Pharmaceutical composition of protease drug and immunomodulator for treating phenylketonuria disease

Abstract

The invention belongs to the field of biological medicines, and relates to a pharmaceutical composition of a protease drug and an immunomodulator for treating phenylketonuria, which has the technical scheme that the protease drug and the immunomodulator for treating phenylketonuria are combined, so that the generation of an anti-protease drug antibody can be effectively reduced, and the curative effect is improved. The in-vivo action time of the protease medicine is prolonged. The invention has the beneficial effects that the combined drug can effectively reduce the immunogenicity of the protease drug, reduce the generation of in-vivo drug antibodies, prolong the action time of the drug, reduce the administration frequency of the protease drug, greatly improve the compliance of patients with phenylketonuria, and reduce the cost. Therefore, the combined medicine can be widely applied to treatment of phenylketonuria.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a drug combination characterized by a combination of a protease drug and an immunomodulator for treating phenylketonuria (PKU). This combination effectively reduces the immunogenicity of the protease drug, decreases the production of drug antibodies in the body, prolongs the duration of drug action, and reduces the frequency of protease drug administration, enabling once-weekly or longer administration. This significantly improves patient compliance with PKU and allows for the widespread application of this combination therapy in the treatment of PKU. Background Technology

[0002] Phenylketonuria (PKU), also known as phenylalanine hydroxylase deficiency, is a metabolic disorder of phenylalanine caused by a deficiency or reduced activity of phenylalanine hydroxylase in the liver. It is a recessive genetic disorder. Phenylalanine is an essential amino acid that participates in various physiological activities, but the human body cannot synthesize it and must obtain it from food. In PKU patients, the deficiency or reduced activity of phenylalanine hydroxylase (PAH) prevents the effective degradation of phenylalanine. This leads to an accumulation of phenylalanine in the plasma. When the plasma concentration of phenylalanine exceeds the renal threshold, it is excreted in the urine, resulting in phenylalanineuria. Furthermore, the accumulated phenylalanine is metabolized through other secondary metabolic pathways to produce substances such as phenylpyruvic acid and phenyllactic acid, which are toxic to the brain and other nervous systems.

[0003] Pegvaliase is the first drug to treat phenylketonuria using the phenylalanine ammonia-lyase (PAL) method. Developed by BioMarin Pharmaceuticals in the United States, Pegvaliase is a PEG-modified product of PAL derived from *Anabaena variegata* by mutating individual amino acids. In 2018, the FDA approved pegvaliase for the treatment of patients whose blood phenylalanine concentrations could not be controlled below 600 μmol·L⁻¹. -1 Adult patients with phenylketonuria within the specified age range.

[0004] Immunogenicity of therapeutic protein drugs refers to the ability of a therapeutic protein and / or its metabolites to induce an immune response or immune-related events against itself or related proteins. Immunogenicity often leads to undesirable immune reactions. During biotherapy, the formation of anti-drug antibodies (ADAs) can neutralize the activity of the biopharmaceutical or accelerate its clearance, alter its pharmacokinetics and biodistribution, cause hypersensitivity reactions, and even endanger life.

[0005] Pegvaliase is a recombinant protease drug used to treat phenylketonuria. Due to its heterologous protein, almost all patients rapidly develop drug-resistant antibodies (ADAs) upon using the drug, leading to various adverse reactions. Some patients even have to discontinue the medication due to hypersensitivity reactions. Because of the development of ADAs, patients need to inject large doses of the drug daily to maintain its efficacy, placing a significant financial burden and causing considerable inconvenience.

[0006] To address the impact of anti-drug antibodies (ADAs) on patient safety and drug efficacy in biotherapy, the combination of highly immunogenic drugs with immunomodulators can be considered in clinical practice to reduce the production of ADAs. In the treatment of rheumatoid arthritis patients, methotrexate is often combined with biologics such as etanercept, infliximab, and adalimumab to reduce ADA production and improve the efficacy of biologics. In 2022, the FDA approved methotrexate combined with pegloticase for the treatment of refractory gout, reducing ADA production and improving patient response rates. This patent provides a drug combination using a special PEGylated phenylalanine lyase mutant and an immunomodulatory agent, which can more effectively reduce the immunogenicity of protease drugs, reduce the production of drug antibodies in the body, prolong the duration of drug action, and reduce the frequency of protease drug administration, achieving once-weekly or longer dosing, greatly improving patient compliance in phenylketonuria (PKU) and enabling the widespread application of this combination therapy in the treatment of PKU. Summary of the Invention

[0007] This invention provides a combination of a protease drug and an immunomodulator for treating phenylketonuria (PKU). Utilizing a special PEGylated phenylalanine lyase mutant and an immunomodulator, this combination effectively reduces the immunogenicity of the protease drug, decreases the production of drug antibodies in the body, prolongs the duration of drug action, and reduces the frequency of protease drug administration, allowing for once-weekly or longer dosing. This significantly improves patient compliance with PKU, enabling the widespread application of this combination therapy in the treatment of PKU.

[0008] The present invention provides a combination of a protease drug and an immunomodulator for treating phenylketonuria, comprising: (1) a polyethylene glycol-modified phenylalanine lyase mutant, characterized in that amino acid residues at positions 72, 240, 467 and 544 of the phenylalanine lyase mutant are replaced with lysine, and one or more of the following amino acid sites in the phenylalanine lyase mutant are modified with polyethylene glycol: K72, K240, K467 and K544, namely lysine residues at positions 72, 240, 467 and 544 of the phenylalanine lyase mutant.

[0009] Furthermore, the mutation site is located using SEQ ID NO:1 or an amino acid sequence having at least 80% homology with it.

[0010] Compared to the naturally derived phenylalanine lyase (SEQ ID NO:2), the recombinant phenylalanine lyase mutant shown in SEQ ID NO:1 contains four amino acid mutation sites, namely K72, K240, K467 and K544, and one or more of these amino acids are covalently modified.

[0011] (2) Immunomodulators are drugs that suppress the immune system, including inhibitors of dihydrofolate reductase, such as methotrexate (MTX).

[0012] In some embodiments, the protease drug and immunomodulator in the drug combination can be prepared as a compound preparation for administration.

[0013] In other embodiments, the protease drug and the immunomodulator in the drug combination are administered separately.

[0014] The polyethylene glycolated phenylalanine lyase mutant provided by the present invention is characterized in that it is a chemical conjugate obtained by activating polyethylene glycol (PEG) and the lysine residues of the phenylalanine lyase mutant by means of an amide bond.

[0015] According to an embodiment of the present invention, the molar ratio of PEG to the phenylalanine lyase mutant is (12-15):1, that is, one molecule of phenylalanine lyase subunit binds to 12-15 polyethylene glycol molecules.

[0016] In other embodiments, the molar ratio of the PEG to the phenylalanine lyase mutant is (6-20):1, meaning that one molecule of phenylalanine lyase subunit binds to 6-20 polyethylene glycol molecules.

[0017] According to an embodiment of the present invention, the PEG is polyethylene glycol succinimide propionate with a molecular weight of 5 kDa.

[0018] In other embodiments, the activating group of the polyethylene glycol is succinimide butyrate, succinimide acetate, succinimide carbonate, or nitrobenzene carbonate, with a molecular weight of 2kDa to 20kDa.

[0019] In some embodiments, the immunomodulator in the drug combination is methotrexate, and pegylated phenylalanine lyase and methotrexate are administered separately. Methotrexate can be administered via various routes such as subcutaneous, oral, intravenous, and intramuscular, with subcutaneous and oral administration being preferred. Each administration is given at a dose of 5 to 25 mg.

[0020] When any of the above compositions is administered, the polyethylene glycol-modified phenylalanine lyase and any immunomodulator are administered separately, wherein the polyethylene glycol-modified phenylalanine lyase is administered subcutaneously, and each administration is at a dose of 5 to 40 mg.

[0021] In one embodiment of any of the provided methods, the composition comprising polyethylene glycol-modified phenylalanine lyase is administered to the subject at least once without immunosuppressants, following one or more administrations of an immunomodulatory agent.

[0022] In one embodiment of any of the provided methods, the immunosuppressant composition and the composition containing polyethylene glycol-modified phenylalanine lyase are administered within 2 hours of each other during each concomitant administration.

[0023] In some embodiments, the method comprises administering an immunosuppressant prior to each concomitant administration of the composition comprising polyethylene glycolated phenylalanine lyase.

[0024] In one embodiment of any of the provided methods, the immunosuppressant methotrexate is pre-treated with PEGylated phenylalanine lyase at least twice, once a week, prior to administration.

[0025] The drug combination of the present invention is used to treat diseases related to high phenylalanine, including but not limited to phenylketonuria and hyperphenylalanineemia, and can effectively inhibit the generation of anti-drug antibodies against biological drugs such as polyethylene glycol-modified phenylalanine lyase, and has good prospects for clinical application. Attached Figure Description

[0026] Figure 1 The results of the modification site analysis of the phenylalanine lyase mutant directed coupling compound in Example 2 of this invention.

[0027] Figure 2 Pah in Embodiment 3 of the present invention enu The change curve of serum phenylalanine concentration after multiple administrations of PL54 and Pegvaliase to mice.

[0028] Figure 3 The figures represent the levels of PL54 and anti-antibody antibodies (Anti-PEG-IgM / IgG antibodies) in rats after multiple administrations of PL54 monotherapy and in combination with different methotrexate (MTX) regimens in Example 4 of this invention.

[0029] Figure 4The values ​​of PK and anti-drug antibody (Anti-PEG-IgM / IgG antibody) in rats after multiple administrations of Pegvaliase, PL5, and PL54 in combination with methotrexate (MTX) in Example 5 of this invention are given. Detailed Implementation

[0030] The present invention will be further illustrated below by means of embodiments. It should be understood that the embodiments of the present invention are only for illustrating the present invention and not for limiting the present invention. Simple improvements to the present invention under the premise of the present invention are all within the scope of protection of the present invention.

[0031] Example 1: Preparation of polyethylene glycol-modified recombinant phenylalanine lyase and mutants

[0032] 1. Preparation of recombinant phenylalanine lyase and mutants

[0033] The gene sequences encoding the amino acid sequences SEQ ID NO:1 (PAL4) and SEQ ID NO:2 (PAL) were synthesized. Expression plasmids pET-30a-PAL4 and pET-30a-PAL were constructed by restriction endonuclease digestion and T4 DNA ligase ligation. The expression plasmids were transformed into *E. coli*, and recombinant engineered expression bacteria were obtained through antibiotic resistance screening. After fermentation in YT medium, expression was induced by IPTG, and the bacterial cells were collected by centrifugation.

[0034] After fermentation, the bacterial cells were lysed physically or enzymatically, and insoluble lysate fragments were removed by centrifugation. The pH of the supernatant was adjusted, and (NH4)2SO4 was added for fractional precipitation to enrich phenylalanine lyase. The phenylalanine lyase precipitate was obtained by centrifugation again. After dissolution, host protein impurities were purified by hydrophobic chromatography, and the eluent was purified by ion exchange chromatography to remove high- and low-molecular-weight related proteins. The purity of the recombinant phenylalanine lyase and mutant was assessed by SDS-PAGE and SEC-HPLC, respectively, and both were no less than 95%. The specific activity of the enzyme was determined by substrate method, and the specific activity of the recombinant phenylalanine lyase mutant was no less than 3.0 U / mg.

[0035] Phenylalanine lyase specifically catalyzes the conversion of phenylalanine to trans-cinnamic acid, releasing ammonia. Trans-cinnamic acid exhibits maximum light absorption at a wavelength of 290 nm. One unit of enzyme activity is defined as the amount of enzyme required to generate 1 μmol of trans-cinnamic acid per minute at room temperature (25°C) and in a 100 mmol / L Tris-HCl buffer solution (pH 8.5).

[0036] Specific assay method: Take an appropriate amount of sample and dilute it to 0.4 mg / ml with activity buffer (100 mmol / L Tris-HCl, pH 8.0); place 2.9 ml of substrate solution (100 mmol / L Tris-HCl, 45 mmol / L Phe, pH 8.0) in a cuvette, add 0.1 ml of diluted sample solution and mix immediately; record the change in absorbance value per minute at a wavelength of 290 nm, and calculate the specific enzyme activity according to the formula.

[0037] Enzyme activity (U / mg) = (A S -A O VN×10 6 / εLTV1

[0038] Among them, A S A O V represents the absorbance of trans-cinnamic acid at the start and end of the reaction, respectively; V is the total volume of the reaction system (L); L is the optical path length of the cuvette (cm); ε is the molar extinction coefficient of trans-cinnamic acid (10238.1L / (mol·cm)); T is the reaction time (min); N is the sample dilution factor; V1 is the sample volume (ml).

[0039] 2. Preparation of PEGylated recombinant phenylalanine lyase and mutants

[0040] The recombinant phenylalanine lyase (PAL) and phenylalanine lyase mutant (PAL4) prepared above were prepared in a modification buffer of 100 mmol / L Na2HPO4-NaH2PO4, pH range 7.5–9.5, at a concentration of not less than 10 mg / ml. 5K-SPA-PEG (polyethylene glycol succinimide propionate with a molecular weight of 5000 Da) was dissolved in the buffer, and both were added at a phenylalanine lyase (mutant) to polyethylene glycol mass ratio of 1:10–1:25. The modification reaction was carried out with stirring at room temperature. Free PEG was removed by ultrafiltration, and the modified product was concentrated to above 10 mg / ml by ultrafiltration (protein content determined by high performance liquid chromatography). The average degree of modification was determined by high performance liquid chromatography with a tandem differential detector (refer to patent CN2019101542414), and the enzyme activity was determined according to the method described in this embodiment. The results showed that the average degree of modification of polyethylene glycol-modified phenylalanine lyase (PL5, corresponding to the unmodified protein PAL) was 9.0±1.0, and the specific enzyme activity was 1.3 U / mg; the average degree of modification of polyethylene glycol-modified phenylalanine lyase mutant (PL54, corresponding to the unmodified protein PAL4) was 13.0±1.0, and the specific enzyme activity was about 1.2 U / mg.

[0041] Other polyethylene glycols with different activating groups (such as N-hydroxysuccinimide, N-hydroxysuccinimide carbonate, N-hydroxysuccinimide acetate, N-hydroxysuccinimide butyrate, N-hydroxysuccinimide succinate, and p-nitrophenyl carbonate) were prepared by following the steps described above for the preparation of polyethylene glycolated phenylalanine lyase and mutants.

[0042] Example 2: Study on the polyethylene glycol modification sites of polyethylene glycolated phenylalanine lyase mutants

[0043] PEGylated phenylalanine lyase and its mutants involve the covalent binding of PEG to the amino side chain of lysine residues or the amino group of N-terminal amino acids in the amino acid sequence. Trypsin and lysyl endopeptidase cannot recognize PEG-modified lysine residues; therefore, the modification site can be inferred by comparing the peptides before and after modification. The inventors used a combination of Trypsin and Lys-C enzymes to enzymatically digest PEGylated phenylalanine lyase and its mutants (PL54 prepared in Example 1) before and after PEG modification. The modification sites of the PEGylated phenylalanine lyase mutants were analyzed by LC-MS, as detailed in Table 1. Figure 1 .

[0044] The results showed that PL54, obtained by coupling PAL4 protein with polyethylene glycol, had its lysine-containing peptides or adjacent peptides disappear or have reduced response after cleavage by enzymes at the mutation sites. It could be determined that all four mutation sites (K72, K240, K467 and K544) were modified, with a modification rate of over 90%.

[0045] Table 1: Comparison of peptide maps before and after PL54 modification

[0046]

[0047]

[0048]

[0049] Example 3: Application of PL54 in an animal model of phenylketonuria

[0050] Pegvaliase, a polyethylene glycol-modified phenylalanine lyase, was purchased from BioMarin Pharmaceuticals, USA (lot number: L161293, 20 mg / mL); PL54 was the sample prepared in Example 1. Eight Pegvaliase samples were selected. enuMice were divided into two groups of four each, and were given PL54 and Pegvaliase injections, respectively, at a dose of 3.0 mg / kg, once a week subcutaneously for four consecutive weeks. Blood samples were collected from each group of mice before administration, and 3 and 7 days after each administration to measure blood phenylalanine concentration (method reference: Judy Peat et al., Determination of Phenylalanine and Tyrosine by HighPerformance Liqiud Chromatography-Tandem Mass Spectrometry).

[0051] Pah enu Changes in serum phenylalanine concentration in mice after repeated administration of PL54 and Pegvaliase, such as Figure 2 As shown in the results, at the same dose (3 mg / kg), PL54 and Pegvaliase had similar phenylalanine-lowering effects on Day 3 after the first administration, and both returned to initial levels on Day 7. However, on Day 3 of the 2nd to 4th administrations, PL54 could lower phenylalanine levels similar to the effect of the first administration, while Pegvaliase lost its phenylalanine-lowering effect. Long-term administration of PL54-5 showed a significantly better phenylalanine-lowering effect than Pegvaliase.

[0052] Example 4: Immunogenicity study of PL54 monotherapy and multiple-dose administration in rats with different methotrexate (MTX) regimens.

[0053] Fifteen male SD rats were randomly divided into three groups: a PL5 administration group, a PL54 combined with MTX group 1, and a PL54 combined with MTX group 2, with five rats in each group. In the PL54 combined with MTX group 1, no MTX pretreatment was given before PL54 administration. In the PL54 combined with MTX group 2, MTX was pretreated before PL54 administration (2 mg / kg subcutaneously twice a week for four weeks). All experimental groups received 2 mg / kg PL54 subcutaneously once a week. In the PL54 combined with MTX groups 1 and 2, an additional 2.0 mg / kg MTX was administered subcutaneously twice a week. The interval between the administration of PEGylated phenylalanine lyase and MTX in the combined administration groups did not exceed 2 hours. All experimental groups were administered the drugs for three consecutive weeks. Blood samples were collected venously before administration and 3 and 7 days after each administration. Plasma was separated, and PL54 blood was detected using a double-antibody sandwich ELISA method. Serum was collected before the first administration and 7 days after each administration for ADA detection. The titers of Anti-PEG-IgM / IgG antibodies in the serum of rats in each group were detected by indirect ELISA (coated with PEG-BSA, added with positive rat Anti-PEG Abs / test serum sample, and HRP-Anti Rats IgM / IgG reaction for color development).

[0054] Subcutaneous administration of PL54 combined with methotrexate injection (MTX) reduced the production of PL54 anti-antibody antibodies in rats and prolonged the duration of PL54 in rat blood, with the best effect observed after 4 weeks of MTX pretreatment. Antibody detection showed that pretreatment with MTX before PL54 combined with MTX administration significantly reduced anti-PEG antibodies in animals; see details. Figure 3 .

[0055] Example 5: Pharmacokinetic and immunogenicity studies of multiple-dose administration of Pegvaliase, PL5, and PL54 in combination with methotrexate (MTX) in rats.

[0056] Pegvaliase is a polyethylene glycol-modified phenylalanine lyase, purchased from BioMarin Pharmaceuticals, USA (batch number: L161293, 20 mg / mL); PL5 and PL54 were samples prepared in Example 1. Thirty male SD rats were randomly divided into six groups: PL5 administration group, PL54 administration group, Pegvaliase administration group, PL5 combined with MTX group, PL54 combined with MTX group, and Pegvaliase combined with MTX group, with five rats in each group. The MTX-treated groups were pre-treated with MTX (2 mg / kg subcutaneously twice a week for four weeks), while the PL5, PL54, and Pegvaliase groups received the corresponding volumes of physiological saline. After four weeks of administration, PL5, PL54, and Pegvaliase were added to the treatment regimen, respectively, while the PL5, PL54, and Pegvaliase groups received the corresponding drugs. PL5, PL54, and Pegvaliase were administered subcutaneously at a dose of 2 mg / kg PL54 once weekly. MTX was administered subcutaneously at a dose of 2.0 mg / kg twice weekly. In the combination therapy group, the interval between the administration of PEGylated phenylalanine lyase and MTX did not exceed 2 hours. All experimental groups were administered the drugs continuously for 3 weeks. Blood samples were collected intravenously before administration and 3 and 7 days after each administration. Plasma was separated, and PL54 and Pegvaliase levels were detected using ELISA. Serum was collected before the first administration and 7 days after each administration to detect Anti-PEG-IgM / IgG antibodies. The detection methods are described in the above embodiments of the present invention.

[0057] PL54 combined with subcutaneous methotrexate injection (MTX) significantly reduced the production of PL54 anti-antibody antibodies in rats and prolonged the maintenance time of PL54 in rat blood, with significantly better effects than PL5, pegvaliase combined with subcutaneous methotrexate injection (MTX), and single drug administration. The anti-PEG-IgM / IgG antibody titer of PL54 combined with MTX in animals was lower than that of PL5, pegvaliase combined with subcutaneous methotrexate injection (MTX), and single drug administration. See details. Figure 4 .

[0058] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A combination of protease drugs and immunomodulators for the treatment of phenylketonuria, comprising: (1) A polyethylene glycol-modified phenylalanine lyase mutant, characterized in that amino acid residues at positions 72, 240, 467 and 544 of the phenylalanine lyase mutant are replaced by lysine, and one or more of the following amino acid sites in the phenylalanine lyase mutant are modified with polyethylene glycol: K72, K240, K467 and K544. Furthermore, the mutation site is located using SEQ ID NO:1 or an amino acid sequence having at least 80% homology with it. (2) Immunomodulators are drugs that suppress the immune system, including inhibitors of dihydrofolate reductase, such as methotrexate (MTX).

2. The polyethylene glycolated phenylalanine lyase mutant of claim 1 is characterized in that it is a chemical conjugate obtained by amide bond binding of activated polyethylene glycol and lysine residues of the phenylalanine lyase mutant.

3. The polyethylene glycol-modified phenylalanine lyase mutant according to claim 1, characterized in that, The molar ratio of polyethylene glycol to the phenylalanine lyase mutant is (6-20):1, preferably (12-15):

1.

4. The polyethylene glycol of claims 1 and 2, including but not limited to polyethylene glycol succinimide butyrate, polyethylene glycol succinimide propionate, polyethylene glycol succinimide acetate, polyethylene glycol succinimide carbonate and polyethylene glycol nitrobenzene carbonate, having a molecular weight of 2kDa to 40kDa, preferably polyethylene glycol succinimide propionate with a molecular weight of 5kDa.

5. The pharmaceutical combination of claim 1, characterized in that, The immunomodulatory agent methotrexate can be an injection, an oral formulation, or a nanoformation, etc.

6. The composition according to claims 1 and 5 is administered, characterized in that, The protease drug and the immunomodulator are administered separately, wherein methotrexate is administered subcutaneously, orally, intravenously, or intramuscularly, with subcutaneous and oral administration being preferred, and each administration is at a dose of 5 to 25 mg.

7. The composition according to any one of claims 1 to 6 is administered, characterized in that, The protease drug and immunomodulator are administered separately, with the PEGylated phenylalanine lyase administered subcutaneously at a dose of 5 to 40 mg per administration.

8. The method of any one of the preceding claims, wherein the immunosuppressant methotrexate is pretreated with PEGylated phenylalanine lyase at least twice, once a week, before administration.

9. The method of any one of the preceding claims, wherein the immunosuppressant composition and the composition comprising polyethylene glycol-modified phenylalanine lyase are administered within 2 hours of each other in each concomitant administration.

10. Use of the method of any one of the preceding claims in the preparation of a medicament for the treatment of diseases related to the prevention and / or treatment of hyperphenylalaninemia, including but not limited to at least one of phenylketonuria and hyperphenylalaninemia.

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