Protein Avail Research
With the rapid development of biotechnology, protein drugs has become the main varieties of new drugs, such as Erythropoietin (EPO), Granulocyte colony-stimulating factor (G-CSF), Interferon, Interleukin, Insulin and a variety of vaccines. So far, more than 40 kinds of important therapeutic drugs listed, and more than 720 kinds of biotechnology drugs are in PhaseⅠor Ⅲ clinical trials or receiving FDA review, of which more than 200 kinds of drugs are into the final approval stage. However, protein drugs will be rapidly cleared in vivo. It is due to the existence of a large number of protease in human tissue, which can hydrolyze protein drugs and inactivate it. At the same time as a result of glomerular filtration, the molecular of less than 69kDa is easily removed in the metabolic process. Liver also plays an important role in the process of drug metabolism. Thus the half-life of protein drugs is very short. In order to maintain a certain degree of therapeutic efficiency, patients need high-dose medication frequently. Long-term and repeated injections not only increase the suffering of the patient but trigger a series of side effects easily. Therefore clinic need development of the long-term protein drugs is needed. At present, the established technology of improving the half-life of protein drugs include: the chemical modification of protein drugs, protein fusion, microencapsulation/nanoparticles, glycosylation, and construction of anti-protease mutant.
1. chemical modification
Chemical modification is an effective way of extending the half-life of protein drugs. It covalently connected the proteins drugs of small molecular weight to the greater molecular, such as polyethylene glycol and albumin. Small molecular weight proteins are covalently connected to the drug with greater molecular weight, such as polyethylene glycol and albumin. Covalent connection to the macromolecules can reduce immunogenicity and improve soluble and biological availability, as well as increase anti-protein hydrolysis and extend the half-life. However, the covalent modification of bioactive molecules is limited by the loss of intrinsic biological activity. For example, receptor-binding is reduced due to steric effect. Non-active molecules are integrated into one or more locus of peptides and cause products heterogeneity. The structure heterogeneity of the final product is usually accompanied by functional heterogeneity, which results in reduction of biological activity or enzyme activity. Polyethylene glycol is a polymer derived from ethylene oxide polymer macromolecules. In comparison with other modifiers, PEG-type modifiers take the advantages of non-toxic, good solubility, low immunogenicity, wide relative molecular weight and more options. PEG itself can give many excellent properties to modified biological molecules. By covalent modification of polyethylene glycol, the relative molecular weight of protein drugs will increase, which will decrease drug excretion and reduce the immunogenicity. These changes are conducive to the extension of the half-life of drug in the body. Accordingly, the pharmacokinetics and pharmacodynamic properties of protein drugs will improve obviously. For example, PEG-modified interferon-α(IFN-α) compared to non-modified IFN-α the half-life is extended to 10 to 20 times; before PEG-modified the superoxide dismutase’s half-life is 5min, after PEG modified to half-life extend the half-life to 4.2h. PEG5000 modified with glucagon, and then test such a modification of glucagon conformation and biological stability. Detect the secondary structure of glucagon samples and modified PEG glucagon, and to assess its physical stability. Found that glucagon without modification is formed a number of intermolecular β-sheet structure, the delay time of forming fiber is short; and PEG modified glucagon is only a small number of intermolecular β-sheet, no fiber, the physical stability significantly higher than that of the former.
To date, there are a variety of PEG-modified proteins drug used in clinical. At present, PEG-modified protein drugs on market are: PEG-modified L-asparaginase (Enzon's Oncaspar), PEG-modified interferon α, β (IFN-α, β) (Schering's PEGIntron), PEG-modified interferon of αa (IFNα-a) (Roche's Pegasys) and PEG-modified of granulocyte colony-stimulating factor (G-CSF) (Amgen's Neulasta).
2. Fusion protein
Using fusion protein can construct biofunctional target protein, this fusion protein is head-to-tail ligation of two or more of the coding region, and the gene expression product consist with the same regulatory sequence. At the molecular level, this technology has the characteristics of simple and flexible design, because when the construction of mutant sequences do not need to inserted target sequence, however it can ligate directly in the C or N-terminal. Therefore, this technology can be widely used in construction of bioactive molecules, and its application will not be limited to the size of biological molecules or activity. It is often selected the molecular with longer half-life, larger molecular weight as the vector to construct fusion protein, and human serum albumin (HSA) or immunoglobulin (IgG) of the Fc fragment is most widely used. Therefore, here the examples given are the integration technology of human serum albumin and the Fc fragment.
Human serum albumin is the major content of plasma protein, molecular weight 67kDa, distributed widely in the circulation of blood-vessels and outside blood-vessel, the half-life is up to 19 days, and the structure is very stable, no significant immunogenicity, and it is an important blood material transport and drug transport vector, so that it is designed to be an ideal drug carrier protein to improve half-life of drugs, delayed drug clearance, increased drug exposure and reduce the frequency of injections, improving patient compliance to treatment and tolerability. Currently, it is confirmed that after the experimental animals apply a fusion protein of HAS with various proteins is half-life extension. For example, albumin is successfully integrated with hormones (insulin, glucagon-natural, human growth hormone), cytokines (interferon α-2b, IL2), obtained a more stable circulating protein, the fusion proteins have a long half-life and biological activity and therapeutic properties as human serum albumin. Many proteins have been modified by using this technology. The half-life of human insulin / albumin fusion protein (Albulin) in mice is 42-fold increased than the natural insulin. The half-life in mice of glucagon-like peptide -1 Su-fusion protein (GLP-1/HSA) expressed in Pichia pastoris is 4 times longer than the GLP-1 alone. The half-life of interferon-α fusion protein (IFN-α/HSA) is prolonged for approximately18 times than non-integration of IFN-α (IFN-α). Take the advantage of antibodies Fc paragraph specific biological function to integrated with some peptide may also increase the plasma half-life of the protein. The human immunoglobulin IgG is the main antibody in vivo, its half-life is approximately 20 days. The construction method of IgG fusion proteins is mostly integrated the N-end of IgG Fc fragment or IgG fragments with the C-end of activity protein. Syntonix Pharmaceuticals developed for EPO-Fc inhalation agents, is conducting clinical Phase I testing to patients with anemia is currently, the preparation may be extended for half-life and alternative injection current, convenient delivery. The significant advantage of agents is without repeated injections, especially for patients with chronic kidney that without dialysis or peritoneal dialysis disease. The preparation showed good results in this experiment.
3. Microencapsulation
Microencapsulation technology has been widely used to enhance the characteristics of pharmacokinetics of therapeutic factors, improve drug stability and the target-dependent release of drugs. Embedded with microcapsules of protein and peptide drugs, the role is not only through the realization of a long-term subcutaneous injection of sustained-release formulations, but also the microcapsule surface and particle size design, the realization of oral mucosal drug delivery, as well as inhalation and other new formulations. The principle of this technology is to partly embed the drug activity in the polymer layer of encapsulation, drug delivery through the skin or muscle, to reduce the aggregate level over time, so that slowly the drug from the sustained-release microcapsules, which is conducive to the stability of the drug, reducing the gastrointestinal tract destruction of the enzyme, to change the course of its operation in vivo, to extend the role of drugs in the body of time.
Now protein microsphere injection drug formulations has been studied and applied to clinical successfully. It is a biodegradable polymer used as a reinforcing material made of protein drugs parcels 1 ~ 250μm diameter of injected microspheres, bio-degradable polymers include starch, gelatin, dextran, albumin, polylactic acid ( PLA), polylactic acid glycolic acid copolymer (PLGA), poly o-ester, poly anhydride, such as caprolactone and polyethylene. Currently used for preparation of sustained-release microspheres of skeleton material is mainly PLGA and PLA, which is more commonly used PLGA.
In addition, Medusa sustained-release system is also being used for protein drug delivery process, it is poly-L-glutamic acid skeleton composed of a hydrophobic α-tocopherol molecules, forming to nanosphere colloidal particle suspended liquid. Tablets as a result of nanosphere hydrophobic interaction of drugs in the region have had the effect of sustained-release drugs. In the body, protein drugs is instead of the endogenous protein in physiological solution, lead to drug control-release. The highest concentration of protein drugs significantly decreased, drug release was significantly prolonged. Medusa sustained-release technology has been used in intravenous injection of IL-2 and IFN-α (2b), and it has been applied to renal cell carcinoma clinical hepatitis C and hepatitis C treatment.
At present, sustained-release preparations of polylactic acid and its copolymers are appeared on market, thyrotropin-releasing hormone TRH drugs - triptorelin slow-release PLGA microspheres, for the treatment of prostate cancer, it may be on a slow release for a month, and it is the first product peptide microspheres. Leuprorelin PLGA slow-release microspheres is mainly used for prostate cancer and endometriosis, and it is also release for a month. After that there are variety of sustained-release microsphere injection of LHRH analogues come into the market. There are a number of protein drug sustained-release microsphere injection are laboratory studies or animal testing stage.
4. Anti-protease mutant
Enzymatic Hydrolysis of biological drugs is the major obstacle need to breakthrough in the development of drug delivery technology. In view of the protein can be easily decomposited by endogenous serum protease or tissue protease, even more the protein degradation of protease can occur in the every stage of absorption, distribution and excretion, and this directly affects the in vivo half-life of drugs, bioavailability, plasma clearance rate parameters. Introduced one or more anti-mutant protein hydrolysis on biologically active molecules, is the research focus of drug design and development. The stability in vivo is also need to introduced artificial amino acid analogues to accomplish. These modifications have a small conformational various, resulting in enhanced anti-proteolysis. For example, the peptides mimetic design target is by replacing the primary structure of protein of any peptide bond, protein drugs improve the stability of resistance to protease degradation, basis on the biological activity of peptides mimetic, to change the main structure of peptides through modification of peptide framework. In order to improve the ribonuclease A (RNaseA) proteinase K resistance and the ability of subtilisin hydrolysis, by using site-directed mutagenesis, replaced Pro with Ala20, after detection it was found hte mutant enzyme (Ala20Pro RNaseA) conformation has no obvious changes, the biological activity is the same as wild-type activity RNaseA, but the ability of resistance to protease hydrolysis increased significantly. According to SDS-PAGE electrophoresis results, the capacity of protease hydrolysis is decreased by 2 times.
5. glycosylation
Protein surface sugar chain can affect the properties of protein pharmacokinetics, biological activity and stability, and glycosylation is necessary for certain protein to develop the biological activity. Compare the glycosylation and non-glycosylated form of therapeutic proteins, on the one hand, an increase of surface side chains will improve protein stability, and interfere the protease with the degradation of protein drugs, on the other hand, it will increase in molecular weight of protein drugs, and reduced glomerular filtration. Erythropoietin (EPO) is a highly glycosylated sialic acid containing glycoprotein, which contains three N sugar chain and an O-chain sugar. Construction of recombinant human erythropoietin protein, respectively, 33 and 88 in the addition of a N glycosylation sites, researchers found that recombinant human EPO in vivo half-life is 3 times the EPO. Recombinant human EPO glycosylation of O is not concerned in the activity in vitro and in vivo role and clearance rate, and the activity of recombinant human EPO with incomplete N glycosylation is normal in vitro, in vivo activity is reduced to 1 / 500 of in vitro activity, and in vivo clearance rate markedly accelerated. Therefore, N glycosylation of protein drugs play an important role in the modification. Recombinant human EPO mutant (Amgen's Aranesp) has been developed and came to the market.
Tissue plasminogen activator (tPA) in the systemic circulation has a high clearance rate, half-life is only 6min. Keyt, et.al constructed tPA mutant and found that possess reducing of tPA plasma clearance rate, and maintaining the normal protein-binding capacity and activity of dissolve blood clots. Effective to extend the half-life of tPA mutant was carried out by Department Kringle1 modified glycosylation, Thr103 with Asn alternative form, that is, TtPA. Then the 296-299 location take place the subsequent four-allele replacement reaction [KNRR (296-299) AAA] mutant to form (TKtPA). TKtPA demonstrated anti-plasminogen activator inhibitor 1 than tPA high 80 times. TKtPA extense the time of in vivo clearance, and reduce the clearance rate, while maintaining normal coagulation activity. Therefore，can serve as an effective low-dose thrombolytic agent.
To sum up, untill now there are develop a number of effective technology to enhanced the half-time of protein drugs, a variety of methods of long-acting protein drugs can significantly improve the stability of biological products, to improve the bioavailability of drugs, and to extend the half-life in vivo.