The Effects of Oral Sirolimus on Insulin Dynamics in Horses with Naturally-occurring ID

1. Hypothesis: Laminitis associated with underlying endocrine disease is arguably the most important cause of morbidity and mortality in horses and ponies worldwide. We now understand the importance of insulin dysregulation (ID) in these cases and hyperinsulinemia appears to be the key event that drives the development (and recurrence) of laminitis: high blood insulin concentrations directly damage the lamellae. Successful prevention and treatment of hyperinsulinemia-associated laminitis (HAL) therefore depends on our ability to control hyperinsulinemia in horses and ponies with ID. Although there are some mechanistic parallels between equine and human ID, treatments for human metabolic syndrome and type II diabetes are generally aimed at improving insulin sensitivity and/or augmenting the production and function of endogenous insulin and therefore have not translated to useful therapies in the treatment of equine ID and HAL. The development of medications specifically to control hyperinsulinemia in horses requires novel approaches. Since hyperinsulinemia is generally most profound in association with feeding, targeting the post-prandial insulin response is critical for prevention of HAL. Sirolimus (rapamycin) is a drug used in humans for the prevention of organ rejection and treatment of cancer, due primarily to its potent anti-proliferative effects. Sirolimus blocks mechanistic target of rapamycin (mMTOR), a nutrient-sensing signaling pathway that has a major role in regulating growth, proliferation and metabolism in mammals. Sirolimus suppresses insulin production in multiple species, therefore its side effects when administered at high doses can include glucose intolerance, hyperglycemia and (reversible) type II diabetes; however, its effects on insulin dynamics appear to vary with species and are dose-dependent. Our preliminary data in horses demonstrates that a single intravenous dose of sirolimus can suppress insulin production in response to an oral sugar challenge for at least 24 hours. We also have preliminary pilot data from horses with experimentally-induced ID showing that once daily oral sirolimus normalizes the insulin response to an oral sugar challenge. Furthermore, horses remain normoglycemic with no apparent adverse effects during 7 days of sirolimus treatment. Sirolimus is a potentially useful therapy for ID and HAL in horses and warrants further investigation. The purpose of the proposed study is to confirm the therapeutic effects of sirolimus in horses with ID; establish an effective dose rate; and gather further information on the safety profile of this drug in the horse. First Hypothesis (H1): Treatment with sirolimus will prevent hyperinsulinemia in horses with experimentally-induced ID by suppressing insulin production in response to an oral non-structural carbohydrate challenge. Second Hypothesis (H2): Treatment with sirolimus will control hyperinsulinemia in horses with naturally-occurring ID challenged with a carbohydrate rich diet. Third Hypothesis (H3): The administration of sirolimus for control of ID will not be associated with clinically-important adverse effects We will use the combined expertise and resources of the collaborators to accomplish the following aims: Aim 1: Evaluate the effects of oral sirolimus on insulin dynamics in an experimentally-induced ID model. We will use a placebo-controlled trial to determine the effects of oral sirolimus at 2 different dose rates (0.03 mg/kg and 0.06 mg/kg once a day) on insulin dynamics by measurement of blood insulin, c-peptide and incretin (GLP-1 and GIP) responses to oral sugar challenge (H1) Aim 2: Evaluate the effects of oral sirolimus on insulin dynamics in horses with naturally-occurring ID. The effects of oral sirolimus will be evaluated in a heterogeneous group of horses with naturally-occurring ID: using a randomized, placebo-controlled crossover study the effect of treatment on insulin dynamics will be determined by measurement of blood insulin, c-peptide and incretin responses to oral sugar and carbohydrate-rich meal challenge (H2) Aim 3: Characterize any adverse effects associated with sirolimus administration in the horse The effects of sirolimus on blood glucose, triglycerides, hematological parameters, laboratory parameters of renal and liver function/damage as well as clinical parameters will be monitored in conjunction with treatment in Aim 1 and Aim 2 (H3) The Research Problem Laminitis remains a major cause of morbidity and mortality in horses and endocrinopathic laminitis is the most common form, with underlying endocrine disorders identified in over 90% of horses presenting with laminitis in one study [1]. Endocrinopathic laminitis has been associated with both pituitary pars intermedia dysfunction (PPID) and equine metabolic syndrome (EMS)[1]; however, studies have shown that only those horses with hyperinsulinemia develop laminitis, supporting the central role of insulin in lamellar injury and leading to the recent terminology change from “endocrinopathic laminitis” to “hyperinsulinemia-associated laminitis” (HAL) [2, 3]. Multiple studies have experimentally confirmed the central role of hyperinsulinemia in laminitis development through induction of laminitis in healthy horses with exogenous insulin infusion and by correlating the severity of clinical laminitis with insulin concentrations in spontaneous EMS cases [4-6]. Although the exact mechanisms by which insulin damages the lamellae are not fully understood [7] it is clear that control of hyperinsulinemia can prevent laminitis [8] and therefore efforts to develop novel therapeutics for HAL are best aimed at the regulation of blood insulin concentrations in horses and ponies with ID. Hyperinsulinaemia in horses and ponies with ID is exacerbated by the ingestion of feed and pasture [9-11]. Because peak blood insulin concentrations occur within 2-4 hours of feeding/access to pasture [10], it follows that control of this post-prandial insulin response is critical in order to effectively prevent and control HAL. Although dietary restrictions are essential in the long-term management of HAL, limited recognition of the early signs of EMS and HAL (which is frequently subclinical) and poor compliance with dietary and exercise recommendations result in frequent management failures and progressive laminitis in many cases [12, 13]. Medications that can effectively reduce hyperinsulinemia are sorely needed in these cases, however current options for pharmacological control of post-prandial hyperinsulinemia in horses are limited. Metformin, an anti-hyperglycaemic and insulin-sensitising drug used in human diabetes, did demonstrate some effect in blunting the post-prandial glycemic and insulinemic responses to oral dextrose in an experimental ID model [14]; however these effects have not been demonstrated in naturally-occurring ID and metformin has been disappointing as a therapeutic for equine ID and HAL in the clinical setting, despite its widespread use. Sodium-glucose cotransports 2 inhibitors, used in the management of hyperglycaemia in human diabetes, have shown great promise in horses, with a study demonstrating that velaglifozin can limit post-prandial hyperinsulinemia (and prevent laminitis) in EMS ponies challenged with a high non-structural carbohydrate diet [8]; however, these drugs are not currently approved for equine use and their cost may present a barrier to clinical use. As such, there remains a need for potent therapeutic agents that can reduce hyperinsulinemia in the horse, thereby preventing the development of insulin-derived lamellar injury and subsequent HAL. Sirolimus (rapamycin) is a drug used in human organ transplantation and cancer treatment primarily for its anti-proliferative and immunosuppressive effects [15]. Sirolimus inhibits mechanistic target of rapamycin (mTOR), a highly conserved serine-threonine protein kinase which receives numerous signals from growth factors, hormones, nutrients, and cellular energy levels to regulate protein translation and cell growth, proliferation, and survival [16]. The control of downstream events is via two discrete complexes: mTOR complex 1 (mTORC1, primarily responsible for regulation of cell growth) and mTOR complex 2 (mTORC2, primarily cytoskeletal regulation and cell survival) [17]. Treatment with sirolimus mimics starvation and activates autophagy, exerting its effects mostly via inhibition of mTORC1, although inhibition of mTORC2 may be pronounced in specific tissues and also with chronic administration [16, 18]. The mTOR pathway is also a key positive regulator of pancreatic ß-cell function [19] and therefore its inhibition can suppress insulin production and secretion. This is encountered as a side effect of sirolimus treatment, which in humans can cause hyperglycemia and hyperlipidemia with evidence of sirolimus-associated changes in glucose and lipid metabolism, as well as insulin sensitivity [17, 20, 21]. However, this effect has also been harnessed therapeutically: sirolimus has been used successfully in human cases of congenital hyperinsulinemia to reduce the production and secretion of insulin, resulting in improved blood glucose regulation [22, 23]. There are conflicting reports on the effects of sirolimus on insulin and glucose dynamics, particularly with chronic administration, and these effects appear to vary with the dose rate used and with the species [20, 24]. To the authors’ knowledge, there are no published studies investigating the effects of sirolimus (or other mTOR inhibitors/”rapalogs”) in the horse. We have preliminary data demonstrating that sirolimus suppresses insulin production in horses. Furthermore, we have pilot data showing that sirolimus is absorbed orally in horses and appears to effectively control post-prandial hyperinsulinemia without exacerbating hyperglycaemia in an experimental ID model (see preliminary data). Sirolimus is a potentially attractive therapy for horses with ID, since direct suppression of ß-cell function should effectively control hyperinsulinemia regardless of the contribution of the different mechanisms that may lead to excess insulin production in the horse (enhanced glucose absorption, enhanced incretin activity, impaired hepatic insulin clearance, or insulin resistance)[25]. In addition, sirolimus may have additional benefits in the treatment of laminitis: activation of mTORC1 signalling is implicated in the development of laminitis in experimental models of HAL [26] and appears to be a common feature of acute laminitis regardless of cause [7] therefore it is possible that sirolimus may have direct therapeutic effects on the lamellae that could potentially interfere with the development and progression of HAL. Investigation of sirolimus as a therapeutic for ID in horses requires evaluation of its insulin suppressive effects at different dose rates as well as confirmation of its effects on insulin and glucose dynamics in horses with naturally occurring ID. Although sirolimus appears relatively safe at lower doses, even in geriatric human patients when taken daily for up to 2 months [27], it is critical that basic information on the safety of sirolimus in horses is evaluated in order to determine its feasibility as a treatment for equine ID. 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