Composition for the treatment of sanfilippo syndrome type a (mps iiia) comprising heparan n-sulfatase (HNS)

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of Sanfilippo syndrome type A (MPS IIIA) comprising heparan N-sulfatase (HNS), and more particularly to an optimal dose and cycle of administration of heparan N-sulfatase that can effectively reduce the accumulation of heparan sulfate (HS) while improving cognition in patients. According to the present invention, it may be useful as an enzyme replacement therapy (ERT) for the treatment of Sanfilippo syndrome type A.

Description

COMPOSITION FOR THE TREATMENT OF SANFILIPPO SYNDROME TYPE A (MPS IIIA) COMPRISING HEPARAN N-SULFATASE (HNS)

[0001] The present invention relates to a pharmaceutical composition for preventing or treating Sanfilippo syndrome type A (MPS IIIA) comprising heparan N-sulfatase (HNS), and more particularly to a pharmaceutical composition characterized by administering HNS at a specific dose and cycle, and a method of preventing or treating MPS IIIA using the same.

[0002]

[0003] Lysosomal storage diseases (LSDs) are inherited metabolic disorders caused by defects in the function of lysosomes. LSDs are caused by lysosomal dysfunction due to a deficiency of a 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) or mucopolysaccharide deposition is one of LSDs that result from accumulation in lysosome due to a deficiency of lysosomal enzymes required for the degradation of glycosaminoglycans (GAGs).

[0004] Sanfilippo syndrome is a type of MPS, named after Dr. Sanfilippo, an American physician who first discovered this condition in 1963. Sanfilippo syndrome, also known as MPS III, is an autosomal recessive genetic disorder that is clinically characterized by the absence of corneal opacity, mild physical changes such as hepatosplenomegaly and skeletal changes, but very severe and progressive central nervous system (CNS) symptoms.

[0005] Sanfilippo syndrome is caused by a deficiency of four different enzymes required to break down polysaccharides, specifically GAGs. Depending on the deficient enzyme, Sanfilippo syndrome is categorized into MPS IIIA (Sanfilippo A), MPS IIIB (Sanfilippo B), MPS IIIC (Sanfilippo C), and MPS IIID (Sanfilippo D). The deficient enzyme and genetic map locus for each of the Sanfilippo syndromes are listed below.

[0006] Type A (MPS IIIA): Heparan N-sulfatase - Chromosome 17 (17q25.3)

[0007] Type B (MPS IIIB): N-acetyl-α-D-glucosaminidase - Chromosome 17 (17q21)

[0008] Type C (MPS IIIC): Acetyl-CoA:α-glucosaminide-N-acetyltransferase -Chromosome 14

[0009] Type D (MPS IIID): N-acetyl-α-D-glucosaminide-6-sulfatase - Chromosome 12 (12q14)

[0010]

[0011] MPS IIIA is caused by a deficiency of heparan N-sulfatase (HNS), an enzyme involved in the breakdown of heparan sulfate (HS), which hydrolyzes the sulfate portion attached to the amino group of the glucosamine residue of HS. Symptoms of MPS IIIA usually appear between the ages of 2 and 6 years, but some cases are diagnosed after the age of 13. Overall, patients with MPS IIIA have significant developmental delays and are known to have poor long-term survival.

[0012] Currently, there is no approved treatment for MPS IIIA, and only palliative care is available for symptomatic relief. Enzyme replacement therapy (ERT), in which patients with MPS IIIA are given exogenously manufactured HNS, is expected to be very useful in the treatment of this disease.

[0013]

[0014] ERT, which involves the administration of a functional lysosomal enzyme that is deficient to correct a deficiency in the enzyme's function, is one of the main therapies to treat LSDs and has the advantage of reducing symptoms and preventing permanent damage to the body with a simple injection. As a well-known ERT for enzyme storage diseases, intravenous (IV) therapy with glucocerebrosidase (GCase) was first FDA approved for Gaucher disease in 1991 (National Gaucher Foundation. Retrieved 2017-06-08).

[0015] However, given that most of LSDs cause excessive accumulation of GAGs in the CNS, especially in the neurons and meninges of the brain, and lead to a variety of CNS disorders, ERTs administered intravenously cannot effectively treat neurological disorders and diseases caused by lysosomal accumulation, especially in the brain, because the active ingredient, lysosomal enzymes, have difficulty in crossing the blood-brain barrier (BBB), resulting in inadequate delivery of enzymes to the CNS. Therefore, various CNS delivery therapies that deliver drugs directly to the CNS to deliver enzymes that bypass the BBB are being studied.

[0016] A variety of therapies have been developed to deliver drugs to the CNS bypass the BBB. In particular, intracerebral injection (IC), intracerebroventricular injection (ICV), and intrathecal injection (IT) are most common administration route to deliver proteins directly into the brain.

[0017] IT and ICV injections have emerged as methods for delivering enzymes to the CNS for MPS and have shown significant reductions in GAGs and significant improvements in neurologic symptoms in various animal models of MPSs (Molecular Therapy - Methods & Clinical Development, 21, 67-75). However, since direct brain injection therapies are very limited in dosage, development of ERTs having appropriate effective doses and cycles is required to achieve effective levels of therapeutic benefit.

[0018]

[0019] Under this technical background, the inventors of the present invention have identified the optimal dose and cycle of administration of HNS to the CNS, which has a significant effect, and have completed the present invention.

[0020]

[0021] The information provided in the Background Art section is intended solely to enhance the understanding of the background of the present invention and may not include information that constitutes prior art known to one having ordinary skill in the art.

[0022]

[0023] [SUMMARY OF THE INVENTION]

[0024] It is an object of the present invention to provide a method of preventing or treating Sanfilippo syndrome type A (MPS IIIA) by administering heparan N-sulfatase (HNS) at effective dose and cycle that are sustainable and stable.

[0025]

[0026] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of Sanfilippo syndrome type A (MPS IIIA) comprising heparan N-sulfatase (HNS), wherein the heparan N-sulfatase is administered to a patient at a dose of 3 to 150 mg per dose, at intervals of 2 to 4 weeks, via intracerebroventricular injection (ICV), intracerebral injection (IC), or intrathecal injection (IT).

[0027] The present invention also provides a method of preventing or treating Sanfilippo syndrome type A (MPS IIIA), comprising administering the pharmaceutical composition to a patient.

[0028] The present invention also provides the use of the pharmaceutical composition for the prevention or treatment of Sanfilippo syndrome type A (MPS IIIA).

[0029] The present invention also provides the use of the pharmaceutical composition for the manufacture of a medicament for the prevention or treatment of Sanfilippo syndrome type A (MPS IIIA).

[0030]

[0031] FIG. 1 is a graph showing the results of the single-administration efficacy test of GC1130A, wherein FIG. 1A shows the HS content in brain (all data represent as mean ± SEM.**P < 0.01, ***P < 0.0005, ****P < 0.0001compared with vehicle treated MPS IIIA mice), and FIG. 1B is shows the HS content in cerebrospinal fluid (CSF) (all data represent as mean ± SEM.**P < 0.01, ***P < 0.0005, ****P < 0.0001compared with vehicle treated MPS IIIA mice).

[0032] FIG. 2 is a graph showing the results of the low-dose, single-administration efficacy test of GC1130A, wherein FIG. 2A shows the HS content in brain (all data represent as mean ± SEM*** P < 0.001, ****P < 0.0001compared with G2) and FIG. 2B shows the HS content in CSF (all data represent as mean ± SEM *P < 0.05, ** P < 0.002, ****P < 0.0001compared with G2).

[0033] FIG. 3 is a graph showing the correlation of HS in brain and CSF when administering via ICV at a single dose.

[0034] FIG. 4 is a graph showing HS content in brain and CSF upon repeated dose depending on dosage of GC1130A (analyzed by one-way ANOVA Dunnett's multiple comparisons test,* p <0.05 ** p<0.01, *** p<0.001, **** P<0.0001VS Vehicle group, (mean ± S.E.M) using software GraphPad Prism version 9.4.0.).

[0035] FIG. 5 is a graph showing the correlation of HS in brain and CSF upon repeated ICV administration.

[0036] FIG. 6 is a graph showing the results of an open field test to evaluate mouse behavior upon repeated administration of GC1130A.

[0037] FIG. 7 shows the biodistribution of GC1130A conjugated with fluorescent dye after IV or ICV administration in mice (graphs in FIG. 7B are n=4 for each time profile).

[0038] FIG. 8 is a time profile for GC1130A administered ICV in the mouse brain, where error bars represent the standard deviation of the mean (n=4).

[0039] FIG. 9 is a graph showing the results of a quantitative assay for GC1130A administered repeatedly ICV into the mouse brain.

[0040] FIG. 10 is a graph showing the results of quantitative IHC analysis of brain pathologic changes and LAMP2 analysis after repeated ICV administration.

[0041] FIG. 11 is a graph showing the results of quantitative IHC analysis of brain pathologic changes, CD68 analysis after repeated ICV administration.

[0042]

[0043] [DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS OF THE INVENTION]

[0044] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by those skilled in the art. In general, the nomenclature used herein is well known and in common use in the art.

[0045]

[0046] In an example of the present invention, to determine the effective dosage and interval of HNS as an ERT, it was observed that administering a single dose of 15 to 60 μg at intervals of 2 to 4 weeks resulted in significant reductions in HS levels and improvements in behavioral indicators in a mouse model of Sanfilippo syndrome type A (MPS IIIA).

[0047] Furthermore, by comparing allometric scaled values based on brain weight and CSF, it was determined that when administered at intervals of 2 to 4 weeks, the single dose for human patients is 3 to 150 mg, preferably 6 to 100 mg, and even more preferably 6 to 60 mg.

[0048]

[0049] Accordingly, in one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of Sanfilippo syndrome type A (MPS IIIA) comprising heparan N-sulfatase (HNS), wherein the heparan N-sulfatase is administered to a patient at a dose of 3 to 150 mg per dose, at intervals of 2 to 4 weeks, via intracerebroventricular injection (ICV), intracerebral injection (IC), or intrathecal injection (IT).

[0050]

[0051] HNS is a lysosomal enzyme that catalyzes the hydrolysis of HS and the N-linked sulfate group from the non-reducing terminal glucosamine moiety of HS or heparan (Biochem. Biophys. Res. Commun. 2001, 280, 1251-1257). Mutations in the heparan N-sulfatase gene (SGSH) are well known to cause MPS type A (MPSIIIA, OMIM# 252900), also known as Sanfilippo A syndrome. MPS type A is characterized by a deficiency of the enzyme heparan N-sulfatase (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, GAGs are accumulated in the lysosomes of neurons and glial cells, causing severe neurological damages.

[0052] As used herein, the term "heparan N-sulfatase" may be used interchangeably with N-sulfoglucosamine sulfohydrolase (SGSH).

[0053] In the present invention, the heparan N-sulfatase may have a wild-type or naturally occurring amino acid sequence. For example, the heparan N-sulfatase may be derived from various organisms, more preferably from humans, but not limited thereto. In the present invention, the heparan N-sulfatase may comprise an amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto. In the present invention, the heparan N-sulfatase may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to a wild-type or naturally occurring sequence.

[0054]

[0055] In the present invention, the heparan N-sulfatase can be a recombinantly produced recombinant enzyme. The recombinant production of heparan N-sulfatase can be facilitated by techniques for the production of recombinant cells for the expression of various target proteins known in the art.

[0056] In the present invention, the heparan N-sulfatase may also be comprised in the form of a fusion protein or conjugate. In the present invention, the heparan N-sulfatase may be fused or conjugated with a moiety capable of binding to a receptor on the surface of brain cells and / or a lysosome-targeted formulation to facilitate cellular uptake or lysosomal targeting. Modification of alternative enzymes, such as heparan N-sulfatase, is disclosed in Korean Patent No. 10-2007044.

[0057]

[0058] In the present invention, the heparan N-sulfatase may be administered to a patient at a dose of 3 to 150 mg, preferably 5 to 120 mg, more preferably 6 to 100 mg, most preferably 6 to 60 mg per dose, at intervals of 2 to 4 weeks, but not limited thereto.

[0059] In particular, the heparan N-sulfatase may also be administered in a single dose of 6 to 60 mg at intervals of 2 to 4 weeks, most preferably at intervals of 2 weeks, but not limited to.

[0060] In the present invention, the total administered volume per dose of the pharmaceutical composition may be 10 mL or less, preferably 9 mL or less, more preferably 6 mL or less, but not limited thereto.

[0061]

[0062] In the present invention, the pharmaceutical composition may comprise heparan N-sulfatase 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 not limited thereto.

[0063] The pharmaceutical composition may further comprise 1 to 40 mM of a histidine buffer.

[0064] Although formulations for central nervous system delivery of heparan N-sulfatase comprising phosphate have been reported (e.g., Korean Patent No. 10-2007044), studies have 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, for central nervous system delivery of heparan N-sulfatase, histidine buffer can be used as a stabilizing agent to replace phosphate. Histidine buffers offer several advantages over conventional phosphate buffers, including a dramatic increase in stability due to reduced protein-protein and protein-buffer interactions, and a significant reduction in turbidity.

[0065] In a pharmaceutical composition according to the present invention, the histidine buffer may be comprised, but is not limited to, at about 1 to about 40 mM, preferably about 2 to about 30 mM, more preferably about 3 to about 25 mM, and most preferably about 5 to about 20 mM. The concentration in the histidine buffer above is a concentration calculated based on the concentration of histidine.

[0066] The pH of the pharmaceutical composition according to the present invention may be, but is not limited to, about 7.8 or more, 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.

[0067]

[0068] In the present invention, the pharmaceutical composition may further comprise a saccharide.

[0069] The inclusion of sugars, in particular trehalose, in the pharmaceutical composition results in remarkably high purity (%) and titers, not only when the composition is used in liquid form as it is, but also when it is formulated and reconstituted into lyophilized formulations.

[0070] In the present invention, the saccharide may be at least one selected from the group consisting of trehalose, sucrose, maltose, lactose and sorbitol. In the present invention, the sugars may be included at a concentration of about 0.1% or more, about 0.5% or more, about 1.0% or more, or about 1.3% or more, and more particularly, about 0.1% to about 5.0%, preferably about 0.5 to about 4.0%, and most preferably about 1.0% to about 3.0%.

[0071] In the present invention, the % concentration of each substance refers to w / v% unless otherwise specified.

[0072]

[0073] In the present invention, the pharmaceutical composition may further comprise a salt.

[0074] In the present invention, the salt may be NaCl or KCl. In the present invention, the salt may be comprised in a concentration of about 30 mM to about 500 mM, preferably about 50 mM to about 400 mM, more preferably about 60 mM to about 200 mM, most preferably about 80 mM to 150 mM, but not limited thereto.

[0075] In the present invention, the salt may be comprised in a concentration having an appropriate osmolarity for central nervous system delivery of the pharmaceutical composition of the present invention. Suitable osmotic concentrations of pharmaceutical formulations for central nervous system delivery are well known in the art.

[0076] 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 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 not limited thereto.

[0077]

[0078] In the present invention, the pharmaceutical composition may further comprise a surfactant.

[0079] In the present invention, the surfactant may be a polysorbate-based surfactant, more preferably polysorbate 20 or polysorbate 80, most preferably polysorbate 20.

[0080] In the present invention, the surfactant may be 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%, but not limited thereto.

[0081] However, when the pharmaceutical composition according to the present invention is formulated into a lyophilized dosage form, reconstituted and administered to a patient, the surfactant may be used in the form included in the solution for reconstitution, rather than included in the pharmaceutical composition and dosage form for lyophilization.

[0082]

[0083] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents conventionally used in pharmaceutical compositions.

[0084] In particular, pharmaceutical excipients useful in liquid protein formulations are well known to those skilled in the art. Non-limiting examples include, for example, body solvents or co-solvents; sugars or sugar alcohols, such as mannitol, sucrose, sorbitol, fructose, maltose, lactose, or dextran; buffers; preservatives, such as benzalkonium chloride, benzethonium chloride, tertiary ammonium salts, and chlorohexidinediacetate; carriers, such as poly(ethylene glycol) (PEG); antioxidants, such as ascorbic acid, sodium metabisulfite, and methionine; chelating agents, such as EDTA or citric acid; or biodegradable polymers, such as water-soluble polyester; cryoprotectants; lyophilization protectants; bulking agents; and stabilizing agents, and protein formulations described herein may include 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), provided that they do not adversely affect the desirable characteristics of the formulation.

[0085]

[0086] 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.

[0087] The liquid formulation is preferably, but not exclusively, in the form of an ampoule or pre-filled syringe.

[0088] Preferably, the pharmaceutical composition can be formulated in a lyophilized formulation. Lyophilized formulations have advantages in storage and transportation and can be prepared by various freeze-drying methods known in the art in addition to those described in the examples of the present invention.

[0089] If the composition according to the present invention is formulated in lyophilized form, i.e. as a dry powder, it can be reconstituted into a liquid composition for administration. Non-limiting examples for the solution for reconstitution may include an ordinary aqueous solution, a saline solution, or the like, and if the composition according to the present invention does not contain a surfactant or contains a surfactant in insufficient amounts, the solution for reconstitution may contain a surfactant such as PS20 or PS80.

[0090]

[0091] The composition according to the present invention can be administered to the central nervous system via various methods of administration. They can be administered to the central nervous system via intracerebroventricular injection (ICV), intracerebral injection (IC) or intrathecal injection (IT), most preferably via intracerebroventricular injection (ICV).

[0092] As used herein, intracerebroventricular injection refers to the administration of a drug by injection into the ventricles of the brain, which are the connected hollow spaces of the brain. Intracerebroventricular injections have the advantage over intracerebral injections 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, and are continuously developed and reported. Various other intracerebroventricular injection devices and techniques known or hereafter developed in the art may be used without limitation for intracerebroventricular injection of a pharmaceutical composition of the present invention.

[0093] 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 describing intracerebral injection methods in detail.

[0094] As used herein, intrathecal injection refers to injection into the spinal canal. Various techniques for intrathecal injection are known in the art and are described in detail in, for example, Lazorthes et al. Advances in Drug Delivery Systems and Applications in Neurosurgery, 143-192 and Omaya et al. Cancer Drug Delivery, 1: 169-179, as a representative method.

[0095]

[0096] In the present invention, when a pharmaceutical composition is administered via intracerebroventricular injection, the subject may have a certain amount of cerebrospinal fluid (CSF) drained from the ventricle prior to administration. The evacuation of CSF may prevent an increase in intracerebral pressure due to a change in volume of CSF after ICV administration.

[0097] Preferably, the total administered volume for intracerebroventricular (ICV) administration of a pharmaceutical composition according to the present invention may be 10 mL or less, preferably 9 mL or less, more preferably 6 mL or less, but not limited thereto.

[0098]

[0099] In the present invention, administration of the pharmaceutical composition to the central nervous system can provide delivery of heparan N-sulfatase to various target tissues, such as the brain, spinal cord, and periphery. In the present invention, the target tissue 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.

[0100] In the present invention, administration of the pharmaceutical composition 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 confer 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.

[0101] In the present invention, administration of the formulation or pharmaceutical composition into 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%, 95% of the normal level or activity of heparan N-sulfatase in the target tissue. In the present invention, administration of the pharmaceutical composition to the central nervous system can achieve an enzymatic level or activity that is increased by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to a control group (e.g., endogenous levels or activity without treatment).

[0102] In the present invention, administration of the pharmaceutical composition 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 the subject before treatment or after vehicle-only administration). In the present invention, administration of the pharmaceutical composition 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 compared to a negative control group.

[0103]

[0104] The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in a single dose or multiple doses.

[0105] Furthermore, the pharmaceutical composition according to the present invention is preferably administered at a preferably rapid rate of administration for patient comfort. As one example, the rate of administration of the pharmaceutical composition 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, and most preferably about 5 ml / min or more.

[0106]

[0107] As used herein, the term "prevention" means any act of preventing the onset of a disease or delaying the progression of a disease by administration of a composition. Also, as used herein, the term "treatment" refers to any act by which the administration of a composition ameliorates the symptoms of a disease or reduces or cures the symptoms of a disease.

[0108] As used herein, "patient" means a mammal, preferably a human, suffering from, or at risk of, a condition or disease that can be alleviated, inhibited or cured by administration of a composition according to the present invention.

[0109]

[0110] In another aspect, the present invention relates to a method of preventing or treating Sanfilippo syndrome type A (MPS IIIA), comprising administering the pharmaceutical composition to a patient.

[0111] In another aspect, the present invention relates to use of the pharmaceutical composition for the prevention or treatment of Sanfilippo syndrome type A (MPS IIIA).

[0112] In another aspect, the present invention relates to use of the pharmaceutical composition for the manufacture of a medicament for the prevention or treatment of Sanfilippo syndrome type A (MPS IIIA).

[0113]

[0114] The present invention will now be described in more detail with reference to the following examples. These examples are intended solely to illustrate the present invention, and it will be 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 examples.

[0115]

[0116] The recombinant heparan N-sulfatase (rHNS) used in examples of the present invention is named "GC1130A" or "GC1130A protein".

[0117]

[0118] Example 1: High-dose single-administration efficacy study for GC1130A

[0119] The purpose of this study was to evaluate the efficacy of GC1130A as a single intracerebroventricular (ICV) injection treatment for Sanfilippo syndrome type 2 (MPS IIIA) in the MPS IIIA mouse (C57BL / 6 Sgshmps3a gene WT, homozygous mutant, 2-week, male & female, n=12) model, and to establish the dosing range and interval for repeated ICV dosing efficacy studies.

[0120] The animals were categorized into five different groups to evaluate the efficacy of the targeted drugs, as shown in Table 1 below.

[0121]

[0122] Groups of treatment and control mice received 12 to 110 μg / dose GC1130A (22 mg / mL, GC Biopharma), and vehicle, respectively, and there were no deaths, systemic symptoms, or changes in body weight and organ weights in any group after ICV administration. The animals were divided into three dose groups and necropsied at 7, 14, and 28 days post-dose, and brain and cerebrospinal fluid (CSF) were collected and stored in a cryogenic freezer until analysis. Heparan Sulfate (HS) content in brain and CSF over time was analyzed by LC-MS / MS to determine the trend of HS clearance after each dose (FIG. 1).

[0123] It can be seen that the diseased mice have already accumulated HS on day 7. At the lowest dose of 12 μg, HS decreased on day 7 and increased on day 14, while at the intermediate doses of 37 μg and 110 μg, HS continued to decrease until day 14 and rebounded on day 28. In other words, it was found that there was a dose-dependent effect in both brain and CSF only on day 14 after drug administration, and repeat doses of 15, 30, and 60 μg were selected based on the above results.

[0124] After 14 days of treatment, there was a clear trend toward a decrease in HS, and after 28 days, the therapeutic effect of the drug waned but was maintained except for the low dose group, so further experiments were conducted with dosing intervals of every two weeks (2QW) or every four weeks (4QW).

[0125]

[0126] Example 2: Low-dose single-administration efficacy study for GC1130A

[0127] The purpose of this study was to evaluate the efficacy of GC1130A as a single low-dose intracerebroventricular (ICV) injection treatment for Sanfilippo syndrome type A (MPS IIIA) in a mouse model of MPS IIIA (C57BL / 6 Sgshmps3agene WT, homozygous mutant, 2-week, male & female, n=14).

[0128] The animals were categorized into five different groups to evaluate the efficacy of the targeted drugs, as shown in Table 2 below.

[0129]

[0130] Groups of treatment and control mice received 1.5 to 15 μg / dose GC1130A (15 mg / mL, GC Biopharma), and vehicle, respectively, and there were no deaths, systemic symptoms, or changes in body weight and organ weights in any group after ICV administration. Treatment groups were necropsied at 14 and 28 days post-dose, and brain and cerebrospinal fluid (CSF) were collected and stored in a cryogenic freezer until analysis. Heparan sulfate (HS) content in brain and CSF over time was analyzed by LC-MS / MS to determine the trend of HS clearance after dose-specific administration (FIG. 2).

[0131] HS in brain showed a statistically significant dose-dependent reduction in all treatment groups compared to the control groups on days 14 and 28 (Day 14: -31% for G3, -61% for G4, -80% for G5 compared to the control groups; Day 28: -32% for G3, -32% for G4, -67% for G5 compared to the control groups). HS in cerebrospinal fluid showed a statistically significant dose-dependent reduction in all treatment groups relative to the control group on day 14, but the reduction was insignificant on day 28 (Day 14: G3 -45%, G4 -62%, G5 -57% relative to the control group; Day 28: G3 -15%, G4 -23%, G5 -8% relative to the control group).

[0132] In other words, the GC1130A treatment group (≥1.5 μg / dose) was confirmed to have the dose-dependent depletion of HS in the brain that is typically observed up to 28 days post-mortem, while in the CSF, a significant HS depletion was observed only at day 14 in the GC1130A treatment group at the 1.5 μg / dose dose.

[0133] In conclusion, based on the concentration of action of GC1130A and the analysis of the maximum volume in the mouse ventricle (5 μL / dose), effects on the brain and cerebrospinal fluid were identified at the lowest dose of 1.5 μg / dose.

[0134] Furthermore, strong correlations in both the hourly and overall analyses confirmed a significant correlation between HS levels in brain and CSF in response to GC1130A administration (FIG. 3).

[0135]

[0136] Example 3: Repeated administration efficacy study for GC1130A

[0137] The purpose of this study was to evaluate the efficacy of GC1130A as an ICV injection treatment for the treatment of central nervous system symptoms in MPS IIIA mice.

[0138] A formulation containing GC1130A protein (5 mM histidine, 125 mM NaCl, 1.8% Trehalose, 0.005% PS20, pH 8.11) was used, and the MPS IIIA mouse (C57BL / 6 Sgshmps3agene WT, homozygous mutant, 2-week, male & female) model was used, and each mouse group is shown in Table 3 below.

[0139]

[0140]

[0141] Cerebrospinal fluid (CSF) samples were collected at necropsy, and the isolated brains were stored in a cryogenic freezer until HS measurements.

[0142]

[0143] HS levels in mouse CSF and brain samples were measured by LC-MS / MS, and a biochemical marker, the open-field test behavioral assessment, was performed to validate the improvement in central nervous system function. In addition, MRI analysis and pathological analysis by immunohistochemistry were performed to confirm changes in actual brain structure (Table 4).

[0144]

[0145]

[0146] The biochemical parameter HS was significantly reduced in both brain and CSF in the GC1130A 15-60 μg treatment group, and the degree of HS inhibition in brain was greater than in CSF. Direct injection into the ventricles of the brain is likely responsible for these results. The 2-week interval dosing resulted in greater inhibition than the 4-week interval dosing, but HS was effectively inhibited at the 4-week interval as well, and no dose dependence or gender difference was observed (FIG. 4).

[0147] Furthermore, a significant positive correlation was found between the HS content in CSF and brain tissue (FIG. 5).

[0148]

[0149] Open field behavioral test confirmed that various behavioral parameters were improved in the treatment group of repeated administration of GC1130A. To ensure statistical significance, data for the vehicle-treated groups of mice with WT and MPS IIIA were calculated by summing the biweekly (Q2W) and monthly (Q4W) treatment groups and found no statistical difference between the two groups. The total activity of GC1130A showed a trend toward increased activity compared to the control group (FIG. 6).

[0150] The minimum dose to expect cognitive improvement was found to be 30 μg / dose x1 / month. Even if HS reduction in CSF is 50% or more, cognitive improvement is not expected if CSF HS is at vehicle-treated levels for 2 weeks. For cognitive improvement, CSF HS must remain 50% or less of vehicle-treated for at least 2 weeks (15 μg / dose x2 / month).

[0151]

[0152] As a result, HNS was found to be effective against MPS IIIA at doses of 15-60 μg / dose in mice, administered every 2 to 4 weeks.

[0153]

[0154] Example 4: Biodistribution of GC1130A conjugated with fluorescent dye after IV or ICV administration in mice

[0155] The purpose of this study was to analyze the in vivo pharmacokinetics of GC1130A in mice according to route of administration and to evaluate the distribution profile of GC1130A in different tissues over time.

[0156] After a single dose of GC1130A 10 mg / kg conjugated with fluorescent dye was administered via IV or ICV to mice, the fluorescence intensity was imaged by in vivo imaging system (IVIS) equipment at time profiles of 0.083, 1, 2, 4, 8, 24, 48, 96, and 192 hours to determine the distribution by organ (Table 5).

[0157]

[0158] When GC1130A was administered via IV, it was observed that there was higher fluorescence intensity in the rest of the body compared to the brain, whereas when administered ICV, it was observed that there was higher fluorescence intensity in the brain compared to other organs (FIG. 7A).

[0159] FIG. 7B shows the average radiant efficiency of brain-time curves after IV or ICV administration of GC1130A, with the average radiant efficiency in the brain being highest after ICV injection compared to the other dosing regimens.

[0160]

[0161] Example 5: Pharmacokinetics of GC1130A administered via ICV in mouse brain

[0162] This study was conducted to evaluate the pharmacokinetics of GC1130A in the brain after a single ICV administration of GC1130A in C57BL / 6 5-week-old mice.

[0163] After ICV administration to 5-week-old C57BL / 6 mice, four mice were necropsied and the brains were homogenized for each of the following time profiles: 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, 168, 240, 336, 504, and 672 hours (Table 6). The homogenates were used to measure GC1130A concentrations in the brain by digital ELISA assay and subjected to pharmacokinetic analysis.

[0164]

[0165] After a single ICV administration of GC1130A, dose-dependent drug exposure was observed in the brain, with a half-life of approximately 7 days (FIG. 8).

[0166]

[0167] Example 6: Quantitative analysis of GC1130A in brain according to repeated ICV administration

[0168] After necropsy, brain tissues of one male and one female per group were embedded in paraffin. After immunostaining using staining equipment, the stained slides were scanned to establish a region of interest (ROI), and the expression of each marker was quantified.

[0169] Brain pathological changes were quantitatively analyzed after repeated ICV administration of GC1130A, confirming drug delivery and efficacy through the CNS by brain administration (FIG. 9).

[0170]

[0171] Example 7: Quantitative IHC analysis of brain pathologic changes - LAMP2, CD68 analysis after repeated ICV administration

[0172] After necropsy, brain tissues of one male and one female per group were embedded in paraffin. After immunostaining using staining equipment, the stained slides were scanned to establish a region of interest (ROI), and the expression of each marker was quantified.

[0173] It was found that LAMP2 and CD68, markers of inflammation and microglia, were increased in the MPS IIIA vehicle group and decreased in the GC1130A group (FIGs.10 and 11). Furthermore, the decrease in these markers was observed to be greater at Q2W (2-week interval) than at Q4W (4-week interval). It was also observed that GC1130A was detected in the same brain regions where LAMP2 and CD68 markers were decreased.

[0174]

[0175] Example 8: Analysis on dosage of GC1130A in humans

[0176] Example 8-1: Determination of starting dose of GC1130A in pediatric clinical

[0177] The pediatric doses proposed in this example are based on methods validated in Hammon K, et al, Clin Transl Sci, 14, 1810-1821, 2021, and were used to translate non-clinical data to human application for ultra-rare neurodegenerative pediatric diseases. Allometric scaling and PK modeling methods were developed to predict dosage from non-clinical data. These methods were compared and analyzed to select the most conservative value as the starting dose for clinical.

[0178]

[0179] Example 8-2: Calculation of human equivalent dose (HED) based on brain weight

[0180] The principle of allometric scaling is based on direct administration to a body compartment and is usually based on body surface area, but in the case of GC1130A, it is based on scaling with a proportion of brain weight since it is administered directly to brain tissue via intracerebroventricular administration.

[0181] Data on mouse brain weight were obtained from an in vivo efficacy repeated administration study in the MPSIIIA mouse model. The treatment groups in this study were 15, 30, and 60 μg and were administered once every 2 weeks. All treatment groups showed significant efficacy but did not show a clear dose-response. To calculate brain weight ratios in humans and animals, the average mouse brain weight of 0.4662 g was obtained from the previous study. The average monkey brain weight was obtained from the 28-week safety study and was 70 g. This brain weight was consistent with the reported brain weight of juvenile monkey. Human brain weight estimates were obtained from a paper that studied the relationship between brain weight and body weight depending on age. Brain weights for males and females were used as the average values in each age category (Table 7).

[0182]

[0183] The brain weight ratio was multiplied by the dosage used in the mouse efficacy study and the dosage used in the monkey safety study to obtain the HED (Table 8).

[0184]

[0185] The therapeutic index based on MTD (maximum tolerable dose) and MED (minimum effective dose) can be used to calculate the MRSD (maximum recommended starting dose), which is the maximum dose recommended in initial clinical trials. However, due to the nature of biopharmaceuticals, no toxicity has been identified, so the maximum dose that can be administered was selected as the NOAEL (No-observed-adverse-effect level) for this drug. Upon considering a safety factor, 1 / 10 NOAEL was used as the standard for the clinical starting dose.

[0186] Since the selection is based on the clinical starting dose NOAEL, the NOAEL converted to human dose and divided by a safety factor of 10 equals 23 mg.

[0187] The low single-dose efficacy study (Example 2) confirmed the effects on the brain and cerebrospinal fluid when administered to mice at a dose of 1.5 μg / dose, which is translated to a human dose of 3.5 mg (1 / 10 of the human dose of 35 mg in the 15 μg group in Table 8). This suggests that 23 mg is a safe and effective dose.

[0188]

[0189] Example 8-3: Selecting a clinical starting dose in a pediatric population

[0190] As GC1130A is administered directly into brain tissue via intracerebroventricular administration, it was scaled not only by brain weight but also by cerebrospinal fluid volume. The average cynomolgus monkey (cynomolgus macaque) weighs 3.6 kg and has an average cerebrospinal fluid volume of 11.6 mL. The average 2-year-old child weighs 14.0 kg and has an average cerebrospinal fluid volume of 3 to 4 mL / kg, which is about 42 to 56 mL. The ratio of cerebrospinal fluid volume between monkeys and humans is approximately 1:4 (William Bonadio, J Emerg Med. 2014 Jan;46(1):141-50, Cheryl D Fryar, et al, Natl Health Stat Report. 2021 Aug:(160):1-24, Jenna M Sullivan, et al, J Transl Med. 2020 Aug 8;18(1):309).

[0191] For GC1130A, the NOAEL in the 28-week repeated-administered monkey safety study was 15 mg, and when multiplied by four, a human dose is 60 mg. Dividing this converted value by a safety factor of 10 yields 6 mg.

[0192]

[0193] The present invention relates to an optimal dosage and cycle of heparan-N-sulfatase (HNS) that effectively reduces the accumulation of heparan sulfate (HS) while improving cognition in patients and may be useful as an enzyme replacement therapy (ERT) for the treatment of Sanfilippo syndrome type A (MPS IIIA).

[0194]

[0195] While the foregoing has described in detail certain aspects of the present invention, it will be apparent to one of ordinary skill in the art that these specific descriptions are merely preferred examples and are not intended to limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

[0196]

[0197] Attached as an electronic file.

Claims

1.A pharmaceutical composition for the prevention or treatment of Sanfilippo syndrome type A (MPS IIIA), comprising heparan N-sulfatase (HNS),wherein the heparan N-sulfatase is administered to a patient at a dose of 3 to 150 mg per dose, at intervals of 2 to 4 weeks, via intracerebroventricular injection (ICV), intracerebral injection (IC), or intrathecal injection (IT).2.The pharmaceutical composition according to claim 1, wherein the heparan N-sulfatase is administered at a dose of 6 to 100 mg per dose.3.The pharmaceutical composition according to claim 1, wherein the total administered volume per dose is 10 mL or less.4.The pharmaceutical composition according to claim 1, wherein the composition further comprises 1 to 40 mM of a histidine buffer.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 1, wherein the composition further comprises a salt.9.The pharmaceutical composition according to claim 8, wherein the salt is NaCl or KCl.10.The pharmaceutical composition according to claim 9, wherein the salt is comprised at a concentration of 30 mM to 500 mM.11.The pharmaceutical composition according to claim 1, wherein the composition 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, wherein being administered into the central nervous system via intracerebroventricular injection (ICV).

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