Pegylated asparaginase and its application

Abstract

The present invention discloses a PEGylated asparaginase and its application in drug preparation and clinical treatment. In the PEG-modified asparaginase of the present invention, one molecule of asparaginase is coupled with 13-45 molecules of polyethylene glycol, wherein the polyethylene glycol is a linear polyethylene glycol with an average molecular weight of 2-20 kDa. The asparaginase after PEG modification prepared by the present invention. The PEGylated asparaginase provided by the present invention has the advantages of reducing immunogenicity and significantly extending half-life, and the coupling of polyethylene glycol and asparaginase is more stable. Compared with the original research drug and generic drug products of the PEGylated asparaginase sold on the market, the PEGylated asparaginase provided by the present invention has a more stable structure, a firm PEG bond, is not easy to fall off, and has higher homogeneity and activity.

Description

Technical Field

[0001] The present invention relates to polyethylene glycol-modified protein drugs, in particular to PEGylated asparaginase and its application in drug preparation and clinical treatment. Background Art

[0002] Asparaginase (ASP), also known as L-asparaginase, L-asparaginase, or L-asparaginase, is an enzyme that catalyzes the hydrolysis of asparagine to aspartic acid. Asparaginase is an effective treatment for acute lymphoblastic leukemia (ALL) in children and adults. In recent years, drugs containing L-asparaginase have been used in combination with chemotherapy regimens to treat NK / T-cell lymphoma, achieving good therapeutic results. NK / T-cell lymphoma is a special type of non-Hodgkin's lymphoma that is more common in Asia and Latin America, with a relatively high incidence in my country. Based on the site of tumor occurrence, NK / T-cell lymphoma can be divided into nasal NK / T-cell lymphoma and non-nasal NK / T-cell lymphoma. In addition, L-asparaginase has been used to treat Hodgkin's disease, acute myeloid leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulum cell sarcoma and melanosarcoma (Kotzia and Labrou, J. Biotechnol. 127 (2007) 657-669).

[0003] The active form of L-asparaginase is a homotetramer composed of four subunits, each consisting of 326 amino acids. L-asparaginase was initially purified from several organisms, including Escherichia coli (E. coli) and Erwinia carotovora. In mammals, L-asparaginase is found only at trace levels in guinea pigs (superfamily Cavioidea) and certain New World monkeys. However, because it is derived from foreign organisms and is a foreign protein to humans, it is highly immunogenic. Progressive immune reactions and systemic allergic reactions are common clinically, limiting its clinical application. (Zhang Lina, Gong Daohua. Jiangsu Medicine. Toxic side effects of L-asparaginase in the treatment of acute lymphoblastic leukemia in children. 2005, 31(5): 392; Wang Ningling, Liu Zhizhang, et al. Toxic side effects and prevention of L-asparaginase in the treatment of childhood leukemia. Chinese Journal of Pediatric Hematology, 2005, 10(3): 133).

[0004] Therefore, how to obtain low-immunogenic L-asparaginase and improve the pharmacokinetics of the protease to enhance tolerance and ease of administration has always been the focus of research.

[0005] One of the methods to solve the above problems is to perform appropriate chemical modification on L-asparaginase.

[0006] Polyethylene glycol (PEG) is a linear, uncharged polymer that can freely curl in solution. It is non-toxic, weakly antigenic, and has good biocompatibility. Using it to covalently modify proteins can increase the protein's in vivo circulation half-life and reduce its antigenicity, increase the protein's solubility, and change the protein's biological distribution in the human body. Since Abuchowski, Davis et al. (J.Biol.Chem.1977,252:3578-3581.) first reported the use of PEG to modify proteins in 1977, PEG modification technology has been widely used in the fields of biomedicine and biotechnology. PEG has been widely used in the modification research of proteins and peptide drugs. Currently, protein PEGylation technology has become one of the most effective methods to reduce the immunogenicity of protein biopharmaceuticals and improve their pharmacokinetic / pharmacodynamic properties, and has been approved by the FDA for use in drugs, food, and cosmetics.

[0007] Despite decades of development, PEGylation technology has become relatively mature. However, a universal PEG modifier and modification method cannot be found for all protein drugs. The protein structure, the molecular weight of the PEG used, its shape, and the site of modification significantly influence the biological activity and efficacy of the PEGylated protein. For the modification of specific drugs, the PEG modifier is the most important factor affecting the physicochemical properties, in vitro and in vivo biological activity, pharmacokinetics, pharmacodynamics, and clinical performance of the modified product. Therefore, the selection of the modifier (type and molecular weight) and the control of the modification reaction play a crucial role in PEGylation technology. Protein structural analysis cannot accurately predict the pharmacokinetic behavior of native proteins. However, protein conjugation with PEG introduces many new variables, such as molecular weight and modifier type, making the prediction of the pharmacokinetic behavior of PEG conjugates even more difficult. Therefore, for different protein drugs, it is necessary to select different types and molecular weights of modifiers and determine the optimal solution through physicochemical property testing and animal experimental evaluation.

[0008] For example, in most cases, the activity of PEGylated protein drugs is reduced compared to the unmodified parent protein, typically reaching only 30%-40% or even lower. For example, Schering-Plough's PEG-Intron, which modifies interferon with a molecular weight of 5000, shows only 8% of the activity of the parent protein after modification. Yoshihiro et al. modified Escherichia coli asparaginase with branched activated PEG (2-0-methoxypolyethylene glycol-4,6-dichloro-s-triazine) of molecular weights 750, 1900, and 5000 U, respectively. The 5000 U PEG modified 73 of the 92 free amino groups (88 δ-amino groups and 4 α-amino groups) of asparaginase, retaining 7% of the original enzyme's activity. The modified product completely lost its ability to bind to asparaginase-resistant serum. Furthermore, the activity of the modified protein generally decreases more significantly with increasing PEG molecular weight. For example, after erythropoietin (EPO) was modified with PEG of molecular weights of 20 kDa, 30 kDa, and 40 kDa, its activity decreased significantly with increasing PEG molecular weight (Yin-jue Wang, Journal of Controlled Release, 2010(145):306-313). Bailon et al. modified Interferon-α-2a with branched 40 kDa PEG, and the resulting single modified product had a longer circulation half-life, but only retained 7% of its in vitro activity (Bailon P, Bioconjugate Chem., 2001, 12:195-202).

[0009] Numerous studies have been conducted on the polyethylene glycol modification of L-asparaginase. Oncaspar (Enzon Inc.), a PEGylated L-asparaginase product, was launched as early as 1994 and approved as a first-line treatment for ALL in children and adults in 2006. However, Oncaspar suffers from poor in vitro and in vivo stability, is prone to shedding, and has significant side effects. The only PEGylated L-asparaginase currently available in China is Hengrui Company's "pegaspargase," a generic version of Oncaspar, which also suffers from issues such as PEG degradation and shedding, poor homogeneity, and severely reduced activity.

[0010] At the same time, there are currently some papers or patents related to PEG site-specific modification of L-asparaginase, but these modifiers are more expensive, the modification process is more complicated, the yield is lower, the overall manufacturing cost is high, and they are not suitable for industrial application.

[0011] Therefore, there is an urgent need in the art to provide a polyethylene glycol-modified L-asparaginase with low production cost, greater stability, and PEG that is not easily detached. Summary of the Invention

[0012] Technical problem to be solved: The present invention aims to provide a PEGylated asparaginase with the advantages of long half-life, low immunogenicity, low cost, etc., which is not easy to shed PEG, has high yield and high uniformity, and its use in the preparation of drugs for treating diseases such as acute lymphoblastic leukemia in children or adults.

[0013] Technical solution: The first object of the present invention is to provide a PEGylated asparaginase, wherein one molecule of asparaginase is coupled with 13-45 molecules of polyethylene glycol, and the polyethylene glycol is a straight-chain polyethylene glycol with an average molecular weight of 2-20 KDa.

[0014] Preferably, PEG is covalently linked to the asparaginase via an amide bond or a urethane bond with the free amino groups of the asparaginase, including lysine residues and / or the N-terminal amino group.

[0015] Preferably, the general structural formula after PEG is coupled to asparaginase is as follows:

[0016]

[0017] wherein ASP is L-asparaginase, PEG is a polyethylene glycol moiety, m is an integer from 13 to 45, and n is an integer from 0 to 3.

[0018] A second object of the present invention is to provide a method for preparing the above-mentioned PEGylated asparaginase, comprising the following steps:

[0019] Step 1: Buffer Exchange

[0020] The asparaginase drug substance solution is absorbed into the ion exchange chromatography column, equilibrated with an equilibration buffer, eluted with an elution buffer, and the eluted protein is collected. The elution buffer is also used as the modification buffer in the next modification reaction;

[0021] Step 2: Modification reaction and purification of modified products

[0022] The asparaginase solution collected in the first step is reacted at a molar ratio of asparaginase to PEG modifier of 1:10-1:200 at 4°C-37°C for 1-24 hours; wherein the asparaginase protein concentration in the PEG modification reaction is 5-40 mg / mL;

[0023] After the reaction, the product was purified by ion exchange chromatography.

[0024] The third object of the present invention is to provide a pharmaceutically acceptable salt of PEGylated asparaginase or a complex thereof. The complex is a combination of two or more different substances.

[0025] Pharmaceutically acceptable salts are nontoxic in the amounts and concentrations in which they are administered. Such salts can be prepared to facilitate pharmaceutical applications by altering the physical characteristics of a compound without interfering with its physiological effects. Useful changes in physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate administration of higher concentrations of the drug.

[0026] The pharmaceutically acceptable salts include acid addition salts such as sulfates, hydrochlorides, fumarates, maleates, phosphates, acetates, citrates, lactates, tartrates, methanesulfonates, benzenesulfonates, etc. The pharmaceutically acceptable salts may be derived from acids such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, lactic acid, tartaric acid, etc.

[0027] The fourth object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned PEGylated ASP or a pharmaceutically acceptable salt thereof or a complex thereof and a pharmaceutically acceptable excipient.

[0028] Pharmaceutically acceptable carriers and / or excipients may also be incorporated into the pharmaceutical compositions according to the present invention to facilitate administration of the specific asparaginase. Suitable carriers for practicing the present invention include calcium carbonate, calcium phosphate, various sugars (lactose, glucose, sucrose), or various starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and physiologically compatible solvents (including sterile aqueous solutions for injection, saline solutions, and dextran, etc.).

[0029] Preferably, the above-mentioned pharmaceutical composition is a lyophilized powder injection.

[0030] Preferably, the PEGylated asparaginase and the pharmaceutical composition thereof are administered intramuscularly, intravenously or subcutaneously.

[0031] The fifth object of the present invention is to provide a use of the above-mentioned PEGylated ASP in the preparation of a drug for treating childhood or adult acute lymphoblastic leukemia, NK / T cell lymphoma, Hodgkin's disease, acute myeloid leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulum cell sarcoma or melanosarcoma.

[0032] Beneficial effects: The PEGylated asparaginase provided by the present invention has the advantages of reduced immunogenicity, significantly prolonged half-life, etc., and the coupling of polyethylene glycol and asparaginase is more stable. Compared with the original and generic drugs of PEGylated asparaginase sold on the market, the PEGylated asparaginase provided by the present invention has a more stable structure, firm PEG binding, is not easy to fall off, and has higher uniformity and activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : HPLC-SEC purity analysis of PEG-ASP conjugates

[0034] High-performance gel filtration analysis revealed no significant impurity peaks in the prepared conjugates, with purity exceeding 98%. The molecular weight of SPA5K-ASP is roughly the same as that of Oncaspar, but higher than that of pegaspargase. Since pegaspargase is also modified with a linear PEGylated molecule with a molecular weight of 5K, this suggests that the degree of modification of SPA5K-ASP is significantly higher than that of pegaspargase. Figure 2 :SDS-PAGE electrophoresis detection of SPA5K-ASP conjugate

[0035] Protein electrophoresis of ASP modified with PEG. Lanes 1-4 contain protein marker, Oncaspar, SPA5K-ASP, and pegaspargase, respectively. Protein electrophoresis results indicate that the modification uniformity of SPA5K-ASP is superior to that of pegaspargase, and that SPA5K-ASP has a higher molecular weight than pegaspargase.

[0036] Figure 3: Protein electrophoresis staining (a) and iodine staining (b) results of Oncaspar, SPA5K-ASP and pegaspargase at different time periods after heat treatment.

[0037] Figure 3a In protein electrophoresis staining (a), the samples in lanes 1-19 are protein marker, Oncaspar 0h, Oncaspar 1h, Oncaspar 2h, Oncaspar 3h, Oncaspar 4h, Oncaspar 5h, SPA5K-ASP 0h, SPA5K-ASP 1h, SPA5K-ASP 2h, SPA5K-ASP 3h, SPA5K-ASP 4h, SPA5K-ASP 5h, pegaspargase 0h, pegaspargase 1h, pegaspargase 2h, pegaspargase 3h, pegaspargase 4h, and pegaspargase 5h, respectively.

[0038] Figure 3bIn protein electrophoresis iodine staining (b), the samples in lanes 1-20 are protein marker, SPA5K PEG, Oncaspar0h, Oncaspar 1h, Oncaspar 2h, Oncaspar 3h, Oncaspar 4h, Oncaspar 5h, SPA5K-ASP 0h, SPA5K-ASP 1h, SPA5K-ASP 2h, SPA5K-ASP 3h, SPA5K-ASP 4h, SPA5K-ASP 5h, pegaspargase 0h, pegaspargase 1h, pegaspargase 2h, pegaspargase 3h, pegaspargase 4h, and pegaspargase 5h, respectively.

[0039] The results of iodine staining and calcein staining of protein electrophoresis showed that the PEG of SPA5K-ASP did not fall off, while the PEG of Oncaspar and pegaspargase fell off easily, which indicated that SPA-5K-ASP was more stable than Oncaspar and pegaspargase.

[0040] Figure 3c Activity assay results showed that under high temperature conditions of 55°C, pegaspargase was almost completely inactivated within 1 hour, Oncaspar retained about 30% of its activity, and SPA5K-ASP retained about 70% of its activity, indicating that it had the best stability.

[0041] Figure 4 :Fluorescence spectrum analysis of different PEG-ASP conjugates

[0042] The endogenous fluorescence spectra of asparaginase, Oncaspar, SPA5K-ASP and peparaginase were scanned using a fluorescence spectrophotometer. The results showed that PEG modification did not change the tertiary structure of asparaginase.

[0043] Figure 5: Comparison of pharmacokinetic properties of PEG conjugates and pegaspargase

[0044] Figure 5a The pharmacokinetic results of pegaspargase and SPA5K-ASP samples were obtained by intravenous (iv) injection. Figure 5b Pharmacokinetic results of pegaspargase and SPA5K-ASP samples administered by intramuscular (im) injection.

[0045] use 125 The plasma concentration of PEG-modified asparaginase was studied using I isotope labeling. The results showed that SPA5K-ASP had slightly better pharmacokinetic properties than pegaspargase. DETAILED DESCRIPTION

[0046] definition:

[0047] The abbreviations used in the present invention have the following meanings:

[0048] PEG, polyethylene glycol; PEG modifier, polyethylene glycol modifier.

[0049] Polyethylene glycol (PEG, HO-(CH2CH2O)n-CH2CH2OH) is a linear polymer with hydroxyl groups at both ends. Polyethylene glycol is formed by the polymerization of ethylene oxide and is composed of repeating oxyethylene groups. It can be branched, straight-chain, or multi-armed. PEG is also known as poly(ethylene oxide) (PEO), poly(oxy-ethylene) (POE), or polyoxirane. Generally, those with a molecular weight below 20,000 are called PEG, while those with a larger molecular weight are called PEO. Ordinary polyethylene glycol has a hydroxyl group at each end. If one end is blocked with a methyl group, methoxy polyethylene glycol (mPEG) is obtained. This derivative is the most commonly used in protein PEGylation technology.

[0050] Polyethylene glycol modifier refers to a polyethylene glycol derivative with functional groups, which refers to activated polyethylene glycol. It is currently mainly used for protein and peptide drug modification, and is also called modified polyethylene glycol, modified PEG.

[0051] M-SPA-5000 and M-SPA-2000 are linear polyethylene glycol succinimidyl propionate with molecular weights of 5000Da and 2000Da respectively; M-SC-10K and M-SC-5000 are linear polyethylene glycol succinimidyl carbonate with molecular weights of 10KDa and 5000Da respectively; M-SCM-20KDa is a linear polyethylene glycol succinimidyl acetate with a molecular weight of 20KDa; M-SBA-5000 is a linear polyethylene glycol succinimidyl butyrate with a molecular weight of 5000Da. Their general structural formula is

[0052] When n is 0, the molecular weight of mPEG is 10KDa and 5000Da, and the corresponding PEG types are M-SC-10K and M-SC-5000 respectively; when n is 1, the molecular weight of mPEG is 20KDa, and the corresponding PEG types are M-SCM-20K respectively; when n is 2, the molecular weight of mPEG is 5000Da and 2000Da, and the corresponding PEG types are M-SPA-5000 and M-SPA-2000 respectively; when n is 3, the molecular weight of mPEG is 5000Da, and the corresponding PEG type is M-SBA-5000;

[0053] M-NPC-5000, a linear polyethylene glycol nitrobenzene carbonate with a molecular weight of 5000Da; the structural formula is as follows

[0054] Among them, the molecular weight of mPEG is 5000Da.

[0055] The term "conjugate" as used in this application refers to the modified product obtained after polyethylene glycol modification of asparaginase;

[0056] Several polyethylene glycol-modified asparaginase modification products can be collectively referred to as PEG-ASP or PEG-modified ASP conjugates in this application.

[0057] The polyethylene glycol modifier used in the present invention is preferably the following: ester-activated polyethylene glycol, more specifically, the polyethylene glycol modifier is succinimidyl propionate-activated polyethylene glycol.

[0058] In the present invention, the modified asparaginase protein can be from any source. Asparaginase can be extracted from Escherichia coli, including but not limited to E. coli. It can also be recombinantly expressed. In specific embodiments of the conjugates of the present invention, the asparaginase has at least about 60% sequence identity with a protein comprising the sequence of SEQ ID NO: 1. More specifically, the asparaginase has at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 100% sequence identity with the protein comprising SEQ ID NO: 1.

[0059] In a specific embodiment, the protein is asparaginase derived from Escherichia coli, which has the sequence of SEQ ID NO: 1.

[0060] Fragments of the protein of SEQ ID NO: 1 are also included in the definition of the protein used in the conjugate of the present invention. The "fragment of the protein of SEQ ID NO: 1" refers to a polypeptide sequence that may include fewer amino acids than SEQ ID NO: 1.

[0061] It is well known in the art that polypeptides can be modified by replacement, insertion, deletion and / or addition of one or more amino acids while retaining their enzymatic activity. For example, it is common to replace an amino acid at a given position with a chemically equivalent amino acid without affecting the functional properties of the protein. Therefore, it is expected that the following changes will produce functionally equivalent products: changes that produce one negatively charged residue replacing another or changes that produce one positively charged residue replacing another. In addition, with the development of polyethylene glycol synthesis technology, new branched polyethylene glycol modifiers can be synthesized by changing the connecting group between the activating group and the polyethylene glycol molecule. The branched polyethylene glycol modifier used in the patent of the present invention is not limited to the structural formula described in the present invention.

[0062] Example 1: Preparation and analysis of PEG conjugates of asparaginase

[0063] Preparation Example 1: The PEGylated asparaginase of the present invention was prepared, purified, and identified by the following method:

[0064] Step 1: Buffer Exchange

[0065] Lyophilized asparaginase powder was dissolved in 20 mM Tris-HCl (pH 8.0) buffer to a protein concentration of 5 mg / mL. The sample was then loaded onto a Q ion exchange column (GE HiTrap Q HP 5mL) using the loading pump of an AKTA chromatography system. After loading, the column was equilibrated with equilibration buffer A for 5 column volumes, followed by a single-step elution with elution buffer B. The eluted peak was collected.

[0066] (Solution A: 20 mM phosphate buffer (pH 8.0), Solution B: 20 ​​mM phosphate buffer + 0.2 M sodium chloride (pH 7.5))

[0067] Step 2: Modification reaction and purification of modified products

[0068] The asparaginase solution collected from the first step was treated with M-SPA-5000 (purchased from Beijing Jiankai Technology Co., Ltd.) as a PEG modifier at a molar ratio of 1:50 for PEG modification at 4°C for 12 hours. The asparaginase protein concentration during the PEG modification reaction was 5 mg / mL. After completion of the reaction, the asparaginase modification rate reached 100%.

[0069] After the reaction, the product was purified by ion exchange chromatography. The chromatographic conditions for purification were: Q ion exchange column (purchased from GE, HiTrap Q HP 5 mL), equilibration buffer C: 20 mM Tris-HCl (pH 9.0), elution buffer D: 20 mM phosphate buffer (pH 8.0) containing 0.1 M NaCl, flow rate 2.5 mL / min, and detection wavelength at 280 nm.

[0070] Sample loading: The above modification reaction product was adjusted to pH 9.0 with 0.5 M NaOH solution and bound to a Q ion exchange column.

[0071] Equilibration: Flush with 5 column volumes of Solution C.

[0072] Elution: The mobile phase ratio is 0-50% D solution, the elution volume is 10 column volumes, and the elution time is 20 minutes.

[0073] HPLC detection of asparaginase PEG conjugate samples

[0074] The analysis was performed using a Waters HPLC BEH200 (4.6×300 mm) analytical column. The buffer was 0.02 M phosphate buffer (pH 6.0) containing 0.1 M sodium sulfate. After fully equilibrated with the sample, the column was eluted at a flow rate of 0.3 mL / min. The detection wavelength was 280 nm, and the detection time for one sample was 15 min. The results are shown in Figure 2. Figure 1 shown.

[0075] Depend on Figure 1 As can be seen, the prepared conjugates contain no significant impurities and have a purity greater than 98%. Furthermore, compared to the original drug Oncaspar (launched in the US by Enzon in 1994 and transferred to Sigma Tau Pharmaceuticals in Italy in 2009, the Oncaspar used in this patent is from Sigma Tau Pharmaceuticals), the size of SPA5K-ASP is essentially the same as Oncaspar. However, compared to the generic drug pegaspargase (purchased from Jiangsu Hengrui Medicine Co., Ltd.), the modified product SPA5K-ASP prepared in this example exhibits a peak to the left of the pegaspargase peak, indicating a higher molecular weight than pegaspargase. Since pegaspargase is also modified with 5K PEG, this indicates that the number of PEG moieties conjugated to SPA5K-ASP is greater than that to pegaspargase. Therefore, the degree of modification of SPA5K-ASP and the original drug Oncaspar is higher than that of the domestically marketed pegaspargase. Calculations indicate that the total yield of the product in this preparation is 80%.

[0076] The specific parameters and yields of Preparation Examples 2-7 are shown in the following table. The steps and parameters not listed in Table 1 are the same as those in Preparation Example 1 (the ion exchange chromatography columns used were purchased from GE, and the modifiers used were purchased from Beijing Jiankai Technology Co., Ltd.):

[0077] Table 1

[0078]

[0079] Experiments have shown that the modified products prepared in Preparation Examples 2-7 bind PEG to ASP more firmly than similar products on the market and are less likely to fall off. The PEG-ASP conjugates used in the following examples are all the conjugates obtained in Preparation Example 1.

[0080] Example 2: Comparison of PEGylated ASP Modification Uniformity

[0081] We compared the homogeneity of PEGylated ASP by SDS-PAGE electrophoresis. The protein stacking gel was a 5% gel and the separating gel was an 8% gel. The stacking gel buffer was 0.5 M Tris-HCl buffer (pH 6.8); the separating gel buffer was 1.5 mol / L Tris-HCl buffer (pH 8.8). A 10 μg protein sample was mixed with an equal volume of sample buffer and boiled at 100°C for 5 minutes before loading and running. After electrophoresis, the sample was stained with Coomassie Brilliant Blue R250 (purchased from Sinopharm Group).

[0082] Depend on Figure 2 As can be seen, compared to similar products on the market—pegaspargase (purchased from Jiangsu Hengrui Medicine Co., Ltd.) and Oncaspar (purchased from Sigma Tau Pharmaceuticals), our modified products SPA5K-ASP and Oncaspar exhibit relatively narrow electrophoretic bands, indicating that the modification uniformity of SPA5K-ASP and the original drug is slightly better than that of pegaspargase. However, it is not completely uniform. Although the four subunits of asparaginase are identical and the modifiable sites on each subunit are the same, due to steric hindrance, during the modification process, the sites already coupled to PEG molecules will shield adjacent modifiable sites, preventing modification by other PEG molecules. Furthermore, due to slight differences in the spatial arrangement of the four subunits, the reactivity of the same modifiable sites on different subunits varies. Taking these two factors into consideration, it is essentially impossible to achieve the same number of PEG molecules coupled to each subunit when modifying asparaginase using random modification methods. Figure 2 In the electrophoresis band of SPA5K-ASP, the electrophoresis band is significantly higher than that of pegaspargase, which also indicates that the molecular weight of SPA5K-ASP is significantly higher than that of pegaspargase, which is consistent with the results in Example 1. Since pegaspargase is also modified with PEG with a molecular weight of 5K, the modification degree of SPA5K-ASP in this example should be higher than that of pegaspargase, but basically consistent with Oncaspar.

[0083] Example 3: Determination of the number of PEGylated ASP modification sites

[0084] Accurately measure 40, 80, 120, 200, 300, and 400 μl of the 1 mg / mL asparaginase (ASP) solution purified in the preparation example of Example 1 and place them in test tubes respectively. Add 0.02 mol / L PB solution at pH 8.0 to 1 mL, then add 1 mL of pH 9.16 NaHCO3-Na2CO3 buffer, and then add 1 mL of 0.1% TNBS solution, and shake vigorously on a mixing shaker. Separately take 1 mg / mL Oncaspar solution, the PEG-ASP conjugate of the present application (Preparation Example 1), and pegaspargase and operate as above. The above samples were reacted at 40° C. for 1.5 hours, and then 1 mL of 10% SDS solution was added respectively, and then 0.5 mL of 1M HCl was added respectively. The OD value was measured at 424 nm and a standard curve was drawn. The Oncaspar sample, SPA5k-ASP, and Hengrui pegaspargase samples were analyzed respectively.

[0085] Among them, the calculation of the average modification rate of PEGylation is:

[0086]

[0087] Calculation of the average number of coupled PEGs: average number of coupled PEGs = average degree of PEG modification × modifiable sites (when modified with PEG with a molecular weight of 5000 Da, there are 33 modifiable sites; when modified with PEG with a molecular weight of 2000 Da, there are 55 modifiable sites; when modified with PEG with a molecular weight of 10000 Da, there are 20 modifiable sites). The calculation results are shown in Table 2.

[0088] Table 2 Average degree of modification and number of coupled PEGs

[0089] sample Average modification Number of coupled PEG Oncaspar 65.6％ 21.64 SPA5K-ASP 65％ 21.45 Pegaspargase 57.4％ 18.942

[0090] Table 2 shows that the number of PEG molecules conjugated to SPA5K-ASP was higher than that to the pegaspargase sample. On average, 21.45 PEG molecules were conjugated to each surface of the asparaginase, 2.5 more than the pegaspargase. This result is consistent with the results in Examples 1 and 2, further confirming that the molecular weight of SPA5K-ASP is greater than that of pegaspargase. The degree of modification of SPA5K-ASP and Oncaspar was essentially the same, which is also consistent with the results in Examples 1 and 2.

[0091] The number of PEG coupled in Preparation Examples 2-7 was 13.1, 45.2, 8.5, 20.3, 22.5, and 21.6, respectively. The number of PEG coupled decreased with increasing PEG molecular weight. PEG with the same molecular weight but different activated groups had slightly different numbers of PEG coupled.

[0092] Example 4: Stability Study of the Conjugated Group of PEG-ASP Conjugates

[0093] Pegaspargase, Oncaspar, and SPA5K-ASP were diluted to 1 mg / mL in Tris-HCl (9.0) buffer. Placed in a 55°C water bath, 100 μl of each aliquot was removed at 0, 1, 2, 3, 4, and 5 hours for protein electrophoresis analysis, iodine staining, and enzyme activity assay.

[0094] Depend on Figure 3a As can be seen, compared to the band in lane 2, the band in lane 3 has significantly migrated downward, indicating a decrease in the sample's molecular weight and, consequently, degradation within one hour of incubation at 55°C. The bands in lanes 4-7 continue to migrate downward, indicating continued sample degradation. In lanes 8-13, however, the protein bands show no significant migration, indicating that the SPA5K-ASP sample did not degrade within 5 hours under these water bath conditions. Lanes 14-19 show that, similar to Oncaspar, the pegaspargase sample also exhibited significant degradation within 5 hours, and the degree of degradation was greater than that of Oncaspar.

[0095] Since PEG can form complexes with iodine ions, PEG molecules can be detected by iodine staining. Figure 3b As can be seen from the iodine staining, Figure 3a The results were similar. Oncaspar and pegaspargase samples showed significant degradation within 5 hours, with bands migrating downward and becoming lighter in color, indicating continuous PEG shedding. The darker bands at the bottom of lanes 3-8 and 15-20 are PEG bands, which gradually darken in color, indicating that the number of PEG molecules dissociated from pegaspargase and oncaspar increases with prolonged water bath treatment. However, the SPA5K-ASP sample in lanes 9-14 showed no band degradation, indicating no PEG molecule dissociation.

[0096] In addition to electrophoresis analysis, the samples were also tested for enzyme activity (referring to the method described in the 2005 edition of the Pharmacopoeia, Part II, page 31). The results are shown in Figure 3c As can be seen from the figure, after 1 hour of water bath treatment, the activity of pegaspargase was almost completely lost, and the activity was basically zero. The enzyme activity of the Oncaspar sample was slightly better than that of pegaspargase, and it retained about 30% of its activity after 1 hour of treatment. The SPA5K-ASP sample retained about 70% of its activity after 1 hour of treatment, and still maintained about 30% of its biological activity after 5 hours of treatment. Its stability was significantly better than that of pegaspargase and Oncaspar. Although Figure 3aIn Figure b, the SPA5K-ASP sample did not degrade, but the 55°C heat treatment likely disrupted the tertiary structure of asparaginase, leading to reduced activity. Analysis of the significant decreases in the activity of pegaspargase and Oncaspar suggests that the PEG molecules are susceptible to shedding. This continuous shedding of PEG molecules significantly reduces their protective effect on asparaginase, resulting in significant activity loss. However, the SPA5K-ASP sample did not experience PEG shedding during treatment, suggesting that the PEG molecules significantly enhance the thermal stability of asparaginase, leading to a relatively slow loss of activity.

[0097] The products in Preparation Examples 2-7 were tested for stability and showed no PEG shedding.

[0098] Example 5: Fluorescence spectrum analysis of PEG-ASP conjugate and original protein

[0099] Intrinsic fluorescence detection of modified and unmodified proteins was performed at an excitation wavelength of 280 nm and an emission wavelength range of 300–400 nm. The scan rate was 1200 nm / min. Slit widths for both excitation and emission were 5 nm, and a 0.1 cm sample cell was used. Detection was performed at room temperature. The concentration range of the tested proteins was 0.1–0.2 mg / mL.

[0100] The effect of PEG modification on the tertiary structure of asparaginase was detected by intrinsic fluorescence. Figure 4 As shown, when excited at a wavelength of 280 nm, the fluorescence emission peak of asparaginase and its modified products is at 315 nm. The fluorescence spectra of the SPA5K-ASP sample, asparaginase, and the commercially available products pegaspargase and Oncaspar are essentially identical, with a characteristic absorption peak at 315 nm. This indicates that PEG modification of asparaginase does not affect its tertiary structure. The sample absorbance values ​​vary somewhat, which may be related to differences in protein concentration.

[0101] Example 6: Comparison of the inhibitory effects of asparaginase-PEG conjugates on different tumor cells

[0102] In order to evaluate the inhibitory rate of PEG-ASP conjugate on tumor cells and compare it with pegaspargase and Oncaspar, we selected THP-1 (human monocytic leukemia cell line), Raji (human lymphoma cell line), and L1210 (mouse leukemia cell line) for evaluation. The inhibition rate of cells was detected by MTT assay, and the inhibition rate of different drug concentrations was investigated, and finally the IC 50 The calculation results are shown in Table 3.

[0103] Table 3 IC values ​​of SPA5K-PEG-ASP conjugate and pegaspargase on tumor cells 50 value

[0104] tumor cells Oncaspar SPA5K-ASP Pegaspargase THP-1 7.5 μmol / L 3.05 μmol / L 7.9 μmol / L Raji 8.1 μmol / L 2.4 μmol / L 8.8 μmol / L L1210 10.69 μmol / L 7.305 μmol / L 10.28 μmol / L

[0105] According to the experimental results, the PEG-modified asparaginase of the present invention showed good anti-tumor effects on the above three types of tumor cells, and the anti-tumor activity of the PEG-modified asparaginase of the present invention was significantly higher than that of pegaspargase and Oncaspar.

[0106] Example 7: Pharmacokinetic study of PEG-ASP conjugates

[0107] We compared the pharmacokinetic characteristics of SPA5K-ASP prepared in the present invention and the similar product pegaspargase on the market.

[0108] We investigated the pharmacokinetics of intravenous and intramuscular injections. 125 I-labeled. The labeled test sample was purified and purity verified by SHPLC. The labeled samples were assayed for protein concentration using a BCA protein assay kit. The samples were then mixed with a specific amount of unlabeled sample and diluted to a 1.175 mg / mL injection solution using 1× PBS buffer (10× vehicle diluted to 1× with water for injection). The prepared drug (approximately 5 μL) was then sampled and radioactivity was determined. Specific activity = radioactivity / protein concentration. Samples were collected at specific time points after administration. If the blood drug concentration at the last blood draw time point in each intravenous administration group was not less than 1 / 20 of the blood drug concentration at the 2-minute time point, blood draws were continued once daily until the blood drug concentration was less than 1 / 20 of the blood drug concentration at the 2-minute time point. If the blood drug concentration at the last blood draw time point in each intramuscular administration group was not less than 1 / 10 of the peak blood drug concentration, blood draws were continued once daily until the blood drug concentration was less than 1 / 10 of the peak blood drug concentration. After blood collection, immediately place the blood sample in an EP tube anticoagulated with sodium heparin (1000 IU / mL, 10 μL), invert 5-10 times, and centrifuge at 4000 rpm for 5 minutes to separate the plasma. Take 50 μL of plasma, add an equal volume of 20% trichloroacetic acid (TCA), vortex to mix, and measure the total radioactivity. Then, centrifuge at 4500 rpm for 10 minutes at room temperature. Discard the supernatant, and measure the radioactivity of the precipitate.

[0109] Calculation of metabolic kinetic parameters: WinNonlin6.2 was used to perform non-compartmental model (NCA) fitting and calculation of major metabolic kinetic parameters such as AUC;

[0110] 2) Concentration calculation:

[0111]

[0112] 3) Data processing: Excel 2007 was used to perform statistical description of the mean, standard deviation and other data.

[0113] The calculation results of various pharmacokinetic parameters are shown in Table 3.

[0114] from Figure 5a As can be seen from the pharmacokinetic curves of Figure 2a, the pharmacokinetic curves of pegaspargase and SPA5K-ASP samples after intravenous injection (iv) and intramuscular injection (im) are basically the same.

[0115] After intravenous administration of the same dose, systemic exposure to SPA5K-ASP was slightly greater than that to pegaspargase; after intramuscular administration of the same dose, systemic exposure to SPA5K-ASP was slightly greater than that to pegaspargase. After dose normalization, systemic exposure to SPA5K-ASP was slightly greater than that to pegaspargase after both intravenous and intramuscular administration. Specific pharmacokinetic parameters are shown in Table 4 below.

[0116] Table 4 Comparison of pharmacokinetic parameters of PEG-ASP conjugate and asparaginase

[0117]

[0118] The half-life of SPA5K-ASP after intravenous and intramuscular administration was 55.8 hours and 55.9 hours, respectively. The half-life of pegaspargase after intravenous and intramuscular administration was 58.00 hours and 46 hours, respectively. The intramuscular half-life of pegaspargase was slightly lower than that of SPA5K-ASP. The AUC of SPA5K-ASP samples was slightly greater than that of pegaspargase. Furthermore, the plasma clearance of these two samples was essentially the same.

[0119] Comprehensive analysis shows that since both samples were randomly modified and used the same PEG molecular weight, their pharmacokinetic properties were not significantly different. However, due to the PEG shedding phenomenon of pegaspargase, its in vitro stability was weaker than that of the SPA5K-ASP sample.

Claims

1. A PEGylated asparaginase, characterized in that On average, one molecule of asparaginase is coupled with 21.45 molecules of polyethylene glycol, wherein the polyethylene glycol is a linear polyethylene glycol with an average molecular weight of 5 kDa, and the polyethylene glycol is coupled to the asparaginase by forming an amide bond or a urethane bond with the free amino group of the asparaginase; Its general structural formula is shown below: Wherein n=2, m=21.45, indicating that an average of one molecule of asparaginase is coupled with 21.45 molecules of polyethylene glycol; ASP is L-asparaginase; the PEGylated asparaginase is prepared by the following method: Step 1: Buffer Exchange Asparaginase API was pipetted onto a Q ion exchange column. After loading, the column was equilibrated with equilibration buffer A for 5 column volumes and then eluted in one step with elution buffer B, collecting the elution peak. Buffer A consisted of 20 mM phosphate buffer, pH 8.0; and Buffer B consisted of 20 mM phosphate buffer containing 0.2 M sodium chloride, pH 7.

5. Step 2: Modification reaction and purification of modified products The asparaginase solution collected from the elution peak in the first step was reacted at a molar ratio of asparaginase to PEG modifier of 1:50 at 4°C for 12 h. The asparaginase protein concentration in the PEG modification reaction was 5 mg / mL. After the reaction, the product was purified by ion exchange chromatography. The chromatographic conditions for purification were as follows: Q ion exchange column, equilibration buffer C: 20 mM Tris-HCl, pH 9.0, elution buffer D: 20 mM phosphate buffer containing 0.1 M NaCl, pH 8.0, flow rate 2.5 mL / min, detection wavelength 280 nm; Sample loading: The above modification reaction product was adjusted to pH 9.0 with 0.5 M NaOH solution and bound to a Q ion exchange column; Equilibration: flush 5 column volumes with solution C; Elution: The mobile phase ratio is 0-50% D solution, the elution volume is 10 column volumes, and the elution time is 20 minutes; The general structural formula of the PEG modifier is as follows: Where n is 2 and the molecular weight of mPEG is 5000 Da.

2. A pharmaceutically acceptable salt of the PEGylated asparaginase according to claim 1.

3. A pharmaceutical composition, characterized in that Contains the PEGylated asparaginase according to claim 1 and pharmaceutically acceptable excipients.

4. Use of the PEGylated asparaginase according to claim 1 or the pharmaceutical composition according to claim 3 in the preparation of a medicament for treating chronic lymphocytic leukemia in children or adults.

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