Pharmaceutical compositions containing heparan N-sulfatase with improved stability

Abstract

The present invention relates to a pharmaceutical composition for CNS delivery of high-concentration heparan N-sulfatase (HNS) with improved stability, and a pharmaceutical dosage form containing the same. The pharmaceutical composition and pharmaceutical dosage form containing the same according to the present invention have excellent dosage form stability, such as reduced turbidity and significantly improved purity, and are therefore useful in enzyme replacement therapy (ERT) for the treatment of mucopolysaccharidosis type III.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition containing a high concentration of heparan N-sulfatase (HNS) with improved stability, and a pharmaceutical dosage form containing the same. More specifically, the present invention relates to a pharmaceutical composition containing a high concentration of heparan N-sulfatase and a histidine buffer, and having a pH of 7.8 or higher, and a pharmaceutical dosage form containing the same.

[0002] [Background technology]

[0003] Lysosomal storage diseases (LSDs) are genetic disorders resulting from defective lysosomal function. Lysosomal storage diseases are caused by lysosomal dysfunction due to the deficiency of one or more enzymes required for the metabolism of lipids, glycoproteins, or mucopolysaccharides. Deficiency of lysosomal enzymes leads to systemic abnormalities due to the lysosomal accumulation of lipids, glycoproteins, or mucopolysaccharides (Nature Reviews Disease Primers. 4(1):27; Biochem. Soc. Trans. 28(2):150-4). Mucopolysaccharidoses (MPS) are a type of lysosomal storage disease caused by intralysosomal accumulation due to the deficiency of lysosomal enzymes required for the degradation of glycosaminoglycans. MPSs are generally classified into types I to VII, depending on the type of enzyme deficiency.

[0004] Enzyme replacement therapy (ERT) corrects the functional deficiency of lysosomal enzymes by administering the missing enzymes. It is one of the main treatments used to treat lysosomal storage diseases. A simple injection therapy has the advantage of minimizing symptoms and preventing permanent damage to the body. A well-known enzyme replacement therapy for enzyme storage diseases is intravenous glucocerebrosidase (GCase) therapy, first approved by the FDA in 1991 for Gaucher disease (National Gaucher Foundation. Retrieved 2017-06-08).

[0005] However, many lysosomal storage diseases induce excessive accumulation of glycosaminoglycans (GAGs) in the nervous system, particularly in brain neurons and spinal cord membranes, leading to various central nervous system disorders. However, intravenous enzyme replacement therapy (ELISA) is unable to effectively treat neurological disorders and diseases caused by lysosomal accumulation in the brain because the active ingredient, lysosomal enzymes, are not properly delivered to the central nervous system due to the difficulty of crossing the blood-brain barrier (BBB). Therefore, various CNS delivery therapies that deliver drugs directly to the central nervous system to bypass the BBB are being investigated.

[0006] Various therapies have been developed to deliver drugs to the central nervous system (CNS) through the administration of enzymes that bypass the BBB. In particular, injection therapies that deliver proteins directly to the brain include intracerebral injection (IC), intracerebroventricular injection (ICV), and intrathecal injection (IT).

[0007] Intrathecal (IT) and intracerebroventricular (ICV) injections are emerging as methods for delivering alternative enzymes to the central nervous system for mucopolysaccharidoses (MPS) and have shown significant reductions in glycosaminoglycan (GAG) levels and significant improvements in neurological symptoms in various MPS animal models (Molecular Therapy - Methods & Clinical Development, 21, 67-75). However, direct brain injection therapy is highly dose-limited, and the development of injectable formulations containing highly concentrated enzymes is essential for effective therapeutic effects.

[0008] Many high-concentration enzyme formulations for CNS-based enzyme replacement therapy (ERT) have been reported. The formulations for CNS delivery reported to date have been buffered, and phosphate buffers are commonly used (Korean Patents Nos. 2,007,044 and 2,272,399, etc.).

[0009] However, research has shown that the use of phosphate buffers in compositions for CNS delivery of heparan N-sulfatase (HNS) negatively impacts the activity of the enzyme (J Inherit Metab Dis. 1993;16(2):465-472; Acta Crystallogr D Biol Crystallogr. 2014 May;70(Pt5):1321-1335).

[0010] Therefore, there is a strong need for pharmaceutical compositions and pharmaceutical dosage forms for CNS delivery that contain heparan N-sulfatase, particularly high concentrations of heparan N-sulfatase, and have high stability.

[0011] Under these circumstances, the present inventors have made extensive efforts to develop a pharmaceutical composition for CNS delivery of high-concentration heparan N-sulfatase (HNS) without using a phosphate buffer, and a pharmaceutical dosage form containing the same. As a result, they have found that the stability of HNS is dramatically improved when a histidine buffer is used as the buffer and the pH is set to 7.8 or higher, thereby completing the present invention.

[0012]

[0013] The above information described in this Background Art is merely intended to enhance understanding of the background of the present invention, and therefore may not include information that constitutes prior art already known to those of ordinary skill in the art to which the present invention pertains.

[0014]

[0015] Summary of the Invention

[0016] An object of the present invention is to provide a pharmaceutical composition and pharmaceutical dosage form containing heparan N-sulfatase (HNS) with improved stability.

[0017] Another object of the present invention is to provide a method for treating mucopolysaccharidosis type IIIA using the pharmaceutical composition or pharmaceutical dosage form.

[0018] It is yet another object of the present invention to provide a use of said pharmaceutical composition or pharmaceutical dosage form for the treatment of mucopolysaccharidosis type IIIA.

[0019] It is yet another object of the present invention to provide a use of said pharmaceutical composition or pharmaceutical dosage form for the manufacture of a medicament for the treatment of mucopolysaccharidosis type IIIA.

[0020]

[0021] To achieve the above object, the present invention provides a pharmaceutical composition comprising heparan N-sulfatase (HNS) and a histidine buffer, and having a pH of 7.8 or higher, and a pharmaceutical dosage form comprising the same.

[0022] The present invention also provides a pharmaceutical composition for treating mucopolysaccharidosis type IIIA, which comprises heparan N-sulfatase and a histidine buffer and has a pH of 7.8 or higher, a pharmaceutical dosage form containing the same, and a method for treating mucopolysaccharidosis type IIIA using the same.

[0023] The present invention also provides the use of the pharmaceutical composition or pharmaceutical dosage form for the treatment of mucopolysaccharidosis type IIIA.

[0024] The present invention also provides the use of the pharmaceutical composition or pharmaceutical dosage form for the manufacture of a medicament for the treatment of mucopolysaccharidosis type IIIA.

[0025] [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 shows the stability of pharmaceutical compositions depending on the type and pH of amino acid buffer.

[0027] (Figure 1A) pH 7.5

[0028] (Figure 1B) pH 8.0

[0029] [Figure 2] FIG. 1 is a graph showing the stability of pharmaceutical compositions depending on the type of amino acid buffer, expressed in terms of B22 and kD values.

[0030] (Figure 2A) B22 values

[0031] (Figure 2B) kD values

[0032] [Figure 3]FIG. 1 shows the comparative results of the stability of pharmaceutical compositions when a histidine buffer and a phosphate buffer are used.

[0033] [Figure 4] FIG. 1 shows changes in turbidity of pharmaceutical compositions depending on pH.

[0034] [Figure 5] FIG. 1 shows changes in turbidity of pharmaceutical compositions depending on pH.

[0035] [Figure 6] FIG. 1 shows the results of observing changes in turbidity of pharmaceutical compositions due to the addition of a surfactant and pH.

[0036] (Figure 6A) Comparison of foreign body images after reconstitution of dosage forms containing HNS with and without PS20 and with 0.005% PS20.

[0037] (Figure 6B) Schematic representation of foreign particles by size after reconstitution of dosage forms containing HNS with or without PS20 / with 0.005% PS20.

[0038] (Figure 6C) A graph showing the size of foreign particles after reconstitution of dosage forms containing HNS with or without PS20 and with 0.005% PS20.

[0039] [Figure 7] FIG. 1 shows the stability of pharmaceutical compositions depending on NaCl concentration.

[0040] [Figure 8] FIG. 1 shows the change in purity of HNS in a composition depending on the trehalose concentration.

[0041] [Figure 9] FIG. 1 shows the change in potency (SA) and purity of HNS in a composition depending on the trehalose concentration.

[0042]

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0045]

[0046] Heparan N-sulfatase (HNS) is a lysosomal enzyme that catalyzes the hydrolysis of N-linked sulfate groups from heparan sulfate and the non-reducing terminal glucosamine moiety of heparan (Biochem. Biophys. Res. Commun. 2001, 280, 1251-1257). Mutations in the heparan N-sulfatase gene (SGSH) are well known to cause rumcopolysaccharidosis type IIIA (MPSIIIA, OMIM# 252900), also known as Sanfilippo syndrome. Mucopolysaccharidosis type IIIA (MPS IIIA; Sanfilippo syndrome type A) is characterized by a deficiency of the enzyme heparan sulfate (HNS), which is involved in the lysosomal catabolism of the glycosaminoglycan (GAG) heparan sulfate (Neufeld EF, et al. The Metabolic and Molecular Bases of Inherited Disease (2001) pp. 3421-3452). In the absence of this enzyme, glycosaminoglycans (GAGs) accumulate in the lysosomes of neurons and glial cells, causing severe neurological damage and abnormal symptoms.

[0047] Although phosphate-containing formulations for central nervous system delivery of heparan N-sulfatase have been reported (e.g., Korean Patent No. 2,007,044), research findings have shown that the use of phosphate buffers negatively affects the activity of heparan N-sulfatase (J. Inherit Metab. Dis. 1993;16(2):465-72; and Acta Crystallogr D Biol. Crystallogr. 2014 May;70(Pt5):1321-35). Therefore, there is a need to develop novel formulations for central nervous system delivery of heparan N-sulfatase that use stabilizers other than phosphate.

[0048] In one embodiment of the present invention, the present inventors have confirmed that a high-concentration heparan N-sulfatase formulation prepared using a histidine buffer exhibits significantly increased protein-protein or protein-buffer stability and significantly reduced turbidity compared to a formulation containing phosphate, thereby demonstrating excellent stability, and have completed the present invention.

[0049] Therefore, in one aspect, the present invention provides a pharmaceutical composition comprising 2 mg / mL to 50 mg / mL heparan N-sulfatase (HNS) and 1 to 40 mM histidine buffer, and having a pH of 7.8 to 9.0.

[0050] As used herein, the term "heparan N-sulfatase" may be used interchangeably with the term N-sulfoglucosamine sulfohydrolase (SGSH).

[0051] In the present invention, the heparan N-sulfatase may be characterized by having a wild-type or naturally occurring amino acid sequence, for example, the heparan N-sulfatase may be derived from various organisms, more preferably, but not limited to, human origin.

[0052] The heparan N-sulfatase can be any molecule or portion of a molecule that replaces the protein activity of naturally occurring heparan N-sulfatase (HNS) or that can ameliorate one or more of the phenotypes or symptoms associated with HNS deficiency. A replacement enzyme suitable for the present invention is a polypeptide having N- and C-terminal amino acid sequences substantially similar to or identical to the mature human HNS protein.

[0053] Typically, human HNS is produced as a precursor molecule that is processed to a mature form. This processing generally involves the removal of a 20-amino acid signal peptide. Typically, the precursor form is a full-length precursor or full-length HNS protein containing 502 amino acids. The N-terminal 20 amino acids are truncated to yield a mature form that is 482 amino acids in length. Therefore, the N-terminal 20 amino acids are generally considered dispensable for HNS protein activity. The amino acid sequences of a typical wild-type or naturally occurring mature form of human HNS protein (SEQ ID NO: 1) and a full-length precursor (SEQ ID NO: 2) are as follows:

[0054] Mature form of HNS: RPRNALLLLA DDGGFESGAY NNSAIATPHL DALARRSLLF RNAFTSVSSC SPSRASLLTG LPQHQNGMYG LHQDVHHFNS FDKVRSLPLL LSQAGVRTGI IGKKHVGPET VYPFDFAYTE ENGSVLQVGR NITRIKLLVR KFLQTQDDRP FFLYVAFHDP HRCGHSQPQY GTFCEKFGNG ESGMGRIPDW TPQAYDPLDV LVPYFVPNTP AARADLAAQY TTVGRMDQGV GLVLQELRDA GVLNDTLVIF TSDGIPFPS GRTNLYWPGT AEPLLVSSPE HPKRWGQVSE AYVSLLDLTP TILDWFSIPY PSYAIFGSKT IHLTGRSLLP ALEAEPLWAT VFGSQSHHEV TMSYPMRSVQ HRHFRLVHNL NFKMPFPIDQ DFYVSPTFQD LLNRTTAGQP TGWYKDLRHY YYRARWELYD RSRDPHETQN LATDPRFAQL LEMLRDQLAK WQWETHDPWV CAPDGVLEEK LSPQCQPLHN EL (SEQ ID NO: 1)

[0055] HNS full-length precursor: MSCPVPACCA LLLVLGLCRA RPRNALLLLA DDGGFESGAY NNSAIATPHL DALARRSLLF RNAFTSVSSC SPSRASLLTG LPQHQNGMYG LHQDVHHFNS FDKVRSLPLL LSQAGVRTGI IGKKHVGPET VYPFDFAYTE ENGSVLQVGR NITRIKLLVR KFLQTQDDRP FFLYVAFHDP HRCGHSQPQY GTFCEKFGNG ESGMGRIPDW TPQAYDPLDV LVPYFVPNTP AARADLAAQY TTVGRMDQGV GLVLQELRDA GVLNDTLVIF TSDNGIPFPS GRTNLYWPGT AEPLLVSSPE HPKRWGQVSE AYVSLLDLTP TILDWFSIPY PSYAIFGSKT IHLTGRSLLP ALEAEPLWAT VFGSQSHHEV TMSYPMRSVQ HRHFRLVHNL NFKMPFPIDQ DFYVSPTFQD LLNRTTAGQP TGWYKDLRHY YYRARWELYD RSRDPHETQN LATDPRFAQL LEMLRDQLAK WQWETHDPWV CAPDGVLEEK LSPQCQPLHN EL (SEQ ID NO: 2)

[0056] In the present invention, the heparan N-sulfatase may be characterized as a recombinant enzyme produced by recombinant means. Recombinant production of heparan N-sulfatase may be easily carried out by various recombinant cell production techniques for expressing target proteins known in the art.

[0057] In the present invention, the heparan N-sulfatase may be in the form of a fusion protein or conjugate. In the present invention, the heparan N-sulfatase may be fused or conjugated with a moiety capable of binding to a receptor on the surface of brain cells and / or a lysosomal targeting molecule to facilitate cellular uptake or lysosomal targeting. Improvements of alternative enzymes such as heparan N-sulfatase are disclosed in Korean Patent No. 2,007,044, etc.

[0058]

[0059] In the present invention, the heparan N-sulfatase may be contained at about 2 mg / mL or more, about 5 mg / mL or more, about 10 mg / mL or more, about 15 mg / mL or more, about 20 mg / mL or more, about 25 mg / mL or more, or about 30 mg / mL or more.

[0060] In another example of the present invention, the heparan N-sulfatase may be contained at a concentration of about 2 to about 50 mg / mL, preferably about 3 to about 40 mg / mL, more preferably about 5 to about 30 mg / mL, even more preferably about 8 to about 25 mg / mL, particularly preferably about 10 to about 20 mg / mL, and most preferably about 12 to about 15 mg / mL, but is not limited thereto.

[0061] In yet another example of the present invention, the heparan N-sulfatase may be contained at a concentration of about 2 to about 20 mg / mL, preferably about 2 to about 16.5 mg / mL, and more preferably about 2 to about 15 mg / mL, but is not limited thereto.

[0062]

[0063] Furthermore, histidine buffers offer various advantages over existing phosphate buffers, such as significantly increased stability due to reduced protein-protein and protein-buffer interactions. In the composition of the present invention, the histidine buffer may be present at a concentration of about 1 to about 40 mM, preferably about 1.5 to about 30 mM, more preferably about 2 to about 20 mM, and most preferably about 3 to about 10 mM, but is not limited thereto. The concentration in the histidine buffer is calculated based on the concentration of histidine.

[0064] In the present invention, as another example, the histidine buffer may be contained at about 1 to about 40 mM, preferably about 1 to about 30 mM, more preferably about 1 to about 20 mM, and most preferably about 1 to about 10 mM, but is not limited thereto.

[0065] It has been confirmed that when the pH of the composition of the present invention is about 7.8 or higher, electrostatic repulsion between proteins or between proteins and buffer increases, resulting in a significant decrease in turbidity and a dramatic increase in the stability of the composition.

[0066] Accordingly, the pH of the composition according to the present invention may be about 7.8 or higher, preferably about 7.8 to about 9.0, more preferably about 7.9 to about 8.9, and most preferably about 8.0 to about 8.8, but is not limited thereto.

[0067]

[0068] In the present invention, the composition of the present invention is characterized by exhibiting low turbidity. As used herein, the term "turbidity" refers to the degree to which a composition is cloudy due to soft substances or impurities in the composition. In pharmaceutical compositions, turbidity is a parameter that indicates the stability of a drug. For example, in the case of a dosage form with low stability, aggregates may form due to protein-protein interactions or protein-buffer attraction or self-association, resulting in increased turbidity.

[0069] In the present invention, the turbidity (T) may be calculated by measuring absorbance at a specific wavelength. Turbidity calculation follows the Beer-Lambert law: T = I / I0 (T: transmittance, I: transmitted intensity, I0: incident intensity). In one embodiment of the present invention, the turbidity was measured at 350 nm using a Lunatic (Unchained Labs), but is not limited thereto.

[0070] In the pharmaceutical composition of the present invention, the turbidity may be characterized as being, but not limited to, about 1.0 or less, preferably about 0.8 or less, more preferably about 0.6 or less, and most preferably about 0.4 or less when measured at 350 nm.

[0071]

[0072] Furthermore, it has been confirmed that the inclusion of saccharides, particularly trehalose, in the present invention results in significantly superior purity (%) and specific activity (SA) not only when used as a liquid pharmaceutical composition but also when formulated into a lyophilized formulation and reconstituted for use. Thus, the composition of the present invention is characterized by the inclusion of saccharides.

[0073] In the present invention, the sugar may be trehalose, sucrose, maltose, lactose, or sorbitol.

[0074] In the present invention, the sugars may be contained at a concentration of about 0.1% or more, about 0.3% or more, about 0.5% or more, about 0.8% or more, about 1.0% or more, about 1.35% or more, or about 1.8% or more. Specifically, the sugars may be contained at a concentration of about 0.1% to about 5.0%, preferably about 0.3% to about 4.0%, more preferably about 0.4% to about 3.5%, even more preferably about 0.5% to about 3.0%, and particularly preferably about 1.0% to 2.0%.

[0075] In another example, the present invention may be characterized in that the sugars are contained at a concentration of about 0.1% to about 3%.

[0076] In the present invention, the % concentration of each substance means w / v % unless otherwise specified.

[0077] The pharmaceutical composition according to the present invention contains a salt. In the present invention, the salt may be NaCl or KCl. In the present invention, the salt may be about 30 mM to about 500 mM, preferably about 50 mM to about 300 mM, more preferably about 60 mM to about 200 mM, even more preferably about 70 mM to 150 mM, and most preferably about 70 mM to 120 mM.

[0078] In another example, the present invention may be characterized in that the salt is about 30 mM to about 300 mM.

[0079] In the present invention, the salt may be included in the pharmaceutical composition of the present invention at a concentration that has an appropriate osmolality for central nervous system delivery. The appropriate osmolality of drug dosage forms for central nervous system delivery is well known in the art.

[0080] In the present invention, the osmotic concentration of the pharmaceutical composition may be, for example, but is not limited to, about 400 mOsmol / kg or less, preferably about 350 mOsmol / kg or less, more preferably about 330 mOsmol / kg or less, even more preferably about 300 mOsmol / kg or less, and most preferably about 290 mOsmol / kg or less.In the present invention, the osmotic concentration of the drug dosage form may be, for example, but is not limited to, about 200 to about 400 mOsmol / kg, preferably about 220 to about 360 mOsmol / kg, more preferably about 250 to about 330 mOsmol / kg, and most preferably about 280 to about 300 mOsmol / kg.

[0081]

[0082] In one example of the present invention, it was confirmed that when polysorbate 20 was added as a surfactant, the turbidity was reduced compared to a dosage form without added surfactant.

[0083] Thus, the pharmaceutical composition according to the present invention may be characterized in that it further contains a surfactant. In the present invention, the surfactant may be a polysorbate surfactant, more preferably polysorbate 20 or polysorbate 80, and most preferably polysorbate 20. In the composition according to the present invention, the surfactant is contained at a concentration of about 0.0001% to about 0.1%, preferably about 0.002% to about 0.07%, more preferably about 0.003% to about 0.05%, and most preferably about 0.004 to about 0.01%.

[0084] However, when the pharmaceutical composition according to the present invention is formulated into a lyophilized dosage form, reconstituted, and administered to a patient, the surfactant may be used in a form contained in a solution for reconstitution, rather than being contained in the pharmaceutical composition and dosage form for lyophilization.

[0085]

[0086] The pharmaceutical composition of the present invention may further contain suitable carriers, excipients, and diluents typically used in pharmaceutical compositions, in addition to the heparan N-sulfatase (HNS), histidine buffer, sugars, salts, and / or surfactants.

[0087] Pharmaceutical excipients useful in particular for liquid protein formulations are well known to those of ordinary skill in the art. Non-limiting examples include specific or universal solvents; sugars or sugar alcohols, such as mannitol, sucrose, sorbitol, fructose, maltose, lactose, or dextran; buffers; preservatives, such as benzalkonium chloride, benzethonium chloride, tertiary ammonium salts, and chlorhexidine diacetate; carriers, such as poly(ethylene glycol) (PEG); antioxidants, such as ascorbic acid, sodium metabisulfite, and methionine; chelating agents, such as EDTA or citric acid; biodegradable polymers, such as water-soluble polyesters; cryoprotectants; lyoprotectants; bulking agents; and stabilizers, as described in Remington: "The Science and Practice of Pharmacy," 20th edition, Alfonso R. Gennaro, Ed., Lippincott Williams & Co., Inc. Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Wilkins (2000), may also be included in the protein dosage forms described herein, but should not negatively affect the desirable characteristics of the dosage form.

[0088]

[0089] In one preferred embodiment, the composition of the present invention comprises:

[0090] 5-30 mg / mL heparan N-sulfatase

[0091] 2–20 mM histidine buffer;

[0092] 0.5 to 3.0 w / v% trehalose; and

[0093] containing 70-150 mM NaCl;

[0094] Optionally, further containing 0.003 to 0.05% polysorbate 20;

[0095] It may be characterized by, but is not limited to, a pH of 8.0 to 8.8:

[0096]

[0097] The composition according to the present invention is characterized in that it is used for the treatment of mucopolysaccharidosis type IIIA (MPS IIIA).

[0098]

[0099] In the present invention, the pharmaceutical composition may be formulated into a pharmaceutical dosage form such as a liquid dosage form or a lyophilized dosage form.

[0100] The liquid dosage form is preferably, but not limited to, an ampoule-type dosage form or a pre-filled syringe-type dosage form.

[0101] Preferably, the pharmaceutical composition may be formulated into a lyophilized dosage form, which is convenient for storage and transportation, and may be prepared by various lyophilization methods known in the art, in addition to the methods described in the examples of the present invention.

[0102] After being formulated, the pharmaceutical composition of the present invention may be reconstituted before administration to adjust the concentration of the active ingredient, heparan-N-sulfatase, or the like.

[0103] In the present invention, even when the pharmaceutical composition is in a liquid dosage form, it may be reconstituted for use. However, when the pharmaceutical composition is formulated in a lyophilized dosage form, it is preferably further reconstituted into a liquid dosage form before administration.

[0104] In the present invention, "reconstituted solution" refers to the solution used for reconstitution, and "reconstituted dosage form" refers to the final composition or dosage form after reconstitution of the pharmaceutical composition of the present invention.

[0105] In the present invention, the reconstitution solution may be an aqueous solution commonly used in the art, such as TAPS buffer, Bicine buffer, Tris buffer, Tricine buffer, TAPSO buffer, or HEPES buffer, or distilled water, but is not limited thereto.

[0106] Furthermore, the reconstituted solution may further contain a surfactant, salt, sugar, or amino acid to adjust the stability of the active ingredient contained in the dosage form after reconstitution, and when the reconstituted solution contains a surfactant, it may be characterized by containing it at a concentration of about 0.0001% to about 0.1%, preferably about 0.002% to about 0.07%, more preferably about 0.003% to about 0.05%, and most preferably about 0.004 to about 0.01%.

[0107] In the present invention, the surfactant is preferably, but not limited to, a polysorbate surfactant such as, for example, PS20 or PS80.

[0108] In the present invention, the ratio of the reconstitution solution can be adjusted to reconstitute the composition in the same, diluted, or concentrated form as before reconstitution.

[0109] In the present invention, the pharmaceutical composition of the present invention may be reconstituted into a composition in the same form as before reconstitution by adding a reconstitution solution so that the ratio (v:v) of the pharmaceutical composition to the dosage form after reconstitution is 1:1 by volume.

[0110] Alternatively, the pharmaceutical composition of the present invention may be reconstituted as a composition in a diluted form compared to before reconstitution by adding a reconstitution solution so that the ratio (v:v) of pharmaceutical composition to dosage form after reconstitution is 1:1.001 or more, 1:1.01 or more, 1:10.1 or more, 1:2 or more, 1:5 or more, or 1:10 or more by volume.

[0111] Alternatively, the pharmaceutical composition of the present invention may be reconstituted as a composition in a concentrated form compared to before reconstitution by adding a reconstitution solution so that the ratio (v:v) of pharmaceutical composition to the dosage form after reconstitution is 1.001:1 or less, 1.01:1 or less, 1.1:1 or less, 2:1 or less, 5:1 or less, or 10:1 or less by volume.

[0112] In the present invention, the dose or ratio of the reconstituted solution may be used based on the final concentration of heparan-N-sulfatase, which is the active ingredient in the dosage form after reconstitution.

[0113] In the present invention, the heparan-N-sulfatase concentration in the reconstituted dosage form may be about 2 mg / mL or more, about 5 mg / mL or more, about 10 mg / mL or more, about 15 mg / mL or more, about 20 mg / mL or more, about 25 mg / mL or more, or about 30 mg / mL or more. The heparan-N-sulfatase concentration in the reconstituted dosage form may be, but is not limited to, about 2 to about 60 mg / mL, preferably about 3 to about 40 mg / mL, more preferably about 5 to about 30 mg / mL, even more preferably about 8 to about 25 mg / mL, particularly preferably about 10 to about 20 mg / mL, and most preferably about 12 to about 15 mg / mL.

[0114] In yet another example of the present invention, the heparan N-sulfatase may be contained at a concentration of about 2 to about 30 mg / mL, preferably about 2 to about 20 mg / mL, and more preferably about 2 to about 15 mg / mL, but is not limited thereto.

[0115] The pharmaceutical dosage form of the present invention may be administered to the central nervous system by various administration methods. In the present invention, the pharmaceutical dosage form for central nervous system administration may be administered to the central nervous system by intracerebroventricular injection (ICV), intracerebral injection (IC), or intrathecal injection (IT), and is most preferably administered to the central nervous system by intracerebroventricular injection (ICV).

[0116] In the present invention, intraventricular injection refers to the administration of a drug by injection into the ventricles, which are connected open spaces in the brain. Intraventricular injection has the advantage of being able to inject into a wider area and deliver a larger amount of drug than intracerebral injection. Various techniques for intraventricular injection are known in the art, for example, the Ommaya reservoir developed by Ayub Ommaya as a traditional intraventricular injection device, which is continuously developed and reported, but is not limited thereto. Various intraventricular injection devices and techniques known in the art or developed in the future may be used without limitation for the intraventricular injection of the pharmaceutical composition of the present invention.

[0117] In the present invention, intracerebral injection refers to the injection of a drug into the brain tissue itself. Various methods for intracerebral injection are known in the art, for example, Mathon et al. 2015 describes intracerebral injection methods in detail.

[0118] In the present invention, intrathecal injection refers to injection into the spinal canal. Various techniques for intrathecal injection are known in the art, and representative intrathecal injection methods are described in detail in, for example, Lazorthes et al., Advances in Drug Delivery Systems and Applications in Neurosurgery, 143-192 and Omaya et al., Cancer Drug Delivery, 1:169-179.

[0119]

[0120] In the present invention, when the pharmaceutical composition or pharmaceutical dosage form is administered by intraventricular injection, the subject may have a certain amount of cerebrospinal fluid (CSF) drained from the ventricles before administration, which can prevent an increase in intracranial pressure due to a change in CSF volume after ICV administration.

[0121] Preferably, the total administration volume of the pharmaceutical composition or pharmaceutical dosage form of the present invention upon intracerebroventricular (ICV) administration may be, but is not limited to, 10 ml or less, preferably 5 ml or less, more preferably 3 ml or less, and most preferably 2 ml or less.

[0122] In the present invention, administration of the pharmaceutical composition or pharmaceutical dosage form to the central nervous system can provide delivery of heparan N-sulfatase to various target tissues, such as the brain, spinal cord, and periphery. In the present invention, the target tissue can include any tissue affected by the lysosomal storage disease being treated, for example, the target tissue can be a brain target tissue, a spinal cord target tissue, and / or a peripheral target tissue, and administration to the central nervous system can provide systemic delivery of heparan N-sulfatase.

[0123] In the present invention, administration of the pharmaceutical composition or pharmaceutical dosage form to the central nervous system can achieve therapeutically or clinically effective levels or activity in various target tissues described herein. As used herein, therapeutically or clinically effective levels or activity refer to levels or activity sufficient to provide a therapeutic effect in a target tissue. For example, a therapeutically or clinically effective level or activity may be an enzymatic level or activity sufficient to ameliorate a symptom associated with a disease (e.g., GAG accumulation) in a target tissue.

[0124] In the present invention, administration of the dosage form or pharmaceutical composition to the central nervous system can achieve an enzymatic level or activity that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the normal level or activity of heparan N-sulfatase in the target tissue. In the present invention, administration of the dosage form or pharmaceutical composition to the central nervous system can achieve an enzymatic level or activity that is increased by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to a control group (e.g., endogenous level or activity without treatment).

[0125]

[0126] In the present invention, administration of the pharmaceutical composition or pharmaceutical dosage form to the central nervous system can induce a decrease in GAG (e.g., heparan sulfate) storage in brain target tissues, spinal cord neurons, and / or peripheral target tissues. In the present invention, the GAG ​​storage can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, or 2-fold compared to a negative control group (e.g., GAG storage in a subject before treatment or after vehicle-only administration). In the present invention, administration of the pharmaceutical composition or pharmaceutical dosage form to the central nervous system can induce reduced vacuolization in neurons. For example, it can induce a decrease of at least 20%, 40%, 50%, 60%, 80%, 90%, 1-fold, 1.5-fold, or 2-fold or more compared to a negative control group.

[0127]

[0128] The pharmaceutical composition or pharmaceutical dosage form of the present invention may be administered in a pharmaceutically effective amount, where "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. The effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route, and excretion rate, treatment duration, concomitant drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, which may be administered sequentially or simultaneously with conventional therapeutic agents, and in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that will achieve maximum efficacy at the minimum dose without side effects, which can be easily determined by one of ordinary skill in the art.

[0129] Furthermore, the pharmaceutical composition or pharmaceutical dosage form of the present invention may be administered to a patient at an appropriate administration interval, preferably at least once a week, more preferably once a week, and most preferably once every two weeks, but is not limited thereto, and for improved patient convenience, it is preferable to administer at the fastest possible administration rate. For example, the administration rate of the pharmaceutical composition or pharmaceutical dosage form of the present invention may be, but is not limited to, about 0.1 ml / min or more, or about 0.5 ml / min or more, preferably about 1 ml / min or more, more preferably about 2 ml / min or more, and most preferably about 5 ml / min or more.

[0130] In yet another aspect, the present invention relates to a method for treating mucopolysaccharidosis IIIA, which comprises administering the pharmaceutical composition or pharmaceutical dosage form of the present invention to a patient in need of treatment, particularly a patient with mucopolysaccharidosis IIIA.

[0131] In yet another aspect, the present invention relates to the use of the pharmaceutical composition or pharmaceutical dosage form for the treatment of mucopolysaccharidosis type IIIA.

[0132] In yet another aspect, the present invention relates to the use of the pharmaceutical composition or pharmaceutical dosage form for the manufacture of a medicament for the treatment of mucopolysaccharidosis type IIIA.

[0133]

[0134] The present invention will be described in more detail below using examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and are not to be construed as limiting the scope of the present invention.

[0135]

[0136] Example 1. Stability of heparan N-sulfatase with buffer

[0137] Example 1-1: Comparison of stability depending on the type of amino acid buffer (DLS evaluation)

[0138] Based on previous reports that phosphate buffers negatively affect the activity of heparan N-sulfatase, DLS analysis was performed to evaluate the presence or absence of protein aggregation depending on the protein concentration when amino acid buffers, specifically histidine buffer, arginine buffer, or glutamate buffer, were used instead of phosphate buffer.

[0139] The amino acid concentration in each amino acid buffer was set to 20 mM, and stability was evaluated while varying the heparan N-sulfatase concentration from 2.52 to 12.6 mg / mL in 200 mM NaCl, pH 7.5 and 8.0.

[0140] The PDI value is calculated as follows:

[0141] PDI=σ 2 / d 2

[0142] σ: Standard deviation of the particle size distribution

[0143] d: The Average Hydrodynamic Particle

[0144]

[0145] The measured mean protein radius (Z-Ave) was determined by dynamic light scattering (DLS) of liquid samples of each formulation using an Uncle instrument, and 8.8 μL of sample was injected three times into a Uni sample loader (Unchained Labs).

[0146] As a result, as shown in Table 1, when histidine buffer was used, all dosage forms, from those containing low to high concentrations of HNS, showed polydispersity index (PDI) values, indicating monodisperse properties and stability of the protein without aggregation. However, when arginine and glutamate buffers were used, low-concentration HNS dosage forms of approximately 2.5 mg / mL were stable, but as the concentration increased above approximately 7.5 mg / mL, the PDI value showed a rapid increase, confirming that the higher the concentration of the dosage form, the greater the level of aggregation and the more the particle sizes became polydispersed, resulting in instability.

[0147] Furthermore, a pH of 8.0 showed a better effect than a pH of 7.5.

[0148] [Table 1]

[0149] Classification by PDI value:

[0150] <0.1: Low polydispersity (low degree of aggregation)

[0151] 0.1-0.25: Medium polydispersity

[0152] >0.25: High polydispersity (high degree of aggregation)

[0153]

[0154] In particular, as seen in Table 1 and Figures 1A and 1B above, when an arginine or glutamate buffer is used, if the heparan N-sulfatase concentration is about 7.5 mg / mL or higher, a Z-Ave value of more than 1,000 nm is shown, which indirectly confirms that protein aggregation occurs. This means that arginine or glutamate buffers are unsuitable for the composition of the present invention.

[0155] In contrast, when histidine buffer was used, a small Z-ave value of approximately 9 nm was observed even when the heparan N-sulfatase concentration was 7.5 mg / mL or higher, indicating that histidine buffer is highly suitable for the composition of the present invention and prevents protein aggregation.

[0156] The Z-Ave value may be expressed as the average protein radius and is measured using DLS equipment. In this patent, liquid samples of each formulation were analyzed using the Uncle instrument. Using the Uncle analysis method, 8.8 μL of sample was injected three times into a Uni sample loader (Unchained Labs). The histidine buffer consistently showed low Z-Ave values.

[0157]

[0158] Example 1-2: Comparison of stability depending on the type of amino acid buffer (KD and B22 evaluation)

[0159] In addition, when histidine buffer, arginine buffer, or glutamate buffer was used according to the composition shown in Table 2, protein-protein interaction and protein-buffer interaction were confirmed using KD and B22 (second virial coefficient) values ​​from the Uncle test, and the dosage form stability was evaluated.

[0160] The KD value is a numerical value that indicates the degree of interaction between proteins, with negative values ​​indicating instability and positive values ​​indicating stability. The B22 value is a variable that indicates colloidal stability (B22), with a positive value indicating a stronger repulsion force between proteins, reducing the probability of aggregation and resulting in a more stable formulation.

[0161] [Table 2]

[0162] As a result, as shown in Figures 2A and 2B, when arginine buffer or glutamate buffer was used, low kD and B22 values ​​were observed, indicating significantly poor stability, while when histidine buffer was used, relatively high kD and B22 values ​​were observed at both pH 7.5 and 8.0, indicating that the dosage form using histidine buffer has excellent stability.

[0163]

[0164] Examples 1-3: Stability of histidine buffers compared to phosphate buffers

[0165] Based on previous reports that phosphate buffer negatively affects the activity of heparan N-sulfatase, when phosphate buffer was replaced with histidine, the protein-buffer interaction was confirmed by Uncle test to evaluate the formulation stability.

[0166] The composition used in the test contained HNS 8 mg / ml; histidine 20 mM, NaCl 154 mM, phosphate 6.7 mM, and NaCl 200 mM.

[0167]

[0168] As a result, it was confirmed that the B22 value significantly increased in the dosage form using histidine buffer, as shown in Figure 3. These results suggest that when histidine buffer is used, protein-buffer interactions are significantly reduced compared to when phosphate buffer is used, resulting in a more stable dosage form.

[0169]

[0170] Example 2. Stability evaluation by pH

[0171] The stability of heparan N-sulfatase compositions containing histidine buffer was evaluated by determining turbidity as a function of pH.

[0172] In the present invention, turbidity was analyzed using Lunatic (Unchained Labs). 2.0 μL of sample was injected into a Lunatic plate (Unchained Labs), and the turbidity was measured at 350 nm. The increase in the turbidity value of the sample was calculated based on the turbidity value of the placebo buffer.

[0173]

[0174] As a result, as can be seen from Tables 3 to 5 and FIG. 4, the compositions containing a low concentration of heparan N-sulfatase (5 mg / mL) had low turbidity over the entire pH range.

[0175] In the low pH range, compositions containing high concentrations of heparan N-sulfatase (13.5 mg / mL) showed a significant increase in turbidity compared to compositions containing low concentrations of heparan N-sulfatase (5 mg / mL).

[0176] However, in compositions containing high concentrations of HNS, the turbidity tended to decrease as the pH increased, and the turbidity decreased significantly particularly when the pH was 7.8 or higher, preferably 7.9 to 8.0 or higher, demonstrating that the pH of the composition has a significant effect on reducing turbidity.

[0177] [Table 3]

[0178] [Table 4]

[0179] [Table 5]

[0180] In particular, in the case of a composition containing a very high concentration of HNS (16 to 17 mg / mL) and 120 mM NaCl, as shown in Figure 5, it was confirmed that the turbidity decreased as the pH increased, and that the turbidity decreased significantly especially when the pH was 7.9 to 8.0 or higher.

[0181]

[0182] Example 3. Evaluation of stability with and without the addition of surfactant

[0183] Example 3-1: Confirmation of changes due to addition of surfactant using Aura equipment

[0184] To more specifically confirm the decrease in turbidity when a lower concentration of surfactant was added, the pH was set to 8.2, 0.005 w / v% polysorbate 20 was added, and the turbidity was measured using an Aura device.

[0185] The composition used in Example 3-1 contained 15 mg / mL HNS, 5 mM histidine buffer, 125 mM NaCl, and 1.8 w / v% trehalose. Experiments were conducted on the composition after it was freeze-dried and then reconstituted with a solution containing PS20 (0.005%), and on the composition after it was freeze-dried and then reconstituted with PS20 (0.005%).

[0186] Turbidity was measured using an Aura device as follows:

[0187] After placing an empty plate and performing "Acquire Background" to measure the background, 30-50 μl of sample was loaded into each well of the plate in triplicate. The cassette with the drying paper was placed on the manifold, and the plate that had undergone the primary vacuum process was placed on top of it. The vacuum valve was then opened to perform secondary drying. Once the vacuum process was complete, the plate was placed in the Aura equipment for measurement. This equipment is based on a 96-well plate and can digitize and graph information on particles 1 μm or larger in size within 30-50 μL of sample compared to the blank. To do this, the plate was vacuum treated and sampled using drying paper.

[0188] As a result, as shown in Figures 6A to 6C, it was confirmed that the turbidity was significantly reduced both when 0.005% polysorbate 20 was added before freeze-drying and when it was added at the time of reconstitution after freeze-drying.

[0189] Example 3-2: Confirmation of the number of insoluble particles by adding a surfactant

[0190] To further verify and confirm the stability of the dosage form due to the addition of a surfactant, the change in the number of insoluble fine particles was confirmed when 0.005% polysorbate 20 was added as a surfactant.

[0191] Each composition used in Example 3-2 contained 15.3 mg / ml HNS, 5 mM histidine, 125 mM NaCl, and 1.8 w / v% trehalose, and had a pH of 8.0. The only difference between the compositions tested was the presence or absence of added PS20 (0.005 w / v%).

[0192]

[0193] As a result of the experiment, it was confirmed that the number of insoluble particles having sizes of 10 μm or more and 25 μm or more was significantly reduced when a surfactant (PS20) was added, as shown in Table 6 below. This result indirectly indicates that the addition of a surfactant improves the stability of the pharmaceutical composition according to the present invention.

[0194] [Table 6]

[0195]

[0196] Example 4. Evaluation of stability depending on salt concentration

[0197] To evaluate the stability of the composition depending on the salt concentration contained in the composition, the pH was set to 7 to 8, and the stability of the composition was confirmed while changing the salt (NaCl) concentration from 100 to 200 mM.

[0198] The composition used in the test in Example 4 contained 12.6 mg / mL HNS and 20 mM histidine. The kD value was used as an evaluation measure of stability, and the kD value was measured in the same manner as in Example 1-1 of the present specification.

[0199] As a result, as shown in Figure 7, it was confirmed that the stability of the composition was low at all NaCl concentrations at pH 7.0, but when the NaCl concentration was 150 mM, it was evaluated as having excellent stability at pH 8, and when the NaCl concentration was 200 mM, it was confirmed that the stability was significantly improved at pH 7.0 or above, especially at 7.5 or above. This result indicates that the stability of high-concentration HNS formulations increases as the NaCl concentration and pH increase.

[0200]

[0201] Furthermore, the change in osmotic pressure due to NaCl concentration was investigated. The average osmotic pressure was 286 and 294 mOsmol / kg when NaCl was added at 125 mM and 130 mM, respectively, confirming that the change in osmotic pressure due to NaCl concentration was not significant. This range of osmotic pressure is generally within the acceptable range for pharmaceutical compositions (Table 7).

[0202] [Table 7]

[0203]

[0204] Example 5. Evaluation of stability based on sugar concentration

[0205] Example 5-1: Stability evaluation (purity) when trehalose is added at 1% or less

[0206] Sugars such as trehalose are primarily used as stabilizers during freeze-drying. In this study, the stability of the composition, particularly after reconstitution in a freeze-dried dosage form, was evaluated from the purity perspective, depending on the sugar concentration in the composition. The stability of the freeze-dried dosage form after reconstitution was confirmed by varying the trehalose concentration from 0 to 1% (v / w).

[0207] Prior to lyophilization, the concentrations of the components in the prepared pharmaceutical composition are as shown in Table 8 below:

[0208] [Table 8]

[0209] For freeze-drying, the liquid composition solution prepared according to the present invention was dispensed into glass vials (3 ml size) in 1.3 ml portions, semi-sealed with rubber stoppers, and then loaded onto the shelves of a freeze-dryer (Lyostar 3, SP Scientific). Freeze-drying was then carried out under the conditions listed in Table 9, and the freeze-dried formulations thus prepared were capped with aluminum caps after completion of aluminum freeze-drying.

[0210] [Table 9]

[0211] The stability of the lyophilized formulations was evaluated by reconstitution with distilled water (DW) and then purity analysis. The pharmaceutical compositions (5-1-a to -f) in Table 8 were lyophilized and reconstituted using 0.286 mL to 0.75 mL of distilled water according to the volume ratio (reconstitution ratio) of pharmaceutical composition to reconstituted dosage form to produce reconstituted dosage forms (5-1-A to -F) (Table 10).

[0212] [Table 10]

[0213] Purity was assessed using size exclusion liquid chromatography (SE-HPLC), a standard method for confirming and quantifying aggregation and fragment levels. Specifically, for size exclusion chromatography, 1 mg / mL HNS was first diluted to 1.0 mg / mL using a mobile phase (40 mM sodium phosphate, 300 mM NaCl, pH 7.5) (no dilution was required for concentrations below 1.0 mg / mL), followed by sterile filtration. 200 μL of the filtered sample was then injected into a vial insert and inserted into a screw-top vial.

[0214] After connecting the mobile phase to the pump, an analytical column (TSKgel G3000SWXL, Tosoh) was attached to a Waters e2695 and Waters 2489 instrument (Waters Japan) while the mobile phase was flowing at a flow rate of 0.5 mL / min. The mobile phase was flowed at a rate of 0.5 mL / min for over 30 minutes to allow equilibration until the detector signal stabilized. Once the autosampler temperature had dropped to 4°C, the sample was inserted into the sampler. After injecting 30 μL of the sample, the mobile phase was flowed for 35 minutes, and the detection peak was confirmed at 280 nm. Analysis was then performed using Empower Pro software on a PC.

[0215] As a result, as shown in Table 11 and Figure 8, the purity was approximately 88% when no trehalose was present, whereas when the trehalose concentration before reconstitution was 0.1 w / v% or higher, the purity improved to approximately 95% or higher.

[0216] [Table 11]

[0217]

[0218] Example 5-2: Stability evaluation (potency and purity) when trehalose is added at 1% or more

[0219] To evaluate the stability of lyophilized formulations containing 1% (w / v) or more trehalose after reconstitution, the trehalose concentration was set to 1.35 w / v% based on the concentration before lyophilization, and the specific activity (SA) and purity of HNS were evaluated at various pH levels.

[0220] The compositions of the prepared dosage form samples are shown in Table 12 below.

[0221] [Table 12]

[0222] To the DS (stock solution), HNS, histidine, NaCl, and trehalose were added to produce pharmaceutical compositions 5-2-a to 5-2-e, which were then lyophilized and reconstituted to produce reconstituted dosage forms 5-2-A to E. The volume ratio of the pharmaceutical composition to the reconstituted dosage form (v:v) was 4:3.

[0223] In the present invention, enzyme activity analysis was carried out by the following method.

[0224] - In the first step, the dosage form sample was reacted with the synthetic substrate 4MU-α-GlcNS to release the terminal sulfate of the substrate (4MU-α-GlcNH2 formation).

[0225] One-step reaction: 4MU-α-GlcNS + HNS → 4MU-α-GlcNH2

[0226] In the second step, α-glucosidase was used to release fluorescent 4MU from 4MU-α-GlcNH2. The fluorescence intensity of the released 4MU was measured using a fluorescence reader, and the enzymatic activity of heparan N-sulfatase in the sample was determined.

[0227] Two-step reaction: 4MU-α-GlcNH2 + α-glucosidase → 4MU

[0228]

[0229] The specific methods for the one-step and two-step reactions are as follows:

[0230] The dosage form sample was diluted to 100 μg / mL with substrate diluent (Michaelis barbital sodium acetate buffer: 29 mM sodium barbital / 29 mM sodium acetate / 0.68% NaCl / 0.02% NaN3, pH 6.5), and the substrate was diluted with the substrate diluent.

[0231] - One-step reaction: 20 μL of serially diluted substrate was dispensed into each well of a 96-well black plate. 10 μL of the sample diluted to 100 μg / mL and blank (sample dilution solution) were added to opposite sides of the wells, taking care not to mix with the substrate solution. One side of the plate was then gently tapped (to prevent the solution from splashing out of the well) to ensure the solutions were mixed thoroughly. The plate was then sealed using a plate sealer and incubated in a 37°C incubator for 17 hours.

[0232] - Two-step reaction: After the first-step reaction (17 hours), 6 μL of the first-step reaction stop solution was added per well to stop the first reaction. One side of the plate was picked up and gently tapped on the other side (to prevent the solution from splashing out of the wells) to mix the solution well. α-glucosidase solution, prepared at 100 U / mL, was diluted 10 times to 10 U / mL using ultrapure water. After adding 10 μL per well, one side of the plate was picked up and gently tapped on the other side (to prevent the solution from splashing out of the wells) to mix the solution well. The plate was sealed using a plate sealer and incubated in a 37°C incubator for 24 hours.

[0233] - Fluorescence measurement: 15 minutes before fluorescence measurement, 1.5 mL of substrate dilution, 300 μL of primary reaction stop solution, 500 μL of ultrapure water, and 10 mL of secondary reaction stop solution were mixed in order to prepare a 4MU diluted solution. 4MU stock was diluted with the 4MU diluted solution as shown in Table 6 to prepare a 4MU standard solution. After the secondary reaction, 200 μL of stop solution was added to each well to terminate the reaction. 4MU standards (standards 1-8) were loaded in duplicate, each at 246 μL per well. Fluorescence was measured using a fluorometer at Ex. 355 nm / Em. 460 nm.

[0234] The purity was measured by size exclusion liquid chromatography (SE-HPLC) in the same manner as above.

[0235]

[0236] As a result, as shown in Table 13 below and Figures 9A and 9B, when the trehalose concentration in the composition, i.e., the lyophilized dosage form, was 1 w / v% or more, the HNS titer was measured to be about 400 (pmol / min / μl) or more, and the purity was confirmed to be about 98% or more in all cases. This confirmed that the composition maintained high stability even when manufactured into a lyophilized dosage form, and also after reconstitution.

[0237] [Table 13]

[0238]

[0239] Example 6. Potency and Purity Verification of Compositions After Reconstitution of Various Compositions

[0240] Various compositions within the range of the present invention were prepared and their stability was confirmed in terms of potency and purity after lyophilization and reconstitution.

[0241] The concentrations of each component in the pharmaceutical composition (before lyophilization) used in the test are as shown in Table 14 below:

[0242]

[0243]

[0244] [Table 14]

[0245] *6-1 and 6-2 are pharmaceutical composition:reconstituted dosage form ratio (v:v) = 1:0.75

[0246] 6-3 to 6-10 are pharmaceutical compositions:reconstituted dosage forms ratio (v:v) = 1:1

[0247] The purity was measured by the same method as described in Example 5-1, and the titer was measured by the same method as described in Example 5-2.

[0248] The composition and measurement results are shown in Table 15 below.

[0249] [Table 15]

[0250] It was confirmed that all of the samples 6-1 to 6-10 exhibited a high purity of 98% or more and a high titer of 690 pmol / min / ug or more.

[0251] [Industrial Applicability]

[0252] The pharmaceutical composition containing a high concentration of heparan N-sulfatase (HNS) of the present invention and the pharmaceutical dosage form containing the same are useful for enzyme replacement therapy (ERT) for treating mucopolysaccharidosis type IIIA. This is because the phosphate buffer, which is known to inhibit the activity of the active ingredient heparan N-sulfatase, is replaced with a histidine buffer, thereby reducing protein-protein or protein-buffer interactions and turbidity, thereby significantly improving dosage form stability.

[0253]

[0254] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. 2 to 50 mg / mL heparan N-sulfatase (HNS); a 1-40 mM histidine buffer; A pharmaceutical composition characterized by having a pH of 7.8 to 9.

0.

2. 2. The pharmaceutical composition according to claim 1, wherein the heparan N-sulfatase is contained at a concentration of 5 mg / ml to 30 mg / ml.

3. 2. The pharmaceutical composition according to claim 1, wherein the histidine buffer is contained at a concentration of 2 to 20 mM.

4. The pharmaceutical composition according to claim 1, wherein the pH is 8.0 to 8.

8.

5. The pharmaceutical composition of claim 1 , further comprising a sugar.

6. 6. The pharmaceutical composition according to claim 5, wherein the sugar is at least one selected from the group consisting of trehalose, sucrose, maltose, lactose, and sorbitol.

7. The pharmaceutical composition according to claim 6, wherein the sugar is contained at a concentration of 0.1% to 5.0% w / v.

8. The pharmaceutical composition of claim 5, further comprising a salt.

9. 2. The pharmaceutical composition according to claim 1, wherein the salt is at least one selected from the group consisting of NaCl and KCl.

10. 10. The pharmaceutical composition according to claim 9, wherein the salt is contained in a concentration of 30 mM to 500 mM.

11. 10. The pharmaceutical composition of claim 1, further comprising a surfactant.

12. 12. The pharmaceutical composition according to claim 11, wherein the surfactant is polysorbate 20 or polysorbate 80.

13. The pharmaceutical composition according to claim 12, wherein the surfactant is contained at a concentration of 0.0001 to 0.1 w / v %.

14. 5-30 mg / mL heparan N-sulfatase; 2-20 mM histidine buffer; 0.5 to 3 w / v% trehalose; 70-150 mM NaCl; 2. The pharmaceutical composition according to claim 1, characterized in that the pH is 8.0 to 8.

8.

15. 2. The pharmaceutical composition of claim 1, characterized in that it has a turbidity of less than 1.0 when measured at 350 nm.

16. 2. The pharmaceutical composition according to claim 1, characterized in that it is used for the treatment of mucopolysaccharidosis type IIIA.

17. A pharmaceutical dosage form comprising the pharmaceutical composition of any one of claims 1 to 16.

18. 18. Pharmaceutical dosage form according to claim 17, characterized in that it is a liquid dosage form or a lyophilized dosage form.

19. 18. The pharmaceutical dosage form of claim 17, which is administered to the central nervous system by intracerebroventricular injection (ICV), intracerebral injection, or intrathecal injection.

20. 19. The pharmaceutical dosage form of claim 18, wherein the lyophilized dosage form contains 2 to 60 mg / mL of heparan N-sulfatase after reconstitution.

21. 20. The pharmaceutical dosage form of claim 19, characterized in that the total dose volume is 10 ml or less.

22. 22. The pharmaceutical dosage form according to claim 21, characterized in that the administration rate is 0.5 ml / min or more.

Images