Pharmaceutical composition comprising heparan n-sulfatase with improved stability

Abstract

The present invention relates to a high concentration heparan N-sulfatase (HNS) pharmaceutical composition with enhanced stability for CNS delivery and a pharmaceutical formulation comprising the same. The pharmaceutical composition and the pharmaceutical formulation comprising the same according to the present invention have excellent formulation stability, such as reduction of turbidity and significant improvement of purity, which can be used in enzyme replacement therapy (ERT) for the treatment of type III mucopolysaccharide storage disease.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition comprising a high concentration of heparan N-sulfatase (HNS) with improved stability, and a pharmaceutical preparation comprising the same. More specifically, the present invention relates to a pharmaceutical composition having a pH of at least 7.8 and comprising a high concentration of heparan N-sulfatase and a histidine buffer, and a pharmaceutical preparation comprising the same. Background Art

[0002] Lysosomal storage diseases (LSDs) are genetic metabolic diseases caused by defects in lysosomal function. Lysosomal storage diseases are caused by lysosomal dysfunction due to the absence of one or more enzymes required for lipid, glycoprotein, or mucopolysaccharide metabolism. The absence 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). Mucopolysaccharidosis (MPS) is one type of lysosomal storage disease, which results from the accumulation within lysosomes due to the absence of lysosomal enzymes required for glycosaminoglycan degradation. Generally, mucopolysaccharidosis is classified into types I to VII according to the type of enzyme deficiency.

[0003] Enzyme replacement therapy (ERT) is one of the main treatment approaches for treating lysosomal storage diseases, which involves administering a defective lysosomal enzyme to correct the enzyme functional defect. The advantage of this simple injection therapy is to reduce symptoms and thus prevent permanent damage to the body. As a well-known enzyme replacement therapy for enzyme accumulation diseases, intravenous (IV) therapy with glucocerebrosidase (GCase) for Gaucher disease was first approved and used by the FDA in 1991 (National Gaucher Foundation. Retrieved 2017-06-08).

[0004] However, in view of the fact that many lysosomal storage diseases cause excessive accumulation of glycosaminoglycans (GAGs) in the nervous system (especially in neurons of the brain and the meninges of the spinal cord), leading to various central nervous system diseases, and the enzyme replacement therapy administered intravenously cannot sufficiently deliver the enzyme to the central nervous system because it is difficult for the active ingredient, i.e., the lysosomal enzyme, to cross the blood-brain barrier (BBB), especially it cannot effectively treat the nervous system disorders and diseases caused by lysosomal accumulation, especially the nervous system disorders and diseases in the brain. Therefore, in order to bypass the BBB to deliver the enzyme, various CNS delivery therapies are being studied to directly deliver drugs to the central nervous system.

[0005] A variety of therapies have been developed to bypass the BBB and deliver enzymes to the central nervous system. In particular, injection therapies for directly delivering proteins to the brain include intracerebral injection (IC), intracerebroventricular injection (ICV), and intrathecal injection (IT).

[0006] Intrathecal (IT) and intracerebroventricular (ICV) injections have emerged as methods for delivering replacement enzymes to the central nervous system for mucopolysaccharidosis (MPS) and have shown significant reduction of glycosaminoglycans (GAG) and significant improvement of neurological symptoms in various animal models of mucopolysaccharidosis (Molecular Therapy-Methods & Clinical Development, 21, 67 - 75). However, the dosing capacity of therapies that are directly injected into the brain is very limited, and thus the development of injectable formulations containing high concentrations of enzymes is crucial for achieving effective levels of therapeutic benefit.

[0007] Numerous high-concentration enzyme formulations for enzyme replacement therapy (ERT) via CNS delivery have been reported, and the formulations reported to date for CNS delivery generally utilize phosphate buffer as a buffer (Korean Patent Nos. 2,007,044, and 2,272,399).

[0008] However, in compositions for CNS delivery of heparan N-sulfatase (HNS), studies have consistently reported that the use of phosphate buffer has a negative impact on the activity of heparan N-sulfatase (J Inherit Metab Dis. 1993; 16(2):465 - 472; Acta Crystallogr D Biol Crystallogr. 2014 May; 70(Pt 5):1321 - 1335).

[0009] Therefore, there is an urgent need for pharmaceutical compositions and pharmaceutical formulations for CNS delivery that contain heparan N-sulfatase, particularly heparan N-sulfatase with high concentration and high stability.

[0010] Against this background, the present inventors sincerely endeavored to develop a pharmaceutical composition for CNS delivery of high-concentration heparan N-sulfatase (HNS) without using phosphate buffer and a pharmaceutical formulation containing the same, and found that using histidine buffer as a buffer and setting the pH to 7.8 or higher significantly improved the stability of HNS.

[0011] The above description of the background art is only intended to enhance the understanding of the background of the present invention and may not include information in the prior art known to those of ordinary skill in the art. Summary of the Invention

[0012] One object of the present invention is to provide a pharmaceutical composition comprising heparan N-sulfatase (HNS) and a pharmaceutical preparation having improved stability.

[0013] Another object of the present invention is to provide a method for treating mucopolysaccharidosis type IIIA using the pharmaceutical composition or the pharmaceutical preparation.

[0014] Another object of the present invention is to provide the use of the pharmaceutical composition or the pharmaceutical preparation for treating mucopolysaccharidosis type IIIA.

[0015] Another object of the present invention is to provide the use of the pharmaceutical composition or the pharmaceutical preparation in the manufacture of a medicament for treating mucopolysaccharidosis type IIIA.

[0016] To achieve the above object, the present invention provides a pharmaceutical composition and a pharmaceutical preparation comprising heparan N-sulfatase (HNS) and a histidine buffer, having a pH of at least 7.8.

[0017] The present invention also provides a pharmaceutical composition for treating mucopolysaccharidosis type IIIA, comprising heparan N-sulfatase and a histidine buffer, having a pH of at least 7.8, and a pharmaceutical preparation comprising the same, and a method for treating mucopolysaccharidosis type IIIA using the same.

[0018] The present invention also provides the use of the pharmaceutical composition or the pharmaceutical preparation for treating mucopolysaccharidosis type IIIA.

[0019] The present invention also provides the use of the pharmaceutical composition or the pharmaceutical preparation in the manufacture of a medicament for treating mucopolysaccharidosis type IIIA. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 is a graph showing the change in the stability of the pharmaceutical composition according to the type of amino acid buffer and the following pH values:

[0021] (a) pH 7.5

[0022] (b) pH 8.0

[0023] FIG. 2 is a graph showing the change in the stability of the pharmaceutical composition according to the type of amino acid buffer, using the B22 value or the kD value.

[0024] (a) B22 value

[0025] (b) kD value

[0026] Figure 3 is a graph showing the comparison results of the stability of the pharmaceutical composition when using a histidine buffer and a phosphate buffer.

[0027] Figure 4 It is a graph showing the change in turbidity of the pharmaceutical composition with pH.

[0028] Figure 5 It is a graph showing the change in turbidity of the pharmaceutical composition with pH.

[0029] Figure 6 is a graph depicting the results of observing the change in turbidity of the pharmaceutical composition according to the addition of surfactant and pH.

[0030] (a) Comparison results of foreign object images after reconstructing the HNS-containing formulation in the absence of PS20 / presence of 0.005% PS20.

[0031] (b) Foreign objects drawn according to size after reconstructing the HNS-containing formulation in the absence of PS20 / presence of 0.005% PS20.

[0032] (c) Foreign objects drawn according to size after reconstructing the HNS-containing formulation in the absence of PS20 / presence of 0.005% PS20.

[0033] Figure 7 It is a graph showing the stability of the pharmaceutical composition according to the NaCl concentration.

[0034] Figure 8 It is a graph showing the change in the purity of HNS in the composition according to the trehalose concentration.

[0035] Figure 9 is a graph showing the change in specific activity (S.A.) and purity of HNS in the composition with the trehalose concentration. Detailed Description

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the terms used herein are well-known and commonly used in the art.

[0037] Heparan-N-sulfatase (HNS) is a lysosomal enzyme that catalyzes the hydrolysis of heparan sulfate and cleaves the N-linked sulfate group from the non-reducing terminal glucosamine moiety of heparan (Biochem. Biophys. Res. Commun. 2001, 280, 1251-1257). It is well known that mutations in the heparan N-sulfatase gene (SGSH) cause mucopolysaccharidosis 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 heparan sulfatase (HNS), an enzyme 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, leading to severe nerve damage and abnormalities.

[0038] Although formulations for central nervous system delivery of heparan N-sulfatase containing phosphate have been reported (e.g., Korean Patent No. 2,007,044), studies have consistently reported a negative impact of phosphate buffer on 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 of heparan N-sulfatase for central nervous system delivery using alternative phosphate stabilizers.

[0039] In one embodiment of the present invention, the inventors have confirmed that a high-concentration heparan N-sulfatase formulation prepared using a histidine buffer has better stability compared to formulations containing phosphate, with significantly increased protein-protein or protein-buffer stability and significantly reduced turbidity.

[0040] Accordingly, in one aspect, the present invention provides a pharmaceutical composition comprising 2 mg / mL to 50 mg / mL of heparan N-sulfatase (HNS) and 1 to 40 mM of a histidine buffer having a pH of 7.8 to 9.0.

[0041] As used herein, "heparan N-sulfatase" may be used interchangeably with N-sulfo-glucosamine sulfohydrolase (SGSH).

[0042] In the present invention, heparan N-sulfatase can be characterized by having a wild-type or naturally occurring amino acid sequence. For example, heparan N-sulfatase can be characterized by being derived from various organisms, more preferably from humans, but is not limited thereto.

[0043] Heparan N-sulfatase can be any molecule or part of a molecule that can substitute for the protein activity of naturally occurring heparan N-sulfatase (HNS) or rescue one or more phenotypes or symptoms associated with HNS deficiency. Alternative enzymes suitable for the present invention are polypeptides having an N-terminus and a C-terminus and an amino acid sequence that is substantially similar or identical to the mature human HNS protein.

[0044] Typically, human HNS is prepared as a precursor molecule that is processed to form its mature form. This processing is usually accomplished by removing 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 20 N-terminal amino acids are cleaved to form the mature form, which is 482 amino acids in length. Thus, the 20 N-terminal amino acids are generally not considered essential for HNS protein activity. The amino acid sequences of the mature form (SEQ ID NO.1) and full-length precursor (SEQ ID NO.2) of a typical wild-type or naturally occurring human HNS protein are shown below.

[0045] Mature form of HNS: RPRNALLLLA DDGGFESGAY NNSAIATPHL DALARRSLLF RNAFTSVSSCSPSRASLLTG LPQHQNGMYG LHQDVHHFNS FDKVRSLPLL LSQAGVRTGI IGKKHVGPET VYPFDFAYTEENGSVLQVGR NITRIKLLVR KFLQTQDDRP FFLYVAFHDP HRCGHSQPQY GTFCEKFGNG ESGMGRIPDWTPQAYDPLDV LVPYFVPNTP AARADLAAQY TTVGRMDQGV GLVLQELRDA GVLNDTLVIF TSDNGIPFPSGRTNLYWPGT AEPLLVSSPE HPKRWGQVSE AYVSLLDLTP TILDWFSIPY PSYAIFGSKT IHLTGRSLLPALEAEPLWAT VFGSQSHHEV TMSYPMRSVQ HRHFRLVHNL NFKMPFPIDQ DFYVSPTFQD LLNRTTAGQPTGWYKDLRHY YYRARWELYD RSRDPHETQN LATDPRFAQL LEMLRDQLAK WQWETHDPWV CAPDGVLEEKLSPQCQPLHN EL(SEQ ID NO.1)

[0046] Full-length HNS precursor: MSCPVPACCA LLLVLGLCRA RPRNALLLLA DDGGFESGAY NNSAIATPHLDALARRSLLF RNAFTSVSSC SPSRASLLTG LPQHQNGMYG LHQDVHHFNS FDKVRSLPLL LSQAGVRTGIIGKKHVGPET VYPFDFAYTE ENGSVLQVGR NITRIKLLVR KFLQTQDDRP FFLYVAFHDP HRCGHSQPQYGTFCEKFGNG ESGMGRIPDW TPQAYDPLDV LVPYFVPNTP AARADLAAQY TTVGRMDQGV GLVLQELRDAGVLNDTLVIF TSDNGIPFPS GRTNLYWPGT AEPLLVSSPE HPKRWGQVSE AYVSLLDLTP TILDWFSIPYPSYAIFGSKT IHLTGRSLLP ALEAEPLWAT VFGSQSHHEV TMSYPMRSVQ HRHFRLVHNL NFKMPFPIDQDFYVSPTFQD LLNRTTAGQP TGWYKDLRHY YYRARWELYD RSRDPHETQN LATDPRFAQL LEMLRDQLAKWQWETHDPWV CAPDGVLEEK LSPQCQPLHN EL(SEQ ID NO.2)

[0047] In the present invention, heparin N-sulfatase can be characterized by a recombinantly produced recombinant enzyme. The recombinant production of heparin N-sulfatase can be readily achieved using techniques known in the art for producing recombinant cells expressing various target proteins.

[0048] In the present invention, heparin N-sulfatase can also be included in the form of a fusion protein or conjugate. In the present invention, heparin N-sulfatase can be fused or conjugated with a moiety capable of binding to a receptor on the surface of a brain cell and / or a lysosome-targeting molecule to facilitate cellular uptake or lysosome targeting. Modifications of alternative enzymes such as heparin N-sulfatase are disclosed in Korean Patent No. 2,007,044, etc.

[0049] In the present invention, the amount of heparin N-sulfatase included can 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.

[0050] In another example of the present invention, the concentration of heparan N-sulfatase included is about 2 to about 50 mg / mL, preferably about 3 to about 40 mg / mL, more preferably about 5 to about 30 mg / mL, more preferably about 8 to about 25 mg / mL, more preferably about 10 to about 20 mg / mL, and most preferably about 12 to about 15 mg / mL, but is not limited thereto.

[0051] In another example of the present invention, the concentration of heparan N-sulfatase included is about 2 to about 20 mg / mL, preferably about 2 to about 16.5 mg / mL, more preferably about 2 to about 15 mg / mL, but is not limited thereto.

[0052] In addition, histidine buffer is a buffer that offers many advantages compared to conventional phosphate buffers, such as significantly increased stability due to reduced protein-protein and protein-buffer interactions. In the composition according to the present invention, the amount of histidine buffer included can be 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 of the histidine buffer is the concentration calculated based on the concentration of histidine.

[0053] In another example according to the present invention, the amount of histidine buffer included can be 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.

[0054] In the composition according to the present invention, it has been found that when the pH is higher than about 7.8, the electrostatic repulsion between protein-protein or protein-buffer increases, and the turbidity also significantly decreases, thus significantly increasing the stability of the composition.

[0055] Therefore, the pH of the composition according to the present invention can be at least about 7.8, 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.

[0056] In the present invention, the composition according to the present invention exhibits low turbidity. As used herein, the term "turbidity" refers to the degree to which the composition is clouded by soft substances or impurities in the composition. In pharmaceutical compositions, turbidity is a parameter indicating the stability of the drug. For example, in less stable formulations, aggregates can form due to protein-protein interactions or protein-buffer attraction and self-association, which can lead to an increase in turbidity.

[0057] In the present invention, the turbidity (T) can be calculated from the absorbance measured at a specific wavelength. The calculation of turbidity follows the Beer-lambert law. T = I / I0 (T: transmittance, I: transmitted light intensity, I0: incident light intensity). In one example of the present invention, the turbidity is measured at 350 nm using Lunatic (Unchained Labs), but is not limited thereto.

[0058] In the pharmaceutical composition according to the present invention, the turbidity measured at 350 nm is 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, but is not limited thereto.

[0059] Furthermore, it has been found in the present invention that the inclusion of saccharides (especially trehalose) causes significantly higher purity (%) and specific activity (S.A.) not only when the pharmaceutical composition is used as a liquid preparation but also when formulated and reconstituted into a lyophilized preparation. Accordingly, the composition according to the present invention contains saccharides.

[0060] In the present invention, the sugar can be trehalose, sucrose, maltose, lactose, or sorbitol.

[0061] In the present invention, the concentration of the included sugar is 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, and more specifically is about 0.1% to about 5.0%, preferably about 0.3% to about 4.0%, more preferably about 0.4% to about 3.5%, more preferably about 0.5% to about 3.0%, and more preferably about 1.0% to 2.0%.

[0062] In another example of the present invention, the concentration of the included saccharides is about 0.1% to about 3%.

[0063] In the present invention, unless otherwise specified, the % concentration of each substance refers to w / v%.

[0064] The composition according to the present invention contains salt. According to the present invention, the salt is NaCl or KCl. In the present invention, the amount of the included salt is about 30 mM to about 500 mM, preferably about 50 mM to about 300 mM, more preferably about 60 mM to about 200 mM, more preferably about 70 mM to 150 mM, and most preferably about 70 mM to 120 mM

[0065] In another example of the present invention, the amount of the included salt is about 30 mM to about 300 mM.

[0066] In the present invention, the concentration of the salt comprised can be such that it has an appropriate osmolarity for central nervous system delivery of the pharmaceutical composition of the present invention. Appropriate osmolarities for pharmaceutical formulations for central nervous system delivery are well known in the art.

[0067] In the present invention, the osmolarity of the pharmaceutical composition can be, for example, about 400 mOsmol / kg or lower, preferably about 350 mOsmol / kg or lower, more preferably about 330 mOsmol / kg or lower, more preferably about 300 mOsmol / kg or lower, and most preferably about 290 mOsmol / kg or lower, but is not limited thereto. In the present invention, the osmolarity of the pharmaceutical formulation can be, for example, 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, but is not limited thereto.

[0068] In one embodiment of the present invention, it was found that adding polysorbate 20 as a surfactant reduced the turbidity compared to a formulation without a surfactant.

[0069] Accordingly, the pharmaceutical composition according to the present invention can be characterized in that it further comprises a surfactant. In the present invention, the surfactant can be characterized in that it is a polysorbate-based surfactant, more preferably polysorbate 20 or polysorbate 80, and most preferably polysorbate 20. In the composition according to the present invention, the concentration of the surfactant comprised is from about 0.0001% to about 0.1%, preferably from about 0.002% to about 0.07%, more preferably from about 0.003% to about 0.05%, and most preferably from about 0.004% to about 0.01%.

[0070] However, when the pharmaceutical composition according to the present invention is formulated as a lyophilized formulation, reconstituted and administered to a patient, the surfactant can be used in a form not included in the lyophilized pharmaceutical composition or formulation, but in the solution used for reconstitution.

[0071] In addition to heparan N-sulfatase (HNS), histidine buffer, saccharides, salts and / or surfactants, the pharmaceutical composition according to the present invention can further comprise suitable carriers, excipients or diluents conventionally used in pharmaceutical compositions.

[0072] In particular, pharmaceutical excipients for liquid protein formulations are well known to those of ordinary skill in the art. Non-limiting examples thereof include specific solvents or common solvents; sugars or sugar alcohols, such as mannitol, sucrose, sorbitol, fructose, maltose, lactose or dextran; buffers; preservatives, such as benzalkonium chloride, benzethonium chloride, quaternary ammonium salts or chlorhexidine diacetate; carriers, such as polyethylene glycol (PEG); antioxidants, such as ascorbic acid, sodium metabisulfite or methionine; chelating agents, such as EDTA or citric acid; biodegradable polymers, such as water-soluble polyesters; cryoprotectants; lyoprotectants; fillers; or stabilizers, and other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington: "The Science and Practice of Pharmacy" 20th edition, Alfonso R Gennaro, Ed., Lippincott Williams & Wilkins (2000) can also be included in the protein formulations described herein, provided that they do not adversely affect the desired properties of the formulation.

[0073] In a preferred embodiment, the composition according to the invention comprises, but is not limited to:

[0074] Heparin N-sulfatase at 5 to 30 mg / mL;

[0075] Histidine buffer at 2 to 20 mM;

[0076] Trehalose at 0.5 to 3.0 w / v%; and

[0077] NaCl at 70 to 150 mM,

[0078] Optionally, it further comprises 0.003 to 0.05% of polysorbate 20,

[0079] having a pH value of 8.0 to 8.8.

[0080] The composition according to the invention is used for the treatment of mucopolysaccharidosis type IIIA (MPS IIIA).

[0081] In the present invention, the pharmaceutical composition can be formulated into a pharmaceutical dosage form, such as a liquid dosage form or a lyophilized dosage form.

[0082] The liquid preparation is preferably but not limited to in the form of an ampoule or a prefilled syringe.

[0083] Preferably, the pharmaceutical composition can be formulated into a lyophilized preparation. The lyophilized preparation has advantages in storage and transportation and can be prepared by various lyophilization methods known in the art in addition to the methods described in the embodiments of the present invention.

[0084] After formulation, the pharmaceutical composition of the present invention can be reconstituted before administration to adjust the concentration of the active ingredient, i.e., heparin-N-sulfatase.

[0085] In the present invention, even when the pharmaceutical composition is in liquid formulation, it can be reconstituted and used, but when it is formulated in lyophilized form, it is preferably reconstituted into a liquid formulation before administration.

[0086] In the present invention, the "reconstitution solution" refers to the solution for reconstitution, and the "reconstituted preparation" refers to the final composition or preparation after reconstitution of the pharmaceutical composition of the present invention.

[0087] In the present invention, the reconstitution solution can be an aqueous solution of TAPS buffer, Bicine buffer, Tris buffer, Tricine buffer, TAPSO buffer or HEPES buffer, or distilled water conventionally used in the art, but is not limited thereto.

[0088] In addition, the reconstitution solution can further contain surfactants, salts, sugars or amino acids to adjust the stability of the active ingredient contained in the reconstituted preparation, and if the reconstitution solution contains surfactants, the concentration of the surfactants contained is 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%.

[0089] In the present invention, the surfactant is preferably but not limited to polysorbate surfactants, such as PS20 or PS80.

[0090] 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.

[0091] In the present invention, the pharmaceutical composition of the present invention can be reconstituted into the same form as before reconstitution by adding the reconstitution solution, so that the pharmaceutical composition and the reconstituted preparation have a volume ratio of 1:1 (v:v).

[0092] Alternatively, the pharmaceutical composition of the present invention can be reconstituted into a diluted form compared to the preparation before reconstitution by adding the reconstitution solution, so that the volume ratio (v:v) of the pharmaceutical composition and the reconstituted preparation is 1:1.001 or greater, 1:1.01 or greater, 1:1.1 or greater, 1:2 or greater, 1:5 or greater, or 1:10 or greater.

[0093] Alternatively, the pharmaceutical composition of the present invention can be reconstituted by adding a reconstitution solution to form a composition in a concentrated form compared to the composition before reconstitution, such that the volume ratio (v:v) of the pharmaceutical composition to the reconstituted composition 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.

[0094] In the present invention, the dose or ratio of the reconstitution solution can be used based on the final concentration of the active ingredient (i.e., heparan-N-sulfatase) in the reconstituted preparation.

[0095] In the present invention, the concentration of heparan-N-sulfatase in the reconstituted preparation included is about 2 mg / mL or higher, about 5 mg / mL or higher, about 10 mg / mL or higher, about 15 mg / mL or higher, about 20 mg / mL or higher, about 25 mg / mL or higher, or about 30 mg / mL or higher. The concentration of heparan-N-sulfatase in the reconstituted preparation includes about 2 to about 60 mg / mL, preferably about 3 to about 40 mg / mL, more preferably about 5 to about 30 mg / mL, more preferably about 8 to about 25 mg / mL, more preferably about 10 to about 20 mg / mL, and most preferably about 12 to about 15 mg / mL, but is not limited thereto.

[0096] In another example of the present invention, the concentration of heparan N-sulfatase included is about 2 to about 30 mg / mL, preferably about 2 to about 20 mg / mL, more preferably about 2 to about 15 mg / mL, but is not limited thereto.

[0097] The pharmaceutical preparation according to the present invention can be administered to the central nervous system by various administration methods. In the present invention, the pharmaceutical preparation for central nervous system administration can be administered to the central nervous system via intracerebroventricular injection (ICV), intracerebral injection (IC), or intrathecal injection (IT), most preferably intracerebroventricular injection (ICV).

[0098] As used in the present invention, intracerebroventricular injection refers to administering a drug by injecting it into the cerebral ventricle, which is a connected hollow space in the brain. Compared with intracerebral injection, the advantage of intracerebroventricular injection is that it can deliver a larger volume of drug over a larger area. Various techniques of intracerebroventricular injection are known in the art, such as, for example but not limited to, the Ommaya reservoir developed by Ayub Ommaya as a traditional intracerebroventricular injection device, which continues to be developed and reported, and various other intracerebroventricular injection devices and techniques known in the art or developed in the future can be used for intracerebroventricular injection of the pharmaceutical composition of the present invention without limitation.

[0099] As used herein, intracerebral injection refers to injecting a drug into the brain tissue itself. Various techniques for intracerebral injection are known in the art, for example, the intracerebral injection method described in detail by Mathon et.al. 2015.

[0100] As used herein, intrathecal injection refers to injecting into the spinal canal. Various techniques for intrathecal injection are known in the art, for example, the intrathecal injection methods described in detail in Lazorthes et al. Advances in Drug Delivery Systems and Applications in Neurosurgery, 143 - 192 and Omaya et al. Cancer Drug Delivery, 1:169 - 179.

[0101] In the present invention, when a pharmaceutical composition or a pharmaceutical preparation is administered via intracerebroventricular injection, a certain amount of cerebrospinal fluid (CSF) can be drained from the cerebral ventricle in the subject before administration. Due to the change in the volume of CSF, the drainage of CSF can prevent the increase in intracranial pressure after ICV administration.

[0102] Preferably, the total administration volume when intracerebroventricular (ICV) administering a pharmaceutical composition or a pharmaceutical preparation according to the present invention can 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.

[0103] In the present invention, administering a pharmaceutical composition or a pharmaceutical preparation to the central nervous system can deliver heparan N - sulfatase to various target tissues, such as the brain, spinal cord or periphery. In the present invention, the target tissue includes any tissue affected by the lysosomal storage disease to be treated. For example, the target tissue can be a brain target tissue, a spinal cord target tissue and / or a peripheral target tissue, and the administration to the central nervous system can provide systemic delivery of heparan N - sulfatase.

[0104] In the present invention, administering a pharmaceutical composition or a pharmaceutical preparation to the central nervous system can achieve a therapeutic or clinically effective level or activity in various target tissues described herein. As used herein, a therapeutic or clinically effective level or activity means a level or activity sufficient to achieve a therapeutic effect in the target tissue. For example, a therapeutic or clinically effective level or activity can be an enzyme level or activity sufficient to improve symptoms associated with a disease (such as GAG accumulation) in the target tissue.

[0105] In the present invention, administration of a preparation or pharmaceutical composition to the central nervous system can achieve an enzyme level or activity in the target tissue 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 present invention, administration of a preparation or pharmaceutical composition to the central nervous system can achieve an increase in enzyme level or activity of 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., untreated endogenous level or activity).

[0106] In the present invention, administration of a pharmaceutical composition or drug preparation to the central nervous system can result in a reduction in the storage amount of GAG (e.g., heparan sulfate) in brain target tissue, spinal cord neurons and / or peripheral target tissues. In the present invention, the storage amount of GAG 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., the storage amount of GAG in a subject before treatment or after administration of only the excipient). In the present invention, administration of a pharmaceutical composition or drug preparation to the central nervous system can cause a reduction in vacuole formation in neurons. For example, compared to a negative control group, it can cause a reduction of at least 20%, 40%, 50%, 60%, 80%, 90%, 1-fold, 1.5-fold or 2-fold or more.

[0107] The pharmaceutical composition or drug preparation according to the present invention can be administered in a pharmaceutically effective amount, where "pharmaceutically effective amount" means an amount sufficient to treat a disorder with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined by factors including the type and severity of the patient's disorder, drug activity, sensitivity to the drug, administration time, administration route and elimination rate, treatment duration, concomitant drug therapy and other factors well known in the medical field. The pharmaceutical composition according to the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in a single dose or multiple doses. Taking into account all of the above factors, it is important that the amount administered can achieve the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0108] In addition, the pharmaceutical composition or pharmaceutical preparation according to the present invention can be administered to a patient at appropriate dosing intervals, preferably but not limited to, at least once a week, more preferably once a week, most preferably once every two weeks, and preferably as soon as possible to facilitate the patient. As an example of the present invention, the dosing rate of the pharmaceutical composition or pharmaceutical preparation according to the present invention can be but is not limited to, about 0.1 ml / min or faster, or about 0.5 ml / min or faster, preferably about 1 ml / min or faster, more preferably about 2 ml / min or faster, most preferably about 5 ml / min or faster.

[0109] In another aspect, the present invention relates to a method for treating mucopolysaccharidosis type IIIA, wherein the pharmaceutical composition or pharmaceutical preparation according to the present invention is administered to a patient in need thereof, particularly a patient suffering from mucopolysaccharidosis type IIIA.

[0110] In another aspect, the present invention relates to the use of the pharmaceutical composition or pharmaceutical preparation for treating mucopolysaccharidosis type IIIA.

[0111] In another aspect, the present invention relates to the use of the pharmaceutical composition or pharmaceutical preparation in the preparation of a medicament for treating mucopolysaccharidosis type IIIA.

[0112] Hereinafter, the present invention will be described in detail by providing embodiments. These embodiments are only intended to illustrate the present invention, and it is obvious to those of ordinary skill in the art that the scope of the present invention should not be construed as being limited by these embodiments.

[0113] Embodiment 1. Stability of heparan N-sulfatase varying with buffer

[0114] Embodiment 1-1: Comparison of stability of different amino acid buffer types (DLS evaluation)

[0115] According to previous reports, phosphate buffer has a negative impact on the activity of heparan N-sulfatase. When phosphate buffer is replaced with amino acid buffer, especially histidine buffer, arginine buffer or glutamate buffer, DLS measurement is performed according to the protein concentration to evaluate protein aggregation.

[0116] The amino acid concentration in each amino acid buffer was set to 20 mM, with 200 mM of NaCl, pH 7.5 or 8.0, and the heparan N-sulfatase concentration was changed from 2.52 to 12.6 mg / mL, and the stability was evaluated.

[0117] The PDI value is calculated as follows

[0118] PDI = σ 2 / d 2

[0119] σ: Standard deviation of the particle size distribution

[0120] d: Average hydrodynamic particle

[0121] The measured average protein radius (Z-Ave) was analyzed by analyzing liquid samples of each formulated composition, where formulation was done by dynamic light scattering (DLS) using an Uncle instrument and measurements were made after injecting 8.8 μL of the sample three times into a Uni sample loader (Unchained Labs).

[0122] As a result, as shown in Table 1, it was found that when using histidine buffer, all formulations containing HNS from low to high concentrations showed low polydispersity index (PDI) values, indicating monodispersity characteristics, thus indicating that the protein was stable and did not aggregate. However, when using arginine or glutamate buffer, the PDI values were stable at low concentrations of HNS of about 2.5 mg / ml, but increased sharply with increasing concentration at about 7.5 mg / mL or higher concentrations, indicating an increase in the level of aggregation and the particle size becoming polydisperse and unstable at higher concentrations.

[0123] In addition, better results were obtained at pH 8.0 compared to pH 7.5.

[0124] [Table 1]

[0125] Stability of Amino Acid Buffers

[0126]

[0127] Classified by PDI value:

[0128] <0.1: Low polydispersity (low aggregation)

[0129] 0.1 to 0.25: Medium polydispersity

[0130] >0.25: High polydispersity (high aggregation)

[0131] In particular, for the Z-Ave values, as shown in Table 1 and Figure 1a and Figure 1b as shown, it was indirectly confirmed that when using arginine or glutamate buffer, heparan N-sulfatase concentrations of about 7.5 mg / mL or higher showed Z-Ave values greater than 1,000 nm, indicating that protein aggregation occurred, thus indicating that arginine or glutamate buffer is not suitable for the compositions of the present invention.

[0132] On the other hand, even when the concentration of heparin N-sulfatase is higher than 7.5 mg / mL, a small Z-ave value of about 9 nm is obtained using histidine buffer. This indicates that histidine buffer is very suitable for the composition of the present invention and prevents protein aggregation.

[0133] The Z-Ave value can be expressed as the average protein radius and is measured using a DLS instrument. In this patent, a Uncle instrument is used to analyze liquid samples of each formulated composition. After injecting 8.8 μL of the sample into the Uni sample loader (Unchained Labs) three times repeatedly, Uncle determination was performed. The histidine buffer continued to exhibit a low Z-Ave value.

[0134] Embodiment 1-2: Comparison of stabilities of different amino acid buffer types (KD and B22 evaluations)

[0135] In addition, when using histidine buffer, arginine buffer, or glutamate buffer according to the compositions in Table 2, the Uncle test for protein-protein interactions and protein-buffer interactions is used to evaluate the formulation stability by measuring the KD and B22 (second virial coefficient) values.

[0136] The KD value indicates the degree of interaction between proteins, where a negative number indicates instability and a positive number indicates stability. The B22 value is a variable indicating colloidal stability (B22), where the more positive the value, the more stable the formulation because the repulsive force between proteins is stronger and the possibility of aggregation is lower.

[0137] [Table 2]

[0138] Composition of the composition for KD or B22 test

[0139]

[0140] As a result, as Figure 2a and 2b shown, when using arginine buffer or glutamate buffer, the stability is significantly reduced due to low kD or B22 values, while when using histidine buffer, the kD or B22 values are relatively high at both pH 7.5 and 8.0. This indicates that the formulation containing histidine buffer has excellent stability.

[0141] Embodiment 1-3: Stability of histidine buffer compared to phosphate buffer

[0142] Based on previous reports that phosphate buffer has a negative impact on the activity of heparin N-sulfatase, when histidine buffer is used instead of phosphate buffer, the Uncle test is used to examine protein-buffer interactions to evaluate the formulation stability.

[0143] The composition used in the test contained 8 mg / ml of HNS; 20 mM of histidine, 154 mM of NaCl or 6.7 mM of phosphate, and 200 mM of NaCl.

[0144] As a result, it was found that the B22 value increased significantly in the formulation using the histidine buffer, as Figure 3 shown. These results indicate that when using the histidine buffer, the phosphate buffer significantly reduced the protein-buffer interaction, resulting in a more stable formulation.

[0145] Embodiment 2. Evaluation of stability varying according to pH value

[0146] The stability was evaluated by determining the dependent turbidity of the heparin N-sulfatase composition containing the histidine buffer according to the pH value

[0147] The turbidity in the present invention was analyzed using Lunatic (Unchained Labs). 2.0 μL of the sample was injected into the Lunatic plate (Unchained Labs), and the turbidity at 350 nm was measured. Based on the turbidity value of the placebo buffer, the degree of increase in the turbidity value of the sample was measured.

[0148] As a result, the composition containing a low concentration of heparin N-sulfatase (5 mg / mL) had a lower turbidity in all pH ranges, as shown in Tables 3-5 and Figure 4 shown.

[0149] In the low pH range, compared with the composition containing a low concentration of heparin N-sulfatase (5 mg / mL), the composition containing a high concentration of heparin N-sulfatase (13.5 mg / mL) had a significantly increased turbidity.

[0150] However, in the composition containing a high concentration of HNS, the turbidity tended to decrease with an increase in pH. In particular, when the pH was higher than 7.8, preferably higher than 7.9 to 8.0, the turbidity decreased significantly, thus confirming that the pH of the composition had a significant effect on the reduction of turbidity.

[0151] [Table 3]

[0152] Change in turbidity of the composition containing a low concentration of HNS according to pH value

[0153]

[0154] [Table 4]

[0155] Change in turbidity of the composition containing a high concentration of HNS according to pH value

[0156]

[0157] [Table 5]

[0158] The turbidity of the composition containing high concentration of HNS varies according to the change of pH value

[0159]

[0160] In particular, for the composition containing a very high concentration of 16 to 17 mg / mL of HNS and 120 mM of NaCl, it was found that the turbidity decreased with the increase of pH, especially when the pH was higher than 7.9 to 8.0, as Figure 5 shown

[0161] Embodiment 3. Evaluation of whether stability varies with the addition of surfactant

[0162] Embodiment 3-1: Confirming the changes caused by the addition of surfactant using an Aura instrument

[0163] To more specifically confirm that the addition of a lower concentration of surfactant is accompanied by a decrease in turbidity, the pH was set to 8.2, and then the turbidity was measured using an Aura instrument when 0.005 w / v% polysorbate 20 was added

[0164] The composition used in Embodiment 3-1 contained 15 mg / mL of HNS, 5 mM of histidine buffer, 125 mM of NaCl, and 1.8 w / v% of trehalose, and experiments were conducted for both cases: where the composition was freeze-dried as it was and then reconstituted in a solution containing PS20 (0.005%); and where PS20 (0.005%) was added to the composition and reconstituted after freeze-drying

[0165] The turbidity was measured using an Aura instrument as described below

[0166] Insert an empty plate, run "acquire background" to measure the background, and then load 30 - 50 μL of the sample into each well of the plate in triplicate. Place a box containing drying paper on the manifold, place the plate completed by the first vacuum treatment on it, and open the vacuum valve for secondary drying. Once the vacuum process is completed, place the plate in the Aura instrument for measurement. Based on a 96-well plate, compared with the blank group, the instrument can quantify and plot the information of particles 1 μm or larger in a 30 - 50 μL sample. For this purpose, the plate is evacuated and sampled with drying paper

[0167] It was found that, as Figures 6a to 6c shown, the turbidity decreased significantly in both cases where 0.005% polysorbate 20 was added before freeze-drying and when it was added during reconstitution after freeze-drying

[0168] Embodiment 3-2: Verification that the number of insoluble particles varies according to the addition of surfactant

[0169] In order to further verify and confirm that the stability of the preparation varies according to the addition of surfactant, the change in the number of insoluble particles was confirmed when 0.005% polysorbate 20 was added as the surfactant.

[0170] Each composition used in Embodiment 3-2 contained 15.3 mg / ml of HNS, 5 mM of histidine, 125 mM of NaCl, and 1.8 w / v% of trehalose, had a pH of 8.0, and the experiment was conducted by only changing the addition amount of PS20 (0.005 w / v%).

[0171] As shown in Table 6 below, it was found that the addition of surfactant (PS20) significantly reduced the number of subvisible particles with sizes of 10 μm and 25 μm or larger. This result indirectly indicates that the addition of surfactant improves the stability of the pharmaceutical composition according to the present invention.

[0172] [Table 6]

[0173] Whether the number of subvisible particles changes with the addition of 0.005 w / v% PS20

[0174]

[0175] Embodiment 4. Evaluation of stability according to salt concentration

[0176] In order to evaluate the stability of the composition according to the salt concentration contained in the composition, the pH was set to 7 to 8, and the concentration of salt (NaCl) was varied between 100 and 200 mM to examine the stability of the composition.

[0177] Each composition used in the test of Embodiment 4 contained 12.6 mg / mL of HNS and 20 mM of histidine. The kD value was used for the measurement of stability, and the measurement of the kD value was performed in the same manner as in Embodiment 1-1 of the specification.

[0178] As a result, as Figure 7 shown, at pH 7.0, it was found that the stability of the composition was low at all NaCl concentrations, but when the NaCl concentration was 150 mM, the stability was evaluated as excellent at pH 8, and when the NaCl concentration was 200 mM, it was found that the stability was significantly higher above pH 7.0, especially above 7.5. The above results indicate that the stability of the high-concentration HNS preparation increases with the increase in NaCl concentration and pH value.

[0179] In addition, as a result of the study on the change in the osmolality according to the NaCl concentration, it was found that when the amount of NaCl contained was 125 mM or 130 mM, the average osmolality was 286 or 294 mOsmol / kg, respectively, thus confirming that the change in the osmolality with the NaCl concentration was not significant. The above osmolality was generally within the acceptable range of the pharmaceutical composition (see Table 7).

[0180] [Table 7]

[0181] Change in osmolality according to NaCl concentration

[0182]

[0183] Embodiment 5. Evaluation of stability according to sugar concentration

[0184] Embodiment 5-1: Evaluation of stability (purity) when adding 1% or less trehalose

[0185] Sugars such as trehalose are often used as stabilizers in lyophilization. In the test, the stability of the composition depending on the concentration of the sugar, particularly the stability of the lyophilized preparation after reconstitution, was evaluated according to the purity. The stability of the lyophilized preparation after reconstitution was determined by varying the concentration of trehalose from 0 to 1% (v / w).

[0186] Before lyophilization, the concentration of each component of the prepared pharmaceutical composition was as shown in Table 8 below:

[0187] [Table 8]

[0188] Concentration of components of the pharmaceutical composition of Embodiment 5-1

[0189]

[0190] Lyophilization was carried out by dispensing 1.3 ml of the liquid solution of the composition prepared according to the present invention into a vial (3 ml size), semi-sealing it with a rubber stopper, and loading it onto the shelf of a freeze dryer (Lyostar 3, SP Scientific). Subsequently, lyophilization was carried out under the conditions listed in Table 9, and after the aluminum lyophilization was completed, the prepared lyophilized preparation was covered with an aluminum cap.

[0191] [Table 9]

[0192] Production conditions of the lyophilized preparation

[0193]

[0194] After reconstitution with water for injection (WFI), purity analysis was performed on the prepared freeze-dried preparations. According to the volume ratio of the pharmaceutical composition to the reconstituted preparation (reconstitution ratio), the pharmaceutical compositions in Table 8 (Compositions 5-1-a to f) were freeze-dried and reconstituted with 0.286 mL to 0.75 mL of distilled water to prepare the reconstituted preparations (Preparations 5-1-A to F) (see Table 10).

[0195] [Table 10]

[0196] Concentrations of the components of the reconstituted preparations of Embodiment 5-1

[0197]

[0198] Assessment of purity using size exclusion liquid chromatography (SE-HPLC). For size exclusion liquid chromatography, this is a standard method for determining and quantifying the levels of aggregation and fragmentation. Specifically, for size exclusion chromatography, first, 1 mg / ml HNS was diluted to 1.0 mg / mL with the mobile phase (40 mM sodium phosphate, 300 mM NaCl, pH 7.5), followed by sterile filtration (if the concentration was less than 1.0 mg / ml, the process was carried out without dilution), and 200 μL of the filtered sample was injected into a vial insert and inserted into a screw top vial.

[0199] After connecting the mobile phase to the pump, an analytical column (TSKgel G3000SWXL, Tosoh Corporation) was assembled while the mobile phase flowed at a rate of 0.5 mL / min to Waters e2695 and Waters 2489 instruments (manufactured by Waters Corporation, Japan). The mobile phase flowed at a speed of 0.5 mL / min for more than 30 minutes for equilibration until the detector signal was stable, and when the temperature of the autosampler dropped to 4 °C, the sample was inserted into the injector. 50 μL of the sample was injected, and the mobile phase flowed for 35 minutes to identify the detection peak at 280 nm. Then, analysis was performed using Empower Pro software on a PC.

[0200] As a result, as shown in Table 11 and Figure 8 as shown, it was found that compared with a purity of approximately 88% in the absence of trehalose, when all trehalose concentrations were higher than 0.1 w / v% before reconstitution, the purity increased to approximately 95% or higher.

[0201] [Table 11]

[0202] Assessment results of the purity after reconstitution according to the concentration of trehalose

[0203]

[0204] HMW: High Molecular Weight

[0205] LMW: Low Molecular Weight

[0206] Embodiment 5-2: Stability Evaluation (Specific Activity and Purity) When Adding More than 1% Trehalose

[0207] In addition, in order to evaluate the stability after reconstitution of the lyophilized preparation containing more than 1% (w / v) trehalose, the trehalose concentration was set to 1.35 w / v% relative to the concentration before lyophilization, and the specific activity (S.A.) and purity of HNS at different pH values were evaluated.

[0208] The composition of the prepared preparation samples is shown in Table 12 below.

[0209] [Table 12]

[0210] Composition of the Preparation Samples Containing More than 1% Trehalose

[0211]

[0212] HNS, histidine, NaCl, and trehalose were added to DS (undiluted) to prepare the pharmaceutical compositions of 5-2-a to 5-2-e, which were reconstituted after lyophilization to prepare the preparations of 5-2-A to E, respectively. At this time, the ratio (v:v) of the pharmaceutical composition to the reconstituted preparation was reconstituted at a volume ratio of 4:3.

[0213] The activity assay of the enzyme of the present invention was carried out as follows.

[0214] - In Step 1, the preparation sample was reacted with the synthetic substrate (4MU-α-GlcNS) to release the sulfate group at the end of the substrate (producing 4MU-α-GlcNH2).

[0215] Reaction in Step 1: 4MU-α-GlcNS + HNS → 4MU-α-GlcNH2

[0216] - In Step 2, α-glucosidase was treated to release fluorescent 4MU from 4MU-α-GlcNH2. The fluorescence value of the free 4MU was measured using a fluorescence reader to determine the enzyme activity of heparin N-sulfatase in the sample.

[0217] Reaction in Step 2: 4MU-α-GlcNH2 + α-glucosidase → 4MU

[0218] The reaction methods in Steps 1 and 2 are detailed as follows.

[0219] Dilute the preparation sample to 100 μg / mL with a 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 dilute the substrate with the substrate diluent.

[0220] - Reaction in Step 1: In a 96-well plate (black), dispense 20 μL of the serially diluted substrate into each well. Add 10 μL each of the sample diluted to 100 μg / mL and the blank group to the other side of the well to avoid mixing with the substrate solution, and then mix the solution simultaneously by holding one side of the plate and gently tapping the other side (to prevent the solution from splashing out of the well). Then seal the plate with a plate sealer and react in an incubator at 37 °C for 17 hours.

[0221] - Reaction in Step 2: After the reaction in Step 1 (17 hours), add 6 μL of the termination solution of the reaction in Step 1 to each well to terminate the primary reaction. Mix the solution thoroughly by holding one side of the plate and gently tapping the other side (to prevent the solution from splashing out of the well). Dilute the α-glucosidase solution prepared at 100 U / mL 10-fold to 10 U / mL with ultrapure water. After adding 10 μL of the solution to each well, hold one side of the plate and gently tap the other side (to prevent the solution from splashing out of the well) to mix the solution thoroughly. Seal the plate with a plate sealer and incubate in an incubator at 37 °C for 24 hours.

[0222] - Fluorescence measurement: 15 minutes before fluorescence measurement, prepare a 4MU diluent by mixing in the order of 1.5 mL of the substrate diluent, 300 μL of the primary reaction termination solution, 500 μL of ultrapure water, and 10 mL of the secondary reaction termination solution. As shown in Table 6, dilute the 4MU stock solution with the 4MU diluent to prepare a 4MU standard solution. After the secondary reaction, add 200 μL of the reaction termination solution to each well to finally terminate the reaction. Load 246 μL of the 4MU standard (standards 1 - 8) in duplicate into each well. Measure the fluorescence with a fluorescence spectrometer at Ex. 355 nm / Em. 460 nm.

[0223] Measure the purity using size exclusion liquid chromatography (SE-HPLC) as described above.

[0224] As a result, as shown in Table 13 below, Figure 9a and 9b when the concentration of trehalose in the composition (i.e., the freeze-dried preparation) is greater than or equal to 1 w / v%, the measured HNS specific activity is greater than or equal to approximately 400 (pmol / min / μg), and the purity is also confirmed to be greater than or equal to approximately 98%, indicating that the composition is highly stable even when prepared in the form of a freeze-dried preparation and remains highly stable even after reconstitution.

[0225] [Table 13]

[0226] Specific activity (S.A.) and purity of the preparation samples containing more than 1% trehalose

[0227]

[0228] Example 6. Confirmation of the specific activity and purity of different reconstituted compositions

[0229] A variety of compositions within the numerical range of the present invention were prepared, and their stability was confirmed based on specific activity and purity through lyophilization and reconstitution.

[0230] The concentration of each component of the pharmaceutical preparation (before lyophilization) used in the test is shown in Table 14 below.

[0231] [Table 14]

[0232] Concentration of the components of the pharmaceutical composition and preparation in Embodiment 6

[0233]

[0234]

[0235] * The ratio (v:v) of the pharmaceutical compositions of 6-1 and 6-2 to the reconstituted preparation is 1:0.75

[0236] The ratio (v:v) of the pharmaceutical compositions of 6-3 to 6-10 to the reconstituted preparation is 1:1

[0237] The purity was measured by the same method as described in Embodiment 5-1 above, and the specific activity was measured by the same method as described in Embodiment 5-2 above.

[0238] The composition and measurement results are shown in Table 15 below.

[0239] [Table 15]

[0240] Measurement results of the specific activity and purity of the samples

[0241]

[0242] All samples from 6-1 to 6-10 were found to show a high purity of more than 98% and a high specific activity of more than 690 pmol / min / μg.

[0243] Industrial applicability

[0244] The pharmaceutical composition and pharmaceutical preparation of the present invention containing a high concentration of heparan N-sulfatase (HNS), and the pharmaceutical preparation containing the same can be used in enzyme replacement therapy (ERT) for treating mucopolysaccharidosis type IIIA by replacing the phosphate buffer known to inhibit the activity of the active ingredient (heparan N-sulfatase) with a histidine buffer (exhibiting significantly improved preparation stability due to reduced protein-protein or protein-buffer interactions and reduced turbidity).

[0245] Although certain aspects of the present invention have been described in detail above, it will be apparent to those of ordinary skill in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Accordingly, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

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

0.

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

3. The pharmaceutical composition according to claim 1, wherein, The concentration of the histidine buffer comprised is 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 according to claim 1, wherein, The composition further comprises a saccharide.

6. The pharmaceutical composition according to claim 5, wherein, The saccharide 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 concentration of the saccharide comprised is 0.1% to 5.0 w / v%.

8. The pharmaceutical composition according to claim 5, wherein, The composition further comprises a salt.

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

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

11. The pharmaceutical composition according to claim 1, wherein, It further comprises a surfactant.

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 concentration of the surfactant comprised is 0.0001 to 0.1 w / v%.

14. The pharmaceutical composition according to claim 1, which comprises heparan N-sulfatase at a concentration of 5 to 30 mg / mL; a histidine buffer at a concentration of 2 to 20 mM; 0.5 to 3 w / v% trehalose; and 70 to 150 mM of NaCl, having a pH value of 8.0 to 8.

8.

15. The pharmaceutical composition according to claim 1, which has a turbidity of less than 1.0 when measured at 350 nm.

16. The pharmaceutical composition according to claim 1, which is used for the treatment of mucopolysaccharidosis type IIIA.

17. A pharmaceutical preparation comprising the pharmaceutical composition according to any one of claims 1 to 16.

18. The pharmaceutical preparation according to claim 17, which is a liquid preparation or a lyophilized preparation.

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

20. The pharmaceutical preparation according to claim 18, wherein, The lyophilized preparation comprises heparan N-sulfatase at a concentration of 2 to 60 mg / mL after reconstitution.

21. The pharmaceutical preparation according to claim 19, wherein, The total administration volume is 10 ml or less.

22. The pharmaceutical preparation according to claim 21, wherein, The administration rate is at least 0.5 ml / min.

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