Myristic acid conjugation of [D-Leu-4]-OB3, a biologically active leptin-related synthetic peptide amide, significantly improves its pharmacokinetic profile and efficacy
Introduction
Reports from our laboratory [5], [6], [7], [8], [19], [23], and a growing number of other laboratories [4], [12], [13], [14], [15], [16], [17], [21], [22], [24], have consistently shown that the entire leptin molecule is not required for the expression of its biological activity. Utilizing in vitro and in vivo approaches, peripheral and intracerebroventricular (ICV) delivery systems, different physiological endpoints and animal models, these studies provide convincing evidence that synthetic peptide analogs that encompass the functional epitope of leptin contain sufficient information to influence leptin-modulated physiologies by pathways that complement, augment, or diverge from those of endogenous leptin.
Our preclinical studies with mouse [D-Leu-4]-OB3 and its analogs have shown that IP, oral, or intranasal delivery of biologically active leptin-related synthetic peptides significantly influences body weight gain, food and water intake, blood glucose, insulin sensitivity, and serum osteocalcin, a sensitive and specific marker of bone turnover, in leptin-deficient ob/ob and leptin-resistant db/db mouse models [5], [6], [7], [8], [19], [24]. More recently, we have shown that oral delivery of [D-Leu-4]-OB3, when given in combination with a number of FDA-approved anti-diabetes drugs, exenatide, pramlintide, and metformin, augments the effects of these therapeutics in both insulin-resistant and insulin-deficient mouse models [10], [20]. Given the fact that the majority of clinically obese humans are leptin-resistant because of defects in transport of leptin across the blood–brain barrier (BBB) [3], the relevance of these results in leptin-resistant rodent models of obesity and diabetes to the clinical management of human metabolic disease may be highly significant.
In two previous studies [9], [18], we described the pharmacokinetcs of [D-Leu-4]-OB3 uptake following delivery by SC, IP, and IM injection, and by oral gavage and intranasal instillation in dodecyl maltoside (DDM, trade name Intravail®), a patented transmucosal absorption enhancing agent. In all cases, the half-life of [D-Leu-4]-OB3 was under one hour, and the physiologically effective dose was in the milligram range. In view of the possible application of [D-Leu-4]-OB3 to the treatment of human obesity and/or diabetes, it was of great importance to improve the pharmacokinetic profile of [D-Leu-4]-OB3, primarily to prolong serum half-life and reduce the optimal effective dose. To address these limitations, i.e., short half-life and high dose, the 14-carbon fatty acid, myristic (tetradecanoic) acid, was conjugated to the N-terminal of [D-Leu-4]-OB3. This new analog was named MA-[D-Leu-4]-OB3. Myristoylation is the same approach that was used to develop detemir insulin (Levemir®, Novo Nordisk), an analog of human insulin with a half-life of 7–8 h, which is commonly used in management of type 2 diabetes mellitus (T2DM) in the clinic. In the present study, we show that conjugation of myristic acid to [D-Leu-4]-OB3 significantly improves its pharmacokinetic profile by (a) extending its half-life from less that one hour to as long as 28 h, depending on the route of delivery; (b) increasing its uptake; (c) reducing the rate at which it is cleared form the plasma; and (d) enabling the minimal effective dose to be reduced.
Section snippets
Housing
3–4 weeks-old male Swiss Webster mice weighing between 12 and 15 g were obtained from Charles River Laboratories (Wilmington, MA, USA). The animals were housed three per cage in polycarbonate cages fitted with stainless steel wire lids and air filters, and supported on ventilated racks (Thoren Caging Systems, Hazelton, PA, USA) 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, and allowed food
Uptake profiles
The uptake profiles of MA-[D-Leu-4]OB3 following SC, IP, IM, oral and intranasal delivery are shown in Fig. 1, Fig. 2. Maximum uptake (Cmax) of MA-[D-Leu-4]-OB3 following SC, IP, and IM administration of 0.1 mg of peptide occurred at 2 h (tmax) and rapidly decreased with time. After 18 h, the concentration of MA-[D-Leu-4]-OB3 in the serum was reduced to near basal levels (Fig. 1). The uptake profiles following oral and intranasal administration were conspicuously different from those observed for
Discussion
On-going efforts in the design, development, and preclinical application of lepin-related synthetic peptide agonists and antagonists [4], [5], [6], [7], [8], [9], [10], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23] indicates that the apparent failure of leptin in the clinic to satisfy the therapeutic needs of the majority of obese humans has acted as a catalyst in efforts to develop novel peptide therapeutics targeted at reducing the pandemic proportions of this disease
Conclusion
Although caution must always be taken in relating the results from animal models to the treatment of human disease, the findings of the present study clearly indicate that myristic acid conjugation of [D-Leu-4]-OB3 significantly improves its pharmacokinetic profile and efficacy. They further suggest that MA-[D-Leu-4]-OB3 may have the potential for development as an oral, noninvasive, and safe therapeutic approach to the management of obesity and/or diabetes in humans.
Acknowledgments
This research was supported by a grant from the Willard B. Warring Memorial Fund, Albany Medical College. The authors also wish to express their thanks to HaiAn Zhang, Ph.D. Siji Thomas, Ph.D. and Khadijat Audu, B.S. of the Albany College of Pharmacy and Health Sciences for their encouragement and technical expertise.
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