Study On PEGylation Of Recombinant Human Epidermal Growth Factor (rhEGF) And Benflumetol

Pegylation of drug is an important method to improve drug action and compliance. The shortcoming in stability, half life, toxicity and immunogenicity was a constraint for the drug delivery and effect.rhEGF is a polypeptide composed of 53 amino acid residues (Mr=6045). It has been used for the treatments of gastric and intestinal ulcers, burns and corneal wound, diabetic foot ulcers and so on. But its unstability in ulcers and wounds limits its effect by common preparation. Benflumetol is a new antimalarial drug developed in the Institute of Microbiology and Epidemiology. It was absorbed and eliminated slowly in vivo. While artemether was absorbed and eliminated quickly in vivo. So Co-artemether, which combinate of 20 mg artemether and 120 mg benflumetol in fixed tablet, was suggested as a first drug for the treatment of Plasmodium falciparum malaria by the WHO experts. In order to improve the absorption and bioavailability of benflumetol, it was PEGylated for the raise of oral absorption.It could give some good suggests for these problems through Pegylation. There were there parts of this study: (1)synthesis of PEG derivatives (2)Pegylation of rhEGF and (3)Pegylation of benflumetol and bioavailability research of PEG-benflumetol.In the part one: There PEG derivatives, which included mPEG-NH₂, mPEG_5k-SSA and mPEG_5k-propionaldehyde, were synthesized. mPEG-OH was esterized by p-toluene sulfonyl chloride and then got mPEG-NH2 through hydrogenising; mPEG-NH₂ reacted with succinic anhydride and N-hydroxy succinimide, at last got mPEG_5k-SSA; mPEG_5k-OH reacted with 3-chloropropionaldehyde diethyl acetal and got acetalational compound. mPEG_5k-propionaldehyde was gotten through this compound decomposed in weak acid. These compounds were identified by NMR and IR.In the part two: the PEGlytion of rhEGF. The conjugation of rhEGF and Esters derivatives (mPEG_5k-SSA and mPEG20k-NHS, 9-fold molar excess) was carried out in phosphate buffer (50mM,pH 8.0) . The reaction between mPEG_5k-SSA and rhEGF produced a heterogeneous mixture of PEGylated rhEGF. The compounds in the conjugation of mPEG_20k-NHS and rhEGF conjugated same number of PEG derivatives. Aldehyde derivatives (mPEG_5k-propionaldehyde and mPEG_5k-ALD. 3-fold molar excess) and rhEGF were dissolved in sodium acetate buffer(50mM,pH 5.5) in the present of sodium cyanoborohydride as a reducing agent and forming a hydrazone linkage between rhEGF and PEG. In coincidence with expect, only mono-PEGylation rhEGF was gotten in the reaction between rhEGF and aldehyde derivatives. But the site of the conjugation has not been identified.In the part there, benflumetol was PEGylated for the improvement of water solubility and the PEG-benflumetol was used for the absorption research in intestine. At first, benflumetol was esterized by succinic anhydride and we got a acid compound. PEG-benflumetol, which could be degrdned, was synthesized through a dehydrolysis reaction between the acid compound and mPEG- NH₂. The structure of PEG-benflumetol was identified by MADIL-TOF. And the purity of the product is 86.71% according to property analysis, which including 2.37% free benflumetol and raw material PEG for the rest. The stability of the product is not good from the 53.83% degradation after three hours in methanol.Absorption of benflumetol and PEG-benflumetol was carried out in 8 SD rats in vivo. Each group had four rats (two female and two male) . A single dose of benflumetol (15mg/kg) and PEG-benflumetol (151mg/kg) was administered orally by enteric capsule. Blood samples were drawn at time points of 0.5h, 0.75h, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, 72h after the administration.After the calculation of k, K_a, t_1/2, AUC0-72, T_peak, C_max, CL, V, MRT. we could decide that PEG-benflumetol get a good bioavailability. And the comparative bioavailability was 317.95%.