Pharmaceutical composition comprising heparan n-sulfatase with improved stability

Abstract

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

Description

Title of Invention: PHARMACEUTICAL COMPOSITION COMPRISING HEPARAN N-SULFATASE WITH IMPROVED STABILITY Technical Field [1] The present invention relates to a pharmaceutical composition comprising a high concentration of heparan N-sulfatase (HNS) with improved stability and a pharmaceutical formulation comprising the same, more particularly, a pharmaceutical composition comprising a high concentration of heparan N-sulfatase and a histidine buffer with a pH of at least 7.8, and a pharmaceutical formulation comprising the same. [2] Background Art [3] Lysosomal storage diseases (LSDs) are inherited metabolic diseases caused by defects in the function of lysosomes. Lysosomal storage diseases are caused by lysosomal dysfunction due to deficiencies in single or multiple enzymes required for the metabolism of lipids, glycoproteins, or mucopolysaccharides. Deficiencies of lysosomal enzymes cause systemic abnormalities due to lysosomal accumulation of lipids, glycoproteins, or mucopolysaccharides (Nature Reviews Disease Primers. 4 (1): 27; Biochem. Soc. Trans. 28 (2): 150-4). Mucopolysaccharidoses (MPS) are one of the lysosomal accumulation diseases that result from intra-lysosomal accumulation due to a deficiency of lysosomal enzymes required for the degradation of glycosaminoglycans. In general, mucopolysaccharidoses are categorized into types I to VII, depending on the type of enzyme deficient. [4] Enzyme replacement therapy (ERT), which involves the administration of a deficient lysosomal enzyme to correct the enzyme's functional deficiency, is one of the main therapeutic approaches used to treat lysosomal storage diseases. This simple injection therapy has the advantage of minimizing symptoms and preventing 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 by FDA in 1991 and used (National Gaucher Foundation. Retrieved 2017-06-08). [5] However, whereas many lysosomal accumulation diseases cause excessive accumulation of glycoaminoglycans (GAGs) in the nervous system, particularly in the neurons and spinal cord membranes of the brain, and lead to various central nervous system disorders, intravenously administered enzyme replacement therapies cannot inadequate deliver the enzyme to the central nervous system because it is difficult for the active ingredient, lysosomal enzymes, to cross the Blood-Brain Barrier (BBB), and especially, cannot effectively treat neurological disorders and diseases caused by lysosomal accumulation, especially in the brain. Therefore, various CNS delivery therapies are being investigated to directly deliver drugs to the central nervous system for the delivery of enzymes that bypass the BBB. [6] Various therapies have been developed to deliver enzymes that bypass the BBB into the central nervous system. In particular, injection therapies that deliver proteins directly to the brain include intracerebral injection (IC), intracerebroventricular injection (ICV), and intrathecal injection (IT). [7] Intrathecal (IT) and intraventricular (ICV) injections have emerged as methods for delivering alternative enzymes to the central nervous system for mucopolysaccharidosis (MPS), and have shown significant reductions in glycoaminoglycans (GAGs) and significant improvements in neurological symptoms in various animal models with mucopolysaccharidosis (Molecular Therapy - Methods & Clinical Development, 21, 67-75). However, therapies that are injected directly into the brain have very limited dosing capabilities, and the development of injectable formulations containing high concentrations of enzymes is essential to achieve effective levels of therapeutic benefit. [8] Many high-concentration enzyme formulations for performing enzyme replacement therapy (ERT) via CNS delivery have been reported, and the formulations reported so far for CNS delivery typically utilize a phosphate buffer as a buffer (Korean Patent Nos. 2,007,044, and 2,272,399). [9] However, in compositions for CNS delivery of heparan N-sulfatase (HNS), studies have consistently reported that the use of phosphate buffers negatively affects the activity of the heparan-N-sulfatase enzyme (J Inherit Metab Dis. 1993;16(2):465-472; Acta Crystallogr D Biol Crystallogr. 2014 May;70(Pt 5):1321-1335).

[10] Therefore, there is an urgent need for a pharmaceutical composition and a pharmaceutical formulation for CNS delivery comprising heparan N-sulfatase, particularly heparan N-sulfatase with a high concentration and a high stability.

[11] With this background, the present inventors have made a good faith effort to develop pharmaceutical compositions for CNS delivery of heparan N-sulfatase (HNS) with a high concentration without the use of phosphate buffers, and pharmaceutical formulations comprising the same, and have found that the use of histidine buffer as a buffer and setting the pH to 7.8 or higher dramatically improve the stability of HNS.

[12]

[13] The description above about the related art is intended solely to enhance the understanding of the background of the present invention and may not include information in prior arts known to one of ordinary skill in the art.

[14]

[15] [S ummary of Invention]

[16] It is an object of the present invention to provide a pharmaceutical composition comprising heparan N-sulfatase (HNS) and a pharmaceutical formulation with improved stability.

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

[18] Another object of the present invention is to provide use of the pharmaceutical composition or pharmaceutical formulation for the treatment of mucopolysaccharidosis type IIIA.

[19] Another object of the present invention is to provide the use of the pharmaceutical composition or pharmaceutical formulation for the preparation of an agent for the treatment of mucopolysaccharidosis type IIIA.

[20]

[21] To achieve the above objectives, the present invention provides a pharmaceutical composition and a pharmaceutical formulation comprising heparan N-sulfatase (HNS) and a histidine buffer, with a pH of at least 7.8.

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

[23] The present invention also provides use of the pharmaceutical composition or pharmaceutical formulation for the treatment of mucopolysaccharidosis type IIIA.

[24] The present invention also provides the use of the pharmaceutical composition or pharmaceutical formulation for the preparation of an agent for the treatment of mucopolysaccharidosis type IIIA.

[25] Brief Description of Drawings

[26] FIG. 1 is a diagram illustrating the stability of a pharmaceutical composition varying depending on the type of the amino acid buffer and the following pHs:

[27] (a) pH 7.5

[28] (b) pH 8.0

[29] FIG. 2 is a diagram illustrating the stability of a pharmaceutical composition varying depending on the type of the amino acid buffer with B22 value or kD value.

[30] (a) B22 value

[31] (b) kD value

[32] FIG. 3 is a diagram illustrating the results of comparison of the stability of a pharmaceutical composition when using a histidine buffer and a phosphate buffer.

[33] FIG. 4 is a diagram illustrating the variation of turbidity of a pharmaceutical composition with pH.

[34] FIG. 5 is a diagram illustrating the variation of turbidity of a pharmaceutical composition with pH.

[35] FIG. 6 is a diagram depicting the results of observing the variation of turbidity of a pharmaceutical composition varying depending on the addition of surfactant and pH.

[36] (a) The results of comparison of images of foreign objects after reconstitution of HNS containing formulation without PS20 / with 0.005% PS20.

[37] (b) Foreign objects plotted by size after reconstitution of HNS containing for mulation without PS20 / with 0.005% PS20.

[38] (c) Foreign objects plotted by size after reconstitution of HNS containing formulation without PS20 / with 0.005% PS20.

[39] FIG. 7 is a diagram illustrating the stability of a pharmaceutical composition according to NaCl concentration.

[40] FIG. 8 is a diagram illustrating the change in purity of HNS in the composition according to trehalose concentration.

[41] FIG. 9 is a diagram of the variation of the specific activity (S.A.) and purity of HNS in the composition according to trehalose concentration.

[42]

[43] [Detailed description of the invention and preferred embodiments]

[44] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by a person skilled in the art. In general, the nomenclature used herein is well known and in common use in the art.

[45]

[46] Heparan-N-sulfatase (HNS) is a lysosomal enzyme that catalyzes the hydrolysis of heparan sulfate and the N-linked sulfate group from 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 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 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) is accumulated in the lysosomes of neurons and glial cells, causing severe neurological damage and abnormalities.

[47] Although formulations for central nervous system delivery of heparan N-sulfatase comprising phosphate have been reported (e.g., Korean Patent No. 2,007,044), studies have been consistently reported 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(Pt 5):1321-35). Therefore, there is a need for the development of novel formulations of heparan N-sulfatase for central nervous system delivery using stabilizing agents that replace phosphate.

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

[49] 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 histidine buffer, with a pH of 7.8 to 9.0.

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

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

[52] The heparan N-sulfatase may be any molecule, or portion of a molecule, capable of replacing the protein activity of naturally occurring heparan N-sulfatase (HNS) or rescuing one or more of the phenotypes or symptoms associated with HNS-deficiency. Suitable replacement enzymes for the present invention are polypeptides having N-terminal and C-terminal, and an amino acid sequence substantially similar or same as the mature human HNS protein.

[53] Typically, human HNS is produced as a precursor molecule that is processed into its mature form. This processing is typically accomplished by removing 20 amino acid signal peptides. 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. Therefore, the 20 N-terminal amino acids are not generally considered necessary 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.

[54] The mature form of HNS: RPRNALLLLA DDGGFESGAY NNSAIATPHL DALARRSLLF RNAFTSVSSC SPSRASLLTG LPQHQNGMYG LHQDVHHFNS FDKVRSLPLL LSQAGVRTGIIGKKHVGPET 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. 1)

[55] 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)

[56] In the present invention, the heparan N-sulfatase can be characterized as a recom-binantly produced recombinant enzyme. Recombinant production of heparan N-sulfatase can be readily accomplished using techniques for the production of recombinant cells for the expression of various target proteins known in the art.

[57] In the present invention, the heparan N-sulfatase may also be included 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 a brain cell and / or a lysosomal targeting molecule to facilitate cellular uptake or lysosomal targeting. Modifications of alternative enzymes, such as heparan N-sulfatase, are disclosed in Korean Patent No. 2,007,044, et al.

[58]

[59] In the present invention, the heparan N-sulfatase may be comprised in an amount of 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.

[60] In another example of the present invention, the heparan N-sulfatase is comprised 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, more preferably about 8 to about 25 mg / mL, more preferably about 10 to about 20 mg / mL, most preferably about 12 to about 15 mg / mL, but is not limited thereto.

[61] In another example of the present invention, the heparan N-sulfatase is comprised at a concentration of 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.

[62]

[63] Furthermore, a histidine buffer is a buffer that provides a number of advantages over conventional phosphate buffers, such as a dramatic increase in stability due to reduced protein-protein and protein-buffer interactions. In the composition according to the present invention, the histidine buffer may be comprised in an amount 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 to. The concentration in the histidine buffer is a concentration calculated based on the concentration of histidine.

[64] In another example of the present invention, the histidine buffer may be comprised in an amount of 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.

[65] In the compositions according to the present invention, it was found that when the pH is above about 7.8, the electrostatic repulsion between protein-protein or proteinbuffer is increased, and the turbidity is also significantly reduced, thereby dramatically increasing the stability of the composition.

[66] Accordingly, the pH of the composition according to the present invention may 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.

[67]

[68] 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 matter or impurities in the composition. In a pharmaceutical composition, turbidity is a parameter that indicates the stability of the drug, for example, in less stable formulations, aggregates may form due to protein-protein in teractions or protein-buffer attraction, self-association, which can lead to increased turbidity.

[69] In the present invention, the turbidity (T) can be calculated by absorbance measurements at specific wavelengths. The calculation of turbidity follows the Beerlambert law. T= 1 / 10 (T: transmittance, I: transmitted intensity, 10: incident intensity). In one embodiment of the present invention, the turbidity is measured at 350 nm using Lunatic (Unchained Labs), but is not limited thereto.

[70] In the pharmaceutical composition according to the present invention, the turbidity, as measured at 350 nm, is about 1.0 or less, preferably about 0.8 or less, more preferably about 0.6 or less, most preferably about 0.4 or less, but is not limited thereto.

[71]

[72] Furthermore, it has been found in the present invention that the inclusion of saccharides, in particular trehalose, results in significantly higher purity (%) and specific activity (S.A.) not only when used as a liquid formulation in a pharmaceutical composition, but also when formulated and reconstituted into a lyophilized formulation. Accordingly, the composition according to the present invention comprises a saccharide.

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

[74] In the present invention, the saccharides are comprised in 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, and more particularly 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%, more preferably about 1.0% to 2.0%.

[75] In another example of the present invention, the saccharides are comprised at a concentration of about 0.1% to about 3%.

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

[77] The pharmaceutical composition according to the present invention comprises a salt. According to the present invention, the salt is NaCl or KC1. In the present invention, the salt is comprised in an amount of 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, most preferably about 70 mM to 120 mM.

[78] In another example of the present invention, the salt is comprised in an amount of about 30 mM to about 300 mM.

[79] In the present invention, the salt may be included in a concentration having an ap propriate osmolarity for central nervous system delivery of the pharmaceutical composition of the present invention. Suitable osmolarity of pharmaceutical formulation for central nervous system delivery is well known in the art.

[80] In the present invention, the osmotic concentration of the pharmaceutical composition may be, for example, about 400 mOsmol / kg or less, preferably about 350 mOsmol / kg or less, more preferably about 330 mOsmol / kg or less, more preferably about 300 mOsmol / kg or less, most preferably about 290 mOsmol / kg or less, but is not limited thereto. In the present invention, the osmotic concentration of the drug formulation may 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, most preferably about 280 to about 300 mOsmol / kg, but is not limited thereto.

[81]

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

[83] Accordingly, the pharmaceutical composition according to the present invention may be characterized as further comprising a surfactant. In the present invention, the surfactant may be characterized as being a polysorbate-based surfactant, more preferably polysorbate 20 or polysorbate 80, most preferably polysorbate 20. In the composition according to the present invention, the surfactant is comprised in 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%, most preferably about 0.004% to about 0.01%.

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

[85]

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

[87] In particular, pharmaceutical excipients useful in a liquid protein formulation are well known to those of ordinary skill in the art. Non-limiting examples thereof include specific solvents or universal solvents; saccharides or saccharide alcohols, such as mannitol, sucrose, sorbitol, fructose, maltose, lactose or dextran; buffers; preservatives, such as benzalkonium chloride, benzethonium chloride, tertiary ammonium salts, or chlorohexidinediacetate; carriers, such as poly(ethylene 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 polyester; cryoprotectants; lyophilization protectants; bulking agents; or stabilizing agents, 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) may also be included in the protein formulations described herein, provided that they do not adversely affect the desirable characteristics of the formulation.

[88]

[89] In one preferred embodiment, the composition according to the present invention comprises, but is not limited to:

[90] 5 to 30 mg / mL of heparan N-sulfatase;

[91] 2 to 20 mM of histidine buffer;

[92] 0.5 to 3.0 trehalose; and

[93] 70 to 150mMofNaCl,

[94] optionally, further comprising 0.003 to 0.05% polysorbate 20,

[95] with a pH of 8.0 to 8.8.

[96]

[97] The composition according to the present invention is for use in the treatment of mucopolysaccharidosis type IIIA (MPS IIIA).

[98]

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

[100] The liquid formulation is preferably, but not limited to, in the form of an ampoule or a pre-filled syringe.

[101] Preferably, the pharmaceutical composition can be formulated in a lyophilized formulation. The lyophilized formulation 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.

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

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

[104] In the present invention, "reconstitution solution" means a solution used for reconstitution, and "formulation after reconstitution" means the final composition or for- mulation after reconstitution of a pharmaceutical composition of the present invention.

[105] In the present invention, the reconstitution solution can be 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.

[106] Furthermore, the reconstituted solution may further comprise surfactants, salts, saccharides, or amino acids to modulate the stability of the active ingredient included in the formulation after reconstitution, and if the reconstituted solution comprises a surfactant, it is comprised in 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%.

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

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

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

[110] Alternatively, the pharmaceutical composition of the present invention may be reconstituted into a diluted form of the composition compared to the formulation before reconstitution by adding the reconstitution solution so that the pharmaceutical composition and the formulation after reconstitution have a volume ratio (v:v) of 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. [Ill] Alternatively, the pharmaceutical composition of the present invention can be reconstituted into a concentrated form of the composition compared to the composition before reconstitution by adding the reconstitution solution so that the pharmaceutical composition and the composition after reconstitution have a volume ratio (v:v) of 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.

[112] In the present invention, the dosage or proportion of the reconstitution solution may be used based on the final concentration of heparan-N-sulfatase, the active ingredient of the formulation after reconstitution.

[113] In the present invention, the heparan-N-sulfatase concentration in the formulation after reconstitution is comprised at a concentration of 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 of the formulation after reconstitution includes a con centration of 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, most preferably about 12 to about 15 mg / mL, but is not limited thereto.

[114] In another example of the present invention, the heparan N-sulfatase is comprised at a concentration of 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.

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

[116] As used in the present invention, intraventricular injection refers to the administration of a drug by injection into the ventricles of the brain, which are connected hollow spaces in the brain. Intraventricular injection has the advantage over intracerebral injection of being able to deliver a larger volume of drug over a larger area. Various techniques for intracerebroventricular injection are known in the art, 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 to be developed in the future may be used without limitation for intracerebroventricular injection of the pharmaceutical composition of the present invention.

[117] As used herein, intracerebral injection refers to the injection of a drug into the brain tissue itself. Various techniques for intracerebral injection are known in the art, for example, Mathon et. al. 2015 describes intracerebral injection methods in detail.

[118] As used herein, intrathecal injection refers to injection into the spinal canal. Various techniques for intrathecal injections are known in the art, for example, intrathecal injection methods are 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.

[119]

[120] In the present invention, when a pharmaceutical composition or pharmaceutical formulation is administered via intracerebroventricular injection, the subject may have a certain amount of cerebrospinal fluid (CSF) drained from the brain ventricle prior to administration. The drainage of CSF may prevent an increase in intracerebral pressure after ICV administration due to a change in the volume of CSF.

[121] Preferably, the total administered volume upon intracerebroventricular (ICV) administration of a pharmaceutical composition or pharmaceutical formulation according to the present invention 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.

[122] In the present invention, administration of the pharmaceutical composition or pharmaceutical formulation to the central nervous system can provide delivery of 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 may be a brain target tissue, a spinal cord target tissue, and / or a peripheral target tissue, and administration to the central nervous system may provide systemic delivery of heparan N-sulfatase.

[123] In the present invention, administration of a pharmaceutical composition or pharmaceutical formulation to the central nervous system can achieve therapeutically or clinically effective levels or activities in various target tissues described herein. As used herein, a therapeutically or clinically effective level or activity means a level or activity sufficient to achieve 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 symptoms associated with a disease (e.g., GAG accumulation) in a target tissue.

[124] In the present invention, administration of the formulation 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 formulation or pharmaceutical composition to the central nervous system can achieve an enzymatic level or activity that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increased compared to control group (e.g., endogenous levels or activities without treatment).

[125]

[126] In the present invention, administration of the pharmaceutical composition or pharmaceutical formulation to the central nervous system can cause a decrease in GAG (e.g., heparan sulfate) storage in brain target tissue, spinal cord neurons, and / or peripheral target tissue. In the present invention, the GAG storage may 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 formulation to the central nervous system can cause reduced vacuolization in neurons. For example, it can cause a reduction 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.

[127]

[128] The pharmaceutical composition or pharmaceutical formulation according to the present invention may be administered in a pharmaceutically effective amount, wherein "pharmaceutically effective amount" means an amount sufficient to treat a condition with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined by factors including type and severity of the patient's condition, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of elimination, duration of treatment, concomitant medications, and other factors well known in the medical field. The pharmaceutical composition according to the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount in which maximum effect in a minimal amount can be achieved without side effects, which can be readily determined by those skilled in the art.

[129] Furthermore, the pharmaceutical composition or pharmaceutical formulation according to the present invention may be administered to the 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 is preferably administered as quickly as possible for patient convenience. As one example of the present invention, the rate of administration of a pharmaceutical composition or pharmaceutical formulation according to 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, most preferably about 5 ml / min or more.

[130] In another aspect, the present invention relates to a method of treating mucopolysaccharidosis type IIIA, wherein the pharmaceutical composition or pharmaceutical formulation according to the present invention is administered to a patient in need thereof, in particular a patient with mucopolysaccharidosis type IIIA.

[131] In another aspect, the present invention relates to use of the pharmaceutical composition or pharmaceutical formulation for the treatment of mucopolysaccharidosis type IIIA.

[132] In another aspect, the present invention relates to use of the pharmaceutical composition or pharmaceutical formulation for the preparation of an agent for the treatment of mucopolysaccharidosis type IIIA.

[133]

[134] Hereinafter, the embodiments are provided to describe the present invention in detail. These embodiments are intended solely to illustrate the present invention, and it is apparent to one of ordinary skill in the art that the scope of the present invention is not to be construed as limited by these embodiments.

[135]

[136] Embodiment 1. Stability of heparan N-sulfatase varying depending on buffer

[137] Embodiment 1-1: Stability comparison of different amino acid buffer types (DLS evaluation)

[138] Following previous reports that phosphate buffer negatively affects the activity of heparan N-sulfatase, DLS assays were performed to assess protein aggregation in accordance with protein concentration when phosphate buffer was replaced with amino acid buffers, specifically histidine buffer, arginine buffer or glutamate buffer.

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

[140] The PDI value was calculated as follows

[141] PDI = o2 / d2

[142] o : The standard deviation of the particle size distribution

[143] d : The average hydrodynamic particle

[144]

[145] The measured average protein radius (Z-Ave) was analyzed by analyzing liquid samples of each formulated composition where the formulation was completed by dynamic light scattering (DLS) by using Uncle instrument, and was measured after three repeated injections of 8.8 qL of sample into a Uni sample loader (Unchained Labs).

[146] As a result, as shown in Table 1, it was found that when using a histidine buffer, all formulations containing low to high concentrations of HNS showed low polydispersity index (PDI) values, indicating a monodisperse characteristic, suggesting that the protein was stable without aggregation. Whereas, when using an arginine or glutamate buffer, the PDI values were stable at low concentrations of HNS of about 2.5 mg / ml, but was sharply increased as the concentration increased at a concentration of about 7.5 mg / mL or more, indicating that the aggregation level increased and the particle size became polydisperse and destabilized at higher concentrations.

[147] Also, a better effect was achieved at a pH of 8.0 rather than at a pH of 7.5.

[148] [Table 1] Stability of amino acid buffers <A) pH 7.50 8.00 Sample Protein con. (mg / ml) ZAve. Dia. (nm) PDI Z Ave. Dia. (nm) 20 mM Histidine .......2.52....... .........8587........... 1152 8540 0.059 5.04 ........ 8.807....... 0.070 ......."W..... 0.071 7.56 8943..... 0.069 8.777 0.051 10.08 8.950 0.032 ».060 0.097 12.6 9.120 0.060 8.920 0.063 (BJ pH 7.50 8.00 Sample Protein con. (mgftnl) ZAve. Dia. (nm) PDI ZAve. Dia. (nm) PDI 20 mM Arginine 2.52 ...................................1347............................................ 0115 8.577 0.062 5.04 22.513 0.155 9.067 0.117 7.56 >1000 0.287 >1000 0.314 10.08 >1000 0.375 >1000 0.463 12.6 >1000 0,419 >1000 0,398 O pH 7.50 8.00 Sample- Protein con. (mg / ml) Z-Ave. Dia. (nm) PDI Z Ave. Dia. (nm) pa 20 mM Glutamate- 2.52 8.857 0.091 8.587 0.098 5.04 9.117 0.116 8.977 0.095 7.56 >1000 0.403 >1000 0.344 10.08 ..... >1000..... 0.483 >1000 "0.317' " 12.6 >1000 1434 >1000 0.305

[149] Classification by PDI value:

[150] < 0.1 : Low polydispersity (low aggregation)

[151] 0.1 to 0.25 : moderate polydispersity

[152] > 0.25 : High polydispersity (high aggregation)

[153]

[154] In particular, for the Z-Ave values, as shown in Table 1 and FIGs. 1(a) and 1(b), it was indirectly confirmed that when an arginine or glutamate buffer was used, heparan N-sulfatase concentrations of about 7.5 mg / mL or more exhibited Z-Ave value of more than 1,000 nm, indicating that aggregation of proteins occurs, suggesting that an arginine or glutamate buffer is not suitable for the composition of the present invention.

[155] On the other hand, the use of histidine buffer resulted in a small Z-ave value of about 9 nm, even when the heparan N-sulfatase concentration was above 7.5 mg / mL. This indicates that the histidine buffer is very suitable for the composition of the present invention, preventing protein aggregation.

[156] The Z-Ave value can be expressed as the average protein radius and is measured with a DLS instrument. In this patent, liquid samples of each formulated composition were analyzed using the Uncle instrument. The Uncle assay was performed after three repeated injections of 8.8 pL of sample into a Uni sample loader (Unchained Labs). The histidine buffer continued to exhibit low Z-Ave values.

[157]

[158] Embodiment 1-2: Stability comparison of different amino acid buffer types (KD and B22 evaluation)

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

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

[161] [Table 2] Composition of compositions for KD or B22 testing PH Amino acH Buffer Remark# 7.5 20 mM His NaCl: 200 mM HNS concentration 12.6 mg / ml 20 mM Arg 20mMGIu 8.0 20 mM His 20 mH Arg 20mMGta

[162] As a result, as shown in FIGs. 2(a) and 2(b), when an arginine buffer or glutamate buffer was used, the stability was significantly lower due to low kD or B22 value, and when a histidine buffer was used, the kD or B22 value was relatively high at both pH of 7.5 and 8.0. This indicates that the formulation with a histidine buffer has excellent stability.

[163]

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

[165] Based on previous reports that a phosphate buffer negatively affects the activity of heparan N-sulfatase, when a phosphate buffer was replaced with a histidine buffer, protein-buffer interactions were checked using the Uncle test to assess formulation stability.

[166] 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.

[167]

[168] As a result, it was found that the B22 value was significantly increased in the formulation using a histidine buffer, as shown in FIG. 3. These results suggest that protein-buffer interactions are significantly reduced by a phosphate buffer when using a histidine buffer, resulting in a more stable formulation.

[169]

[170] Embodiment 2. Evaluation of stability varying depending on pH

[171] Stability was assessed by determining a dependent turbidity in a heparan N-sulfatase composition containing a histidine buffer depending on pH.

[172] Turbidity in the present invention was analyzed with Lunatic (Unchained Labs). 2.0 p,L of the sample was injected into the Lunatic plate (Unchained Labs) and the turbidity at 350 nm was measured. Based on the turbidity of placebo buffer value, the degree of increase in the turbidity value of the sample was measured.

[173]

[174] As a result, compositions containing low concentrations of heparan N-sulfatase (5 mg / mL) had lower turbidity in all pH ranges, as shown in Tables 3 to 5 and FIG. 4.

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

[176] However, in the composition comprising a high concentration of HNS, the turbidity tended to decrease as the pH increased, and in particular, the turbidity decreased significantly when the pH was above 7.8, preferably above 7.9 to 8.0, confirming that the pH of the composition had a significant effect on the decrease in turbidity.

[177] [Table 3] Turbidity changes in compositions containing a low concentration of Hi S depending on pH HNS (mgM.) ...........msudrne (mM) NaCI (mM) Trehalose (wA / %) pH TurbWity 5 3.75 90 1.35 ................'W..................... 0.019 781 0.019 7 94 0.018 818 0.017 8.33 0015 8 44 0.017

[178] [Table 4] TUrbidity changes in compositions containing a high concentration of HNS depending on pH HNS (mg / mL) Histidine (mM) NaCI (mM) Trehalose (w / v%) pH Turbidity 13.5 375 90 1.35 7.53 1.654 7.77 0.947 8.00 0.143 8.17 0.057 8.27 0.045 8 45 0.067

[179] [Table 5] Turbidity changes in compositions containing a high concentration of HNS depending on pH HNS (mg / mL) Histidine (mM) NaCl (mM) Trehalose (w / v %) pH Turbidity 16.5 5 120 18 7.60 1.701 7.84 0.939 8.02 0.290 8.17 0.077 8.42 0.075

[180] In particular, for compositions containing HNS at very high concentrations of 16 to 17 mg / mL and 120 mM of NaCl, it was found that the turbidity decreased with increasing pH, especially pH above 7.9 to 8.0, as shown in FIG. 5.

[181]

[182] Embodiment 3. Evaluation of stability depending on surfactant addition or not

[183] Embodiment 3-1: Confirmation of changes due to surfactant addition using Aura instruments

[184] To more specifically confirm the reduction in turbidity with the addition of lower concentrations of surfactant, the pH was set to 8.2, and then the turbidity in the case of the addition of 0.005 w / v% polysorbate 20 was checked by the Aura instrument.

[185] 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 performed for both of the cases where the composition was lyophilized as it is and then reconstituted in a solution containing PS20 (0.005%), and where PS20 (0.005%) was added to the composition and reconstituted after lyophilization.

[186] Turbidity was measured using an Aura instrument, as described below.

[187] An empty plate was inserted and 'Acquire Background' was run to measure the background, then the plate was loaded with 30-50 pl of samples per well in triplicate. The cassette with drying paper was placed on the manifold, and the plate finished by the first vacuum process was placed on top of it, and the vacuum valve was opened for secondary drying. Once the vacuum process was complete, the plate was placed in the Aura instrument for measurement. Based on a 96-well plate, this instrument can quantify and plot the particle information of 1 um or more within 30-50 uL of sample compared to blank. For this purpose, the plates were vacuumed and sampled using drying paper.

[188] As a result, it was found that the turbidity was significantly reduced both when 0.005% polysorbate 20 was added before lyophilization and when it was added in the reconstitution after lyophilization, as shown in FIGs. 6(a) to 6(c).

[189] Embodiment 3-2: Confirmation of the number of insoluble particulates varying depending on surfactant addition

[190] To further verification and confirmation of the stability of the formulation varying depending on the addition of surfactant, the change in the number of insoluble particulates with the addition of 0.005% polysorbate 20 as a surfactant was confirmed.

[191] 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, at a pH of 8.0, and the experiments were performed by varying only the addition of PS20 (0.005 w / v%).

[192]

[193] As shown in Table 6 below, it was found that the addition of surfactant (PS20) significantly reduced the number of sub visible particulates having sizes of 10 pm and 25 pm or more. This result indirectly shows that the addition of surfactant improves the stability of the pharmaceutical composition according to the present invention.

[194] [Table 6] Change In the number of sub visible particulates depending on the addition of 0.005 w / v% PS20 or not Whether a surfactant is added Components of composition Determination criteria Insoluble Particulate Test Results Surfactant-tee 5 mM histidine 125 mM NaCl 1.8% Trehalose 10 um or more units / vial s 6000 units / vial 3718.5 units / vial 25 um or more units / vial £ 600 units / vial 754 3 units / vial Surfactant added 5 mM histidine 125 mM NaCl 1.8%Trehalose 0,005% PS20 10 um or more units / vial < 6000 units 1 vial 196.56 units / vial 25 um or more units / vial s 600 units / vial 10.17 units / vial

[195]

[196] Embodiment 4. Evaluation of stability depending on salt concentration

[197] To evaluate the stability of the composition according to the salt concentration included in the composition, the pH was set to 7 to 8 and the concentration of salt (NaCl) was varied from 100 to 200 mM to check the stability of the composition.

[198] 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.

[199] As a result, as shown in FIG. 7, at pH 7.0, the stability of the compositions was found to be low at all NaCl concentrations, but when the NaCl concentration was 150 mM, the stability was evaluated to be excellent at pH 8, and when the NaCl concentration was 200 mM, the stability was found to be significantly higher above pH 7.0, especially above 7.5. The above results indicate that the stability of high concentration HNS formulations increases as NaCl concentration and pH increase.

[200]

[201] Furthermore, as a result of investigation of the variation of osmolality according to NaCl concentration, it was found that the average osmolality when NaCl was comprised in an amount of 125 mM or 130 mM was 286 or 294 mOsmol / kg, respectively, confirming that the variation of osmolality according to NaCl concentration was not significant. The above osmolarities are generally in the acceptable range for pharmaceutical compositions (see Table 7).

[202] [Table 7] Change in osmolarity depending on NaCl concentration HNS Concentration (mg / ml) Buffer Average Osmolarity (mOsmol / kg) 13.5 5 mM histidine 125 mM NaCl 18%Trehalose 286 5 mM histidine 130 mM NaCl 18%Trehalose 294

[203]

[204] Embodiment 5. Evaluation of stability depending on saccharide concentration

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

[206] saccharides such as trehalose are often used as stabilizers in lyophilization. In the test, the stability of the compositions depending on the concentration of the saccharides in the compositions was evaluated, particularly the stability after reconstitution in lyophilized formulations, in terms of purity. The stability of the lyophilized formulations after reconstitution was determined by varying the concentration of trehalose from 0 to 1% (v / w).

[207] Prior to lyophilization, the concentration of each component of the prepared pharmaceutical composition is shown in Table 8 below:

[208] [Table 8] Concentration of the components of a pharmaceutical composition of Embodiment 5-1 Pharmaceutical composition Identification number HNS Con. (mg / mL) histidine (mM) NaCI (mM) Trehalose (%) 5-1-a 11 2.5 75 0.5 5-1-b 10 1.65 50 0.35 5-1-c 13 1.1 33.3 0 5-1-d 13 1.1 33.3 0.11 5-1-e 13 1.1 33.3 0176 5-1-f 13 1.1 33.3 0.22

[209] Lyophilization was performed by dispensing 1.3 ml of the liquid solution of the composition prepared according to the present invention into a glass vial (3 ml size), semisealing 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 the prepared lyophilized formulation was capped with an aluminum cap after completion of aluminum lyophilization.

[210] [Table 9] Manufacturing conditions for lyophilized formulation Step Classification 1 I Shelf temperature CC) 1 I Vacuum degree (mlorr) 1 Loading Hold 4 30 N / A 2 Freezing Rate -60 64 N / A 3 Freezing Hold -60 156 N / A 4 Primary Warming Rate -25 60 40 5 Primary Drying Hold -25 2100 40 7 Secondary Warming Rate 20 60 40 8 Secondary Drying Hold 20 1200 30

[211] The stability of the prepared lyophilized formulation was analyzed for purity after reconstitution with water for injection (WFI). According to the volume ratio of the pharmaceutical composition to the formulation after reconstitution (reconstitution ratio), 0.286 mL to 0.75 mL of distilled water was used to lyophilize and reconstitute the pharmaceutical compositions in Table 8 (Compositions 5-1-a to f) to prepare the formulations (Formulations 5-1-A to F) after reconstitution (see Table 10).

[212] [Table 10] Concentration of the components of a formulation after reconstitution of Bnbodiment 5-1 Composition after reconstitution Identification number HNS Con. (mg / mL) histidine (mM) NaCl (mM) Trehalose (*) Reconstitution Rate 5-1-A 22 5 150 1 1:0.5 5-1-B 30 3030303 5 151.5151 515 1 1:0.33 5-1-C 59.09090909 5 151.3636 364 0 1 : 0.22 5-1-D 0.5 1 :0.22 5-1-E 0.8 1 : 0.22 5-1-F 1 1 :0.22

[213] Evaluation of purity was performed using size exclusion liquid chromatography (SE-HPLC). For size exclusion liquid chromatography, it is a standard method for determining and quantifying aggregation and fragment levels. Specifically, for size exclusion chromatography, 1 mg / ml HNS was first diluted to 1.0 mg / mL using mobile phase (40 mM sodium phosphate, 300 mM NaCl, pH 7.5), followed by sterile filtration (if the concentration is below 1.0 mg / ml, the process was conducted without dilution), and 200 pL of the filtered sample was injected into a vial insert and inserted into a screw top vial.

[214] After connecting the mobile phase to the pump, an analytical column (TSKgel G3000SWXL, Tosoh) was equipped while flowing the mobile phase at a rate of 0.5 mL / min to Waters e2695 and Waters 2489 instruments (manufactured by Waters, Japan). The mobile phase was flowed at a speed of 0.5 mL / min for more than 30 minutes to equilibrate until the detector signal was stabilized, and when the temperature of the autosampler drops to 4°C, the sample was plugged into the sampler. 50 pL of the sample was injected, and the mobile phase was flowed for 35 minutes to identify the detection peak at 280 nm. The analysis was then performed using Empower Pro software on a PC.

[215] As a result, as shown in Table 11 and FIG. 8, the purity was found to be improved to about 95% or more for all trehalose concentrations above 0.1 w / v% prior to reconstitution, compared to a purity of about 88% in the absence of trehalose.

[216] [Table 11] Purity assessment results after reconstitution depending on trehalose concentratton HNS Con. (formulation after reconstitution) (mg / mL) Trehalose concentration (before lyophilization) HMW (%) Main peak (%) LMW (%) 22 0.35 0.83 98.31 0.87 30.3030303 0 11.28 88.09 0.63 59.09090909 0.11 4 54 94.53 0.92 0.176 1.92 9713 0.94 0.22 2.05 96.99 0.96 0.5 0.39 99.61 N / A HMW: high molecular weight LMW: tow molecular weight

[217]

[218] Embodiment 5-2: Stability evaluation (specific activity and purity) when adding more than 1% trehalose

[219] Furthermore, to evaluate the stability after reconstitution of lyophilized formulations 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 various pH were evaluated.

[220] The composition of the prepared formulation samples is shown in Table 12 below.

[221] [Table 12] Composition of formulation samples containing more than 1% trehalose Identification Number (Sample) HNS (mg / ml) Histidine mM NaCl mM Trehalose (w / v%) pH PS20 (W / V%) DS 15.21 3 75 90 0 7.47 0 5-2-a 12 375 90 1 35 7.52 5-2-b 375 90 1.35 7.83 5-2-c 3.75 90 1.35 8.00 5-2-d 375 90 1.35 819 5-2-e 3.75 90 1.35 8.43 5-2-A 16.84 5 120 1.8 7.60 5-2-B 15.98 5 120 1.8 7.84 5-2-C 16.36 5 120 1.8 8.02 5-2-D 17.00 5 120 18 8.17 5-2-E 16.34 5 120 1.8 842

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

[223] The activity assay of the enzyme of the present invention was performed as follows.

[224] - In step 1, the formulation sample was reacted with the synthetic substrate, 4MU-a-GlcNS, to free the sulfate on the substrate end (generating 4MU-a-GlcNH2).

[225] Reaction in step 1: 4MU-a-GlcNS + HNS^4MU-a-GlcNH2

[226] - In step 2, a-glucosidase was treated to free 4MU having fluorescence from 4MU-a-GlcNH2. The fluorescence value of the free 4MU was measured using a fluorescence reader to determine the enzymatic activity of heparan N-sulfatase in the sample.

[227] Reaction in step 2: 4MU-a-GlcNH2 + a-glucosidase—>4MU

[228]

[229] The method of the reactions in steps 1 and 2 in detail is provided as follows.

[230] Formulation samples were diluted to 100 p,g / 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 substrate diluent.

[231] - Reaction in step 1: In a 96-well plate (Black), 20 p.L of serially diluted substrate was dispensed into each well. The sample diluted to 100 p,g / mL and blank were added 10 pL each to the opposite side of the well to avoid mixing with the substrate solution, and then the solutions were mixed simultaneously by holding one side of the plate and gently striking the other side (to prevent the solution from splashing out of the well). The plate was then sealed with a plate sealer and reacted in a 37 °C incubator for 17 hours.

[232] - Reaction in step 2: After the reaction in step 1 (17 hours), 6 pL of stop solution for reaction in step 1 per well was added to stop the primary reaction. The solution was mixed well by holding one side of the plate and gently striking the other side (to prevent the solution from splashing out of the well). The a-glucosidase solution prepared at 100 U / mL was diluted 10-fold to 10 U / mL using ultrapure water. After adding 10 pL of solution per well, one side of the plate was held and the other side was gently stroked (to prevent the solution from splashing out of the well) to mix the solution well. The plate was sealed with a plate sealer and incubated in a 37 °C incubator for 24 hours.

[233] - Fluorescence measurement: 15 minutes before the fluorescence measurement, a 4MU diluent was prepared by mixing 1.5 mL of substrate diluent, 300 pL of primary reaction stop solution, 500 pL of ultrapure water, and 10 mL of secondary reaction stop solution in this order. The 4 MU stock was diluted with the 4 MU diluent as shown in Table 6 to prepare the 4 MU standard solution. After the secondary reaction, 200 pL of reaction stop solution was added to each well for final stopping of the reaction. The 4MU standards (Standards 1-8) were loaded 246 pL into each well in duplicate. Fluorescence was measured at Ex. 355 nm / Em. 460 nm with a fluorescence measurement instrument.

[234] Measurement of purity was performed as described above using size exclusion liquid chromatography (SE-HPLC).

[235]

[236] As a result, as shown in Table 13 below, FIGs. 9(a) and 9(b), when the trehalose concentration in the composition, i.e., the lyophilized formulation, was greater than or equal to 1 w / v%, the HNS specific activity was measured to be greater than or equal to about 400 (pmol / min / pg), and the purity was also confirmed to be greater than or equal to about 98%, indicating that the composition was highly stable even when prepared in the form of a lyophilized formulation and remained highly stable even after reconstitution.

[237] [Table 13] Specific acivi^ (SA) and purity of formuMton samples containing more Ban 1% trehalose TwfeMty pH UV (axp-nHl SA      ’ .............. ............1 V............ ns 2’29 7 47 15 2- 1112 66 rm 5 2.. 1 469 7 52 N / A / 83 100 0 5-2-c 8.00 0.043 8W 100.0 5 2 v 0.041 843 100.0 5 ?A 1 701 I (a 16 84 78MI5 70??- ? 10O0 5-2-B - ..... ||||W 0939 IM 15.98 8 / 0« 100.0 5 26 O2W 1« 935 79 -..... 11.0 J 5-2-0 0077 8 17 1700 / 80 54 ,70’5 • 11)0 0 5-2t 0075 8 4? ’6 34 ....................LdV'j............................ Md

[238]

[239] Example 6. Confirmation of the specific activity and purity of the composition of different compositions after reconstitution

[240] Various compositions within the numerical range of the present invention were prepared, and their stability was confirmed in terms of specific activity and purity by lyophilization and reconstitution.

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

[242] [Table 14] Concentrations of the components of tie pharmaceutical compositions and the formulations in Embodiment 6 Identification Number Formulation HNS WW Histidine mM 11 Trehalose (w / v%) PS20 (wAm) >r Pharmaceutical ■Coiposibn (before lyophilization) 11.25 5 93 75 135 0 00375 Formulation after reconsSWBon- (after lyophilization) 15 6.66 125- 1.8 0.005 6-2* Pharmaceutical Composition (before lyophilization) 11.25 5 300 135 0.05 FSSulaion alef • reconstitulon (after lyophilization) 15 6.66 400 IS 0067 6-3 Pharmaceutical •Composition (before lyophilization) 10 5 300 1.35 0.005 Formulation after •reconstiuion (after lyophilization) 10 5 300 1.35 0005 ••«-4 Pharmaceutical Composition (before>ophifeai(^ 10 5 300 1.35 0.05 Formulation after reconstitution (alter lyophifeation) 10 5 300' 1.35 0.05 6-5 Pharmaceutical 10 5 300 3.0 0.05

[243] Composition (before lyophilization) Formulation after reconstitution (after lyophilization) 10 5 300 3.0 <05 w Pharmaceutical ■Composition (Before lyophilization) 5 5 300 135 0.05 FonStiaion after reconstttBtim (after lyophilization) 5 5 300 1.35 0.05 6-7 Pharmaceutical Composition (before lyophilization) 5 5 200 1.35 0.05 FSmulalonaler reconstitution (aft«>ophilzaia< 5' 5 200 135 0.05 6-8 Pharmaceutical ComposiSon (before lyophilization) 2 5 300 135 0.05 Formulation after recoi^tituttcm (after lyophilization) / 2 5 300 1.35) 0.05 6-9 Pharmaceutical Composition (before lyophilization) 5 200 '1.35: 005 Formulation after ieconstitaticn( (after lyophilization) 2 5 200 13S 0.05

[244] 6-to Pharmaceutical ComposiHon (before lyophilization) 2 5 150 1.35 0.05 Formulation alter reconsBtution (after lyophilization) 2 5 150 1.35 0.05

[245]

[246]

[247]

[248]

[249] * 6-1 and 6-2 have the ratio (v:v) of the pharmaceutical composition to the formulation after reconstitution of 1 : 0.75 6-3 to 6-10 have the ratio (v:v) of the pharmaceutical composition to the formulation after reconstitution of 1 : 1 Purity was measured by the same method as described in Embodiment 5-1 above, and specific activity was measured by the same method as described in Embodiment 5-2 above. The composition and measurement results are shown in Table 15 below. [Table 15] Measurement results of specific activity and purity of samples Veasumment resuts Axspea'arce iafer recor.; pH Sfnal jnAWd soMtitr) pH ■Ceomp eted fwvuiatton: ccr servant formulas on a*ter reoor SIC 00 SA 6-1 »12» 15« 1» .............|ji||i|..................... ■■ aw MS» IM 1» WM 1¾¾ •.197 ».125 IM# 1» ®M 3 ; S MS M2* M7 820.7 «*14 »5 w 102» ■Bill........................ 11111 awi fcOM 191 993 909.1 •w 7.972 SOS 993 790,» ■ MS 7.99» 2.00 M3 »49.1 W IW 7» 202 9243 610 am 7 2 01 930 1042S        \

[250] All of the samples of 6-1 to 6-10 were found to exhibit high purity of greater than 98%, and high specific activity of greater than 690 pmol / min / ug.

[251] Industrial Applicability

[252] A pharmaceutical composition and a pharmaceutical formulation comprising high concentrations of heparan N-sulfatase (HNS) and a pharmaceutical formulation comprising the same of the present invention can be useful in enzyme replacement therapy (ERT) for the treatment of mucopolysaccharidosis type IIIA by replacing the phosphate buffer, which is known to inhibit the activity of the active ingredient, heparan N-sulfatase, with a histidine buffer, resulting in significantly improved formulation stability due to reduced protein-protein or protein-buffer interactions and reduced turbidity.

[253]

[254] Although the foregoing has described in detail certain aspects of the present invention, it is apparent to one 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

Claims

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

0.

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

3. The pharmaceutical composition according to claim 1, wherein the histidine buffer is comprised 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 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 saccharide is comprised at a concentration of 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 KC1.

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

11. The pharmaceutical composition according to claim 1, which 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 surfactant is comprised at a concentration of 0.0001 to 0.1 w / v%.

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

17. the treatment of mucopolysaccharidosis type IIIA. A pharmaceutical formulation comprising a pharmaceutical com

18. position according to any one of claims 1 to 16. The pharmaceutical formulation according to claim 17, which is a

19. liquid formulation or a lyophilized formulation. The pharmaceutical formulation according to claim 17, which is administered into the central nervous system via intracerebroventricular

20. injection (ICV), intracerebral injection, or intrathecal injection. The pharmaceutical formulation according to claim 18, wherein the lyophilized formulation comprises 2 to 60 mg / mL of heparan N-sulfatase after reconstitution.

21. The pharmaceutical formulation according to claim 19, wherein the total administered volume is 10 ml or less.

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

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