Nigel A. Calcutt - US grants

Diabetic peripheral neuropathy may be indicated by sensory disorders including spontaneous pain, hyperalgesia or allodynia, by slowed sensory and motor nerve conduction velocities or by structural pathology. Diabetic rats also develop sensory, electrophysiologic and subtle structural disorders. This supports their use as a model of the early stages of hyperglycemia-induced peripheral nerve disorders in the absence of overt structural pathology and allows study of both the etiologic mechanisms linking hyperglycemia to nerve dysfunction and also development of potential therapeutic agents. Recent evidence suggest that peripheral nerve requires ongoing neurotrophic support and that hyperalglycemia disrupts this. Providing exogenous neurotrophic support that either replaces or supercedes diminished endogenous support mechanisms has been proposed as a therapeutic strategy for treating diabetic neuropathy. Prosaposin is the precursor for intracellular saposins but is also secreted in an unprocessed form which has neurotrophic properties. These neurotrophic properties are shared by prosaposin mimetics, small peptides derived from the prosaposin molecule that lack the other properties of saposins. Our preliminary data suggest that prosaposin mimetics called prosaptides prevent or attenuate electrophysiologic, biochemical and structural disorders in the peripheral nerve of diabetic rats, encompassing indices of both sensory and motor function in both large and small fibers. This broad spectrum of efficacy is beneficial for a potential therapeutic because diabetes affects all divisions of the peripheral nervous system. Prosaptides also rapidly ameliorate hyperalgesia in diabetic rats, suggesting a second action distinct from the neurotrophic properties and which may have additional therapeutic benefits to those diabetic patients who develop painful diabetic neuropathy. We propose to establish the therapeutic profiles of prosaptides for treating electrophysiologic and structural disorders of peripheral nerve in diabetic rats that are associated with developing neuropathy and also for treating disorders that reflect pain states. We will also correlate the therapeutic actions of prosaptides with effects on neurochemical abnormalities present in diabetic rats, including investigation of the effect of diabetes on endogenous prosaposin production. The goal is to establish prosaptides as novel therapeutic agents for treating diabetic neuropathy and to provide mechanistic explanations for why hyperglycemia causes nerve disorders.

[unreadable] DESCRIPTION (provided by applicant): Diabetic neuropathy is a widespread clinical problem for which there is no FDA-approved, mechanistically based treatment and is predicted to develop in over half of the approximately 20 million people currently afflicted by diabetes mellitus in the USA. A significant proportion of both insulin-deficient (type 1) and insulin- resistant (type 2) diabetic patients complain of pain or paresthesias that impair quality of life. The etiology of painful diabetic neuropathy is not known and current treatment strategies are limited to drugs with ill-defined mechanisms of action and with side effect profiles that impede normal daily functions and limit effective dosing. Diabetic rats develop hyperalgesia in response to paw formalin injection and allodynia in response to light touch, and they are widely used to model painful diabetic neuropathy in both mechanistic and drug efficacy studies. Recent findings have implicated spinal oligodendrocytes as a site of a pathogenic lesion that initiates spinally mediated hyperalgesia in diabetes. The pathogenic mechanism involves metabolism of excess glucose by aldose reductase, located exclusively in spinal oligodendrocytes, and the subsequent up-regulation of cyclooxygenase 2 (COX-2) in spinal oligodendrocytes and/or neurons. We propose to define the cellular mechanisms by which glucose metabolism by aldose reductase induces COX-2 expression and activity and also the inter-cellular mechanisms that allow oligodendrocytes to initiate spinal hyperalgesa. This will be accomplished by applying a combination of cellular, biochemical and pharmacological techniques to in vitro studies using mature oligodendrocytes derived from the spinal cord of adult normal and diabetic rats. We also intend to investigate pathogenic mechanisms underlying tactile allodynia and formalin-evoked hyperalgesia in diabetic rats that are not related to spinal aldose reductase and COX-2 activity. We will address the hypothesis that decreased expression of the KCC2 cation cotransporter converts spinal GABAergic pathways from inhibitory to excitatory to promote tactile allodynia. The role of altered primary afferent BDNF production in inducing altered spinal KCC2 expression and GABA function and the convergence or divergence of the pathogenic mechanisms during diabetes that induce COX-2 and GABA mediated spinal hyperalgesia will be investigated in diabetic rat models using a combination of behavioral, electrophysiological, pharmacological, biochemical and morphological techniques. The proposed studies are an extension of our recently published and preliminary data and their goal is to define the mechanisms by which diabetes alters spinal sensory processing in favor of amplified and aberrant signals so that new therapeutic targets may be identified and translated into mechanistically directed treatments that will prevent and ameliorate painful diabetic neuropathy. PUBLIC HEALTH RELEVANCE Our primary aim is to investigate the mechanisms by which diabetes alters the capacity of the spinal cord to transduce sensory information passing from the periphery to the brain. The goal is to understand the role of the spinal cord in the pathogenesis of painful diabetic neuropathy so that new treatments for this debilitating condition can be developed. [unreadable] [unreadable] [unreadable]

Neuropathy is the most common of the secondary complications associated with diabetes, with over half of all patients developing some form of nerve dysfunction in their lifetime. The etiology of diabetic neuropathy is not well understood and animal models of diabetic neuropathy have been widely used to investigate potential etiologic mechanisms. A number of hypotheses have been presented as a result of such studies, including involvement of increased glucose metabolism by aldose reductase, reduced nerve blood flow, oxidative stress and loss of neurotrophic support and it is becoming clear that many of these mechanisms are interconnected. However, conventional drug-based therapies derived from these mechanisms have yet to be validated in clinical trials and there is still no mechanistically targeted, FDA approved, therapy for diabetic neuropathy. There have been recent reports in the clinical literature that local treatment of limbs with near infra red energy (NIRE) can improve symptoms of neuropathy in diabetic patients. The mechanism of action is not clear, although there is a small scientific literature showing that light energy can have a variety of physiologic effects in mammals, including vasodilation and consequent increases in local blood flow. Because the clinical data demonstrating efficacy of NIRE on diabetic neuropathy is limited and further mechanistic studies may be restricted by technical and ethical considerations, we will perform quantitative studies of the efficacy of NIRE in streptozotocin diabetic rats. This animal model exhibits functional disorders of peripheral nerve that resemble early diabetic neuropathy and is useful for screening potential therapeutic approaches for diabetic neuropathy. We will examine the efficacy of NIRE in preventing and reversing structural, functional and neurochemical disorders of peripheral sensory and motor nerves and have already performed a preliminary study that suggests some efficacy is likely to be confirmed. The purpose of the project is to establish (or dispute) the scientific and mechanistic rationale for the use of NIRE in treating diabetic neuropathy: If efficacy is established, our findings will promote future studies designed to identify plausible mechanisms of action.

DESCRIPTION (provided by applicant): Diabetic neuropathy is a widespread clinical problem, for which there is no FDA-approved, mechanistically based treatment. There is considerable interest in the hypothesis that neuropathy is secondary to microvascular disease in diabetic patients and drug therapies intended to induce vasodilation or angiogenesis in nerve are being explored in both pre-clinical and clinical studies. However, these systemic pharmaceutical approaches have not overcome the problem of how to target nerve blood flow without impacting vascular beds in other organs where increasing blood flow may be harmful to diabetic patients. We have therefore become interested in non-pharmacologic approaches to inducing local, rather than systemic, blood flow. Pulsed low intensity ultrasound increases local blood flow as part of its wound healing properties via both vasodilator and angiogenic mechanisms. To our knowledge, low intensity ultrasound has not undergone comprehensive preclinical evaluation of its potential to prevent or alleviate indices of diabetic neuropathy. We will address the general hypothesis that low intensity ultrasound treatment is capable of inducing biochemical and physiologic events in rat models of type and type 2 diabetes that prevent and reverse development of functional and structural indices of neuropathy via an ability to modulate local tissue blood flow. We have performed exploratory studies to investigate the effects of ultrasound treatment on nerve disorders in the streptozotocin-diabetic rat model of type 1 diabetes and found that it ameliorated nerve conduction slowing. We propose to undertake a comprehensive survey of the effects of low-intensity ultrasound on functional and structural nerve disorders in STZ-diabetic rats in both prevention and reversal paradigms and to extend optimal treatment regimens to the ZDF model of type 2 diabetes. This will be our primary goal and, while it represents a somewhat observational and high-risk approach, we believe that this is balanced by the potential for our findings to prompt an unusually rapid translation of positive preclinical observations to clinical use because of the non-invasive, non-systemic and non-drug based nature of the treatments. Our secondary goal will be to begin to investigate a potential mechanism of action, namely that ultrasound treatment induces HIF/VEGF/EPO-mediated reparative responses in the nerve in response to exaggerated nerve ischemic hypoxia induced by acute diversion of blood from nerve to muscle. By investigating a local, non-pharmacologic, approach to treating diabetic neuropathy we hope to avoid the side-effects, systemic effects and cost concerns that are inherent to current pharmaceutical-based approaches to ameliorating neuropathy in patients who are likely to require treatment for the rest of their lives. PUBLIC HEALTH RELEVANCE Our primary aim is to investigate the effect of low-intensity ultrasound to prevent and treat nerve damage in diabetic rats. Our secondary aim is to investigate whether the mechanism of action is related to induction of changes in blood flow local, without there being any general systemic effects. The goal is to determine whether this non-invasive, non-pharmaceutical, therapy has potential for rapid translation to use in patients suffering from diabetic neuropathy, for whom life-long treatment with systemic drugs designed to improve nerve blood flow may be costly and have harmful side-effects.

DESCRIPTION (provided by applicant): Peripheral neuropathy is the most common complication of diabetes and will afflict over half of the 25 million Americans who currently suffe from diabetes. There is no FDA-approved therapy to prevent neuropathy or reverse the distal degenerative neuropathy already present in many newly diagnosed diabetic patients. Recent clinical and experimental studies have emphasized that retraction of the peripheral terminals of small sensory axons as an early feature of diabetic neuropathy. This offers a window of opportunity to halt or reverse the dying-back of peripheral terminals before neuronal death. We have recently discovered that muscarinic M1 receptor antagonists can enhance axonal outgrowth from adult rat sensory neurons grown under defined in vitro conditions and also prevent distal neuropathy in diabetic rodents. This prompts us to propose that adult peripheral neurons are under constant cholinergic constraint that moderates the growth capacity of axon terminals and that manipulating this endogenous system offers a novel therapeutic approach to reversing early diabetic neuropathy. We will investigate the mechanism of cholinergic constraint in adult sensory neurons and determine whether diabetes alters this process as part of the pathogenic mechanism of diabetic neuropathy. We will also demonstrate the therapeutic potential of manipulating this endogenous cholinergic constraint mechanism to reverse established distal neuropathy in diabetic rodents and use corneal confocal microscopy to illustrate that preclinical efficacy of a therapeutic can be rapidly translated to demonstrable efficacy in diabetic subjects with peripheral neuropathy.

DESCRIPTION (provided by applicant): Peripheral neuropathy is the most common complication of diabetes and will afflict over half of the 25 million Americans who currently suffe from the disease. There is no FDA-approved therapy to reverse the distal degenerative neuropathy that is already present in many newly diagnosed diabetic patients and which gets progressively worse over time. Recent clinical and experimental studies have demonstrated that the retraction of peripheral terminals of small sensory axons from the epidermis of the skin is an early feature of diabetic neuropathy. This offers an opportunity to halt or reverse the dying-back of peripheral terminals before neuronal death occurs. Our preclinical studies have demonstrated that structurally diverse muscarinic receptor antagonists such as pirenzepine, VU0255035, MT-7 and oxybutynin promote axonal growth from sensory neurons of adult rats in vitro and that this class of drugs also prevents loss of intra-epidermal nerve fibers (IENF) and other features of neuropathy in rodent models of type 1 and type 2 diabetes. It therefore appears that adult peripheral neurons are under constant endogenous cholinergic constraint of axonal growth. The practical application of this knowledge is that muscarinic receptor antagonists may be viable and novel therapeutics for reversing early diabetic neuropathy. The FDA has recently approved use of the muscarinic receptor antagonist oxybutynin (Gelnique 3%TM) as a daily topical therapy for overactive bladder. The drug is applied daily to a region of the skin as a gel and has a good safety profile, the main side effect being dry mouth, which is predictable for an anti-cholinergic drug. As oxybutynin is one of the drugs that we found to prevent IENF depletion in diabetic rodents, we now propose a pilot clinical trial to determine whether Gelnique 3%TM, used off label in an investigator initiated study, can promote re-growth of IENF in a group of type 2 diabetic subjects with established peripheral neuropathy. We will take advantage of our prior experience using a clinical trial design that successfully showed efficacy of drug therapy in improving IENF density in type 2 diabetic subjects, to power the study appropriately and focus on neuropathy end points that are amenable to recovery. We will also enhance the earlier design by performing a double blind, placebo controlled study in subjects with type 2 diabetes and established peripheral neuropathy that will measure IENF density in skin biopsies collected before and 20 weeks after daily application of Gelnique 3%TM to the proximal leg as the primary end point. Secondary end points will be skin blood flow, quantitative sensory test values and quality of life score. We will also include an exploratory study of sudomotor function and sweat gland innervation that will provide data on autonomic neuropathy and assist development and validation of new biomarkers for this understudied aspect of diabetic neuropathy. Our intent in performing this pilot clinical trial is to illustrate that a therapeutic emerging from a focused preclinical drug discovery program can be rapidly translated to show demonstrable efficacy in diabetic subjects with peripheral neuropathy. The goal is to provide proof-of-concept data to support (or refute) the further development of muscarinic antagonists as a new and safe treatment for diabetic neuropathy.