Druggability Evaluation Of A Protamine-Like Peptide

Objective Protamine (Trade name:Protamine sulfate, PS) is a highly cationic arginine-rich peptide isolated from sperm of the salmonidae or clupeidae family as well as other species. PS which is widely used as an UFH antidote could closely conbine UFH due to its highly cationic property, thus making heparin inactive. Futhermore, PS could serves as an additive to prolong the absorbsion of the insulin, making it widely used in diabetic therapy. PS, which possessed good efficacy, however, has many adverse effects including systematic hypotension, anaphylactic reaction, anaphylactoid reactions and catastrophic pulmonary hypertension by the virtue of its heterogeneous and highly alkaline nature. PS isolated and purified from sperm of fishes by sulfuric acid is heterogenic and be of difference in molecular weight. It is hard to establish a quality control for PS due to its diverse source of fish and the fish of same kind but from different regions. In addition, PS is, so far, the only antidote approved by the FDA and CFDA to reverse UFH activity and once became an orphan drug in 2011 in China. So protamine analogues that are characterized by pure constitution, definite molecular weight, high potency, and absence of immune response are expected. Based on the features that PS are arginine-rich proteins, here a series of small peptides with different amount of arginine residues were previously synthesized and screened in our lab, and we finally presented a promising peptide with fifteen arginine residues (Named as R15). As single molecules, dose-effect relationship in terms of UFH neutralization by R15 is stable and its products would not vary with different batches. So R15 could offer well defined quality standard and identical batches which is of benefits of commercial production. Comparisons were made in this paper between PS and the sulfate form of R15 (Named as R15S) aided by heparin neutralization studies, drug toxicity studies and anaphylactic analysis.Methods First, we prepared the sulfate form of R15 (R15S). Stability of R15S samples in room temperature during 30 days was measured using High performance liquid chromatograph (HPLC). For its UFH neutralizing ability, method of protamine sulfate potency determination on Chinese pharmacopeia was chose to determine the potency of PS, R15 and R15S. Comparisons on UFH neutralization ability, between PS and R15S, were made aided by active partial thromboplastin time (APTT), anti-Xa and anti-IIa assays in vitro and in vivo. For the drug toxicity, MTT tests and acute toxicity assays in Kun Ming mice were performed to measure the cytotoxicity and median lethal dose (LD50) of R15S, separatively. Heparinized Wistar rats were rapidly injected with R15S or PS and then the alterations of mean artery pressure were noted. For the anaphylactic analysis, immunogenicity detections assays were conducted on guinea pigs immunized with PS or R15S respectively for the detection of any antibodies of either PS or R15S. A competitive ELISA method was applied to the determination of cross-reaction between R15S and guinea pig anti-protamine antibodies.Results The R15 sulfate, R15S, was prepared in our lab and its stability for 30 days in room tempreture (25±3℃) was good. For the UFH neutralizaing ability, the results of APTT, anti-Xa assays and anti-IIa assays in vitro and in vivo showed that R15S has equal heparin neutralizing ability to PS. For the aspect of drug toxicity, there was no cytotoxicity for R15S and PS at the concentration of 60μmg-1 or below, but R15S showed lower cytotoxicity than PS at 250μg·mg-1 to 1000 μg·mg-1. The LD50 of R15S was 35.4 mg·kg-1, which is similar to PS (LD50(PS,i.v.)=30 mg·kg-1～38 mg·kg-1), measured by intravenous injection into Kun Ming mice.22 mgHg decreases was observed on heparinized Wistar rats with rapid intravenous injection of either PS or R15S. For the immunogenicity studies,8/10 of guinea pigs immunized with PS demonstrated high level of anti-protamine antibodies (Titer 1:1000) whereas none of 12 guinea pigs immunized with PS exhibited any detectable antibodies. R15S reduced the level of cross-reactivity to guinea pig anti-protamine antibodies, compared to PS (R15S v.s. PS,30% vs.80%). Above all, R15S possessed similar profile on efficacy and drug toxicity (Cytotoxicity, acute toxicity and effects on MAP by rapid intravenous injection of two heparin antidotes), but performed better on immunologic safty evaluationss (Immunogenicity and cross reaction toward anti-protamine antibodies) than PS which showed severe immonoreactions.Conclusion We prepared and evaluated a new UFH antagonist, R15S. R15S showed compatable UFH reversal ability, similar drug toxicity and superior safety in immunology, and it is easy for R15S to establish strict quality control due to its homogeneous feature and definite molecular weight, all of which made it a potential candidate deserving further studies.