmol per liter. 10 sulfonylurea receptor (SUR1) subunit of the ATPsensitive potassium (K ATP ) channel. It is predicted to be pathogenic and has been identified in the homozygous state in a patient with severe hyperinsulinemic hypoglycemia who was not involved in this study. The third infant was homozygous for an ABCC8 splicing mutation (c g A). No mutations in ABCC8, KCNJ11, or HNF4A were found in the fourth infant. No additional mutations were identified on dosage analysis of the ABCC8 gene. (Dosage analysis is used to rule out the deletion of any gene when a heterozygous mutation is identified.) Response to Sirolimus The responses of each of the four infants to treatment with sirolimus are shown in Tables 1 and 2. Owing to the severity of their hypoglycemia, the infants required a combination of glucagon infusion and intravenous fluids with a high concentration of dextrose to maintain normoglycemia at the initiation of treatment with siroli n engl j med 370;12 nejm.org march 20, 2014

5 brief report mus, after which a good glycemic response was noted. Accordingly, the doses of dextrose were gradually tapered and enteral feeding simultaneously increased. Over a period of 2 to 3 weeks, each infant maintained stable blood glucose levels without the need for intravenous glucose infusion. Glucagon and octreotide infusions were then gradually discontinued, since blood glucose levels were stable at more than 63 mg per deciliter. Subsequently, all four infants were able to receive all their nutrition enterally, and each continued to receive oral sirolimus therapy. The infants were also able to fast for 6 to 8 hours without the development of hypoglycemia (i.e., blood glucose levels remained higher than 63 mg per deciliter at the end of the fast) (Table 2). One infant (Patient 4, who had a homozygous ABCC8 mutation and very severe hyperinsulinemic hypoglycemia) required a small dose of octreotide (10 μg per kilogram per day) in order to fast for 6 hours without the development of hypoglycemia. Each patient was discharged when enteral feedings were established and the infant could fast for 6 to 8 hours without the development of hypoglycemia. The measured levels of nonesterified fatty acids and 3β-hydroxybutyrate rose at the end of the fast, suggesting that insulin suppression was present because of treatment with sirolimus (Table 2). Parents were instructed to monitor blood glucose levels at least three times a day before feeding. A review of the records of parental monitoring of blood glucose levels did not reveal any episode of hypoglycemia during treatment with sirolimus. All four patients are currently continuing to receive sirolimus and are being followed closely for assessment of glycemic control, serum trough levels of sirolimus, and any clinical or biochemical adverse events (Table 2). Follow-up assessments conducted until the infants reached 1 year of age indicated good glycemic control. Laboratory assessments have shown normal complete blood counts (without neutropenia) and normal levels of blood urea nitrogen, creatinine, and electrolytes during the follow-up assessments every 3 months. The side effects observed included transient elevation of aminotransferase levels, which resolved spontaneously, and mild elevation of triglyceride levels. The results of liver function studies have otherwise been normal. Neither sepsis nor any other serious infection developed in any of the infants. Sirolimus was discontinued in one of the patients at 7 months of age; within 3 days, severe hypoglycemia developed, requiring intravenous infusion of glucose and subcutaneous administration of octreotide. Sirolimus was reinitiated, and during the next 3 to 4 weeks the glucose infusion and the octreotide were tapered and then discontinued. Discussion The management of diffuse hyperinsulinemic hypoglycemia that is unresponsive to diazoxide poses a major therapeutic challenge. The successful use of sirolimus, either alone or as an adjuvant therapy with octreotide, for severe diffuse hyperinsulinemic hypoglycemia, reported here, appears to be a potential alternative to subtotal pancreatectomy. Sirolimus therapy enabled us to discontinue intravenous infusions of dextrose and glucagon in all four patients and to halt octreotide therapy in three of the four patients. At 1 year of age, the four patients were continuing to receive sirolimus therapy and were normoglycemic, without any apparent major adverse events. Our patients had good glycemic control while receiving sirolimus a finding similar to that in adults with insulinoma who were treated with mtor inhibitors. 7,9,14,15 In one study, everolimus, with or without concomitant administration of octreotide, was shown to be effective in the treatment of pancreatic neuroendocrine tumors. 8 In another study involving three patients with insulinoma, everolimus was associated with normalization of plasma glucose levels within 14 days after initiation of treatment. 16 The authors of that study observed the effect of everolimus on glycemic control and attributed it to the decrease in insulin levels as well as to direct effects on tumor size and peripheral insulin resistance. 16 The mechanism of action of mtor inhibitors in hyperinsulinemic hypoglycemia has not been fully delineated. Although the effect of sirolimus on beta-cell mass is probably achieved through inhibition of the mtor complex 1 (mtorc1) pathway, the chronic insulin resistance induced by sirolimus is thought to be mediated through the subsequent disassembly and inactivation of mtorc2 and the down-regulation of the prosurvival protein kinase B, leading to decreased function and viability of the existing beta cells. 5,17,18 n engl j med 370;12 nejm.org march 20,

6 Functional insulin receptors have been observed on beta cells, where they mediate insulinstimulated insulin production and release in response to increases in intracellular calcium and the activation of protein kinase C. 19,20 Hence, mtor inhibition may affect the number of insulin receptors that are present on pancreatic beta cells, which would reduce insulin secretion. 21 Furthermore, the impairment of glucose-stimulated insulin secretion and proinsulin biosynthesis has been observed in islets treated with sirolimus. 22 In cultures, sirolimus has been shown to decrease the number of ductal cells, which are a potential source of islet cells, and in vivo it has been shown to alter the glucose-stimulated secretion of insulin. 23 Long-term treatment with sirolimus in recipients of renal transplants has been noted to induce peripheral insulin resistance by impairing the activation and signaling of protein kinase B through the insulin-receptor substrate pathway. 24 In a recent study that showed long-term maintenance of normoglycemia with the use of everolimus in a patient with disseminated insulinoma and severe hypoglycemia, the authors noted that the insulin and C-peptide levels remained high for several months after treatment, although the hypoglycemia resolved. 15 Hence, the authors proposed that everolimus acted predominantly by inducing insulin resistance. In another study, longterm treatment with sirolimus was shown to cause glucose intolerance by up-regulating hepatic gluconeogenesis. 25 In short, there are a number of possible mechanisms by which sirolimus could inhibit the functioning of already formed beta cells, induce their apoptosis, and promote insulin resistance. The adverse effects of mtor inhibitors such as everolimus and sirolimus include stomatitis, increased risk of infection, immunosuppression, abnormalities in renal function, fatigue, and pneumonitis. 14 Episodes of transient elevations of aminotransferase levels have been reported in adults; the levels returned to the normal range with a reduction in the dose of everolimus. 15 One of our patients had a transient elevation of liver enzyme levels that resolved spontaneously. An elevation of triglyceride levels has been observed in our patients and warrants close observation. At this time, it appears that in these patients the benefits of treatment (avoiding subtotal pancreatectomy and its associated complications) outweigh the potential risks. Nevertheless, patients with hyperinsulinemic hypoglycemia who are treated with sirolimus must be monitored on a regular basis to assess glycemic control and to watch for the occurrence of adverse events. The fact that severe hypoglycemia developed in one patient when sirolimus was discontinued indicates that the drug was affecting blood glucose levels. We hope to discontinue the use of sirolimus in these patients at the earliest possible opportunity; the timing will be dictated by the blood glucose level of each individual patient. In summary, we report a therapeutic strategy in which we used mtor inhibitors to treat patients with hyperinsulinemic hypoglycemia who otherwise would have required subtotal pancreatectomy. Treatment with mtor inhibitors, alone or in combination with somatostatin analogues, may be a feasible option for selected patients with no contraindication, although the longterm adverse effects and efficacy of such treatment require further study. Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. References 1. Senniappan S, Shanti B, James C, Hussain K. Hyperinsulinaemic hypoglycaemia: genetic mechanisms, diagnosis and management. J Inherit Metab Dis 2012;35: Stanescu DE, Hughes N, Kaplan B, Stanley CA, De León DD. Novel presentations of congenital hyperinsulinism due to mutations in the MODY genes: HNF1A and HNF4A. J Clin Endocrinol Metab 2012;97(10):E2026-E Rahier J, Guiot Y, Sempoux C. Persistent hyperinsulinaemic hypoglycaemia of infancy: a heterogeneous syndrome unrelated to nesidioblastosis. Arch Dis Child Fetal Neonatal Ed 2000;82:F108-F Beltrand J, Caquard M, Arnoux JB, et al. Glucose metabolism in 105 children and adolescents after pancreatectomy for congenital hyperinsulinism. Diabetes Care 2012;35: Alexandrescu S, Tatevian N, Olutoye O, Brown RE. Persistent hyperinsulinemic hypoglycemia of infancy: constitutive activation of the mtor pathway with associated exocrine-islet transdifferentiation and therapeutic implications. Int J Clin Exp Pathol 2010;3: Kwon G, Marshall CA, Pappan KL, Remedi MS, McDaniel ML. Signaling elements involved in the metabolic regulation of mtor by nutrients, incretins, and growth factors in islets. Diabetes 2004;53: Suppl 3:S225-S Kulke MH, Bergsland EK, Yao JC. Glycemic control in patients with insulinoma treated with everolimus. N Engl J Med 2009;360: Yao JC, Lombard-Bohas C, Baudin E, et al. Daily oral everolimus activity in patients with metastatic pancreatic neuroendocrine tumors after failure of cyto n engl j med 370;12 nejm.org march 20, 2014

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