Macromolecular Modifications And Evaluation Of Bioactive Peptides

Bioactive peptides with therapeutic potentials are well known for their high potency and low toxicity, but there are some limitations in their clinical application, such as short serum half-lives, unstable to protease, etc. Calcitonins (CT) are series of peptide hormones with 32 amino acid residues, which are known mainly for their hypocalcemic effect and inhibition of bone calcium absorption. Although there are several differences in individual amino acid in calcitonin sequences from different species, they all have a disulfide bridge between cysteine residues at positions 1 and 7, and a prolinamide at the C-terminal. In general, calcitonins originated from fish have been found to be more potent than the mammalian ones. Synthetic calcitonins have been approved by FDA for the treatment of hypercalcemia, postmenopausal osteoporosis and Paget's disease since 1980's. Natural calcitonins have short biological half-lives in vivo, and they are unstable in neutral or basic solutions. In order to improve its stability, and to further investigate its SAR, CT analogues and their macromolecular modifications were designed, and the stability and biological activity of these compounds were evaluated as well.Based on our previous consequences and literatures, further modifications on CT sequence involved: 1) It was reported that bioactivity was still remained when the disulfide bond was cut off. Thus, a valine or an alanine was designed to substitute the cysteine at the N-terminal respectively to obtain CT analogues without a disulfide bridge; 2) The glycine at positions 10 and 28 was an achiral residue, which give rise to the flexibility of CT and result in the variety of CT conformations. Proline is a steric constraint residue. Then, the glycine was designed to be replaced by proline at the two positions to fix the conformation of CT; 3) Fragment at positions 19 to 22 was designed to be deleted according to the literature in order to obtain CT analogues with shorter sequences; 4) The Citrulline(Cit) was designed to replace the Lys11 to increas the stability to enzymatic degradation.In addition, PEGylation is one of the optimal approaches to change the physiochemical characteristics of therapeutic proteins. Studies have indicated that the pharmacokinetic properties of polyethylene glycol (PEG)-modified proteins have been improved greatly, showing better physical and thermal stability, longer half-lives, increased solubility, greater Cmax (peak concentration), delayed Tpeak (peak time), restricted V (apparent volume of distribution), reduced immunogenicity and antigenicity , decreased toxicity and reduced renal clearance. PEG can be coupled to the side chain groups of a protein or a peptide, such as the amino, carboxyl and thiol groups. Reactive specificity is one of the key factors to PEGylation. Otherwise a heterogeneous mixture containing multiple PEGylated products would probably lead to great difficulty in further purification, structure determination and decreased bioactivity. Now, site-specific PEGylation is believed to be an effective approach to maintain bioactivity and quality control. It can be realized through the highly reactive thiol group with thiol-reactive PEG reagents. PEGylation of CT was shown to significantly increase the stability of CT in tissue homogenates, probably due to protection from proteolytic enzymes by PEG molecules. Also, regarding the hypocalcemic effects after intranasal dministration to rats, the PEGylated CT exhibited significantly prolonged serum calcium lowering effect compared with unmodified CT. The hypocalcemic effect reflects the amounts of CT present in the systemic circulation and the sensitivity of the response to CT, so it can be related to improved effects for the treatment of bone diseases such as Paget's disease and postmenopausal osteoporosis.Objective: To replace some amino acid residues of CT prototype was supposed to obtain some CT analogues with better activity and biological stability. Meanwhile, we can presume the further structure-function relationship of CT. In the other aspect, we use mPEG-MAL to modified the CT analogue with the best activity, in order to investigate internal metabolism.Methods: CT analogues were prepared according to the principle of solid phase peptide synthesis (SPPS). They were assembled on the MBHA or Rink amide resins from the N- to C-terminal. Fmoc/tBu protected amino acids were used as the orthogonally synthetic strategy. The whole protected peptides were cleaved from the resin by TFA or anhydrous HF to get crude peptides. The crude peptides were then purified and analyzed by reversed-phase high-performance liquid(RP-HPLC), and investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). PEGylations were performed in solution.Results: According to the above protocol, 10 CT analogs and 9 corresponding PEGylated products were obtained(the purities 87%~98%,yields 10%~22%). The found molecular weights were in accordance with the theoretical data. And, the MALDI-TOF-MS data indicate that only single PEG is covalent to the CT analogue.Conclusions: The preliminary screening tests showed that 4 compounds showed bioactivity, and all lower than the model compound, but 2 of them are higher than the CT prototype drug. According to the above results, it is found: 1) CT analogues without disulfide bridge at the N-terminal exhibited bioactivity. 2) Gly10,28 and their flexible tendency are necessary for CT bioactivity. The replacement of proline with fixed conformation is unfavorable. 3) How does PEGylation affect the activity is still unclear based on these primary data.