Assessing the Safety of MA-[D-Leu-4]-OB3, a Synthetic Peptide Leptin Mimetic: Two Pre-Clinical Toxicity Studies in Male and Female C57BL/6 Mice.

Although the regulatory influence of leptin on energy balance, glycemic control, immunity, reproduction, and cognition is well established, its clinical application to common obesity and its co-morbidities has been limited by impaired transport across the blood-brain barrier, and tendencies to induce adverse side effects. To circumvent these drawbacks, MA-[D-Leu-4]-OB3, a leptin-related synthetic peptide that mimics the metabolic and neurotrophic effects of leptin in mouse models of genetic and non-genetic obesity, diabetes, and cognitive dysfunction, has been developed. This report presents the results of our initial efforts to assess the safety of orally delivered MA-[D-Leu-4]-OB3. Two pre-clinical studies were done in male and female C57BL/6 mice: a short-term study with a high dose of MA-[D-Leu-4]-OB3 (50 mg/kg/100 µL/day) and a dose-response study with 3 increasing concentrations of MA-[D-Leu-4]-OB3 (16.6, 50, and 150 mg/kg/100 µL/day). Body weight, food and water intake, glucose tolerance, and episodic memory were evaluated. Once-daily cage-side clinical observations were conducted to detect any physical or behavioral indicators of toxicity. Our results indicate that all metabolic and neurologic endpoints tested were either unaffected or improved by MA-[D-Leu-4]-OB3, and no clinical indicators of toxicity were evident. Together with our previously reported efficacy data, these results provide additional evidence supporting further development of this novel synthetic peptide leptin mimetic as a first-in-class peptide drug candidate for the treatment of a number of metabolic and/or cognitive dysfunctions in humans.

MA-[D-Leu-4]-OB3, a small molecule synthetic peptide leptin mimetic, improves episodic memory, and reduces serum levels of tumor necrosis factor-alpha and neurodegeneration in mouse models of Type 1 and Type 2 Diabetes Mellitus.

BACKGROUND: Extracellular beta-amyloid (Aß), intra-neuronal hyper-phosphorylated tau protein, and chronic inflammation are neuropathological hallmarks of Alzheimer's Disease (AD). A link between AD, insulin dysfunction, and tumor necrosis factor-alpha (TNF-α) in promoting both tau and Aß pathologies in vivo has been proposed. METHODS: MA-[D-Leu-4]-OB3 was given, with or without insulin, to streptozotocin (STZ)-treated male Swiss Webster mice, and to male diet-induced obese (DIO) mice. Brains were excised, and coronal sections were imaged with fluoro jade-C (FJC), thioflavin-S, or hematoxylin and eosin (H&E). Serum TNF-α and IGF-1 were measured by ELISA. Histopathological changes in the cerebral cortex (CC) and hippocampus (HC) were correlated with changes in glycemic regulation, episodic memory, and serum levels of TNF-α and IGF-1. RESULTS: In STZ-treated mice, blood glucose and serum TNF-α and IGF-1 were reduced by insulin alone, and normalized when MA-[D-Leu-4]-OB3 was given in combination with insulin. Improvement in episodic memory was inversely correlated with the number of FJC-positive cells in the CC and HC and serum TNF-α and IGF-1. FJC, thioflavin-S and H&E staining indicated no Aß deposition. Similar results were observed in DIO mice treated with MA-[D-Leu-4]-OB3. CONCLUSIONS: The mechanism by which MA-[D-Leu-4]-OB3 improves episodic memory in mouse models of TIDM and T2DM appears to be related to improved insulin sensitivity and reduced TNF-α-induced neurodegeneration. GENERAL SIGNIFICANCE: MA-[D-Leu-4]-OB3 may have application to human pre-clinical and clinical AD and AD-like dementia by interrupting the cascade of insulin resistance, neuro-inflammation, and neurodegeneration, that characterizes these diseases.

[D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3, small molecule synthetic peptide leptin mimetics, improve glycemic control in diet-induced obese (DIO) mice.

We have previously shown that following oral delivery in dodecyl maltoside (DDM), [D-Leu-4]-OB3 and its myristic acid conjugate, MA-[D-Leu-4]-OB3, improved energy balance and glucose homeostasis in genetically obese/diabetic mouse models. More recently, we have provided immunohistochemical evidence indicating that these synthetic peptide leptin mimetics cross the blood-brain barrier and concentrate in the area of the arcuate nucleus of the hypothalamus in normal C57BL/6J and Swiss Webster mice, in genetically obese ob/ob mice, and in diet-induced obese (DIO) mice. In the present study, we describe the effects of oral delivery of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 on glycemic control in diet-induced (DIO) mice, a non-genetic rodent model of obesity and its associated insulin resistance, which more closely recapitulates common obesity and diabetes in humans. Male C57BL/6J and DIO mice, 17, 20, and 28 weeks of age, were maintained on a low-fat or high-fat diet and given vehicle (DDM) alone or [D-Leu-4]-OB3 or MA-[D-Leu-4]-OB3 in DDM by oral gavage for 12 or 14 days. Body weight gain, food and water intake, fasting blood glucose, oral glucose tolerance, and serum insulin levels were measured. Our data indicate that (1) [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 restore glucose tolerance in male DIO mice maintained on a high-fat diet to levels comparable to those of non-obese C57BL/6J wild-type mice of the same age and sex maintained on a low-fat diet; and (2) the influence of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 on glycemic control appears to be independent of their effects on energy balance. These results suggest that [D-Leu-4]-OB3 and/or MA-[D-Leu-4]-OB3 may have application to the management of the majority of cases of common obesity in humans, a state characterized at least in part, by leptin resistance resulting from a defect in leptin transport across the blood-brain barrier. They further suggest that these small molecule synthetic peptide leptin mimetics, through their influence on glycemic control, may prevent the pre-diabetic state associated with most cases of common obesity from escalating into overt type 2 diabetes mellitus.

Oral delivery of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3, synthetic peptide leptin mimetics: Immunofluorescent localization in the mouse hypothalamus.

This study describes the localization of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3, synthetic peptide leptin mimetics, in the hypothalamus of Swiss Webster and C57BL/6J wild-type mice, leptin-deficient ob/ob mice, and leptin-resistant diet-induced obese (DIO) mice. The mice were given [D-Leu-4]-OB3 or MA-[D-Leu-4]-OB3 in 0.3% dodecyl maltoside by oral gavage. Once peak serum concentrations were reached, the mice received a lethal dose of pentobarbital and were subjected to intracardiac perfusion fixation. The brains were excised, post-fixed in paraformaldehyde, and cryo-protected in sucrose. Free-floating frozen coronal sections were cut at 25-µm and processed for imaging by immunofluorescence microscopy. In all four strains of mice, dense staining was concentrated in the area of the median eminence, at the base and/or along the inner wall of the third ventricle, and in the brain parenchyma at the level of the arcuate nucleus. These results indicate that [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 cross the blood-brain barrier and concentrate in an area of the hypothalamus known to regulate energy balance and glucose homeostasis. Most noteworthy is the localization of [D-Leu-4]-OB3 immunoreactivity within the hypothalamus of DIO mice via a conduit that is closed to leptin in this rodent model, and in most cases of human obesity. Together with our previous studies describing the effects of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 on energy balance, glucose regulation, and signal transduction pathway activation, these findings are consistent with a central mechanism of action for these synthetic peptide leptin mimetics, and suggest their potential usefulness in the management of leptin-resistant obesity and type 2 diabetes in humans.

Myristic acid conjugation of [D-Leu-4]-OB3, a biologically active leptin-related synthetic peptide amide, significantly improves its pharmacokinetic profile and efficacy.

We have previously described the pharmacokinetics of mouse [D-Leu-4]-OB3, a synthetic peptide amide with leptin-like activity, following delivery by subcutaneous (SC), intraperitoneal (IP), and intramuscular (IM) injection, and by oral gavage and intranasal instillation. These profiles suggested that the observed efficacy of [D-Leu-4]-OB3 on energy balance, glycemic control, and bone turnover in ob/ob and db/db mice might be improved by efforts directed toward improving its bioavailability, i.e., increasing maximum uptake (Cmax), extending serum half-life (t½), and reducing plasma clearance (CL). To address these issues, myristic (tetradecanoic) acid was conjugated to the N-terminal of [D-Leu-4]-OB3 (designated MA-[D-Leu-4]-OB3), and the pharmacokinetics of MA-[D-Leu-4]-OB3 in male Swiss Webster mice following SC, IP, and IM injection in PBS, and by oral and intranasal delivery in dodecyl maltoside (DDM, trade name Intravail®), a transmucosal absorption enhancing agent, were compared to those of [D-Leu-4]-OB. At a dose of MA-[D-Leu-4]-OB3 10-fold lower than that used previously for [D-Leu-4]-OB3 (0.1 mg vs.1.0 mg, respectively), Cmax of MA-[D-Leu-4]-OB3 was 11.1-, 7.5-, 1.9-, and 1.7-fold higher, t1/2 was 3.5-, 5.0-, 9.1-, and 86.7-fold longer, and CL was 17.0-, 11.6-, 5.7-, and 5.0-fold slower than [D-Leu-4]-OB3 following SC, IP, IM, and oral delivery, respectively. Furthermore, in leptin-resistant obese male db/db mice, oral delivery of MA-[D-Leu-4]-OB3 in DDM at concentrations up to 10-fold lower than those used with [D-Leu-4]-OB3 reduced fasting blood glucose levels in a dose-related manner.

[D-Leu-4]-OB3, an orally bioavailable leptin-related synthetic peptide insulin sensitizer: a study comparing the efficacies of [D-Leu-4]-OB3 and metformin on energy balance and glycemic regulation in insulin-deficient male Swiss Webster mice.

The effects of oral delivery of exenatide or pramlintide acetate in dodecyl maltoside (DDM) on energy balance and glycemic control in insulin-resistant obese db/db mice are enhanced when given in combination with [D-Leu-4]-OB3. To examine the anti-hyperglycemic influence of [D-Leu-4]-OB3 in a non-obese insulin-deficient animal model, we compared the effects of metformin (200mg/kg) and [D-Leu-4]-OB3 (40 mg/kg) on energy balance and glycemic control in streptozotocin (STZ)-induced diabetic male Swiss Webster (SW) mice. Diabetic mice were given insulin (Levemir(®), sc) alone, or in combination with metformin or [D-Leu-4]-OB3 orally in DDM, for 14 days. Body weight and food and water intake were measured daily. Fasting blood glucose levels were determined every other day. Serum C-peptide was measured by ELISA. Diabetic mice receiving insulin alone for 14 days gained significantly more weight than DDM-treated control mice, or mice given insulin in combination with metformin or [D-Leu-4]-OB3. The weight gain seen in mice given insulin alone was accompanied by significant increases in both food and water intake. Mice treated with insulin in combination with metformin or [D-Leu-4]-OB3, consumed significantly less food and water. Blood glucose levels in STZ-treated mice receiving insulin alone were reduced to 65.3% of initial levels, while mice receiving insulin with metformin or [D-Leu-4]-OB3 were reduced to 44.5% and 38.9%, respectively. Our results indicate that [D-Leu-4]-OB3 is as effective as metformin in preventing the body weight gain associated with insulin therapy, and on a molar basis, that the efficacy of [D-Leu-4]-OB3 as an insulin sensitizer may equal or surpass that of metformin.

Intranasal administration of mouse [D-Leu-4]OB3, a synthetic peptide amide with leptin-like activity, enhances total uptake and bioavailability in Swiss Webster mice when compared to intraperitoneal, subcutaneous, and intramuscular delivery systems.

Using a synthetic peptide strategy, we localized an active domain in mouse leptin to a sequence between amino acids 106 and 140. Intraperitoneal (ip) administration of a number of synthetic peptide amides encompassed by this domain reduced body weight gain, food and water intake, blood glucose levels, and increased insulin sensitivity in genetically obese mice. In the present study, we examined the pharmacokinetics of mouse [D-Leu-4]OB3, our most potent peptide, in male Swiss Webster mice following ip, subcutaneous (sc), and intramuscular (im) injection, and after intranasal administration with Intravail, a new class of patented transmucosal delivery enhancement agents. Total uptake (1,072,270, 1,182,498; 1,481,060; ng/ml/min), serum half-life (48.8; 34.0; 30.0 min) and relative bioavailability (1.0, 1.1; 1.4;) of mouse [D-Leu-4]OB3 were similar when the peptide was given by ip, sc, or im injection, respectively. Total uptake and relative bioavailability were enhanced following intranasal delivery (4,336,963 ng/ml/min and 4.0, respectively), and serum half-life was 41.1 min. These results indicate that intranasal delivery of mouse [D-Leu-4]OB3 with Intravail is a more effective method of peptide administration than injection methods, and suggest that it may have potential as a novel, non-invasive approach to the treatment of obesity and its associated metabolic dysfunctions in humans.