Insulin Pegylated And Pegylated Insulin, The Hypoglycemic Effects

Insulin was chosen as a model drug for the study of PEGylation of protein drugs. The physicochemical properties and bioactivity of PEGylated insulin were studied. And the hypoglycemic effect following the oral administration of mono-PEGylated insulin was studied with superporous hydrogel containing interpenetrating polymer networks(SPH-IPN)as a drug carrier.1 Activation of PEGMonomethoxy-poly(ethylene glycol) 5000(MPEG5000) was activated to produce MPEG-succinimidyl succinate(MPEG-SS) and succinimidyl ester of carboxymethyl MPEG(MPEG-SCM) with activity degree both over 90%.The characteristic absorption band at 1740 cm^-1(ester carbonyl),1785 cm^-1 and 1813 cm^-1 (carbonyl on succinimidyl) of the infrared spectrum of MPEG-SS and MPEG-SCM indicated the existence of MPEG succinimidyl active ester.The ¹H-NMR analysis demonstrated that each MPEG derivative contained the corresponding characteristic groups.2 Preparation of PEGylated insulinThe resultant MPEG5000-SS and MPEG5000-SCM were used to modify insulin randomly.Taken MPEG5000-SS as an example,effects of reaction solvents,initial molar ratio of MPEG derivative to insulin and reaction time on synthesis of PEGylated insulin were investigated with TNBS spectrometry and SDS-PAGE analysis.And the optimal synthesis scheme was as follows:insulin was dissolved in the solvent of DMF/0.1 M borate buffer pH 7.4(60:40,v/v),and the concentration of insulin was in the range from 1 mg·ml^-1 to 10 mg·ml^-1.MPEG-NHS active ester powder was added at the ratio of 1:5(mol/mol) of insulin to MPEG-NHS active ester. After stirring at ambient temperature for 4 h,6-Aminocaproic acid about 2 molar times of MPEG-NHS active ester was added to quench the reaction.Then the reaction mixture was dialyzed against 0.01%NH₄HCO₃ solution and lyophilized.Gel filtration chromatography using Sephadex G75,cation exchange chromatography on Protein-Pak^TM SP 8H,C18 reversed-phase high performance liquid chromatography were employed to separate the PEGylated insulin.The results showed that SEC was able to separate PEGylated insulin from intact insulin and impurity,but did not exhibit heterogeneity of MPEG-insulin conjugates or remove the inactive MPEG from the mixture of PEGylated insulin.IEC and RP-HPLC could resolve heterogeneous composition or PEGylated insulin mixture.The elution sequence of PEGylated insulin in IEC was just the opposite in RP-HPLC.On the basis of the above research,SP Sepharose cation exchange chromatography and Q Sepharose anion exchange chromatography were used to separate PEGylated insulin in two steps,and three species of PEGylated insulin, namely mono-,di-,and tri-PEGylated insulin were purified,and the purity were above 90%according to the results of RP-HPLC.3 Characterization of PEGylated insulinThe molecular weights of mono-,di- and tri-PEGylated insulin were determined through SDS-PAGE and MALDI-TOF MS.The apparent molecular weights of mono-, di-,and tri-PEGylated insulin were 11.1,23.5,35.0 KDa through SDS-PAGE,and the apparent molecular weight of mono-PEGylated insulin is closed to its' calculated molecular weight(10.8 KDa).The molecular weights of mono-,di-PEGylated insulin determined by MALDI-TOF MS were 10.8 KDa and 15.9 KDa,which were corresponding to the calculated molecular weights of them.The ultraviolet absorption spectra of three species of PEGylated insulin were similar to that of insulin,which had absorption peaks at about 220 nm and 275 nm.So the concentration of PEGylated insulin could be calculated as the concentration of insulin solution that could be determined by measuring OD₂₇₅.The circular dichroic spectra analysis of PEGylated insulin demonstrated that the attachment of PEG to insulin does not alter the tertiary structure of conjugates as compared to native insulin. The self-association of PEGylated insulin was inhibited and the inhibition correlated with the degree of PEGylation.The physical stability of insulin and three species of PEGylated insulin were studied with accelerated shake test under the condition of 200 rpm of shaking speed at 37℃,the fibrillation of mono-PEGylated insulin was inhibited,and it gave at least 17-fold increase of physical stability as compared to insulin while the physical stability of di- and tri-PEGylated insulin were greatly enhanced more than insulin and mono-PEGylated insulin.The in vitro enzymatic degradation experiment showed that the degradation of insulin by pepsin,trypsin and chymotrypsin could be inhibited through PEGylation, and the inhibition correlated with the degree of PEGylation.4 The hypoglycemic effect of PEGylated insulinThe hypoglycemic effect of PEGylated insulin was compared with insulin after subcutaneous injection(0.5 U·kg^-1) in healthy mouse.In the group of insulin,the blood glucose level reduced to 26%of the control value within 30 min then started rising and approach to 90%of the control value within 3 h.In the groups of mono-, di-,and tri-PEGylated insulin,the blood glucose level reduced to 52%,76%,and 87% of the control value within 90min,120min,and 90min,respectively.After that,the blood glucose level rose to 90%of the control value within 5 h in mono-PEGylated insulin group that is a little slower than di-(3-4 h) and tri-(2 h) PEGylated insulin. Using insulin as a standard,the hypoglycemic effects of mono-PEGylated insulin, di-PEGylated insulin and tri-PEGylated insulin were calculated by comparing the AAC of serum glucose level-time course curve between different groups,which were 93%,46%and 24%of native insulin respectively.The hypoglycemic effect of intravenous administration of insulin and three species of PEGylated insulin in healthy rats showed that mono-PEGylated insulin had an evident hypoglycemic effect and a longer hypoglycemic time comparing to insulin.The activity of di-PEGylated insulin and tri-PEGylated insulin had a great loss and their hypoglycemic effects in vivo were not obvious.A drug loading of(10.52±0.87)%(wt) was achieved by loading the SPH-IPN with mono-PEGylated insulin by adsorption.The result of drug release experiment in vitro showed that mono-PEGylated insulin was released from the SPH-IPN rapidly and the amount of released drug exceeded 80%in 10 min.The enteric-coated capsules for rat were filled with mono-PEGylated insulin loaded SPH-IPN.After oral administration of these capsules for rats,obvious hypoglycemic effect was observed.The blood glucose level declined to the nadir in 8-10 h that was approximately 65%of the initial value.The control groups were orally administered with insulin solution,enteric-coated capsules filled with mono-PEGylated insulin or enteric-coated capsules filled with SPH-IPN respectively and notable hypoglycemic effect failed to be found out in these condition.And a relative pharmacological availability(PA) of 5.1±2.0%was achieved after oral administration of mono-PEGylated insulin loaded SPH-IPN compared with subcutaneous administration of insulin solution.PEGylation of insulin could inhibit the self-association tendency of insulin,and increase the physical stability as well as resistance to proteolysis while retaining most bioactivity of native insulin.And with the functions of enzyme inhibition,permeation enhancing and adhesion to mucous membrane,SPH-IPN could increase the pharmacological availability of PEGylated insulin after oral administration. Combination of PEGylation and SPH-IPN would be a promising technique for oral delivery of protein and peptide drugs.