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

Abstract

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.

Introduction

Leptin, an adipocytokine hormone, has physiologic impacts far beyond its most well-recognized role in body weight maintenance and appetite suppression. Growing evidence from both in vitro and in vivo studies has further elucidated leptin’s role as a common biochemical mediator in multiple physiologic capacities including, but not limited to, glycemic control and cognitive function.^1–3 Harnessing the beneficial effects of leptin will make it a strong candidate for the therapeutic and/or prophylactic treatment for a number of human metabolic and neurologic pathologies.

In this regard, the positive impact of leptin in reversing many metabolic and cognitive dysfunctions in leptin-deficient rodent models and humans is well established. ¹ There are, however, complicating factors in the therapeutic use of leptin in the clinic. These include the pre-existing hyperleptinemia present in common obesity, as well as some adverse side effects associated with chronic leptin therapy: angiogenesis, carcinogenesis, and the potential to aggravate autoimmune disease.^1,4–6

The hyperleptinemia seen in common obesity is the result of the presence of an excess of white adipose tissue (WAT) and increased leptin production. ¹ Abnormally high circulating levels of endogenous leptin induce central leptin resistance by reducing the efficiency of the saturable transport of leptin across the blood-brain barrier (BBB),^7,8 thereby decreasing leptin signaling. Paradoxically, mid-life obesity followed by late-life weight loss, which is also a risk factor for the development of Alzheimer’s disease (AD), ⁹ appears to be a biochemical double-edged sword in which the initial hyperleptinemia responsible for central leptin resistance in mid-life is replaced by leptin deficiency due to the low circulating levels of leptin associated with being underweight.

Overcoming the caveats associated with chronic leptin therapy has been very difficult. Currently, only metreleptin, a recombinant form of human leptin, has received Federal Drug Administration (FDA) approval for clinical use, and is limited to the treatment of lipodystrophy in leptin-deficient individuals. ¹⁰ Application of metreleptin therapy outside of this patient population has met with either marginal or completely negligible results. Notably, in one clinical trial, metreleptin was discontinued due to a significant number of patients developing anti-leptin antibodies. ¹¹ Unfortunately, many potential applications for leptin are limited in that the majority of cases of human obesity that are resistant to elevated levels of endogenous leptin are also resistant to exogenously delivered leptin as well.

For a number of years, our laboratory has focused its efforts on overcoming the limitations of exogenous leptin therapy by developing small molecule synthetic peptide leptin mimetics. Initial efforts resulted in the identification of a small peptide, 15 amino acids long, within the sequence of mouse leptin that showed leptin-like activity in genetically obese ob/ob mice following intraperitoneal delivery. ¹² This peptide has been modified over the years to improve its efficacy, pharmacokinetics, and method of delivery in a number of mouse models of human disease.^13–20 Most worthy of special note is the proven ability of these leptin mimetics to cross the BBB and to localize in the hypothalamus in all mouse models tested, ¹⁷ thus overcoming the central resistance associated with common obesity.

Also worthy of note, in streptozotocin-treated Swiss Webster mice, a model of type 1 diabetes mellitus (T1DM) and AD, on a molar basis, these leptin mimetics were equally as effective as metformin in preventing the body weight gain associated with insulin therapy, and in increasing insulin sensitivity. ¹⁴ Similar results were seen in diet-induced obese (DIO) mice, a model of type 2 diabetes mellitus (T2DM) and vascular dementia (VaD). ¹⁵ In both of these models, MA-[D-Leu-4]-OB3 not only reduced body weight gain and restored glycemic control, but also improved episodic memory. The prophylactic potential of MA-[D-Leu-4]-OB3 was also recently confirmed by its ability to prevent and/or slow the progression of obesity, insulin resistance, and cognitive impairment in normal C57BL/6 mice placed on a high-fat diet immediately after weaning. ²¹

In the 2 studies reported here, we provide evidence that oral delivery of MA-[D-Leu-4]-OB3, previously shown to modulate energy balance, glycemic control, serum lipids, and cognitive function in mouse models of leptin deficiency and leptin resistance, does not induce anorexia, severe weight loss, hypoglycemia, cognitive dysfunction, or any physical or behavioral clinical indicators of toxicity when given to male or female C57BL/6 mice at concentrations significantly higher than the minimal effective dose, or for extended periods of time.

Materials and Methods

Animals and Housing

Three- to four-week old male and female C57BL/6 wild type (B6-DIOCONTROL-M and B6-DIOCONTROL-F) mice, obtained from Taconic Farms (Germantown, NY, USA) were used in these studies. Upon arrival, the mice were randomized by weight into treatment groups (n = 4 or 5 mice per group), and group-housed in polycarbonate cages fitted with stainless steel wire lids and air filters in the Albany Medical College Animal Resources Facility. The mice were maintained at a constant temperature (24°C) with lights on from 07:00 to 19:00 h.

Feeding and Weighing

The mice received approximately 400 g (tared) of standard rodent chow, and tared water bottles containing approximately 400 mL of water were placed on each cage. Food and water (also tared) were added to the cages when needed throughout the course of this study. Because the mice were group-housed in both studies, cumulative food and water intake per mouse was determined by dividing the total intake during the course of each study by the number of mice in the cage. When oral glucose tolerance tests (OGTTs) were done, overnight fasts (16 h) were initiated by removing food from the cages between 17:00 and 18:00 h the afternoon before testing. OGTTs were performed between 09:00 and 10:00 h the following day. The mice were weighed daily between 08:00 and 09:00 h on an Acculab V-333 electronic balance (Cole-Parmer, Vernon Hills, IL, USA).

MA-[D-Leu-4]-OB3 Administration

MA-[D-Leu-4]-OB3, the myristic acid (MA) conjugate of [D-Leu-4]-OB3 (Ser-Cys-Ser-dLeu-Pro-Gln-Thr), ¹⁹ was prepared commercially at >97% purity as a C-terminal amide by Atlantic Peptides (Lewisburg, PA, USA). MA-[D-Leu-4]-OB3 was dissolved in vehicle (.3% dodecyl maltoside, DDM, trade name Intravail®, Aegis Therapeutics, San Diego, CA, USA, reconstituted in sterile deionized water), and delivered by oral gavage (100 μL) once daily between 16:00 and 17:00 h. This time-frame was chosen based on the active feeding time of the mice (after lights out at 19:00 h), and the pharmacokinetics of MA-[D-Leu-4]-OB3: the maximum concentration (C_max) following oral delivery is reached at 4 h (T_max), and the half-life (T_1/2) is 29 h. ¹⁶

Blood Glucose Measurement

Initial blood samples were drawn by nicking the tail of each mouse. Subsequent samples were obtained by gently removing the scab that formed at the site of the nick. The blood droplet formed at the nick was applied to a glucose test strip. Glucose levels were measured with a OneTouch Verio IQ glucose meter (Janssen Pharmaceuticals, Raritan, NJ, USA).

Oral Glucose Tolerance Testing

Following an overnight fast (16 h), mice received a single dose of 100 μL DDM (vehicle) alone or MA-[D-Leu-4]-OB3 in 100 μL DDM. After 60 minutes, blood glucose was measured to establish basal levels, and the mice were challenged with 100 μL d-glucose (2 g/kg) in phosphate-buffered saline (PBS). Additional glucose measurements were taken at 15, 30, and 60 minutes after the glucose challenge.

Novel Object Recognition Behavioral Testing

Novel object recognition testing was done as described by Bevins and Besheer. ²²

Apparatus and environment : In a designated behavioral testing room, a square white plastic chamber with smooth walls and floor, and divided into 4 equal quadrants, each 51 cm long × 51 cm wide × 38 cm high, was thoroughly cleaned with 70% ethanol. Overhead lights were turned off, and floor lamps with red bulbs were placed at the sides of the chamber so that red lighting was evenly distributed over the 4 quadrants. A Logitech HD Pro Webcam C920 (Logitech Inc., Newark, CA, USA) video camera with an adjustable zoom lens was mounted on an overhead bracket facing straight down over the center of the chamber. Habituation, training, and testing phases, each 10 minutes long, were recorded with an automated video tracking system (ANY-maze 6.06, Stoelting, IL, USA).

Protocol: Object selection and placement : Prior to novel object recognition testing, the mice had been handled for approximately 15 minutes daily. To habituate the mice to the testing environment, each mouse was placed in an empty quadrant prior to the training and testing phases, and allowed to freely explore for 10 minutes. During the training phase, the mice were exposed to 2 identical 50 mL serum bottles (familiar objects), approximately 9 cm high and 5 cm in diameter. These objects were placed in the center of each quadrant, equidistant from each other and from the quadrant walls. The mice were placed midway between the 2 objects with their noses pointing forward, and allowed to explore the objects for 10 minutes. In the testing phase, one of the serum bottles was replaced with a rubber duckling (novel object), approximately 7 cm high and 7 cm long. The objects were placed in the center of each quadrant equidistant from each other and from the quadrant walls. As in the training phase, mice were placed midway between the 2 objects with their noses pointed forward, and allowed to explore these objects for 10 minutes. Approximately 1 hour separated the training phase from the testing phase, during which time the mice were returned to their home cages. The chamber was cleaned with 70% ethanol after each habituation/training/testing period.

Scoring: Videos from the training and the testing phases were blinded and manually scored. The exploration time of both novel and familiar objects was defined as the time a mouse interacted with an object, that is, when the mouse’s nose touched the object or was within 1 cm of the object and pointed directly at the object. Not counted as exploration: when the mouse climbed on top of the object or was not approaching the object. For each mouse, the novel object and familiar object exploration times (in seconds) were recorded, and the discrimination index (DI), expressed as percent, was calculated as follows: time (in seconds) exploring the novel object minus time (in seconds) exploring the familiar object divided by the total time exploring both objects, multiplied by 100.

Cage-Side Clinical Observations

Once-daily cage-side clinical observations were made prior to treatment each day. Any physical changes in skin, coat quality, lacrimation, pupil size, mucous membranes, secretions, or excretions in peptide-treated mice compared to vehicle-treated control mice were noted, as were any behavioral changes in gait, activity, grooming, circling, response to handling, self-mutilation, or aggression.

All of these animal procedures were approved by the Albany Medical College Animal Care and Use Committee, and were performed in accordance with relevant guidelines and regulations as outlined in the Guide for the Care and Use of Laboratory Animals, Eighth edition (2011) (http://grants.nih.gov/grant/olaw/guide-for-the-care-and-use-of_laboratory-animals.pdf).

Statistical Analysis

All data are expressed as mean + SEM. SigmaPlot®12.5 for windows (SPSS, Chicago, IL. USA) was used to determine statistical significance. Differences in body weight, glucose tolerance AUC, novel and familiar object exploration times, and discrimination index were compared by t test or one-way analysis of variance (ANOVA), followed by post hoc analysis using the Holm-Sidak method. Differences between groups were considered statistically significant when P < .05.

Results

Short-term acute toxicity study: A time-line describing this protocol based on Sibley et al ²³ is shown in Figure 1.

Figure 1.

Male and female C57BL/6 mice were given MA-[D-Leu-4]-OB3 (50 mg/kg/day) or vehicle by oral gavage in two 4-day cycles separated by a 10-day washout period. Novel object recognition (NOR) testing and oral glucose tolerance testing (OGTT) were done before and after each cycle and after the washout period. Body weight was measured daily; cumulative food and water intake were measured weekly. Cage-side clinical observations for physical or behavioral indicators of toxicity were done daily.

Effects of MA-[D-Leu-4]-OB3 (50 mg/kg/day) on body weight gain in male and female C57BL/6 mice. The effects of MA-[D-Leu-4]-OB3 on body weight gain in male and female C57BL/6 mice are shown in Figure 2

Figure 2.

Effects of MA-[D-Leu-4]-OB3 (50 mg/kg/day) on body weight gain in male and female C57BL/6 mice before and after cycle 1 of treatment (Figure 2(a)), after the 10-day washout period (Figure 2(b)), and before and after cycle 2 of treatment (Figure 2(c)). Each bar and vertical line represents the body weight (mean + SEM, n = 5) in grams for each group of mice. The numbers shown in the white bars represent body weight gain (+) or loss (−) in grams compared to body weight before treatment or washout. No statistically significant (P > .05) difference between mice receiving vehicle or MA-[D-Leu-4]-OB3 was noted at any stage of the study.

: before and after cycle 1 of treatment (Figure 2(a)), after the 10-day washout period (Figure 2(b)), and before and after cycle 2 of treatment (Figure 2(c)). No statistically significant difference (P > .05) in body weight gain was observed between vehicle- and MA-[D-Leu-4]-OB3-treated male or female mice at any stage of the study.

Effects of MA-[D-Leu-4]-OB3 (50 mg/kg/day) on cumulative food and water intake in male and female C57BL/6 mice. The effects of MA-[D-Leu-4]-OB3 on cumulative food and water intake throughout the course of the study are shown in Figures 3a and (b), respectively. No significant difference in food or water intake was observed in either male or female mice receiving vehicle or MA-[D-Leu-4]-OB3.

Figure 3.

Effects of MA-[D-Leu-4]-OB3 (50 mg/kg/day) on cumulative food (Figure 3(a)) and water (Figure 3(b)) intake by male and female C57BL/6 mice. Each bar represents the total amount of food (in grams) and water (in mL) consumed per mouse during the entire course of the study. The numbers shown in the white bars indicate an increase (+) or decrease (−) in food or water intake in cycle 2 compared to cycle 1. No difference in food or water intake between mice receiving vehicle or MA-[D-Leu-4]-OB3 was noted in either males or females.

Effects of MA-[D-Leu-4]-OB3 (50 mg/kg/day) on glucose tolerance in male and female C57BL/6 mice. The effects of MA-[D-Leu-4]-OB3 on glucose tolerance, expressed as area under the curve (AUC), are shown in Figure 4: before (Figure 4(a)) and after (Figure 4(b)) cycle 1, after the 10-day washout period (Figure 4(c)), and after cycle 2 (Figure 4(d)). No statistically significant difference (P > .05) in glucose tolerance between vehicle- and MA-[D-Leu-4]-OB3-treated male or female mice was observed at any stage of the study.

Figure 4.

Effects of MA-[D-Leu-4]-OB3 (50 mg/kg/day) on oral glucose tolerance in male and female C57BL/6 mice before (Figure 4(a)) and after (Figure 4(b)) cycle 1 of peptide treatment, after the 10-day washout period (Figure 4(c)), and after cycle 2 of peptide treatment (Figure 4(d)). Each point and vertical line represents serum glucose level (mean + SEM, n = 5) at base, and 15, 30, and 60 minutes after an oral glucose challenge. Total serum glucose is expressed as area under the curve (AUC). No statistically significant difference (P > .05) in glucose tolerance was observed between vehicle- and MA-[D-Leu-4]-OB3-treated male or female mice at any stage of the study.

Effects of MA-[D-Leu-4]-OB3 (50 mg/kg/day) on episodic memory in male and female C57BL/6 mice. The effects of MA-[D-Leu-4]-OB3 on episodic memory, expressed as discrimination index (DI) are shown in Figure 5: before (Figure 5(a)) and after (Figure 5(b)) cycle 1, after the 10-day washout period (Figure 5(c)), and after cycle 2 (Figure 5(d)). After the first 4 days of MA-[D-Leu-4]-OB3 treatment (cycle 1), episodic memory in both male and female mice was significantly (P < .05) improved compared to vehicle-treated controls (Figure 5(b)). After the 10-day washout period, episodic memory was essentially the same in all 4 groups (Figure 5(c)). After the final 4 days of MA-[D-Leu-4]-OB3 treatment (cycle 2), episodic memory in both male and female MA-[D-Leu-4]-OB3-treated mice was again significantly (P < .05) improved in both males and females when compared to vehicle-treated controls.

Figure 5.

Effects of MA-[D-Leu-4]-OB3 (50 mg/kg/day) on episodic memory in male and female C57BL/6 mice before (Figure 5(a)) and after (Figure 5(b)) cycle 1 of peptide treatment, after the 10-day washout period (Figure 5(c)), and after cycle 2 of peptide treatment (Figure 5(d)). Each bar and vertical line represents mean + SEM, n = 5) discrimination index (DI) of each group of mice. *P < .05 compared to vehicle-treated control mice.

Effects of MA-[D-Leu-4]-OB3 (50 mg/kg/day) on physical and/or behavioral indicators of toxicity in male and female C57BL/6 mice. Once-daily cage-side clinical observations throughout the test period revealed no evidence of any physical or behavioral indicators of toxicity in MA-[D-Leu-4]-OB3-treated male or female C57BL/6 mice. When compared to vehicle-treated control mice, no differences in skin, fur, secretions, stool, lacrimation, gait, activity level, response to handling, grooming time, circling, or aggression were noted in MA-[D-Leu-4]-OB3-treated male or female mice.

Dose range-finding study: A time-line describing this protocol based on Di Martino et al ²⁴ is shown in Figure 6.

Figure 6.

Male and female C57BL/6 mice were given vehicle or MA-[D-Leu-4]-OB3 (16.6, 50, or 150 mg/kg/day) by oral gavage for 10 consecutive days. Novel object recognition (NOR) testing and oral glucose tolerance testing (OGTT) were done before and after the 10-day treatment period. Body weight was measured daily; cumulative food and water intake were measured after the 10-day treatment period. Cage-side clinical observations for physical or behavioral indicators of toxicity were done daily.

Effects of Increasing concentrations of MA-[D-Leu-4]-OB3 (16.6, 50, and 150 mg/kg/day) for 10 days on body weight gain in male and female C57BL/6 mice. The effects of increasing concentrations of MA-[D-Leu-4]-OB3 on body weight gain in male and female C57BL/6 mice are shown in Figure 7(a) and (b), respectively. No statistically significant difference (P > .05) in body weight gain in male or female MA-[D-Leu-4]-OB3-treated mice compared to vehicle-treated control mice was observed at any concentration of MA-[D-Leu-4]-OB3 tested.

Figure 7.

Effects of MA-[D-Leu-4]-OB3 (16.6, 50, or 150 mg/kg/day) on body weight gain in male (Figure 7(a)) and female (Figure 7(b)) C57BL/6 mice before and after 10 days of vehicle or peptide treatment. Each bar and vertical line represents the body weight (mean + SEM, n = 4) in grams for each group of mice. The numbers shown in the white bars represent body weight gain (+) in grams compared to initial body weight. No statistically significant (P > .05) difference between mice receiving vehicle or MA-[D-Leu-4]-OB3 was noted at any concentration of MA-[D-Leu-4]-OB3 tested.

Effects of increasing concentrations of MA-[D-Leu-4]-OB3 (16.6, 50, and 150 mg/kg/day) for 10 days on cumulative food and water intake in male and female C57BL/6 mice. The effects of increasing concentrations of MA-[D-Leu-4]-OB3 on cumulative food and water intake by male and female C57BL/6 mice are shown in Figure 8a and (b), respectively. Compared to vehicle-treated controls, no significant difference in cumulative food or water intake was observed in either male or female mice at any concentration of MA-[D-Leu-4]-OB3 tested.

Figure 8.

Effects of MA-[D-Leu-4]-OB3 (16.6, 50, or 150 mg/kg/day) on cumulative food and water intake by male (Figure 8(a)) and female (Figure 8(b)) C57BL/6 mice. Each bar represents the total amount of food (in grams) and water (in mL) consumed per mouse during the 10-day treatment period. No significant difference in cumulative food intake was observed in either male or female mice given MA-[D-Leu-4]-OB3 at any concentration tested.

Effects of increasing concentrations of MA-[D-Leu-4]-OB3 (16.6, 50, and 150 mg/kg/day) for 10 days on glucose tolerance in male and female C57BL/6 mice. The effects of increasing concentrations of MA-[D-Leu-4]-OB3 on glucose tolerance, expressed as area under the curve (AUC), in male and female C57BL/6 mice after 10 days of treatment are shown in Figure 9. In male C57BL/6 mice, no statistically significant (P > .05) difference in glucose tolerance between vehicle-treated mice and mice receiving MA-[D-Leu-4]-OB3 at all concentrations tested was noted either before (Figure 9(a)) or after (Figure 9(b)) the 10-day test period. Similar results were seen in female C57BL/6 mice before (Figure 9(c)) and after (Figure 9(d)) receiving MA-[D-Leu-4]-OB3 for 10 days.

Figure 9.

Effects of MA-[D-Leu-4]-OB3 (16.6, 50, or 150 mg/kg/day) on oral glucose tolerance in male C57BL/6 mice before and after (Figure 9(a) and (b), respectively) and in female C57BL/6 mice before and after (Figure 9c and (d), respectively) 10 days of peptide treatment. Each point and vertical line represents serum glucose level (mean + SEM, n = 4) at base, and 15, 30, and 60 minutes after an oral glucose challenge. Total serum glucose is expressed as area under the curve (AUC). No statistically significant difference (P > .05) in glucose tolerance was observed between vehicle- and peptide-treated male or female mice at any concentration of MA-[D-Leu-4]-OB3 tested.

Effects of increasing concentrations of MA-[D-Leu-4]-OB3 (16.6, 50, and 150 mg/kg/day) on episodic memory in male and female C57BL/6 mice. The effects of increasing concentrations of MA-[D-Leu-4]-OB3 on episodic memory before and after 10 days of treatment in male and female C57BL/6 mice are shown in Figure 10(a) and (b), respectively. The discrimination index (DI) for each group of mice, before and after peptide treatment, was calculated from the exploration time data. No statistically significant difference (P > .05) between vehicle- and peptide-treated males or females was observed at any concentration of MA-[D-Leu-4]-OB3 tested.

Figure 10.

Effects of MA-[D-Leu-4]-OB3 (16.6, 50, or 150 mg/kg/day) on episodic memory, expressed as discrimination index (DI), in male (Figure 10(a)) and female (Figure 10(b)) C57BL/6 mice, before and after 10 days of treatment. Each bar and vertical line represents the discrimination index (DI) (mean + SEM, n = 4) of each group of mice. Differences in episodic memory between vehicle- and peptide-treated males or females were not statistically significant (P > .05) at any concentration of MA-[D-Leu-4]-OB3 tested.

Effects of increasing concentrations of MA-[D-Leu-4]-OB3 (16.6, 50, and 150 mg/kg/day) on physical and/or behavioral indicators of toxicity in male and female C57BL/6 mice. Once-daily cage-side clinical observations throughout the 10-day test period indicated no evidence of any physical or behavioral changes in MA-[D-Leu-4]-OB3-treated male or female C57BL/6 mice when compared to vehicle-treated controls.

Discussion

In contrast to previous studies in mouse models of full-blown or developing metabolic and/or neurologic disease, the results we report here are from 2 studies which were designed to explore the potential of MA-[D-Leu-4]-OB3, at doses many times higher than the minimal effective dose 8.3 mg/kg/day (unpublished data), to induce adverse side effects, particularly anorexia, weight loss, hypoglycemia, insulin resistance, and cognitive dysfunction, in healthy male and female C57BL/6 mice.

The normal leptin levels in C57BL/6 mice (2-5 ng/mL), when compared to the hyperleptinemia characterizing genetically obese db/db mice and diet-induced obese (DIO) mice used in our previous studies, as well as their normal metabolic and neurologic baselines, make this mouse an appropriate model with which to assess any deleterious effects of MA-[D-Leu-4]-OB3 on metabolic or neurologic functions that may be influenced by central leptin receptors, particularly those in the arcuate nucleus of the hypothalamus where leptin exerts its major effects on appetite and energy expenditure, ²⁵ and in the hippocampus and cortex, the major centers of memory and cognition. ²⁶ Our data clearly indicate that none of the negative metabolic or neurologic outcomes that could potentially be caused by elevated concentrations or prolonged treatment with MA-[D-Leu-4]-OB3 occurred. All parameters measured were either unaffected or improved by MA-[D-Leu-4]-OB3.

In this regard, worthy of special note are the effects of MA-[D-Leu-4]-OB3 on cognitive function, but not on body weight gain, food and water intake, or glucose tolerance, that were observed in both males and females given MA-[D-Leu-4]-OB3 at 50 mg/kg/day in 4-day intervals and in the 10-day study. These results suggest that there may be separate therapeutic thresholds and/or tolerance levels for the regulatory actions of MA-[D-Leu-4]-OB3 on metabolic and neurologic functions. This notion is further supported by the significant improvement in episodic memory seen at 50 mg/kg/day in males and females in treatment cycles one and two of the acute study, when compared to the influence of the same concentration of MA-[D-Leu-4]-OB3, and the higher concentration tested, which was significantly diminished in both males and females, when given for 10 days.

Although the metabolic functions tested were unchanged at all concentrations of MA-[D-Leu-4]-OB3 tested during the course of the 4-day and 10-day test periods, cognitive function appeared to be much more sensitive to both dose and duration of exposure to MA-[D-Leu-4]-OB3. Also worthy of note, the change in efficacy of MA-[D-Leu-4]-OB3 on cognitive function observed in the 10-day study, although not statistically significant, was more pronounced in females than in males, suggesting the possibility of an additional sex-dependent influence on neurologic, but not metabolic, sensitivity to MA-[D-Leu-4]-OB3. These differences are currently under investigation in our laboratory, and include efforts focused on identifying any histopathological changes and/or serological abnormalities that may be associated with high concentrations and/or extended exposure to MA-[D-Leu-4]-OB3.

Although caution must always be taken in translating the results seen in rodent models to human disease, the absence of any significant indicators of metabolic, neurologic, or behavioral toxicity we observed in this study, even at concentrations of MA-[D-Leu-4]-OB3 much higher than the minimally effective concentration, coupled with possible differences in metabolic and neurologic dosing thresholds and tolerance levels, suggests that the application of MA-[D-Leu-4]-OB3 to various human pathologies may have a wide therapeutic index.