Site-Specific PEGylation for High-Yield Preparation of Lys²¹-Amine PEGylated Growth Hormone-Releasing Factor (GRF) (1−29) using a GRF(1−29) Derivative FMOC-Protected at Tyr¹ and Lys¹²

Abstract

PEGylation has been viewed as an effective means of overcoming the therapeutic restriction of growth hormone-releasing factor (1-29) (GRF(1-29)) due to its short biological lifetime caused by severe proteolysis and rapid glomerular filtration. Of three isomers according to the PEGylation sites (Tyr1, Lys12, or Lys21), PEGylated GRF(1-29) at Lys21-amine (Lys21-PEG-GRF(1-29)) was shown to have the highest bioactivity. In this report, we propose a unique two-step site-specific PEGylation method capable of producing only Lys21-PEG-GRF(1-29) with a single composition in high yield using a GRF(1-29) derivative protected at Tyr1 and Lys12 and remained available at Lys21 (FMOC1,12-GRF(1-29)). The first step of this reaction involved the PEG attachment to FMOC1,12-GRF(1-29), and the second step involved the removal of FMOC moieties. This PEGylation process was optimized at the following conditions: 0.2-0.3% (v/v) triethylamine concentration, 5.0-6.0-fold molar amount of PEG, reaction temperature of 25-45 degrees C, and reaction time of 30 min. Under these conditions, the maximum yield of Lys21-PEG-GRF(1-29) produced was ca. approximately 95%, 6.3-fold higher than that by nonspecific PEGylation at pH 8.5. Significantly, this site-specific Lys21-PEG-GRF(1-29) was found to have greatly increased resistance to rat plasma, liver, and kidney homogenates, with 7.0-, 25.4-, and 16.4-fold longer half-lives vs GRF(1-29), respectively. Furthermore, 125I-Lys21-PEG-GRF(1-29) displayed significantly reduced liver and kidney distributions and extended blood presence vs 125I-GRF(1-29) in rats. Due to these benefits, Lys21-PEG-GRF(1-29) displayed an enhanced initial growth hormone release in vivo despite having 15% remaining activity in vitro. This devised PEGylation method using an FMOC-protection/deprotection strategy would provide great usefulness for PEGylating bioactive peptides in terms of improved biological potency, elevated production yield, and a uniform composition.

Emerging PEGylated drugs

“…Recent research in the growth hormone therapy field has focused on the applications of growth hormone-releasing factor (GRF 1 -29), a fragment of growth hormone-releasing hormone (1 -44), which stimulates the release of somatropin from the anterior pituitary [86][87][88][89]. This research was undertaken as an alternative to the development of intravenous hGH (somatropin) for the treatment of growth hormone deficiencyrelated disorders.…”

Site-Specific PEGylated Exendin-4 Modified with a High Molecular Weight Trimeric PEG Reduces Steric Hindrance and Increases Type 2 Antidiabetic Therapeutic Effects

“…The major benefit of site-specificity is its increased production yields and, therefore, significantly reduced production costs. In contrast, nonspecific PEGylation is difficult to control, and additional, often costly, separation procedures must be adopted to obtain the purified product. − …”

“…Peptides have a short biological half-life because they have fast renal filtration and proteolytic degradation. Moreover, peptides display distinct biological activity and proteolytic stability according to the PEGylation sites because they have more specific active or metabolic sites than proteins. ,, Therefore, site-specific PEGylation must be consider for maximizing therapeutic effects of peptide.…”

GHRH

“…Thus, besides CJC-1295, PEGylated or cyclic GHRH analogs as well as fatty body-GHRH analogs with improved biological activity have been designed. 7,34 Recently, GHRH analogs have been assayed with the aim of augmenting GH secretion in individuals with low endogenous GH levels and excess visceral obesity, including patients with HIV receiving highly active antiretroviral therapy. 8 Certainly, the growing development of potent and long-acting GHRH analogs will pave the way to prevent or ameliorate the pathological states associated to GHRH deficiency.…”