Study On Sustained-Release Formulation Of Proteins And Its Application For Human Growth Hormone

Recombinanted proteins drugs, as a group of the most effective and natural therapeutics, have been broadly applied in many therapeutic areas since 1982, the year that first product in this category was launched. Now, there have been about 165 recombinant products clinically applied and about 270 on the pipelines of development, as well as a number of biotech rising stars (players), such as Amgen, Genentech and Genzyme, and EUR40 billion global sales in this field. As more and more proteins have become clinically available, the current dosage regimen, frequent injection due to poor oral availability and short in vivo life of proteins, is no longer satisfactory. In the case of chronic condition treatment, for example, frequent injection for years or even life time create a serious patient compliance problem; while for organ repairs that have probably the chance for only one injection, few hours or even minutes of in vivo life of administrated cell growth factor cytokines is far from sufficient to maintain their effective level during weeks long therapeutic period. Introducing genes to diseased tissue to express cytokines also encouters difficulties such as non-viral delivery and controlled gene expression. To extend the effective period, sustained-release dosage forms possess great advantages over other protein long-acting strategies in terms of efficacy, broadness of indications and safety due to the fact that this approach do not require altering protein sequences and chemical structures.However, developing sustained-release dosage forms has encountered a series of formidable formulationative challenges for which 31 years of research efforts to date have not resulted in a single product commercialized in this category. Due to the fragile tertiary structure (not seen in peptides), the sustained-release technologies successfully used for chemical and peptide drugs are no longer feasible for proteins. Rather, they cause denaturing and immunogenicity of these macromolecules. How to preserved the nature state and bioactivity of proteins during formulation process of sustained-release system is critical to the technology breakthrough, yet, is the objective of the present study.The technical hurdles in developing protein sustained-release technology include:(1) Protein denaturing and deactivation due to exposing to a water/oil or a water/air interfaces;(2) Burst release due to intra-microsphere osmotic pressure generated by protein stabilizers;(3) Incomplete release due to protein aggregation and adsorption onto the degradable polymer which forms the matrix of microspheres;(4) Complicated formulation process in order to avoid protein denaturing and poor reproducibility due to the formulation complexity.(5) Safety for body injection for all excipients used in the system.A successful protein sustained-release technology must resolve all the above problems simultaneously. Thus, following approaches were taken in the present study:(1)Incorporating structurally delicate proteins into fine polysaccharide glass particles resistant to hazardous conditions under a milder condition. Briefly, fragile proteins were added into an aqueous dextran-polyethylene glycol (PEG) mixture followed by freezing-induced phase-separation or emulsification at low temperature. The proteins were partitioned into the dispersed dextran phase which was then lyophilized to glassy particles without contact with water-oil or water-air interfacial tension, factors know to denature proteins. Once loaded into polysaccharide glassy particles, proteins became resistant to organic solvents, thus can easily be formulated into various pharmaceutical dosage forms and medical devices. We also modified the previously reported Zn2+ induced protein precipitation process by carrying out process in a PEG continuous phase. The previously irregular Zn2+-protein particles became spherical and dense when complexed and precipitated in the PEG phase, a morphology preferred in successive formulation steps.(2) Avoiding dissolution of protein-dextran particles and water-oil interface when microencapsulating these particles into PLGA/PLA microspheres. To achieve this goal, the glassy particles were suspended in a dichloromethane solution of PLGA (or PLGA-PLA) and emulsified into a hydrophilic"oil phase"continuous phase which does not dissolve dextran. (We name this continuous phase as"hO"and the process as S/O/hO hereafter). With this process, protein (and polysaccharide) and denaturation can be avoided, and thus high loading efficiency can be achieved. Since the hydrophilic"oil"can be removed by washing with water, this S/O/hO process can also get ried of the safety and environment concerns of using large amount of organic solvent to wash away the oil for previously reported"solid-in-oil-oil"(S/O/O) microencapsulation process.(3) Preventing protein aggregation and adsorption onto PLGA matrix by the nature (Large molecular weight, hydrophilicity and viscosity) of polysaccharide dispersed phase surrounding the protein molecules. The polysaccharide microenvironment stabilizes and isolates proteins from aggregation and adsorption so that incomplete protein release can be minimized. In addition, by adjusting the ratio of PLGA and PLA, microspheres with a"core-shell"morphology can be formed. The protein-dextran particles are encapsulated in the core region, leading a drastically reduced burst release.In this study, we carried out a series examination on the above-discussed procedure using model proteins (myoglobin, BSA andβ-galactosidase). Density, X-ray diffraction (XRD) and differential scanning calometry (DSC) measurements on the protein-dextran particles indicated that the particles were dense and glassy. Electron microscopic measurement and laser scattering measurement showed that the particles were spherical in shape,and smooth and ranging in size range of 1-2 microns in diameter and with smooth surfaces. Reconstitution ofβ-Galactosidase particles at each formulation step of protein-dextran particles, followed by assay of its enzymatic activity, suggested that over 90 % bioactivity were preserved after sounicating the particles in organic solvents,Eximation onβ-Galactosidase was reconstituted after each formulation step to prepare the protein-dextran particles and assayed for enzymatic activity. The suggested that over 90 % bioactivity were preserved above 90% of original protein sample after all the steps were performed including washing the protein-loaded particles in organic solvent,when vortexing polysaccharide glassy particles in organic solvent followed by centrifuging for 25 mins, consisting with our hypothesis for protein protection by glassy polysaccharide. Direct observation using optical and electronic microscopes confirmed the spherical shape and smooth surface the PLGA microspheres, as well as their core-shell morphology (in the cases of such design). Using myoglobin and BSA, we optimized formulation parameters and achieved nearly zero-order release profiles and about 80% protein loading effeciency. Measurements using SEC-HPLC confirmed that protein aggregation caused by our formulation procedures was negligible.With optimized formulation parameters, the new microencapsulation method was applied to formulating sustained-release microspheres for recombinant human growth hormone (rhGH). Our preliminary study showed that the proteins were release nearly linearly from core-shell -structured PLGA-PLA microspheres with minimal burst and incomplete release. A SEC-HPLC assay of the release medium indicated that the monomer content was the same as that of the original protein sample.The (organic) solvent resistance enables the protein-polysaccharide particles be easily incorporated in variety of medical devices for sustained-release protein therapy. In the present study, protein-dextran particles were dispersed in PLA polymer and coated on cardiovascular stents and hemostasia gauze. Proteins (myoglobin, BSA and granulocyte colony stimulating factor i.e. G-CSF) coated on these devices showed nearly linear sustained-release profile and well preserved bioactivity (80% as original G-CSF).In the present study, we have not only proposed and examined solutions for each technical problem, but also address all the issues systematically, so that all the principal difficulties discussed above can be overcome at once. The problems caused by resolutions of other problems were avoided. By optimizing formulation parameters using model proteins and pharmaceutical protein (rhGH), all the required criteria (protein loading efficiency, protein structure integrity, protein release kinetics and protein activity preservation) were satisfied. It may be feasible to conclude that the present study has offered an inclusive solution for all the key issues blocking the way to develop sustained-release dosage forms of protein drugs. The method demonstrated in this study is also useful to fabricating protein-related medical devices.