Pegylated phenylalanine lyase injection and application

Abstract

The invention provides a preparation combination formula of pegylated phenylalanine lyase. According to the method, the physical change and chemical change of the pegylated phenylalanine lyase caused by the influence of salt, oscillation, pH and temperature are solved, particularly the technical problems of high temperature, oscillation and freeze-thaw stability can be solved, and the stability and activity effect of the pegylated phenylalanine lyase protein polymer can be kept for a long time.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme preparations, and particularly to a polyethylene glycolylated phenylalanine lyase injection and its application. Background Art

[0002] Human phenylketonuria (PKU) is an amino acid metabolic disorder. Due to the enzyme defect in the phenylalanine (PA) metabolic pathway, phenylalanine cannot be converted into tyrosine, resulting in the accumulation of phenylalanine and its keto acids, which are excreted in large amounts in urine. High levels of phenylalanine and its breakdown products can cause significant medical problems, including intellectual disability, microcephaly, and seizures.

[0003] L-Phenylalanine ammonia-lyase (PAL, EC4.3.1.5) is an enzyme that catalyzes the hydrolysis of L-phenylalanine to produce trans-cinnamic acid and ammonia. It is widely distributed in plants and has also been identified in fungi and a limited number of bacteria. Since PAL is an exogenous protein with strong immunogenicity, progressive immune reactions and systemic allergic reactions are commonly seen clinically, which limits its clinical application. Polyethylene glycol modification of PAL can significantly reduce antibody levels and improve drug efficacy. Currently, the product of phenylalanine ammonia-lyase modified with polyethylene glycol by BioMarin Pharmaceutical Company was approved by the FDA in May 2018 for PKU adult patients with blood phenylalanine concentration exceeding 600 μmol / L under existing management to reduce blood phenylalanine concentration, but there are still significant problems of high immunogenicity and low stability.

[0004] The inventors of the present invention further modified phenylalanine lyase. By mutating amino acids at specific sites and subjecting them to polyethylene glycol modification, its immunogenicity was improved; there are still problems with the stability of the polyethylene glycolylated phenylalanine lyase mutant, and key factors affecting drug efficacy and quality such as depolymerization, formation of high polymers, and loss of enzyme activity are likely to occur. Through research on different formulation prescriptions and stability investigations, the present invention discovered a drug combination that can improve the stability of polyethylene glycolylated phenylalanine ammonia-lyase. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the present invention provides a polyethylene glycolylated phenylalanine lyase injection and its application. It solves the problems of high immunogenicity and low stability existing in the prior art.

[0006] In the first aspect of the present invention, there is provided a polyethylene glycolylated phenylalanine lyase injection, which comprises a polyethylene glycol-modified phenylalanine lyase mutant, a pharmaceutical excipient, and water;

[0007] (1) The amino acid residues at positions 72, 240, 467, and 544 of the phenylalanine lyase mutant are replaced by lysine, and the amino acid sites modified by polyethylene glycol include one of the following sites: K72, K240, K467, and K544.

[0008] Further, the mutation sites are located based on the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80% homology thereto.

[0009] (2) The pharmaceutical excipients include a protein stabilizer, a protein protectant, and an inorganic salt buffer, wherein the protein stabilizer includes one or two of L-phenylalanine and trans-cinnamic acid;

[0010] The protein protectant includes one or several of trehalose, sucrose, and mannitol;

[0011] The inorganic salt buffer includes one of phosphate buffer and Tris-Hcl.

[0012] Compared with the phenylalanine lyase from natural sources, the SEQ ID NO:1 sequence contains 4 amino acid mutation sites, namely K72, K240, K467, and K544, which can significantly reduce the immunogenicity of phenylalanine lyase. Modification of phenylalanine lyase with polyethylene glycol can further improve the immunogenicity of PAL. In some embodiments, the polyethylene glycolylated phenylalanine lyase with the amino acid sequence of SEQ ID NO:1 has better immunogenicity than the polyethylene glycolylated phenylalanine lyase prepared under the same conditions without the mutation sites of K72, K240, K467, and K544 when administered subcutaneously to rats multiple times at a dose of 2 mg / kg once a week.

[0013] In some embodiments, the molecular weight of the polyethylene glycol is 2 - 40 KDa, preferably 2 - 10 KDa;

[0014] Optionally, the polyethylene glycol is a linear polyethylene glycol or a branched polyethylene glycol;

[0015] Optionally, the polyethylene glycol has a monomethoxy group or a hydroxyl group;

[0016] Optionally, the polyethylene glycol is a polyethylene glycol with a reactive group;

[0017] Optionally, the polyethylene glycol is coupled to the lysine of the phenylalanine lyase through an amide bond;

[0018] Optionally, the reactive group of the polyethylene glycol includes at least one of succinimidyl butyrate, succinimidyl propionate, succinimidyl acetate, succinimidyl carbonate, and nitrobenzene carbonate, and preferably succinimidyl propionate.

[0019] In some embodiments, the molar ratio of the polyethylene glycol to the phenylalanine lyase is (6-20):1. The active form of the phenylalanine lyase is a tetramer. Here, it refers to the phenylalanine lyase subunit, that is, one phenylalanine lyase subunit is covalently bound to 6-20 polyethylene glycols.

[0020] In some embodiments, the concentration of the polyethylene glycol-modified phenylalanine lyase is 5-20 mg / ml.

[0021] In some embodiments, the inorganic salt buffer includes phosphate or Tris-Hcl with an ion concentration of 10-50 mmol / L, preferably 25 mM phosphate.

[0022] In some embodiments, the pH value of the polyethylene glycol-modified phenylalanine lyase injection is 6.5-7.5, preferably pH 7.0±0.3.

[0023] In some embodiments, the content of the protein stabilizer is 0.5-3 mM, preferably 1 mM.

[0024] In some embodiments, the mass percentage content of the protein protectant is 3%-8%, preferably, the mass fraction is 6.5%.

[0025] Preferably, the injection formulation includes: 10 mg / ml polyethylene glycol-modified phenylalanine lyase, 1 mM phenylalanine, 6.5% sucrose, 25 mM PB (pH 7.0±0.3), and the rest is water;

[0026] Preferably, the injection formulation is as follows: 10 mg / ml polyethylene glycol-modified phenylalanine lyase, 1 mM phenylalanine, 3%-4% mannitol, 20 mM PB (pH 7.0±0.3), and the rest is water;

[0027] Preferably, the injection formulation is as follows: 10 mg / ml polyethylene glycol-modified phenylalanine lyase, 1 mM phenylalanine, 7%-8% trehalose, 20 mM PB (pH 7.0±0.3), and the rest is water.

[0028] In the second aspect of the present invention, a preparation method of a polyethylene glycol-modified phenylalanine lyase injection is provided, including the following steps:

[0029] Mix the polyethylene glycol-modified phenylalanine lyase, the protein stabilizer, the protein protectant, and the osmotic pressure regulator, adjust the pH, and filter to obtain the product.

[0030] In the third aspect of the present invention, there is provided the use of the pegylated phenylalanine lyase injection in the preparation of a drug for preventing and / or treating phenylalanine-related diseases, including but not limited to at least one of phenylketonuria and hyperphenylalaninemia.

[0031] The technical principle of the present invention is as follows:

[0032] In the present invention, phenylalanine lyase is modified. The amino acids at positions 72, 240, 467, and 544 in the immunodominant region of phenylalanine lyase (SEQ ID NO: 1) are mutated to lysine, and polyethylene glycol modification is carried out, which can significantly improve the immunogenicity of PAL. However, the stability of the pegylated phenylalanine lyase has problems, and key factors affecting drug efficacy and quality such as depolymerization, formation of high polymers, and loss of enzyme activity are prone to occur. Through research on different formulation prescriptions and stability investigation, the present invention has discovered a drug combination that can improve the stability of pegylated phenylalanine lyase.

[0033] The present invention has the following technical effects:

[0034] The object of the present invention is to provide a formulation prescription composition that can maintain the stability and activity of the protein aggregate for a long time and is more suitable for clinical application by screening more and a wider range of the formulation of pegylated phenylalanine lyase. The pegylated phenylalanine lyase composition provided by the present invention effectively avoids physical and chemical changes of pegylated phenylalanine lyase caused by the effects of salt, shaking, pH, and temperature, and can especially solve the technical problems of high temperature, shaking, and freeze-thaw stability.

[0035] The pegylated phenylalanine lyase composition provided by the present invention effectively avoids physical and chemical changes of pegylated phenylalanine lyase caused by the effects of salt, shaking, pH, and temperature, and can especially solve the technical problems of high temperature, shaking, and freeze-thaw stability. Description of the Drawings

[0036] Figure 1 LC-MS diagrams of pegylated phenylalanine lyase before and after modification by enzymatic digestion;

[0037] Figure 2 SEC-HPLC comparison diagrams before and after freeze-thaw of Prescription 1 in Example 4 of the present invention;

[0038] Figure 3 SEC-HPLC comparison diagrams before and after freeze-thaw of Prescription 2 in Example 4 of the present invention;

[0039] Figure 4 SEC-HPLC comparison diagrams before and after freeze-thaw of Prescription 3 in Example 4 of the present invention;

[0040] Figure 5 This is the SEC-HPLC comparison chart before and after freeze-thawing of Prescription 4 in Example 4 of the present invention;

[0041] Figure 6 This is the SEC-HPLC comparison chart before and after freeze-thawing of Prescription 5 in Example 4 of the present invention; Detailed implementation manners

[0042] The technical solutions in the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0043] The detection indexes and methods in the embodiments of the present invention are as follows:

[0044] The detection method of the average modification degree is to utilize the linear relationship between the concentration of protein (phenylalanine lyase) in the solution and its differential refractive index (RI) and ultraviolet (UV) absorption. Polyethylene glycol has no UV absorption, but its concentration has a linear relationship with the differential refractive index (RI) absorption value, and the differential refractive index and UV absorption of protein and PEG do not affect each other. Through the combination of SEC-HPLC differential-ultraviolet detectors, a standard curve of the content of calibrated protein and PEG content-absorbance peak area can be established, the contents of protein and polyethylene glycol in the test sample can be measured, and the molar content ratio of protein and polyethylene glycol molecules can be calculated by combining the relative molecular weights of protein and polyethylene glycol, which is used as the characterization of the average modification degree.

[0045] Enzyme activity assay method: Phenylalanine lyase can specifically catalyze the conversion of phenylalanine into trans-cinnamic acid and release ammonia. Trans-cinnamic acid has the maximum light absorption at a wavelength of 290 nm. The enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol of trans-cinnamic acid per minute at room temperature (25 °C) under the condition of 100 mmol / L Tris-HCl (pH 8.5) buffer solution.

[0046] High and low molecular weight detection method: Use a TSKgel 5000pwxl 7.8×300mm 10μm chromatographic column, mobile phase: 0.15mol / L Na 2 HPO 4 -NaH 2 PO 4 , 0.5mol / L NaCl, and the pH value is 7.0; analyze under the chromatographic conditions of a wavelength of 280nm.

[0047] Preparation of polyethylene glycolylated phenylalanine lyase in Example 1

[0048] The gene sequence with the amino acid sequence of SEQ ID NO:1 was synthesized by total gene synthesis, and the expression plasmid pET-30a-PAL4 was constructed by restriction endonuclease digestion and ligation with T4 DNA ligase. The expression plasmid was transformed into Escherichia coli, and recombinant expression engineering bacteria were obtained by resistance screening. After fermentation culture in YT medium, IPTG was used to induce expression, and the cells were collected by centrifugation.

[0049] After the cells obtained by fermentation were lysed by physical or enzymatic methods, the insoluble lysis fragments were removed by centrifugation. The pH of the supernatant was adjusted and (NH 4 ) 2 SO 4 was added for fractional precipitation to enrich phenylalanine lyase; the phenylalanine lyase precipitate was obtained by centrifugation again. After the precipitate was dissolved, the host protein impurities in the expression were purified by hydrophobic chromatography, and the eluate was purified by ion exchange chromatography to remove high- and low-molecular-related proteins. The purity of the recombinant phenylalanine lyase mutant (PAL4) was evaluated by SDS-PAGE and SEC-HPLC, and was not less than 95%; the specific activity of the enzyme was determined by the substrate method, and the specific activity of the recombinant phenylalanine lyase mutant (PAL4) was not less than 3.0 U / mg.

[0050] Take the protein solution of the phenylalanine lyase mutant (amino acid sequence as shown in SEQ ID NO:1) prepared above, adjust it to a buffer ionic strength between 10 and 200 mM, a modified pH range of 7.5 to 10.5, and a protein concentration of 5 to 20 mg / ml in any one of the phosphate or carbonate buffer systems. Add the modifier 5K-SPA-PEG (polyethylene glycol succinimidyl propionate with a molecular weight of 5000 Da) to the protein solution at a mass ratio (modifier: protein) of 9:1 to 15:1, and stir and react at room temperature for 1 to 2 h. Ultrafilter through an ultrafiltration column 4 to 8 times to remove free PEG and other by-products in the reaction solution, and concentrate the sample to 6 to 10 mg / ml to obtain polyethylene glycol succinimidyl propionate phenylalanine lyase stock solution (abbreviated as polyethylene glycolated phenylalanine lyase, PL54). After detection, the specific activity of the polyethylene glycolated phenylalanine lyase (PL54) was 2.31 U / mg, and the average modification degree was 13.5.

[0051] Polyethylene glycols with other different activating groups (such as: succinimidyl butyrate, succinimidyl propionate, succinimidyl acetate, and succinimidyl carbonate) were used to prepare polyethylene glycolated phenylalanine lyase according to the above steps.

[0052] Example 2 Identification of PEG Modification Sites of Polyethylene Glycolated Phenylalanine Lyase

[0053] PEGylated phenylalanine lyase is formed by covalently binding PEG to the amino side chain of lysine residues in the amino acid sequence or the amino group of the N-terminal amino acid; trypsin and lysyl endopeptidase cannot recognize lysine modified by PEG. Therefore, the modification sites can be inferred by comparing the peptides before and after modification. In this invention, Trypsin and Lys-C were used to digest the PEGylated phenylalanine lyase before and after PEG covalent coupling in Example 1, and the modification sites of the PEGylated phenylalanine lyase mutant were analyzed by LC-MS. See Figure 1 .

[0054] The results showed that for the PEGylated phenylalanine lyase obtained by PEG modification and coupling of the phenylalanine lyase protein in Example 1, after lysine digestion, the peptides containing the sites of K72 (T6 peptide segment), K240 (T20 peptide segment), K467 (T36 peptide segment) and K544 (T44 peptide segment) or adjacent peptide segments disappeared or the responses were significantly reduced, indicating that 4 lysines were modified by PEG.

[0055] Other PEGs with different activating groups (such as: succinimidyl butyrate, succinimidyl propionate, succinimidyl acetate and succinimidyl carbonate) were all detected by a similar method, and it was found that 4 lysines were partially modified by PEG.

[0056] Preparation of PEGylated phenylalanine lyase injection with different formulations in Example 3

[0057] The stock solution of PEGylated phenylalanine lyase prepared in Example 1 was formulated into a PEGylated phenylalanine lyase injection (referred to as injection) according to the formulations shown in Table 1.

[0058] 1. Add the prescribed amount of the stock solution of PEGylated phenylalanine lyase into the preparation container;

[0059] 2. Add the prescribed amounts of phenylalanine, protective agent and / or osmotic pressure regulator, stir and mix evenly, and adjust the pH to 6.5 - 7.5 with 1 mol / L phosphate (PB buffer);

[0060] 3. Make up the volume to the final volume of the batch preparation with a buffer;

[0061] 4. Filter with two - stage 0.2 μm filters,

[0062] 5. Fill the solution obtained in step 4 according to the filling operation procedure, cap and label to obtain the PEGylated phenylalanine lyase injection with different formulations.

[0063] Table 1 Prescription design table of PEGylated phenylalanine lyase injection

[0064] Prescription Group Prescription Composition Prescription 1 10 mg / ml Active Ingredient, 25 mM PB, pH 7.0 Prescription 2 10 mg / ml Active Ingredient, 150 mM PB, 1 mM L-Phenylalanine, pH 7.0 Prescription 3 10 mg / ml Active Ingredient, 25 mM PB, 1 mM L-Phenylalanine, 100 mM NaCl, pH 7.0 Prescription 4 10 mg / ml Active Ingredient, 25 mM PB, 1 mM L-Phenylalanine, 6.5% Sucrose, pH 7.0 Prescription 5 10 mg / ml Active Ingredient, 25 mM PB, 1 mM L-Phenylalanine, 3.25% Sucrose, pH 7.0 Prescription 6 10 mg / ml Active Ingredient, 25 mM PB, 1 mM L-Phenylalanine, 1.5% Sucrose, pH 7.0 Prescription 7 10 mg / ml Active Ingredient, 25 mM PB, 1 mM L-Phenylalanine, 7.5% Trehalose, pH 7.0 Prescription 8 10 mg / ml Active Ingredient, 25 mM PB, 1 mM L-Phenylalanine, 3.6% Mannitol, pH 7.0 Prescription 9 10 mg / ml Active Ingredient, 25 mM PB, 1 mM L-Phenylalanine, 6.5% Sucrose, pH 8.5 Prescription 10 10 mg / ml Active Ingredient, 25 mM PB, 1 mM L-Phenylalanine, 6.5% Sucrose, pH 7.5 Prescription 11 10 mg / ml Active Ingredient, 25 mM PB, 1 mM L-Phenylalanine, 6.5% Sucrose, pH 6.5 Prescription 12 10 mg / ml Active Ingredient, 25 mM PB, 1 mM L-Phenylalanine, 6.5% Sucrose, pH 4.0 Prescription 13 10 mg / ml Active Ingredient, 25 mM PB, 3 mM L-Phenylalanine, 6.5% Sucrose, pH 7.0 Prescription 14 10 mg / ml Active Ingredient, 25 mM PB, 0.25 mM L-Phenylalanine, 6.5% Sucrose, pH 7.0

[0065] Note: The main drug is polyethylene glycolated phenylalanine lyase

[0066] Example 4 Effect of Freeze-Thaw on Polyethylene Glycolated Phenylalanine Lyase Injection with Different Formulations

[0067] The inventor performed freeze-thaw on Formulations 1 to 14 prepared in Example 3, and then detected the enzyme activity and high and low molecular contents of polyethylene glycolated phenylalanine lyase injection with different formulations

[0068] Freeze-thaw method: After preparing polyethylene glycolated phenylalanine lyase injection with different formulations, perform repeated freeze-thaw (24 hours at -20°C, take out and thaw at 5°C for 1 cycle), cycle 3 times, and sample at the 1st and 3rd times to detect the specific enzyme activity (ultraviolet method) and high and low molecular contents (SEC-HPLC method) of the samples. The detection results are shown in Table 2

[0069] Table 2 Results of the Effect of Freeze-Thaw on Polyethylene Glycolated Phenylalanine Lyase Injection with Different Formulations

[0070]

[0071]

[0072] From Formulations 1 and 3 in Table 1 and Table 2, it can be seen that: the enzyme activity of Formulation 3 containing 100 mM sodium chloride has a significant change after freeze-thaw, and its low molecular protein increases significantly, indicating that the chloride ion concentration in the solution injection has an impact on its stability during repeated freeze-thaw (the freeze-thaw investigation results are similar after replacing 25 mM PB with 25 mM Tris-HCl pH 7.0 ± 0.3 in Formulation 3). And, compared with Formulation 3 containing sodium chloride and Formulation 1 without sodium chloride, it can be shown that the stability of the formulation without chloride ions is better

[0073] From Formulations 2, 4 to 14 in Table 1 and Table 2, it can be seen that: Formulation 2 does not contain chloride ions, and its enzyme activity has a significant change after freeze-thaw, and its low molecular protein increases significantly. There are no significant changes in the low and high molecular contents of Formulations 4, 7, and 8. And, compared with Formulation 2, the enzyme activity and high and low molecular protein contents of the formulations containing sucrose, trehalose, and mannitol are relatively stable after freeze-thaw, which is significantly better than the formulations without sucrose, trehalose, and mannitol. Among Formulations 4 to 6, the low molecular weight increases and the enzyme activity decreases after freeze-thaw in Formulation 6 with a sucrose content of 1.5%, and its stability is significantly worse than that of Formulations 4 and 5. Therefore, using a certain concentration of sucrose, trehalose, and mannitol has a protective effect on proteins

[0074] Example 5 Effect of High Temperature on the Specific Enzyme Activity of Polyethylene Glycolated Phenylalanine Lyase with Different Formulations

[0075] The prescriptions 1 to 14 prepared in Example 3 were examined at high temperature (37°C), and then the enzyme activity and the contents of high and low molecular weights of the pegylated phenylalanine lyase injection with different prescriptions were detected.

[0076] Table 3 Effects of high temperature on pegylated phenylalanine lyase with different preparation prescriptions

[0077]

[0078]

[0079] According to the data in Table 2 and Table 3, for Prescription 1 under high temperature conditions, the enzyme activity showed a downward trend and the high molecular weight increased; for Prescription 6 under high temperature conditions, the change in enzyme activity was not obvious, but the high molecular weight had an increasing trend; for Prescriptions 9 and 12 under high temperature conditions, the changes in high and low molecular weights were not obvious, and the enzyme activity had a decreasing trend. It is speculated that the pegylated phenylalanine lyase may have broken or denatured; for Prescription 14 under high temperature conditions, the enzyme activity showed a downward trend; for Prescriptions 4, 5, 7, 8, 10, 11, and 13 under high temperature conditions, the changes in enzyme activity and high and low molecular weights were not significant.

[0080] Osmotic pressure (mOsmol / Kg) of pegylated phenylalanine lyase injection with different prescriptions in Example 6

[0081] The osmotic molality of each prescription prepared in Example 3 was indirectly determined by measuring the freezing point depression of the solution, and the measurement results are shown in Table 4.

[0082] The osmotic molality range of normal human blood is 285 - 310 mOsmol / kg. The results show that the osmotic pressures of Prescriptions 2 - 4, 7 - 8, and 11 - 13 are within a reasonable range. Combining Examples 4 and 5, Prescriptions 4, 7 - 8, 11, and 13 have high freeze-thaw and high temperature stability, and at the same time, the osmotic pressure is within the isosmotic range of the human body.

[0083] Table 4 Results of the effects of different prescriptions on the osmotic pressure (mOsmol / Kg) of pegylated phenylalanine lyase injection

[0084] Prescription Group Osmotic Pressure (mOsmol / Kg) Prescription 1 63 Prescription 2 307 Prescription 3 298 Prescription 4 304 Prescription 5 196 Prescription 7 306 Prescription 8 304 Prescription 11 315 Prescription 12 335 Prescription 13 310

[0085] Example 7 Scale-up verification of the prescription of pegylated phenylalanine lyase injection

[0086] Based on the experimental results of Examples 4 - 6, Prescription 4 with 25 mM PB, 1 mM L - phenylalanine, 6.5% sucrose, and 10 mg / mL polyethylene glycolated phenylalanine lyase (pH 7.0 ± 0.3) was finally selected for pilot - scale production of three batches of samples (not less than 5000 vials per batch, 1 mL / vial) (for the specific preparation, see Example 2), and the samples were placed under the following conditions for stability investigation.

[0087] 1. Investigation conditions

[0088] The specific investigation contents are shown in Table 5.

[0089] Table 5 Stability investigation plan

[0090]

[0091] 2. Test results

[0092] The results of the influencing factor investigation of polyethylene glycolated phenylalanine lyase injection are shown in Table 6; the results of the accelerated investigation of polyethylene glycolated phenylalanine lyase injection are shown in Table 7.

[0093] Table 6 Results of the influencing factor investigation of polyethylene glycolated phenylalanine lyase injection

[0094]

[0095]

[0096] Table 7 Results of the accelerated stability investigation of polyethylene glycolated phenylalanine lyase injection

[0097]

[0098] From the data in Tables 6 and 7, it can be seen that for the three batches of injections prepared according to Prescription 4, there were no obvious changes in high - and low - molecular substances after 3 cycles of freeze - thawing, 3 times of out - of - cold - chain, 5 days of shaking, 30 days of light exposure investigation, and 15 days of high - temperature investigation. The titer was stable and there was no obvious decrease, indicating that Prescription 4 has good stability.

[0099] Example 8 Pah enu Pharmacodynamics study of repeated administration in mice

[0100] Phenylalanine hydroxylase (PAH) is the core of phenylalanine metabolism in animals, converting phenylalanine into tyrosine. Defects in this gene are the basis of phenylketonuria. By inducing missense mutations with ENU (reference: Lukas Villiger et al., Treatment of a metabolic liver disease by in vivo genome base editing in adult mice; missense mutation F263S), the catalytic activity of PAH in homozygous mice was severely reduced, and the accumulation of phenylalanine in the serum could not be metabolized, which was significantly higher than that in normal wild-type mice. It can be used as a mouse model for phenylketonuria in the study of this preparation (abbreviated as Pahenu mouse or model mouse).

[0101] Six Pahenu mice were selected and divided into 3 groups, with 2 mice in each group. The polyethylene glycolated phenylalanine lyase injection of Prescription 4 in Example 7 was used to administer to the phenylketonuria animal model mice at low, medium, and high doses. The low, medium, and high dose groups were administered 0.5 mg / kg, 1.5 mg / kg, and 4.5 mg / kg respectively, by subcutaneous injection once a week. Among them, blood samples were collected from each group of mice 1 h before administration, and 2, 5, and 7 days after administration, and the phenylalanine content in the serum was detected by LC-MS.

[0102] The results showed that after subcutaneous administration at medium and high doses (1.5 mg / kg and 4.5 mg / kg), the phenylalanine content in the serum could be effectively reduced. The decrease in phenylalanine content in the medium dose group at the first administration was about 40% compared with that before administration. The serum phenylalanine could be reduced below the method detection limit within 2 days after the first administration at the high dose. After the first administration, the drug effect could be maintained for 5 - 7 days. After repeated administration 5 times, the drug effect was maintained. The decrease in phenylalanine content in the serum of the medium dose group was about 75% within 2 days, and the decrease in phenylalanine content in the serum of the high dose group exceeded 90%.

[0103] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. PEGylated phenylalanine lyase injection, characterized in that it contains a PEGylated phenylalanine lyase mutant, pharmaceutical excipients and water; (1) The amino acid residues at positions 72, 240, 467 and 544 of the phenylalanine lyase mutant are replaced by lysine, and the amino acid sites modified by PEG include one of the following sites: K72, K240, K467 and K544. Furthermore, the mutation sites are located based on the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80% homology thereto. (2) The pharmaceutical excipients include a protein stabilizer, a protein protectant and an inorganic salt buffer, wherein the protein stabilizer includes one or two of L-phenylalanine and trans-cinnamic acid; the protein protectant includes one or several of trehalose, sucrose and mannitol; the inorganic salt buffer includes one of phosphate buffer and Tris-Hcl.

2. The PEGylated phenylalanine lyase injection according to claim 1, characterized in that the molecular weight of the PEG used for modification is 2-40 KDa, preferably 2-10 KDa; Optionally, the PEG is a linear PEG or a branched PEG; Optionally, the PEG has a monomethoxy group or a hydroxyl group; Optionally, the PEG is a PEG with a reactive group; Optionally, the PEG is coupled to the lysine of the phenylalanine lyase mutant through an amide bond; Optionally, the reactive group of the PEG includes at least one of succinimidyl butyrate, succinimidyl propionate, succinimidyl acetate, succinimidyl carbonate and nitrobenzene carbonate, preferably succinimidyl propionate.

3. The PEGylated phenylalanine lyase injection according to claim 1 or 2, characterized in that the molar ratio of PEG to phenylalanine lyase in the PEGylated phenylalanine lyase is (6-20):

1.

4. The PEGylated phenylalanine lyase injection according to claim 1, characterized in that the content of the PEGylated phenylalanine lyase mutant is 5-20 mg / ml.

5. The PEGylated phenylalanine lyase injection according to claim 1, characterized in that the inorganic salt buffer includes phosphate or Tris-Hcl with an ion concentration of 10-50 mmol / L, preferably 25 mM phosphate.

6. The PEGylated phenylalanine lyase injection according to claim 1, characterized in that its pH value is 6.5-7.5, preferably pH 7.0±0.

3.

7. The PEGylated phenylalanine lyase injection according to claim 1, characterized in that the content of the protein stabilizer is 0.5-3 mM, preferably 1 mM.

8. The PEGylated phenylalanine lyase injection according to claims 1 and 9, characterized in that the mass percentage content of the protein protectant is 3%-8%, preferably, the mass fraction is 6.5%.

9. The pegylated phenylalanine lyase injection according to claim 1, wherein, the injection formulation is as follows: 10 mg / ml pegylated phenylalanine lyase mutant, 1 mM phenylalanine, 6.5% sucrose, 25 mM PB (pH 7.0 ± 0.3), and the balance is water.

10. Use of the pegylated phenylalanine lyase injection according to any one of claims 1 to 9 in the preparation of a medicament for preventing and / or treating hyperphenylalanine-related diseases, including but not limited to at least one of phenylketonuria and hyperphenylalaninemia.

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