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Therapeutic Management of Burn Induced Hyperglycemia with Insulin and Metformin Thesis The University of Texas Medical Branch Cell Biology Graduate Program By: Arham Ali, MD Mentors: Celeste C. Finnerty, PhD; David N. Herndon, MD FACS Definitions and acronyms: BA- Bone age BMC- Bone mineral content BMD- Bone mineral density BMI- Body mass index BSA- Body surface area BWI- Burn wound infection CA- Chronological age CDC- Centers for disease control and prevention CFU- Colony forming units DEXA- Dual energy X-ray absorptiometry FSR- Fractional synthetic rate (a fraction of the unbound protein pool synthesized over a unit of time) GAM- Generalized additive mixed model HEs- Hyperglycemic episodes ICU- Intensive care unit IRB- Institutional review board ISI- Insulin sensitivity index IU- International units IVGTT- Intravenous glucose tolerance test I/V- Intravenous LBM- Lean body mass OGTT- Oral glucose tolerance test OR- Operating room procedure PO- Per os (by mouth) REE- Resting energy expenditure RTF- Ready-to-feed SD- Standard deviation SEM- Standard error of the mean S/Q- Subcutaneous TBSA- Total body surface area TGC- Tight glycemic control 2 Abstract: Burn injury afflicts approximately 450,000 individuals annually in the United States. Despite efforts to decrease incidence via implementation of public awareness campaigns and drastic improvements in living conditions, nearly 11 million people worldwide required treatment at a major burn center or hospital in 2004. Sustained hypermetabolism and hypercatabolism are notorious changes associated with severe burn injury. Anchored within these pathophysiological changes is the development of hyperglycemia and insulin resistance. These perturbations in metabolism may persist for up to three years following burn injury and are wellestablished contributors to post burn morbidity and mortality. Accordingly, the implementation of tight glycemic control protocols in burn centers across the nation ensued. Insulin administration has been shown to attenuate the hyperglycemic response to burn injury. Moreover, it is thought that insulin may improve auxiliary components of hypercatabolism, indicative of insulin’s anabolic properties. However, a drawback associated with insulin administration is the development of hypoglycemia which has led to the search for alternative glucose modulating agents. Metformin, a well-known anti-hyperglycemic agent, has been shown to decrease burn induced hyperglycemia in adults. Relative euglycemia was attained by use of metformin exclusive of the adverse effects associated with insulin administration (hypoglycemia). However, the safety and efficacy of metformin in pediatric burn patients has not been investigated. Moreover, at large doses metformin is a known complex I inhibitor of the electron transport chain. Whether or not these alterations in mitochondrial respiration persist at clinically relevant dosages in pediatric burn patients remains unknown. We hypothesize that insulin attenuates hypercatabolism independent of glucose modulating effects, and that metformin will safely attenuate hyperglycemia and associated metabolic changes in severely burn injured children. We will test this hypothesis in two specific aims. By use of a prospective study design, in the first aim we will determine the clinical effects of insulin vs. no insulin therapy in children with severe burn injury. Patients will be divided into two groups: those who receive insulin to decrease serum blood glucose levels and those who did not require insulin administration. In the second aim, we will investigate the novel use of metformin or placebo in pediatric burn patients in a prospective, randomized control trial. This IRB-approved clinical trial will be the first of its kind in determining safety and efficacy profiles of metformin in pediatric burn patients. Outcomes to be collected in both aims include (but are not limited to) parameters pertaining to glucose homeostasis and insulin resistance, body composition, metabolic changes, growth, incidence of infections and sepsis, and mortality. Upon completion of this project, we will improve our understanding of glucose homeostasis and the therapeutic modulators which control the hyperglycemic response to burn injury in children. If metformin is found to safely improve clinical outcomes similar to insulin (barring hypoglycemia), we will implement the use of metformin in our pediatric patients to alleviate hyperglycemia following burn injury. 3 A. Specific Aims: Hyperglycemia and hypermetabolism are hallmarks of the pathophysiological response to burn injury. Post burn hyperglycemia can be attributed to an increased rate of glucose release in blood,1 reduced tissue extraction of glucose,2 and impaired insulin receptor signaling.3 This form of stress induced hyperglycemia may be an adaptive and protective response to thermal injury. Conversely, detrimental clinical sequelae ensue during hyperglycemia post burn in the absence of pharmacological intervention.4 Insulin is an anabolic hormone administered intravenously or by subcutaneous injection inducing widespread systemic effects described thoroughly in the literature. In burn injured patients, insulin attenuates the hyperglycemic response following thermal injury.5,6 Outcomes of tight glycemic control (TGC) in burn patients have been studied in several observational and randomized control trials.7-10 Stringent control improved body composition, decreased muscle catabolism, and reduced the incidence of sepsis, multiple organ failure, and mortality. However, despite these improvements,11 patients on insulin therapy are at an increased risk of hypoglycemia.7 Importantly, a single incident of severe hypoglycemia has been associated with an increased risk of mortality in critically ill and burn injured patients.12,13 Moreover, the metabolic changes associated with insulin resistance may persist for up to three years after initial burn injury.14 This has led to the search for alternative glucose modulating agents in the management of burn induced hyperglycemia. Metformin is a biguanide drug administered orally to manage hyperglycemia in patients with type II diabetes mellitus. Metformin has been suggested as a safe alternative in the management of post burn glucose control,15 due to a decreased incidence of hypoglycemia.16 Glucose lowering effects are attributed to a decrease in glucose production (liver) and an increase in peripheral tissue (muscle) glucose uptake.17-19 However, in terms of safety, outcomes following the use of metformin in burn patients remain unknown. Of major concern is lactic acidosis (which may or may not be attributed to mitochondrial complex I inhibition), a side effect linked to medications of the biguanide drug class, but not specific to metformin. Lactic acidosis is commonly exhibited in patients with hepatic or kidney dysfunction and tissue hypoperfusion. Nevertheless, a causal link correlating metformin and lactic acidosis remains yet to be determined.20 We hypothesize that insulin will attenuate hypercatabolism independent of glucose modulating effects, and that metformin will safely attenuate hyperglycemia and associated metabolic changes in severely burned injured children. We will address the aforementioned gaps in our understanding of glucose homeostasis in burn patients in the following specific aims in order to improve care of pediatric burn patients: Specific Aim 1: Investigate alternative clinical effects of insulin in thermally injured children. We will compare clinical outcomes of children with severe burn injury treated with or without insulin to maintain blood glucose levels <180 mg/dL. By dividing groups into ‘insulin’ and ‘no insulin’, we will determine if exogenous insulin administration attenuates hypermetabolism and/or hypercatabolism following burn injury, independent of glucose modulating effects. Specific Aim 2: Elucidate the clinical outcomes in the management of post burn hyperglycemia by placebo and metformin. We will study the use of metformin to decrease hyperglycemia in pediatric burn patients. The current standard of care includes the administration of insulin when blood glucose levels exceed 180 mg/dL (~10.0 mmol/l). This approach will be carried out via an IRB-approved randomized controlled trials implemented at Shriners Hospital for Children- Galveston. Outcomes include glucose homeostasis, wound healing, insulin resistance, hypermetabolism, body composition, organ function, incidence of infections, and mortality. By understanding whether metformin improves clinical outcomes, we may implement a new therapeutic regimen for our patient population. Our findings will help us to determine the therapeutic strategies and the mechanisms by which they act to improve clinical outcomes. If metformin is deemed safe and effective in attaining glucose homeostasis in pediatric burn patients, we will implement its use in our current therapeutic regimen. 4 B. Research Strategy Background and Significance Burn injury There have been an estimated 450,000 incidents of burn injury requiring medical treatment in the United States in 2012.21 Despite implementation of public awareness campaigns, burn injury ranks fourth amongst all injuries.22 Two characteristic features of the pathophysiological response to severe burn injury include hypermetabolism and hyperglycemia. Hypermetabolism is marked by increased cardiac output23,24 and resting energy expenditure25,26 followed by detrimental changes in body composition.24,27-29 This response to burn injury is thought to be attributed to increased endogenous catecholamines and cortisol. Unlike most other forms of trauma, these derangements in metabolic homeostasis remain persistent for up to three years post thermal injury.24,30-32 Hyperglycemia associated with burn injury Stress-induced hyperglycemia is the result of increased gluconeogenesis (hepatic) and decreased peripheral glucose uptake (particularly in muscle).33-35 It is well known that burn injury is one of the most severe forms of trauma, inciting stressinduced hyperglycemia that persists long after initial insult. The cumulative effects of catecholamines, cortisol, and glucagon have been postulated to contribute to the development of stress induced hyperglycemia.36 Investigators from our institution have previously described the relationship of these hormones and associated down-stream effects as depicted in figure 1.3 Collectively, these hormones stimulate gluconeogenesis, foster lipolysis and ketogenesis in the liver, and bolster insulin resistance. The pathophysiological increase in glucose levels serves as an energy source for glucose dependent tissues (such as the brain). This response previously led clinicians to Figure 1:3 Metabolic changes associated with burn believe that stress induced hyperglycemia should be left induced hyperglycemia. unregulated to provide energy for increased metabolic demands observed in critically ill patients.37 However, our laboratory has found in burn injured patients with elevated glucose levels, a high incidence of bacteremia/fungemia and mortality.4,7 Increased incidence of infections may be attributed to impaired immune function as a result of hyperglycemia. Elevated glucose levels alter cytokine production in macrophages resulting in suppressed bactericidal activity in leukocytes, therefore decreasing immune function.38,39 Similarly, immune function is compromised via immunoglobulin glycosylation when plasma blood glucose is >220 B mg/dL, resulting in decreased opsonic A activity (opsonization is a process in which pathogens are marked for ingestion and destruction by phagocytes).40 Alternative to derangements in immune function, investigators from our institution have found that hyperglycemia promotes protein degradation, further enhancing the catabolic response to burn injury.41,42 Furthermore, post burn hyperglycemia has been associated with decreased skin graft survival (regardless of bacterial colonization).43 Our institution has studied changes in glucose homeostasis 24 in over 970 children with burn injury and Figure 2: Following burn injury, patients demonstrate long term fasting hyperglycemia (A) and increased fasting insulin plasma levels (B), signifying insulin resistance (n=970). compared them to non-burn, healthy Bars indicate mean ± SEM. Asterisks indicate statistical significance (p<0.05) compared children (figure 2).24 The results to non-burned controls. 5 demonstrate that aberrations in glucose homeostasis (fasting hyperglycemia and hyperinsulinemia) arise shortly after burn injury, and that insulin resistance persists for up to three years later. Sustained long term hyperglycemia is of great concern as it can impact morbidity and mortality. Figure 3 summarizes the clinical consequences of hyperglycemia following burn injury.4,9,35,40,41,43-45 Interestingly, similar outcomes are also observed in patients with diabetes mellitus, a condition in which patients exhibit impaired insulin function leading to hyperglycemia if left untreated. The subsequent consequences of hyperglycemia have led to the implementation of stringent glucose control regimens in intensive care units across the nation. Notably, large fluctuations in blood glucose of thermally injured patients serve as a precursor for sepsis and mortality.46 Currently, a conclusive target glucose range in pediatric burn patients has not been identified. Proposed target glucose ranges include 80-110 mg/dL7, 90-120 mg/dL5, and 100Figure 3: Hyperglycemia following burn injury augments whole body stress including: increased susceptibility to infections 140 mg/dL9 to name a few. Thus, reaching a consensus attributed to decreased immune function, impaired wound healing, on the safest glucose range for critically ill patients and decreased muscle catabolism. BWI= Burn wound infection. remains yet to be defined.47 Tight glycemic control The practice of maintaining tight glycemic control (TGC) in the intensive care unit (ICU) has been a subject of debate over the past decade. Van den Bergh and colleagues reported TGC (maintaining blood glucose <110 mg/dL) decreased incidence of infections, sepsis organ failure and mortality.48 However, others have reported inconclusive or conflicting data, with some noting detrimental effects associated with TGC.49-51 TGC protocols in burn injured patients have been studied in several observational and randomized controlled trials. Decreased incidence of sepsis, multiple organ failure, and mortality have resulted from TGC.7-10 The results of the first prospective randomized controlled intensive insulin trial in children with burn injury was performed at our institution and published in 2010.7 Daily mean blood glucose levels in intensive insulin treated patients, otherwise known as the TGC treatment arm (target blood glucose 80-110 mg/dL), were significantly decreased during acute admission compared to control (target blood glucose 140-180 mg/dL), indicating improvements in burn induced hyperglycemia (fig 4A). With regard to infections, bacteremia and fungemia were found to be more common in patients with poor glucose control post burn.4 Of note, fungal infections are traditionally found in patients with diabetes and/or immunocompromised states. The presence of such infections in burned patients is indicative of hyperglycemia and immune system depression following thermal injury. Figure 4B summarizes additional significant improvements in morbidity seen in the TGC arm which include a decreased inflammatory response, improved lipid profile, and improved organ function as defined by the Denver2 score.7 (Denver2 is a clinical scoring system used by critical care physicians to classify organ function of the kidney, A B Figure 4:7 Intensive insulin treatment (tight glycemic control) significantly decreases daily mean blood glucose post burn (4A). Intensive insulin therapy decreased morbidity post burn (4B). Children with burn injury treated with intensive insulin therapy demonstrated significant improvements in inflammation, lipid profile, and organ function compared to control. * indicates significant difference between intensive insulin treatment (n=49) vs. control (n=137); P <0.05. 6 liver, heart, and lung.52 Organ failure may be defined when Denver2 scores exceed a value of 3.) In a similar study, the incidence of nosocomial and ventilator associated pneumonia, and urinary tract infections were decreased in patients on TGC therapy (by administration of insulin at high doses) compared to control.9 Despite the beneficial effects of TGC in both critically ill and burn injured patients, insulin administration often results in an increased risk of hypoglycemia. In an effort to clarify discrepancies with TGC, a large, multicenter prospective randomized trial lead by the NICE-SUGAR trial investigators demonstrated that patients on TGC have a higher incidence of mortality in critically ill adults.53 Although mortality in TGC treated patients was decreased compared to controls in the previously described trial at our institution,7 the incidence of hypoglycemia in the TGC group was high. In fact, 26% of patients enrolled in the TGC arm observed episodes of severe hypoglycemia (defined as blood glucose <40 mg/dL) compared to only 9% of control patients.7 It is well known that insulin therapy is associated with a risk of inducing hypoglycemia, increasing both morbidity and mortality.54-56 Of particular concern, even a single incident of hypoglycemia has been associated independently with an increased risk of mortality in critically ill patients.13 Recently, our laboratory reported that greater than one episode of hypoglycemia in children with burn injury results in increased incidence of infections, sepsis, multi-organ failure, and mortality.57 Therefore, hypoglycemia in burn injured patients should be avoided at all costs, making glucose control a variable to be monitored as closely as possible to ensure optimal patient outcomes. Maintaining euglycemia in burn patients is particularly difficult as burn injury induces a state of hypermetabolism, thereby requiring large caloric intake to prevent negative entropy. Quite often, these patients are ordered continuous feeds through enteral feeding tubes in an attempt to maintain body weight and attain nutritional needs. Enteral nutrition occasionally requires intermittent cessation as burn patients undergo weekly surgical intervention. These periodic interruptions lead to a disruption of gastrointestinal tract motility and absorption, resulting in an increased risk of hypoglycemia. Furthermore, younger children are at a greater risk of developing hypoglycemia due to an inability to increase blood flow in this state, limiting serum glucose availability.58,59 The high incidence of hypoglycemia has prompted discussion of the most effective glucose range in the literature. Safe maintenance of blood glucose levels within a target range is paramount in improving morbidity. Insulin and Metformin attenuate burn induced hyperglycemia In severely burned patients, sub-maximal doses of insulin given during acute hospitalization has been reported to accelerate donor site healing time, attenuate lean body mass (LBM) loss, and improve muscle protein synthesis.7,60 In rats, insulin has been shown to improve resistance to burn wound infections.61 Intensive insulin therapy has been shown to improve wound protein synthesis in the early postoperative period following burn injury.62 This phenomenon can be attributed to pro-mitogenic and anti-apoptotic effects on fibroblasts and keratinocytes by insulin downstream.63 This pathway is of great interest in burn patients as autograft skin is the preferred method of wound coverage.64,65 However, the exogenous administration of insulin is associated with hypoglycemia. Therefore, alternative glucose modulating pharmacological interventions have been proposed, of which metformin has been favored due to ease of administration (tablet vs. intravenous/ subcutaneous) and a low risk of hypoglycemia.66 Metformin (Glucophage) is an oral anti-hyperglycemic drug used widely in the management of diabetes mellitus. A member of the biguanide drug class, metformin was introduced as a safe alternative to its predecessor phenformin (which has since been removed from the market due to a high incidence of lactic acidosis).67 Glucose lowering effects are attributed to a decrease in endogenous glucose production, and an increase in glucose clearance and oxidation, likely by augmenting insulin sensitivity.18,68 These actions directly counter derangements of glucose homeostasis in burn patients, suggesting metformin may serve as a viable alternative in place of insulin in the management of hyperglycemia following burn injury.15,19,68 In addition to maintaining euglycemia, metformin also decreases low density lipoprotein cholesterol and triglyceride synthesis. This is of particular importance as triglyceridemia is independently associated with an increased risk of morbidity following burn injury. Adverse outcomes include increased length of hospital stay, hepatomegaly, inflammation, multiple organ failure, and mortality.69 On the grounds that metformin has been widely prescribed to individuals with diabetes, extensive safety and efficacy profiles have been reported. It has been suggested that there is no causal relationship between metformin and hypoglycemia, a phenomenon clearly associated with the administration of insulin, therefore eliminating this concern.15,17,70 7 A B C We have conducted several clinical studies to investigate the effect of metformin on glucose kinetics in severely burned adults.15,19,68 The first was conducted in 10 adult patients (age: 36±4 years) with >60% of the total body surface area (TBSA) burns. After 8 days of metformin or placebo administration, glucose kinetics were measured using a combination of intravenous glucose tolerance tests (IVGTT) and isotopic dilution using radiolabeled glucose (6,6-d2 glucose). Measurements taken during the fasting (basal) period demonstrated that metformin lowered endogenous glucose production and glucose oxidation. During intravenous glucose infusion, metformin accelerated glucose clearance, thereby attenuating hyperglycemia (fig 5A). Serum insulin levels were nearly three times higher in patients on metformin compared to placebo during the glucose infusion period (fig 5B). Endogenous insulin production was increased at this time in the metformin group, although not enough to carry statistical significance (fig 5C). This trend demonstrates that either metformin signals hepatic insulin production, or that metformin increases circulating insulin availability. This may also indicate that metformin inhibits the breakdown of insulin by unknown mechanisms. Hyperinsulemic euglycemic clamps are considered the gold standard test to measure insulin resistance/sensitivity. By use of this test, our laboratory has shown that metformin increased glucose uptake into muscle. Conversely, glucose kinetics were not significantly altered in the placebo group.15 Thus, metformin attenuates hyperglycemia via peripheral glucose uptake (muscle) and improves insulin resistance post burn, providing strong support for our proposed studies. The off-label use of metformin to augment burn induced hyperglycemia in children will be the first prospective randomized control trial of its kind. In aim 2 of my thesis, we will investigate the aforementioned clinical outcomes in addition to others described subsequently. Muscle catabolism following burn injury A major component of the hypercatabolic response to burn injury is catabolism of skeletal muscle, a drastic response that persists for up to at least 9 months following thermal injury.71 During starvation, ketosis and lipolysis provide energy for metabolic utilization and to protect muscle reserves. Conversely, following burn injury the body is unable to utilize fat as an efficient source of energy.3 In turn, skeletal muscle is the major fuel source following burn which leads to significant muscle wasting, days after injury.72 Following burn injury, both muscle protein synthesis and degradation are increased. However, a negative net balance in protein metabolism yields overall muscle loss as a result of greater protein breakdown relative to synthesis.73 Skeletal muscle is responsible for the majority (70-80%) of whole body insulin-stimulated glucose uptake via GLUT-4 receptors.74 Therefore, a decrease in muscle mass may indirectly account for insulin resistance exhibited in this patient population. Excessive erosion of lean tissue impairs physical rehabilitation post burn.75 These changes in body composition are measured clinically by use of dual energy x-ray absorptiometry (DEXA) scans. Interestingly, both insulin and metformin have been shown to improve derangements in body composition following burn injury. In figure 6A7 we have shown that children on higher dosages of insulin (intensive insulin therapy) show significant improvements in bone mineral density (BMD), body fat, lean body mass (LBM), and body mass from admission to discharge compared to those on lower insulin dosages. Improvements in body composition and donor site wound protein synthesis62 Figure 5:15 Metformin attenuates hyperglycemia following burn injury by increasing glucose clearance (A) and increasing insulin availability (B). A possible third mechanism includes increasing endogenous insulin production marked by increased C-peptide levels in the metformin group (C). * P<0.05 for metformin-treated (n=5) vs. placebo control patients (n=5). 8 indicate that insulin may have beneficial effects for the burn patient, independent of anti-hyperglycemic effects. We have obtained these results (figs 4 and 5) by investigating the effects of insulin in a doseA dependent manner. However, in specific aim 1, we will investigate the effects of insulin vs. no insulin administration in addition to dose and age dependent effects in children with severe burn injury. In reference to muscle protein synthesis, we have studied muscle fractional synthetic rate (FSR) using isotopic phenylalanine (which is neither synthesized nor secreted in muscle) as a tracer in adult burn patients given metformin (fig 6B).19 It was found that metformin significantly increased skeletal muscle FSR after only one week of administration. Improvements in body composition by insulin and metformin may reduce the incidence of infection in B addition to improving wound healing since a 10-15% loss of lean body mass (LBM) has been associated with a significant increase in infections and delayed FSR wound healing.76 Furthermore, while the infusion of insulin alone did not significantly affect the FSR during baseline evaluation, co-administration of insulin and metformin resulted in a statistically significant increase in the FSR of muscle protein in adult burn patients. The implications of these findings may indicate additive or perhaps even synergistic effects of the combined administration of insulin and Figure 6: Change in body composition as assessed by DEXA metformin in patients with burn injury. The combined scans from admission to discharge in patients on intensive administration of insulin and metformin demonstrated insulin therapy (n=49) vs. conventional insulin therapy (control, n=137) in children with severe burn injury. Intensive insulin improvements in glucose control, lowered insulin therapy significantly improved bone mineral density (BMD), body requirements, and decreased incidence of fat, total lean, and body mass compared to control (A).7 Adult hypoglycemia than administration of insulin alone.77-79 burn patients (n=5) show significant improvements in skeletal In specific aim 2 of our study we will further identify muscle fractional synthetic rate (FSR) after 1 week of metformin 19 the clinical effects of the combined administration of administration (B). *P<0.05; I= Insulin; Met= Metformin insulin and metformin in pediatric burn patients. Growth arrest following burn injury Normal growth, skeletal maturation, and ultimate adult height are dependent on a number of factors including age, sex, race, genetics, nutrition, metabolism, and overall health. As described earlier, severe burn injury incites hypercatabolism, adversely affecting energy utilization which results in long term consequences such as growth arrest in children. Investigators from our institution have tracked growth/height velocities in 80 children (males and females) for three years post severe burn injury.80 At one year post burn injury, approximately 50% of males and 40% of females were greater than 2 SD below expected mean height (fig 7). Furthermore, growth arrest continued for the second year post burn in approximately 1/3 of patients. However, by the third year post burn, growth arrest had resolved in the majority of children to yield a height distribution representative of normal growth patterns. Whether or not ultimate adult height is affected in this population remains yet to be determined. Furthermore, it is currently unknown if 80 insulin, an anabolic hormone, alters growth following Figure 7: Growth Velocity in children with Severe Burn Injury (n=80) is significantly delayed up to two years post burn injury in burn injury. Therefore, we will investigate the effects of males and females. *p<0.05 compared to admission height. insulin on growth in Aim 1 of my thesis, assessed by 9 height velocity and bone maturation over two years. Lactic Acidosis Lactic acidosis may be defined as serum lactic acid > 5 mmol/L and a blood pH of <7.35. 81 Drugs from the biguanide drug class have been associated with causing lactic acidosis. Phenformin, the biguanine predecessor to metformin, was removed from the market due to a high incidence of lactic acidosis. The earliest use of metformin can been dated back to as early as 1957 in Europe. However, it wasn’t until 1995 that the Federal Drug Administration approved its use in the USA.16,82 Phenformin and metformin have different molecular structures and pharmacokinetics83 thereby moderating lactate metabolism through different pathways.84 Contrary to phenformin, metformin is postulated to augment glucose oxidation without significantly altering fasting lactate production in peripheral tissue.85 Despite these stark differences, lactic acidosis has been inappropriately linked to all biguanide drugs. Conditions associated with lactic acidosis may be classified into five categories (fig 8). Drugs associated with lactic acidosis include phenformin, isoniazid, and Figure 8: Conditions associated with lactic potassium cyanide. Genetic causes include disorders in metabolic acidosis enzymes; pyruvate dehydrogenase deficiency, fructose 1,6 phosphatase deficiency, and glucose-6-phosphate dehydrogenase deficiency. Of particular concern in critically ill individuals are hepatic and renal disorders which may result in either impaired metabolism or decreased clearance of lactic acid. Burn injury impairs hepatic and renal function which may contribute to the accumulation of lactic acid irrespective of insulin or metformin administration. For this reason we will closely monitor patients for this severe adverse event in specific aim 2. Similarly, sepsis or conditions associated with decreased tissue perfusion may also contribute to the development of lactic acidosis. However, it should be noted that metformin has been extensively studied and no direct causal relationship to lactic acidosis has been found. An outcomes study was conducted to compare patients taking metformin for one year (n=7,227) to those receiving ‘usual care’ with sulfonylureas (n= 1,505). No incidents of lactic acidosis were observed in either group.86 Likewise, a meta-analysis encompassing 194 trials, spanning nearly 37,000 patient-years of metformin treatment, failed to report any incidents of lactic acidosis.87 Nonetheless, although metformin may not directly contribute to the formation of lactic acidosis, we will diligently monitor our patients for this condition as burn injury results in impairments in hepatic and renal function in conjunction with decreases in tissue perfusion, both of which may independently cause lactic acidosis. Innovation: Shriners Hospitals for Children (Galveston) treats a unique pediatric burn population. As world leaders in the management of severe burn injury in children, our faculty and ancillary staff provide the highest standard of patient care. Patients remain in the Shriners health care system until the age of 21, and return annually for clinical needs and to participate in research. In this thesis, I will overview how maintenance of glucose homeostasis with insulin and/or metformin improves patient outcomes. Although metformin has long been used to attain euglycemia in diabetic patients, this clinical trial will be the first use of metformin in children inflicted with burn injury. By comparing standard of care (insulin) and metformin therapeutic modalities, we will delineate similarities and differences in insulin and metformin in regard to clinical outcomes. As insulin resistance remains a problem for our patients postdischarge, we will administer metformin for 1 year post-burn. The novel use of metformin in our pediatric burn patients enrolled to a prospective, randomized controlled trial has far reaching implications. As leaders in burn care, the findings of these studies will be reviewed by burn institutes across the world, many of which may implement new therapeutic regimens accordingly. If the reduction in blood glucose is determined to be the sole cause of improved morbidity and mortality, we will administer metformin in place of insulin to reduce the incidence of hypoglycemia and hypoglycemic related consequences. If metformin is found to be ineffective or detrimental to clinical care, insulin will remain the standard of care in this patient population. Approach: Experimental Design for Specific Aim 1 Specific Aim 1: Investigate alternative clinical effects of insulin in thermally injured children. 10 We will compare clinical outcomes of treating children with severe burn injury with either no insulin or insulin designed to maintain blood glucose levels <180 mg/dL. By dividing groups into insulin and no insulin, we will determine if insulin attenuates the hypermetabolic and/or hypercatabolic response to burn injury, independent of glucose modulating effects. Disclaimer: Data from specific Aim 1 was presented nationally at the Southern Surgical Association, 125th Annual Meeting, Hot Springs, Virginia in December, 2013 and published in the Journal of the American College of Surgeons in April, 2014.88 Additionally, data from a subset of patients in this specific aim have previously been reported in 2010. The aim of the study was to investigate the clinical effects of intensive insulin therapy in severely burned children.7 Growth and bone maturation data was presented nationally at the Endocrine Society, 97th Annual Meeting, San Diego, California in March, 2015. The abstract is currently in press in Endocrine Reviews and scheduled to print in May, 2015. Study Population: Well will investigate the effects of no insulin vs. varying dosages of insulin by reviewing data from children with severe burn injury admitted to our institution from 1998-2013. During this period, 6,573 patients were admitted, 295 of which were not randomized to studies of various anabolic agents other than insulin. Exclusion criteria include: presence of preexisting illnesses such as diabetes, hepatitis, HIV, AIDS, or malignancy within the past five years; anoxic brain injury; delayed admission (burn to admit >7 days); decision not to treat due to severity of thermal injury. After exclusion, patients were divided into those that did not receive insulin (‘No Insulin’, n=98) and those who did receive insulin (‘Insulin’, n=145). To avoid confounds of puberty on growth and bone maturation, inclusion criteria for this sub-analysis encompassed age 3.75-12.25 years in males and 3.75-10 years in females. Between these ages, there is a relatively stable growth rate. Therefore, only pre-pubescent children were included for this sub-analysis. Drug Administration: Upon admission, insulin was administered according to hospital standard sliding scales via intravenous or subcutaneous routes to alleviate hyperglycemia. A subset of patients in the ‘Insulin’ group received larger doses of insulin to maintain glucose control between 80-110 mg/dL. Patients in the ‘No Insulin’ group did not attain hyperglycemia severe enough to warrant clinical insulin administration. Blood glucose levels for these patients were <180 mg/dL for the vast majority of in-patient hospital stay. Burn care: All full-thickness burns will be excised within 24 hours of admission. We have previously reported that early excision and wound grafting significantly improved wound closure and reduced incidence of burn wound infections and sepsis, thereby decreasing the number of days admitted in the ICU.89,90 The majority of any unburned skin will be harvested for autologous skin grafting using an electric dermatome (Padgett’s Instruments, Kansas City, MO) set at 0.010 in (Zimmer Inc., Warsaw, IN). Subsequent operations will occur approximately weekly (once donor sites have healed) until the burn wounds are 95% healed at which point patients are discharged from the ICU. Muscle, fat, and skin biopsies for molecular, proteomic, and histologic analyses will be obtained during surgery. With regards to wound care, autologous skin grafts will be placed over areas debrided of burn eschar (eschar is the leather-like slough remnant of devitalized skin after burn tissue necrosis). These areas were covered with gauze saturated with Polysporin Mycostatin ointment to prevent infection. Skin graft donor sites will be covered with Scarlet Red (Sherwood Medical, St. Louis, MO)impregnated fine gauze to allow for quantification of wound healing time. Glucose homeostasis: Daily mean, maximum, minimum, and 6 am glucose will be calculated according to serial blood sample collections during the acute stay in the ICU in addition to follow up appointments in the clinic. The number of moderate and severe hypoglycemic episodes, as defined previously, will be calculated. Oral and/or intravenous glucose tolerance tests will be performed to assess insulin sensitivity. Briefly, an unlabeled bolus (75 grams) of glucose is administered in fasted patients. Thereafter, plasma insulin, c-peptide, and glucose measurements are measured every 30 minutes, over 2 hours, and are compared to baseline levels. There are various criteria for interpreting oral glucose tolerance test results. To diagnose diabetes mellitus, we will use guidelines set forth by the American Diabetes Association.91 Patients will be evaluated with an OGTT at six months to determine if they should continue metformin. Metformin will be discontinued for two weeks before the OGTT to ensure accurate results. If glucose values are above any of the values listed below, patients will continue receiving metformin up to 12 months post burn. An abnormal OGTT will be defined as observing any one of the following: (A) Fasting glucose levels >100 mg/dL or (B) 2 hours glucose levels >140 mg/dL or (C) peak glucose levels >180 mg/dL after eating regardless of time. Manipulations of 11 these results to further assess insulin sensitivity and resistance by means of ISI HOMA and Matsuda, QUICKI, and insulogenic index scores will be calculated as previously described.7 Hypermetabolism: The hypermetabolic response to burn injury may be assessed by measuring pulse, respiratory rate, blood pressure, mean arterial pressure, cardiac output, and cardiac index parameters. Metabolic response to injury and illness will be studied by steady-state REE. This provides the information needed for optimal nutritional management. REE is measured with a Sensor Medics 2900 metabolic measurement cart. REEs were collected during a resting state by trained respiratory therapists in an environmentally controlled room when the patient is in a relaxed state. Subjects were tested in the supine position using a ventilated and clear hood. REE is calculated by measuring the relationship between oxygen consumption and carbon dioxide production. As previously mentioned, REE measurements were used to guide nutritional management. Data gathered was compared with age predicted normal values based on the HarrisBenedict equation. Body Composition and Growth: Anthropometric parameters will be measured weekly until discharge to track changes in BMI. DEXA scans will be used to determine changes in whole-body, spine, leg, and arm lean body mass. Leg scans will be subdivided at the knee into upper and lower regions to allow pre-to post-treatment comparisons of the muscle groups. Arm regions will also be separated from whole-body measures to determine treatment effects on these muscle groups. DEXA measurements will be conducted as previously published.7 Bone age will be determined using the Greulich and Pyle standard atlas for assessment of bone age in children.92 Radiographs were interpreted by a faculty pediatric radiologist well versed in bone age assessment by assessment of ossification centers in radiographic imaged of the left wrist (non-dominant hand). Height/growth velocities were determined using standardized growth charts from the Centers for Disease Control and prevention (CDC) to compare rate of burn patient growth to ageappropriate, normal healthy children.93 Of note, children with a height velocity below the 3rd percentile (>2 standard deviations) of normal are considered to have impaired growth (fig. 9). These children are termed those with ‘growth arrest’. Bone age Figure 9: Growth Charts obtained from the CDC were used to derive height velocity percentiles for boys with and growth velocity were measured at discharge, 6, 12, 18, and burn injury at 12 months post burn. 24 months post burn. Inflammatory response: The inflammatory response will be assessed weekly until discharge. Cytokine concentrations were measured using the Bio-Plex Human Cytokine 17-Plex panel with the Bio-Plex Suspension Array System (Bio- Rad, Hercules, CA). Immunological analyses will be performed using flow cytometry, ELISA, and western blots. Incidence of infection, pneumonia, and sepsis: We will inspect patients daily for infection while admitted to the ICU. Episodes of infection, pneumonia, and sepsis will be recorded. Episodes of infection will be defined as >105 colony forming units (CFU)/gram tissue. Pneumonia will be defined by the following criteria: new progressive and persistent infiltrate, consolidation, or cavitations, in light of the baseline evaluation for inhalational injury on chest X-ray, along with signs of sepsis, worsening gas exchange (decreased PiO2/FiO2 ratio), increased O2, and change in the sputum (e.g., purulent or increased sputum production). Sepsis in burn patients has been defined by the American Burn Association consensus conference in 2007.94 12 Overview of study assessments: Blood will be drawn at admission and once each week until discharge for hormone measurements, cytokine profiling, and acute-phase-protein analysis. Patients will undergo metabolic stable isotope infusions between 4 and 5 days following the first operative debridement and grafting. Additional infusions will be performed again at 4 and 5 days after the fourth operation. During the stable isotopic studies, skin, wound, and muscle biopsies will be performed to assess protein fractional synthetic and fractional breakdown rates, lipolysis, and glucose production. Beginning on day 3 after the first operation, assessment of wound healing will begin. On days 4 and 5 after the first and fourth operations, studies will be performed to assess REE and body composition. Indirect calorimetry and DEXA will be performed to assess REE, substrate utilization, and body composition. These studies will be repeated on days 4 and 5 following the fourth operation to allow for assessment of treatment-induced effects. We will minimize patient invasion by coordinating time points for studies and tissue collection with scheduled visits to the operative Figure 10: Study schematic identifying treatment groups and summary of time points of surgery room to address surgical needs. A and data collection from first admission to first discharge. Note, data collection will continue after discharge up to one year post-burn. OR= Operating room procedure; POD= Post operative day. figurative summary of data collection is outlined in figure 10. Statistical Analysis: Data were analyzed by our statistician, Clark R. Andersen, MS using R statistical software (R Core Team, 2013, version 3.0.1). Since significant differences were noted in demographic and burn injury characteristics (table 1) a generalized additive mixed (GAM) model was used to account for nonlinear effects on the response due age and time post-burn, and to account for repeated measures correlation by blocking on subject. Treatment group (‘no insulin’ or ‘insulin’), age, gender, TBSA, time post-burn, inhalation injury, and number of infections were factored into the GAM model. Log or power transformations to better approximations of normality were utilized as appropriate, then reverse-transformed for presentation. All statistical tests assumed a 95% level of confidence. Mortality was analyzed by an independent statistician, Kristofer Jennings, PhD, using a Bayesian model to predict mortality. Demographics and burn injury characteristics were similar between groups (No Insulin vs. Insulin) in the sub-analysis investigations effects of insulin on growth and bone maturation. Therefore, the GAM model was not used in this analysis. Data with normal distribution was analyzed using student’s t-test continuous variables, and chi-squared tests for categorical variables using SigmaStat statistical software integrated with SigmaPlot 10 (SigmaStat, 2007 version 3.5.1). Data in support of Specific Aim 188: A total of 295 patients were eligible to participate in this study. Significant differences in demographic and burn injury characteristics were noted between groups (table 1). Generally, patients who received insulin were older and suffered from larger and more severe (third degree) burns with concomitant inhalation injury. However, we accounted for these differences in the GAM statistical model, allowing us to interpret outcomes data according to treatment group. Table 1: Demographics and Burn Injury Characteristics. D= Days; LOS= Length of stay; NS= Not significant; TBSA= Total body surface area. Data presented as means ± SD or counts (percentages). Glycemic Control Daily mean, maximum, and minimum blood glucose levels were collected and analyzed. Mean glucose was significantly elevated in Insulin vs. No Insulin groups (data not shown; p<0.01). Daily maximum (p<0.0001) and minimum (p<0.01) glucose levels were significantly different between groups (fig. 11). Upon further examination, we found that blood glucose levels were directly proportional with age, an important finding that has not been 13 reported in the literature. Furthermore, with each year increase in age, there was a 9% increase in the number of hyperglycemic episodes (HEs) defined as blood glucose values >180 mg/dL (p<0.0001). This may explain why children in the No Insulin group were significantly younger than those in the Insulin group as they were less likely to develop hyperglycemia. Alternative determinants of hyperglycemia included percent TBSA burned (each percent increase corresponded in a 3% increase in HEs; p<0.0001) and burn wound infections (BWI) (each additional BWI corresponded in a 6% increase in HEs; p<0.01). Patients in the insulin group received a daily dose of 7 ± 13 international units (IU), a daily total of 90 ± 140 IU, a cumulative total of 1570 ± 3160 IU from ICU admission to discharge, and were on insulin for 15 ± 16 days (means ± SD). As previously mentioned, insulin was administered according to an insulin sliding scale (as per hospital protocol) to maintain blood glucose levels within the desired target range. Although insulin administration was associated with a 35% reduction in the Figure 11: Daily maximum and minimum blood glucose. Patients in the no insulin group had lower maximum blood glucose levels (P<0.0001). Patients in the insulin group had lower minimum blood glucose measures, placing them at increased risk for hypoglycemia (P<0.01). Figure 12: Insulin: Dose response effects on glucose. Daily mean blood glucose levels generally decrease as increasing doses of insulin are administered. However, after ~200 IU of insulin, no effect is seen, indicating either decreased utilization or severe insulin resistance. Red bar indicates approximately 200 units of insulin administration. IU= International units. number of HEs (p=0.002), it also resulted in profound hypoglycemia. 52 mild hypoglycemic episodes in 43% of patients on insulin vs. 10 episodes in 8% of patients in the No Insulin group were noted (p<0.0001). Similarly, 42 severe hypoglycemic episodes in 14% of patients on insulin vs. only 3 episodes in 2% of patients in the No Insulin groups were observed (p<0.001). Children on insulin experienced 5.7 times more hypoglycemic episodes compared to children in the no insulin group (p<0.0001). As expected, glucose levels varied according to insulin dosage. Overall, blood glucose levels decreased as more insulin was administered throughout the day up to an extent. Once patients received approximately 200 IU of exogenous insulin, further attenuation of hyperglycemia was lost (fig.12). We speculate either decreased insulin availability or overt insulin resistance in these patients may account for decreased efficacy. We may be able to predict which patients require closer monitoring using this marker. Nearly identical findings were seen when we examined cumulative insulin administration from admission to discharge (data not shown), with a marked change in glucose levels noted at 200 IU of insulin administration, a cautionary marker of poor glucose control. Resting Energy Expenditure The administration of insulin resulted in a significant increase in the metabolic rate from first admission to first discharge (figure 13). Patients treated with insulin demonstrated a 9.4% increase in REE compared to those not treated with insulin (p=0.009). Both the amount of insulin administered during the 24 hours prior to measuring REE in addition to the total amount administered from first admission to first discharge were directly proportional to an increase in REE and percent predicted REE. In fact, a 1% increase in insulin administration was associated with a 0.02% increase in REE (p=0.002). REE continued to increase significantly beyond 200 14 IU of insulin administration, even though no effect on glucose was observed at this dose (fig. 11). Finally, age was also found to play a significant role in the determination of the metabolic rate. Younger children were less likely to become hypermetabolic (p<0.0001), which corroborates with earlier findings reported from our laboratory.95 Figure 13: Insulin administration significantly increased resting energy expenditure over time (p=0.009). Data presented as means ± SEM. Figure 14: Insulin: Dose response effects on lumbar spine bone mineral content. Bone mineral content increases as total insulin administration increases from ~10-200 IU (p=0.03). Red bar indicates ~200 IU of insulin administration, at which point effect is halted. Data presented as means ± SEM. Body Composition Patients on insulin therapy showed marked improvements in body composition (assessed by DEXA scans) compared to patients not receiving insulin. Favored outcomes were noted in: Whole body bone mineral content, lean mass, and percentage of fat mass (p<0.001); Lumbar spine bone mineral content (p<0.05) and bone mineral density (p<0.01); and visceral adipose tissue fat mass (p<0.05) during the first 30 days following burn injury. Many of these parameters (but not all) remained significantly elevated up to one year post-burn injury. However, once we made adjustments using the GAM model, only lumbar spine bone mineral content remained significantly elevated in a dose dependent manner (p=0.03). A striking observation was noted in the dose response curve (fig. 14). Insulin progressively increased lumbar spine bone mineral content as dosing increased from ~10-200 IU. However, upon further administration, bone mineral content decreased, indicating a loss of effect. These results suggest that the ability of insulin to augments body composition following severe burn injury plateaus once ~200 IU have been administered. Bone Maturation and Growth Bone age and height velocities were determined in 62 pre-pubescent children (No Insulin, n=21; Insulin, n=41). No significant differences in terms of demographics or burn injury characteristics were noted between groups in this sub-analysis (data not shown). Patients in the Insulin group received on average, 800±680 IU of insulin from admission to discharge for 14±22 days. Bone age (BA) was divided by chronological age (CA) to obtain BA:CA ratios (normal= 0.951.05). No significant difference was noted between groups over two years post burn injury in terms of BA:CA ratios (fig. 15). However, insulin administration significantly improved height velocity at 12 and 18 months post burn (fig. 16A; p≤0.05). Height velocity was plotted Figure 15: Bone Age over two years post on growth charts provided by the CDC to identify height/growth burn. No difference in BA:CA ratios were velocity percentiles as described previously (fig. 9). These charts detected. Data presented as mean ± SEM. were derived for males and females at 12 and 24 months (data not shown). Children were then divided into three groups based on height velocity percentiles compared to normal, healthy children: Normal, < 2 SD (growth arrest), and >2 SD. We are most interested in identifying those 15 children who are <2SD from the norm as these children show stunted growth. Over twice as many children in the No Insulin group were below 2 SD compared to children who received insulin at 12 months post burn (78 vs. 38%; p=0.02; fig. 16B). Although this pattern of growth arrest continued at 24 months post burn in the No Insulin group, statistical significance was not found (40 vs. 21%; p=0.06; fig. 16C). A B C Figure 16: Insulin administration significantly improved height velocity at 12 and 18 months post burn (a). Height velocity stratification in children at 12 (b) and 24 (c) months post burn. Children not treated with insulin were twice as likely to exhibit growth arrest for two years following severe burn injury. Data presented as mean ± SEM *p ≤0.05 Infections No significant difference between groups in the percentage of patients with burn wound infections, pneumonia, or sepsis was found. However, each additional burn wound infection was associated with a 6% increase in the number of hyperglycemic episodes (p=0.004) and significant increases in both serum creatinine and total bilirubin (p<0.01), regardless of group. Using logistic regression no significant relationship in the species of infection cultured (gram positive, gram negative or fungal), was found. Mortality Notably, 17 deaths were observed in patients on insulin therapy vs. no deaths in children not receiving insulin (p=0.001). Patients who expired were inflicted with severe burn injury (mean % TBSA burned= 80 ± 14%). The combined effect of insulin treatment and % TBSA burned were found to be significant (p=0.02), with the administration of insulin leading to earlier death. The hazard ratio was calculated to be approximately 1.92. We tried to identify the mechanisms by which insulin may have been related to mortality. However, no relationship between mortality and insulin dose (p=0.18), day post burn insulin was first received (p=0.29), or the number of hypoglycemic episodes in patients who expired (p=0.098) was found. Summary: The advent of intensive insulin therapy to maintain tight glycemic control in patients inflicted with burn injury has been described in several prospective, randomized control trials.5,7,9 However, in these studies all patients were treated with insulin and divided into groups based upon differing target blood glucose ranges. We’ve observed that the administration of insulin to decrease blood glucose <180 mg/dL is often required, but not universal in pediatric burn patients. Here, we have investigated two cohorts amongst pediatric burn patients: those administered insulin to maintain relative euglycemia; and patients never treated with insulin. By comparing these two polarizing groups, we were able to clearly examine the clinical impact of insulin on patient outcomes following burn injury. Although the administration of insulin improved body composition and growth, patients in this group were also noted to undergo significantly more episodes of hypoglycemia. Since hypoglycemia has been associated with poor outcomes including increased risk of death, the benefits of insulin therapy do not outweigh the risks. Therefore, the implementation of tight glycemic protocols at our institution and others have been abrogated. Furthermore, once patients have received ~200 IU of exogenous insulin, further administration may prove to be futile. Perhaps administering ~200 IU of insulin may serve as a surrogate marker of patients with severe illness, and this finding requires further review. We will continue to investigate the effects of other glucose modulating agents to attenuate burn induced hyperglycemia. As outlined in specific aim 2, metformin may emerge to be a safe and effective alternative, either in combination with insulin or as standalone therapy. 16 Specific Aim 2- Elucidate the clinical outcomes in the management of post burn hyperglycemia by insulin and metformin. By directly comparing endpoints in clinical care between treatment groups, we will have a better understanding of whether insulin or metformin more significantly improves whole-body and organ function post burn. Experimental Design for Specific Aim 2: Study population and patient accrual: Patients between 10–18 years of age with greater than 20% TBSA burns will be enrolled into an institutional review board (IRB) -approved, prospective randomized control trial. Exclusion criteria include: Age <10 or >18 years, presence of preexisting illnesses such as diabetes, hepatitis, HIV, AIDS, or malignancy within the past five years; prevalent or acquired allergy to metformin; impaired hepatic or renal function; anoxic brain injury; delayed admission (burn to admit >7 days); decision not to treat due to severity of thermal injury. Insulin and metformin administration: On admission, all patients will be resuscitated with Ringer's Lactate solution to account for the massive fluid loss and third spacing exhibited by patients following burn injury. Immediately after admission, patients or their legal guardian will be asked for consent to participate in the study. After obtaining consent, we will randomize patients into one of the following two groups: (1) Control- Patients will receive a placebo twice a day in addition to standard burn care for up to one year following burn injury. Conventional insulin therapy will be given to maintain blood glucose levels between 140-180 mg/dL. (2) Metformin- Patients will receive 250-500 mg twice a day in addition to standard burn care to maintain blood glucose levels between 80-140 mg/dL. Drug administration will be initiated as soon as possible after enrollment and will be continued until patients are discharged from the intensive care unit (ICU). After discharge, patients on placebo or metformin will continue to receive the respective drug twice a day for up to one year post burn injury. Drugs will be administered for at least 14 days. Insulin will be given intravenously or subcutaneously and metformin via a duodenal feeding tube (in-patient) or per os (out-patient). As metformin is started in the ICU shortly after admission, it is expected that a stable dose will be obtained before discharge. In addition it is common for patients to remain in the community for 6-12 weeks post burn injury as they participate in an exercise program (both treatment groups) to improve mobility and strength lost following burn injury. Before discharge home, patients will have an OGTT performed. During the 6-12 week time period in the community, glycemic control will continue to be monitored. Patients and their families will receive education and training during this period regarding study drug administration, glucose monitoring, urine monitoring and safety reporting. By the time the family returns to their home, they will be knowledgeable about all aspects of metformin, when to hold a dose for glucose <70 mg/dL and when to contact a physician if needed. If glucose is above 180 mg/dL after discharge home, the patient will be instructed to contact their family physician. In addition they will be asked to call the hospital to speak to a physician to provide further instructions. In the control group, insulin infusion will be started if blood glucose exceeds 180 mg/dl. The infusion will be adjusted according to a sliding scale to maintain blood glucose levels to 140–180 mg/dl. These glucose ranges have been selected to comply with guidelines set forth by the Surviving Sepsis Campaign.96 Insulin dose will be based on measurements of blood glucose from arterial blood performed at 15-120 minute intervals using a glucose analyzer (AccuCheck Advantage, Roche Diagnostics, Indianapolis, IN). The dose of insulin will be adjusted according to a standardized insulin sliding scale. We will administer metformin to decrease blood glucose to 80-140 mg/dL. Our preliminary data show that the dose of metformin needed to decrease glucose levels to this range in adults is 850 mg every 8 hours. In children, the dose will be based on body surface area (BSA), taking into account both the height and weight of the child (dose will be ~250–500 mg every 12 hours). All patients will receive enteral feeding of Vivonex Ready-to-Feed (RTF) (Novartis, Minneapolis, MN) at a rate of 1.4 times their REE (determined by indirect calorimetry), beginning the second day after admission. One liter of Vivonex RTF provides 1,000 kcal (composition: 50 g of proteins, 175 g of carbohydrates, 12 g of fat) and multiple essential vitamins and electrolytes. All patients will receive the same caloric amount to avoid differences in nutrition and metabolism between groups. When patients go to the operating room for surgical procedures, metformin will be discontinued the morning of surgery. Patients will be reassessed following surgery for renal function and lactic acid accumulation. After these parameters are determined to be within normal ranges, metformin will be restarted. For patients that receive contrast studies with iodine materials, metformin will be discontinued for 48 hours following the 17 procedure, assessment will be done for renal failure and lactic acidosis between 24-48 hours, metformin will be restarted after renal function and lactic levels are determined to be normal. While in the ICU, metformin will be held for any lactic acid levels outside normal limits. Acidosis may ensue following burn injury due to periods of sepsis or ventilator status. Lactic acidosis has been described previously. If this occurs, metformin will be discontinued until the condition resolves. At that time the physician and/or the safety committee will re-evaluate the patient’s condition and if the lactic acid level has returned to normal, metformin may be restarted. During the time the subject is an inpatient or an outpatient, if there is a single episode of a lactic acid value above normal limits, metformin will be temporarily suspended. Re-administration of metformin will occur only once lactic acid levels return to normal and a physician has reviewed the events leading to the incident. Data supporting specific aim 2 We have successfully enrolled 33 patients into a prospective, randomized control trial to investigate the effects of metformin (n=13) vs. control/placebo (n=20). No significant differences in patient demographics and burn injury characteristics were found between groups (table 3). Glycemic Control Metformin was initiated on average six days after burn injury in the treatment group. Metformin significantly reduced mean blood glucose levels for the first two weeks following burn injury compared to control/placebo (fig. 17). The intended reduction in glucose was not accompanied with an increased incidence of hypoglycemic episodes. In fact, children on metformin did not experience hypoglycemia at all. Of the 2472 recorded glucose Table 3: Demographics and Burn Injury Characteristics. D= Days; LOS= Length of stay; TBSA= Total body surface area. Data presented as means ± SD or counts (percentages). Figure 17: Weekly mean blood glucose. Metformin significantly attenuated hyperglycemia following burn injury in children compared to placebo/control. Data presented as mean ± SEM; *p<0.05. measurements, a single measurement of 60 mg/dL was recorded and found to be the absolute minimum in all children treated with metformin. For reference, mild or severe hypoglycemia is diagnosed when plasma blood glucose levels are <60 mg/dL or <40 mg/dL respectively. Conversely, there were seven minor and three major episodes of hypoglycemia in 25 and 20% of children in the control group respectively. This data suggests that metformin can be used to attenuate burn induced hyperglycemia in children without risking the development of hypoglycemia. Furthermore, the data corroborates the response to metformin in burned adults we have previously reported.15 Hypoglycemia in the control group is attributed to significantly larger exogenous insulin administration. Patients in the control group required over three times the amount of exogenous insulin administration in order to maintain relative euglycemia (blood glucose <180 mg/dL) compared to children on metformin (table 4). Small increases in insulin sensitivity assessed by oral glucose tolerance tests (OGTT) were noted in the metformin group: Insulin sensitivity index (ISI) Matsuda scores were higher in metformin patients compared to control/placebo (5.0 ± 3.3 vs. 3.8 ± 3.3; p=0.13), as were ISI HOMA (0.58 ± 0.64 vs. 0.40 ± 0.28; p=0.31), and QUICKI scores (0.34 ± 0.04 vs. 0.32 ± 0.04; p=0.31). Currently, these differences are not significant between groups. However, Freemark et al. reported Table 4: Exogenous insulin administration. Metformin significantly decreased significant improvements in ISI HOMA and the need for insulin administration to maintain blood glucose levels <180 QUICKI scores in obese adolescents randomized mg/dL. IU- International units. Data represented as medians ± SD. 18 to metformin treatment for 6 months,97 a response we anticipate to observe as the total number of patients enrolled in the clinical trial increases. We then compared differences in fasting glucose levels from OGTTs taken close to admission to those performed approximately four months post burn. Metformin significantly decreased fasting glucose levels compared to control, indicating marked improvements in long term glucose homeostasis (-20 ± 14 vs. -4 ± 11 mg/dL; p=0.04). It is postulated that metformin inhibits gluconeogenesis and/or glycogenolysis, thereby reducing fasting blood glucose levels.85,98 Resting Energy Expenditure At our institute, daily caloric needs and dietary intake are based on data measured from resting energy expenditure (REE)/indirect calorimetry. Metformin significantly reduced percent predicted REE by hospital discharge compared to control/placebo (116±36 vs. 157±37 %, p=0.04; fig. 18). An alternative method of measuring REE is to divide actual REE by weight. This method also showed a significant reduction in the hypermetabolic state by hospital discharge in children treated with metformin vs. control (33±14 vs. 48±10 Kcal/day/kg; p=0.03). We hypothesize this reduction in REE to be attributed to reduced insulin requirements in metformin vs control patients (table 4). In specific aim 1, we found that Figure 18: Hypermetabolism. Metformin significantly insulin administration was associated with a 9% increase in reduced percent predicted resting energy expenditure (REE) by hospital discharge compared to control. REE following burn injury in children (fig. 13). Therefore, with Data presented as mean ± SEM; *p<0.05. reduced insulin requirements to maintain relative euglycemia, metformin indirectly attenuates hypermetabolism by hospital discharge. Safety (lactic acidosis) One of the major outcomes of this trial was to identify whether or not metformin is safe to administer in children (age 10-18 years) following severe burn injury. As discussed previously, metformin has been linked to the development of lactic acidosis. Therefore, we closely monitored for this adverse effect. To date, there have been no incidents of lactic acidosis (defined as blood pH <7.25 and lactic acid >2.1 mmol/L) while on metformin as assessed by serial measurements of lactic acid and pH in arterial blood gases (fig. 19). Summary: In this preliminary study, we investigated the effects of metformin on the hyperglycemic response to severe burn injury in children. To the best of our knowledge, this is the first prospective, randomized control trial of its kind in this population. B A Our findings indicate that metformin is a safe and effective Figure 19: Lactic acidosis. Lactic acid (a) and pH (b) were similar between groups. Metformin did not cause lactic acidosis in children with severe burn injury. Red bar alternative to insulin indicates maximum normal level. Green bars indicate normal range. Data presented as administration in this population. mean ± SEM. Metformin significantly reduced mean blood glucose levels, insulin requirements, and hypermetabolism compared to control/placebo during acute hospitalization. Most importantly, metformin was not related to the advent of hypoglycemia or lactic acidosis. Long term effects remain yet to be determined. We will continue to monitor these patients for 1 year following burn injury, and thereafter for safety and efficacy. Limitations: We would like to address a few limitations in our specific aims and findings thereof. In specific aim 2, loss to follow up may potentially hinder our ability to measure long term variables as we have designed to follow up with these patients for at least 1 year post burn injury. However, burn injured patients are unique in that 19 treatment includes serial surgical operations in order to obtain cosmetic and motor function. Following severe burn injury, patients typically undergo 10-20 surgical procedures within 5 years. Therefore, our patients return periodically, at which time we collect data to meet our proposed protocol. Lack of compliance may be another drawback seen with even the best designed clinical trials. Before discharge from the ICU, we thoroughly explain to patients and their families the benefits of maintaining blood glucose within a particular range and the consequences which may occur if euglycemia is not achieved. During follow up visits in our outpatient clinic, we will identify causes for reduced compliance and all efforts will be made to correct any misinterpretations. Parents are asked to document drug administration on a day to day basis in a log book which is examined during follow up. This provides an audit to assure proper drug dosing has been achieved. In the event that metformin fails to attenuate hyperglycemia or adverse events are noted, the dosage may be titrated accordingly. Temporary or permanent cessation of the drug may be implemented if needed. We have noted the current metformin dose of 500 mg given twice a day, although safe, may not be the most effective dose. No significant differences were noted in mean blood glucose levels between groups at 3 and 4 weeks post burn which prompts us to believe that a higher dose may be required to further attenuate hyperglycemia. As this is the first clinical trial approved to administer metformin to children with severe burn injury, we are very cautious and elected a conservative dosing approach. Since no serious adverse events were noted in the treatment group, we may increase the dose to 850-1000 mg administered twice a day. These dosages have been reported to be safe and effective in children and adolescents with obesity,99-103, type II diabetes mellitus,104,105 and in girls with hyperandrogenism106 and polycystic ovarian syndrome.107 Furthermore, long term accumulation of metformin in blood and tissue over 1 year of administration may have profound effects in the outcomes measured. 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