Elsevier

Peptides

Volume 62, December 2014, Pages 176-182
Peptides

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

https://doi.org/10.1016/j.peptides.2014.10.007Get rights and content

Highlights

  • Myristic acid conjugation of [D-Leu-4]-OB3, a biologically active leptin-related synthetic peptide amide, increases its bioavailability.

  • Myristic acid conjugation of [D-Leu-4]-OB3 extends its serum half-life.

  • Myristic acid conjugation of [D-Leu-4]-OB3 slows its clearance from the plasma.

  • Myristic acid conjugation of [D-Leu-4]-OB3 enables the minimal effective dose to be reduced 10-fold.

Abstract

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.

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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