PEGylated substance P augments therapeutic angiogenesis in diabetic critical limb ischemia

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Abstract

Substance P (SP) is a therapeutic peptide that has been widely used to induce angiogenesis and tissue regeneration. However, its therapeutic efficacy is often limited due to rapid degradation in vivo and a short half-life (˜1 min) after systemic administration. In the present study, we chemically modified SP with polyethylene glycol (PEG) to generate long-lasting formulations with increased stability and extended retention time in vivo and evaluated their ability to enhance therapeutic angiogenesis. Compared to the unmodified SP, PEGylated SP (PEG-SP) exhibited significantly increased half-life in vivo (˜360-fold increase in normal mouse and ˜120-fold increase in diabetic mouse). Systemic injection of PEG-SP led to a marked increase in therapeutic efficacy and angiogenesis in diabetic hindlimb ischemia, as evident from the remarkable improvement in salvage of ischemic limb and recovery of blood perfusion in diabetic mice with limb ischemia. These formulations increased endogenous stem cell mobilization to the injured site and modulated the immune system to increase microvessel formation and reduce systemic inflammation. Importantly, PEGylation efficiently reduced the injection doses of SP while still maintaining its therapeutic efficacy. To conclude, our study demonstrated that PEG-SP formulations exhibited an improved in vivo pharmacokinetic profile with enhanced efficacy and clinically acceptable treatment regimens, thus acting as a potent therapeutic for diabetic ischemia and inflammation.

Introduction

Substance P (SP), a short peptide composed of 11 amino acids (RPKPQQFFGLM), is a member of the neuropeptide family. It has been demonstrated to execute a novel function in tissue repair by stimulating the mobilization of stem cells to the injury site and contributing to vessel formation [1]. During tissue injury, SP activates the endogenous nociceptive signaling pathway, thereby facilitating mobilization of stem or progenitor cells, such as mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs), from bone marrow into the blood circulation and subsequently to the injured site to promote angiogenesis and tissue healing [2]. Owing to the wound-healing function of SP, its exogenous administration has been demonstrated to accelerate tissue repair in various tissue defective and injury models [1], [3], [4], [5], [6], [7], [8]. In addition, SP contributes to tissue healing and enhances recovery from injury by modulating the immune system through the suppressed secretion of pro-inflammatory factors (e.g., tumor necrosis factor-α [TNF-α] and interferon-γ [IFN-γ]), enhanced secretion of anti-inflammatory factors (e.g., interleukin-10 [IL-10] and transforming growth factor-β [TGF-β]), and increased polarized migration of M2 type macrophage to the injured site [3], [9], [10].

Despite the huge potential of SP as a therapeutic drug for tissue repair, its contribution to regenerative medicine is limited owing to its rapid degradation in vivo and the resultant short half-life. Short peptide drugs such as SP usually undergo rapid enzymatic degradation and denaturation by proteases in the circulatory system [11], [12]. For instance, in vivo half-life of SP under diabetic environment has been found to be less than 1 min after systemic injection [13]. Chronic diabetic conditions are associated with increased levels of plasma proteases including neutral endopeptidase (NEP) that rapidly degrades SP [14]. This drawback, in turn, renders SP ineffective in inducing complete tissue repair and functional recovery of the defective tissue. Therefore, the need of the hour is to develop methods to prolong the half-life and in vivo retention of SP to ensure its increased therapeutic efficacy for tissue injuries and complications associated with diabetes. Recently, our group demonstrated a high-density lipoprotein-mimicking nanodisc formulation to increase in vivo stability of SP and validated its therapeutic efficacy in diabetic hindlimb ischemia [13]. However, more clinically relevant modification strategies, with a low risk factor, are required to efficiently use SP as a therapeutic drug.

In the present study, we employed a synthetic approach to modify SP with polyethylene glycol (PEG) and checked various treatment regimens to maximize the therapeutic efficacy of PEG-conjugated SP (PEG-SP) with an aim to minimize the risk of potential toxicity in mice with diabetic critical limb ischemia. PEG, a biologically inert, non-immunogenic, and water-soluble polymer, has been widely used to chemically modify several biomolecules, such as clinically approved proteins and peptide drugs, to extend their therapeutic effects. These include molecules such as phenylalanine ammonia-lyase (phenylketonuria) [15], interferon β-1a (multiple sclerosis) [16], and antihemophilic factor VIII (hemophilia A) [17]. PEGylation has been demonstrated to improve the in vivo half-life of proteins and peptides by protecting them from active sites of serum proteases and interrupting their in vivo clearance, as well as reducing immunogenicity without compromising their activity [18], [19]. In the current study, we demonstrated that PEGylation significantly prolonged the in vivo half-life of SP (˜360-fold increase in normal mouse and ˜120-fold increase in diabetic mouse), thus improving the potential of SP-mediated therapeutic angiogenesis for treating diabetic hindlimb ischemia by boosting stem cell mobilization and immune system modulation. PEGylation of short peptides such as SP might be more difficult to produce therapeutically effective peptide drugs with prolonged half-life and improved stability but without impairment of bioactivity, compared to PEGylation of protein drugs with larger molecular weight, due to limited reaction sites for PEGylation and shielding of active sites in small peptides by PEG moieties. In this study, we demonstrate for the first time that SP could be successfully modified with PEG moieties without interrupting binding of SP to target ligands, improving its therapeutic efficacy for diabetic ischemia treatment. Importantly, PEGylation led to a decrease in the frequency of SP administration, without affecting the effective dose of SP in vivo. Therefore, the present study may provide useful insights for developing efficient drug-delivery strategies and providing clinically acceptable therapeutic regimens.

Section snippets

Synthesis and characterization of PEG-SP

A scheme for PEG-SP synthesis is illustrated in Fig. 1A. Briefly, 250 mg/mL of 10 kDa methoxy PEG-succinimidyl succinate (10 kDa mPEG-SS; SunBio, Anyang, Korea) and 10 mg/mL of SP (Anygen, Kwang-ju, Korea) dissolved in 10 mM Tris buffer (pH 7.5) were mixed in equal volume and stirred at 4 °C overnight. The PEGylated SP was then isolated from the reaction mixture using the Amicon ultra centrifugal filter system (NMWL 10 kDa; EMD Millipore, Billerica, MA, USA).

The isolated PEG-SP was characterized using

PEG-SP synthesis and characterization

To increase the stability and half-life of SP in vivo, SP peptides were chemically modified by PEGylation, resulting in the conjugation of PEG moieties to the N-terminal arginine (R) and lysine (K) via coupling between carboxylic acids and amines (Fig. 1A). GPC analysis confirmed the fraction of di-PEGylated SP in the reaction product to be approximately 78.5% (Supplementary Fig. 1). We did not separate di-PEGylated SP and mono-PEGylated SP, and the mixture of both was used for the experiments.

Discussion

Diabetes often results in severe pathological complications that are untreatable using conventional remedies. In particular, no treatment is available for diabetic peripheral angiopathy owing to constant vascular damage and inflammatory dysfunction, ultimately leading to limb amputation [34]. Moreover, diabetic conditions impair the activity of endogenous stem cells that are involved in tissue healing and reduce their reparative capacity [35], [36], [37], [38]. Thus, a therapeutic strategy

Conclusions

To summarize, we demonstrated the potential of PEG-SP as a peptide drug inducing therapeutic angiogenesis in diabetic peripheral ischemia with enhanced efficacy and within clinically acceptable regimens. Considering that no treatment exists to fundamentally address the diabetes-associated critical limb ischemia and amputation, PEG-SP would be a promising therapeutic candidate for diabetic patients suffering from peripheral ischemia. In addition, PEG-SP could be evaluated for treating other

Acknowledgements

The present work was supported by grants (HI13C1479 and HI18C1492) from the Korea Health Technology R&D Project funded by the Ministry of Health and Welfare and a grant (2017R1A2B3005994) from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (MSIT), Republic of Korea. We thank Prof. Byung-Soo Kim and Dr. Geunjae Jeong (School of Chemical and Biological Engineering, Seoul National University, Republic of Korea) for their help with Doppler imaging analysis.

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