Metal Ion-Protein Chelates For Sustained Release Of Interferon

Interferon (IFN) refers to a set of highly pleiotropic cytokines in management of various neoplastic disorders and chronic viral diseases. It is of a short serum half-life when injected subcutaneously (-2-6 hours) and frequent administration must be made to maintain the therapeutic efficiency. Several formulations with protracted effect have been developed, including biodegradable microspheres, multivesicular liposomes and molecular modification with polyethylene glycol (PEGylation). During these formulations, process of PLGA microspheres often incorporates conditions that denature and inactivate many proteins, such as elevated temperatures, surfactants or aqueous/organic solvent interfaces; also, the polymers still had to been dissolved in organic solvents (such as methylene chloride) which would affect the biological activity of the target protein. Other techniques either include complicated manufacturing processes which result in relatively low loadings of the active drug substances, or involve chemical modification and redesign of the molecules which lead to creation of new molecular entities.Alternatively, metal ion-protein chelation technology which conjugates metal ion with protein molecule provides us another promising approach. This study, by chelating recombinant human interferon-a-2b (rhIFN) with zinc ions, develops carrier-free delivery systems for sustained release of rhIFN.Amorphous zinc-rhIFN chelate microparticles were prepared by co-precipitating rhIFN with zinc acetate in neutral solutions. The prepared chelate microparticles were of a mean diameter of 0.46μm and of negative charges (mean value of Zeta potential was-14mV). In vitro release results showed that the release of rhIFN from the chelates mainly depended on the kinds and concentrations of anions added in the dissolution buffer. The amorphous chlates provided a prolonged PK profile characterized as t1/2 of 4.5h, compared to that of 2.5h for soluble rhIFN. The relative bioavailability based on serum rhIFN levels was 92.3%.The amorphous zinc-rhIFN chelate microparticles can be modified by protamine. Introduction of protamine (2.5-20 mg/mL) into the chelation system had several prominent effects. First, percentage of chelated rhIFN was lowered (from >99% to-90%); second, particle size was gradually increased (from～0.45μm to～2μm); last but important, it extended the release period of the chelate both in vitro (complete release was retarded from 8h to 48h) and in vivo(t1/2 was prolonged from 4.5 h to 15.5 h and mean residence time from 9.4 h to 29.6 h). Size-exclusion liquid chromatography and cytopathic effect inhibition assay indicated rhIFN preserved its structural and functional integrity in these chelates with or without modification by protamine.Then crystalline zinc-rhIFN chelates were prepared. From initial screening by hanging drop evaporation method, conditions for producing crystalline chelates that well reserved the activity and possessed good slow-release potential were obtained. Based on the obtained conditions, batch production of crystalline chelates and in vitro release study were carried out. It was found that increasing the zinc concentrations in crystallization solutions from 100 mM to 400 mM would result in smaller crystalline chelates sizes that presented a gradually slower dissolution speed. The changes in anions for zinc salts from acetate to sulfate or chloride led to production of larger chelates, which answered for much slower release profiles and longer extension in release period. Rising in equilibration temperatures from 4℃to 22℃or lowering in pH values of the crystallization solutions from 5.5 to 5.0 would not only bring down the chelates yields but also make the chelates more fragile to be eroded by other ions in the dissolution buffers. The in vivo pharmacokinetic behaviors of crystalline chelates and soluble rhIFN were investigated by single subcutaneous administration in rabbits. The crystalline zinc-rhIFN chelates, which were composed of a nonregular core and radically aligned amylase fibers, presented "Maltese cross" when inspected under a polarized microscopy. The crystalline formulation provided a significantly prolonged PK profile characterized as t1/2 of 13.2h, compared to that of 2.5h for soluble rhIFN. Its mean residual time (MRT) was prolonged from 4.5h to 26.8h. The relative bioavailability based on serum IFN levels of about 177.4%.The crystalline zinc-rhIFN chelates can also be modified by protamine by simultaneous incubation of rhIFN with zinc acetate and protamine. The yields of protamine modified crystalline zinc-rhIFN chelates were～75%, and the mean diameters of the obtained chelates decreased as the concentration of protamine added (0.1-0.5 mg/mL). The protamine modified crystalline zinc-rhIFN chelates were similar to those crystalline chelates without modification by protamine in terms of structure, microscopic view properties and X-ray diffraction patterns. Addition of protamine into the crystallization systems extended the release period of the crystalline chelates both in vitro and in vivo, and this extending potential was protamine concentration dependent. As 0.2mg/mL protamine was added, the t1/2 and MRT was extended to 25.8h and 52.8h from 13.2h and 26.8h, repectively; while 0.5mg/mL protamine was added, the t1/2 and MRT was extended to 25.6h and 70.1h from 13.2h and 26.8h, repectively. Size-exclusion liquid chromatography and cytopathic effect inhibition assay indicated rhIFN preserved its structural and functional integrity in these crystalline chelates with or without modification by protamine.