Granulocyte colony stimulating factor (G-CSF) is one of the colony stimulating factors that contribute to the formation of the colonies of bone marrow cells. It can specifically stimulate and regulate the proliferation, differentiation, survival and activation of granulocytes, and is promising in treating granulopenia of various causes.
Human G-CSF gene is located in the q21-22 region on chromosome 17 with a full length of 2.5 kb. The gene is consisted of 5 exons and 4 introns, and the corresponding mature protein comprises 174 amino acids. G-CSF expressed by E. coli has a Met on its N-terminus, and is consisted of 175 amino acids, as shown in FIG. 1 (SEQ ID NO:1). The molecule comprises 5 cystein residues, wherein Cys37-Cys43 and Cys75-Cys85 form two pairs of disulfide bonds, and comprises 4 lysine residues, located on positions 17, 24, 35 and 41 separately.
Neutropenia caused by chemotherapy, especially febril neutropenia (FN) is the most common and usually the most serious side effect after chemotherapy of cancer patients, especially in the first cycle of chemotherapy. FN will lead to enforced hospitalization and use of antibiotics, causing extra economic burden and high fatality. Another damaging result is that due to the reduction of neutrophils, the scheme for the treatment of cancer patients has to be changed, for example reducing the dosage for chemotherapy and postponing the next therapeutic cycle, which are directly related to the final therapeutic result. Since the approval of rHuG-CSF by FDA in 1991, millions of cancer patients subjected to chemotherapy have benefited therefrom. The medicament has been listed as one of top 10 sellers of the world's biotech medicament, and is believed to be very promising.
However, recombinant human G-CSF has shortened in vivo half life (t1/2 of about 1.3-4.2 h) and activity, and tends to be hydrolyzed by enzymes and cleared from the kidney, therefore needs to be injected many times, which is very inconvenient for the patient and may cause some undesired responses which will affect the efficacy.
Recently, the development of polyethylene glycol (PEG) modification technique has provided an alternative for solving the above-mentioned technical problem.
PEG is a nontoxic and dissolvable neutral polymer. It is biocompatible and hemocompatible, and has been approved by FDA for tropical and enteral use and intraveneous injection. PEG modification of a protein is achieved by activating one or both terminal groups on both ends of PEG to create a functional group, which is reactive with at least one group in the protein to be bound, so as to bind PEG with the N or C terminal of the protein or a specific amino acid, wherein the site for PEG modification is universal.
PEG is the polymer of ethylene glycol and ethylene oxide, also called carbowax, with the structure shown in the following formula:CH2(OH)—(CH2CH2O)n—CH2OH
The appearance of conventional PEG changes as its molecular weight increases: it appears from colorless viscous liquid (190-630 Dalton), white paste (950-1050 Dalton) to white waxy or flaky solid (>1200 Dalton). Usually, PEG for modifying proteins or other drugs has a large molecular weight (Table 1), such as the U-shaped double-stranded PEG with a molecular weight of 40 KD employed in Pegasys (PEGylated interferon α2a injection, PEGASYS®, Roche, Shanghai), or the linear PEG with a molecular weight of 12 KD employed in Peg-Intron (PEGylated interferon α2b injection, PEG-INTRON®, Schering-Plough, US), or the linear PEG with a molecular weight of 20 KD employed in Neulasta® (PEGylated granulocyte colony-stimulating factor, Amgen US). The in vivo metabolic process of conjugated PEG is quite clear, indicating that PEG is a good, safe drug modifier with no side effects.
TABLE 1The parameters for basic physicochemical properties of currently marketedPEGylated protein drugs[18]t1/2 beforet1/2 afterProduct (Trade name)Average MWMW of PEGpegylationpegylationManufacturePegylated L-asparaginase (Oncaspar)143 kDa 5 kDa20h2 weeksEnzonPegylated IFNα2b (PEGIntron) 31 kDa12 kDa4-12h40hSchering(22-60h)Pegylated IFNα2a (Pegasys) 60 kDa40 kDa5.1h80hRoche(3.7-8.5h)(50-140h)Pegylated G-CSF (Neulasta) 39 kDa20 kDa3.5h15-80hAmgren
Proteinaceous drugs after polyethylene-glycolation (PEGylation) will have significantly improved properties, including prolonged pharmacokinetic half-life (Table 1), reduced immunogenicity, improved safety, increased efficacy, decreased frequency of administration, increased solubility, enhanced protease resistance, which facilitate the controlled release of the drugs. For example, as disclosed in U.S. Pat. No. 4,179,337, after the conjugation of PEG with enzymes and insulin, the immunogenicity of the proteins is reduced, while a certain percentage of the original activity of the proteins still remains. Another unique effect of pegylation is that the in vitro activity of the protein is decreased, but the in vivo activity is increased. For example, as disclosed in U.S. Pat. No. 4,179,337, after the conjugation of PEG with an enzyme or insulin, the immunogenicity of the protein is reduced, and the activity of the protein is significantly decreased, but the protein still retains a certain percentage of the original activity.
PEGs used to modify drugs are divided into two types according to the structure: the linear type and the branched type. For example, Pegasys® (PEGylated interferon α2a injection, PEGASYS®, Roche US) employs a U-shaped branched double-stranded PEG derivative, with an average molecular weight ranging from 26 KD-66 KD (U.S. Pat. No. 5,382,657—Filed Aug. 26, 1992—Hoffmann-La Roche Inc.), which is represented by the following formula:
wherein, R and R′ are independently low molecular weight alkyl groups, n and n′ are from 600 to 1500.
Linear PEG molecule having a molecular weight of 20kD is used in NEULASTA® (pegylated granulocyte colony-stimulating factor, Amgen) approved by U.S. FDA in 2002 (U.S. Pat. No. 5,824,784—Filed Oct. 12, 1994—Amgen Inc.). The reaction for modification is as follows:

Amgen's Neulasta® employs PEG with aldehyde group on the end. The modification is on the N-terminal amino acid of the protein and PEG-G-CSF modified at a single point is obtained. It is characterized in that PEG is conjugated with G-CSF via C—N bond.
PEGs with different configurations are used, to modify proteins, resulting in products with apparently different features. Prior art literature (Monfardini C, Schiavon O, Caliceti P, et al. Bioconjugate Chem, 1995, 6 (1):62-69) reports that comparing to linear PEG, branched PEG improves the protein's pH resistance, thermal stability and resistance to protease digestion.
Chinese Patent ZL 03801105.0 reported a new double-stranded Y-shaped PEG derivative, which has the following basic structure:
wherein, Pa and Pb are the same or different hydrophilic polymers, which can be polyethylene glycol, polypropylene glycol, polyvinyl alcohol, polyacrylmorpholine or their copolymers, preferably is polyethylene glycol and its copolymers;
j is an integer from 1 to 12;
Ri is H, a substituted or unsubstituted C1-12 alkyl group, a substituted aryl, an aralkyl or a heteroalkyl;
X1 and X2 are independently a linking group, wherein X1 is (CH2)n, and X2 is selected from the group consisting of (CH2)n, (CH2)nOCO, (CH2)nNHCO, and (CH2)nCO; n is an integer from 1 to 10; and
F is a terminal group selected from the group consisting of a hydroxyl group, a carboxyl group, an ester group, acyl chloride, hydrazide, maleimide, pyridine disulfide, capable of reacting with amino, hydroxyl or mercapto group of a therapeutic agent or a protein to form a covalent bond.
When Pa and Pb are preferably PEG or its copolymer, the basic molecular structure thereof is

The Y-shaped PEG modifies the protein on the free amino group of the protein, wherein the modification site is not fixed.
In prior art, N-hydroxysuccinimide activation can be used to synthesize Y-shaped branched NHS-PEG which is used for modification. NHS-PEG is characterized in that it can form amide bond with the free amino group on lysine or the free terminal amino group of rhG-CSF, and the amide bond can be hydrolyzed in vivo slowly, so that the activity of rhG-CSF is restored. However, the currently used Y-shaped branched NHS-PEG generally have the defect of high activity and poor selectivity, and cannot be used to achieve directional selection of modification sites, therefore it is difficult to obtain products with modification on a single fixed-site.