Compositions for treating Sanfilippo syndrome type A (MPS IIIA) comprising heparan N-sulfatase (HNS)

Abstract

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 an optimal dose and period of administration of heparan N-sulfatase, which can effectively reduce the accumulation of heparan sulfate (HS) while improving the cognition of patients. According to the present invention, it can be used as enzyme replacement therapy (ERT) for treating Sanfilippo syndrome type A.

Description

Technical Field

[0001] This invention relates to a pharmaceutical composition comprising heparin N-sulfatase (HNS) for the prevention or treatment of Santa Felipe syndrome type A (MPS IIIA), and more specifically, to a pharmaceutical composition characterized by administration of HNS at specific doses and cycles, and a method of using the pharmaceutical composition for the prevention or treatment of MPS IIIA. Background Technology

[0002] Lysosomal storage diseases (LSDs) are inherited metabolic disorders caused by defects in lysosomal function. LSDs result from lysosomal dysfunction caused by a deficiency of one or more enzymes required for the metabolism of lipids, glycoproteins, or mucopolysaccharides. The deficiency of lysosomal enzymes leads to the accumulation of lipids, glycoproteins, or mucopolysaccharides in lysosomes, resulting in systemic abnormalities (Nature Reviews Disease Primers. 4 (1): 27; Biochem. Soc. Trans. 28 (2): 150-4). Mucopolysaccharide storage disease (MPS), or mucopolysaccharide deposition, is a type of LSD caused by the accumulation of glycosaminoglycans (GAGs) in lysosomes due to a deficiency of lysosomal enzymes required for the degradation of GAGs.

[0003] Santa Felipe syndrome is a type of MPS, named after Dr. Santa Felipe, an American physician who first discovered the condition in 1963. Also known as MPS III, Santa Felipe syndrome is an autosomal recessive genetic disorder clinically characterized by the absence of corneal opacity, mild physical changes such as hepatomegaly and skeletal changes, but with very severe and progressive central nervous system (CNS) symptoms.

[0004] San Phillips syndromes are caused by a deficiency of four different enzymes required to break down polysaccharides (especially GAG). Based on the deficient enzymes, San Phillips syndromes are classified into MPS IIIA (San Phillips A), MPS IIIB (San Phillips B), MPS IIIC (San Phillips C), and MPS IIID (San Phillips D). The deficient enzymes and their corresponding genetic loci are listed below for each type of San Phillips syndrome.

[0005] Type A (MPS IIIA): Heparin N-sulfatase - Chromosome 17 (17q25.3)

[0006] Type B (MPS IIIB): N-acetyl-α-D-glucosidase - Chromosome 17 (17q21)

[0007] Type C (MPS IIIC): Acetyl-CoA: α-glucosidase-N-acetyltransferase-Chromosome 14

[0008] Type D (MPS IIID): N-acetyl-α-D-glucosinolate-6-sulfatase - Chromosome 12 (12q14)

[0009] MPS IIIA is caused by a deficiency of heparin N-sulfatase (HNS), an enzyme involved in the breakdown of heparin sulfate (HS), which hydrolyzes the sulfate moiety linked to the amino group of the glucosamine residue of HS. Symptoms of MPS IIIA typically appear between the ages of 2 and 6, but some cases are diagnosed after the age of 13. In general, patients with MPS IIIA have significant developmental delays and are known to have low long-term survival rates.

[0010] Currently, there is no approved treatment for MPS IIIA, and only palliative care is available to relieve symptoms. Enzyme replacement therapy (ERT), which involves administering exogenously produced HNS to MPS IIIA patients, shows promise in treating this disease.

[0011] ERT, which involves administering a functional lysosomal enzyme to correct the deficiency of enzyme function, is one of the main therapies for LSD, and its advantage lies in relieving symptoms and preventing permanent damage to the body through simple injection. As a well-known ERT for enzyme storage diseases, intravenous (IV) therapy using glucocerebrosidase (GCase) was first approved by the FDA for Gaucher disease in 1991 (National Gaucher Foundation. Retrieved 2017-06-08).

[0012] However, given that most LSDs accumulate in the CNS, particularly in neurons and meninges of the brain, leading to excessive GAG ​​accumulation and various CNS disorders, intravenously administered ERTs are not effective in treating neurological disorders and diseases caused by lysosomal accumulation, especially in the brain, because the lysosomal enzymes, as the active ingredients, have difficulty crossing the blood-brain barrier (BBB), resulting in insufficient enzyme delivery to the CNS. Therefore, various CNS delivery therapies are being investigated, which involve direct drug delivery to the CNS to bypass the BBB and deliver the enzymes.

[0013] Several therapies have been developed to deliver drugs to the CNS by bypassing the BBB. In particular, intracerebral injection (IC), intraventricular injection (ICV), and intrathecal injection (IT) are the most common routes of administration for delivering proteins directly to the brain.

[0014] Intraocular injection (IT) and intracerebral transurethral injection (ICV) have become methods for delivering enzymes to the central nervous system (CNS) for multiple myeloid leukemia (MPS), demonstrating significant reductions in gamma globulin (GAG) and significant improvements in neurological symptoms in various animal models of MPS (Molecular Therapy - Methods & Clinical Development, 21, 67-75). However, due to the highly dose-dependent nature of direct brain injection therapy, there is a need to develop ERTs with appropriate effective doses and cycles to achieve effective levels of therapeutic benefit.

[0015] Against this technical background, the inventors of the present invention have determined the optimal dose and cycle for administering HNS to the CNS, which has significant effects, and have completed the present invention.

[0016] The information provided in the background section is intended only to enhance the understanding of the background of the invention and may not include information constituting prior art known to those skilled in the art. Summary of the Invention

[0017] The purpose of this invention is to provide a method for preventing or treating San Phillips syndrome type A (MPS IIIA) by administering heparin N-sulfatase (HNS) at a sustainable and stable effective dose and periodicity.

[0018] To achieve the above objectives, the present invention provides a pharmaceutical composition comprising heparin N-sulfatase (HNS) for the prevention or treatment of Santa Felipe syndrome type A (MPS IIIA), wherein the heparin N-sulfatase is administered to the patient at a dose of 3 to 150 mg per dose at intervals of 2 to 4 weeks via intraventricular injection (ICV), intracerebral injection (IC), or intrathecal injection (IT).

[0019] The present invention also provides a method for preventing or treating Santa Felipe syndrome type A (MPS IIIA), the method comprising administering the pharmaceutical composition to a patient.

[0020] The present invention also provides the use of the pharmaceutical composition for the prevention or treatment of San Phillips syndrome type A (MPS IIIA).

[0021] The present invention also provides the use of the pharmaceutical composition in the preparation of a medicament for the prevention or treatment of San Phillips syndrome type A (MPSIIIA). Attached Figure Description

[0022] Figure 1 This is a graph showing the single-dose efficacy test results of GC1130A, in which... Figure 1A shows the HS content in the brain (all data are expressed as mean ± SEM. Compared with vector-treated MPS IIIA mice, **P < 0.01, ***P < 0.0005, ***P < 0.0001), and Figure 1 B shows the HS content in cerebrospinal fluid (CSF) (all data are expressed as mean ± SEM. Compared with vector-treated MPS IIIA mice, **P < 0.01, ***P < 0.0005, ***P < 0.0001).

[0023] Figure 2 This is a graph showing the results of low-dose, single-dose efficacy tests of GC1130A, in which... Figure 2 A shows the HS content in the brain (all data are expressed as mean ± SEM, *** P < 0.001, **** P < 0.0001 compared to G2), and Figure 2 B shows the HS content in CSF (all data are expressed as mean ± SEM. Compared with G2, * P < 0.05, ** P < 0.002, **** P < 0.0001).

[0024] Figure 3 This is a graph showing the correlation between HS in the brain and CSF when administered as a single dose via ICV.

[0025] Figure 4 This is a graph showing the changes in HS content in the brain and CSF with GC1130A dose after repeated administration (using GraphPad Prism software version 9.4.0, analyzed by one-way ANOVA Dunnett's multiple comparison test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 VS vector group, (mean ± SEM)).

[0026] Figure 5 This is a graph showing the correlation of HS in the brain and CSF after repeated ICV administration.

[0027] Figure 6 is a graph showing the results of an open field experiment evaluating the behavior of mice after repeated administration of GC1130A.

[0028] Figure 7 The biodistribution of GC1130A conjugated with a fluorescent dye after IV or ICV administration in mice is shown. Figure 7 The chart in B is for each time curve (n=4).

[0029] Figure 8The curves show the time curves of ICV administration of GC1130A to the mouse brain, where the error bars represent the standard deviation of the mean (n=4).

[0030] Figure 9 This is a graph showing the quantitative results of repeated ICV administration of GC1130A to the mouse brain.

[0031] Figure 10 This is a graph showing the results of a quantitative IHC analysis, or LAMP2 analysis, of brain pathological changes after repeated ICV administration.

[0032] Figure 11 This is a graph showing the results of a quantitative IHC analysis, or CD68 analysis, of brain pathological changes after repeated ICV administration. Detailed Implementation

[0033] Unless otherwise defined, all technical and scientific terms used herein will have the same meaning as commonly understood by those skilled in the art. Generally, the terms used herein are those well-known and commonly used in the art.

[0034] In embodiments of the present invention, in order to determine the effective dose and interval of HNS as an ERT, it was observed that in a San Philippo syndrome type A (MPS IIIA) mouse model, administration of a single dose of 15 to 60 μg at intervals of 2 to 4 weeks resulted in a significant reduction in HS levels and improvement in behavioral indicators.

[0035] Furthermore, by comparing allometric growth scale values ​​based on brain weight and CSF, a single dose of 3 to 150 mg, preferably 6 to 100 mg, and even more preferably 6 to 60 mg, was determined for human patients when administered at 2 to 4-week intervals.

[0036] Therefore, in one aspect, the present invention relates to a pharmaceutical composition comprising heparin N-sulfatase (HNS) for the prevention or treatment of San Phillips syndrome type A (MPS IIIA), wherein the heparin N-sulfatase is administered to the patient at a dose of 3 to 150 mg per dose at intervals of 2 to 4 weeks via intraventricular injection (ICV), intracerebral injection (IC), or intrathecal injection (IT).

[0037] HNS is a lysosomal enzyme that catalyzes the hydrolysis of HS and the N-linked sulfate groups of the non-reducing terminal glucosamine moiety from HS or heparin (Biochem. Biophys. Res. Commun. 2001, 280, 1251-1257). Mutations in the heparin N-sulfatase gene (SGSH) are known to cause MPS type A (MPSIIIA, OMIM#252900), also known as San Phillips A syndrome. MPS type A is characterized by a deficiency of heparin N-sulfatase (HNS), an enzyme involved in the lysosomal catabolism of glycosaminoglycan (GAG) heparin sulfate (Neufeld EF, et al. The Metabolic and Molecular Bases of Inherited Disease (2001) pp. 3421-3452). In the absence of this enzyme, GAG accumulates in the lysosomes of neurons and glial cells, leading to severe neurological damage.

[0038] As used herein, the term "heparin N-sulfatase" is used interchangeably with N-sulfoglucosylhydrogenase (SGSH).

[0039] In this invention, the heparin N-sulfatase can have a wild-type or naturally occurring amino acid sequence. For example, the heparin N-sulfatase can be derived from various organisms, more preferably from humans, but is not limited thereto. In this invention, the heparin N-sulfatase can include, but is not limited to, the amino acid sequence represented by SEQ ID NO: 1. In this invention, the heparin N-sulfatase can include 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.

[0040]

[0041] In this invention, heparin N-sulfatase can be a recombinant enzyme produced through recombinant synthesis. Techniques known in the art for generating recombinant cells to express various target proteins can facilitate the recombinant production of heparin N-sulfatase.

[0042] In this invention, heparin N-sulfatase may also be included in the form of a fusion protein or conjugate. In this invention, heparin N-sulfatase may be fused or conjugated with portions capable of binding to receptors and / or lysosomal targeting agents on the surface of brain cells to promote cellular uptake or lysosomal targeting. Korean Patent No. 10-2007044 discloses modifications of alternative enzymes such as heparin N-sulfatase.

[0043] In this invention, heparin N-sulfatase is administered to the patient at doses of 3 to 150 mg, preferably 5 to 120 mg, more preferably 6 to 100 mg, and most preferably 6 to 60 mg, at intervals of 2 to 4 weeks, but is not limited thereto.

[0044] In particular, heparin N-sulfatase can also be administered in a single dose of 6 to 60 mg at intervals of 2 to 4 weeks, preferably at intervals of 2 weeks, but is not limited thereto.

[0045] In this invention, the total volume of the pharmaceutical composition administered per dose may be 10 mL or less, preferably 9 mL or less, more preferably 6 mL or less, but is not limited thereto.

[0046] In this invention, the pharmaceutical composition may include heparin N-sulfatase at concentrations 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, and most preferably about 12 to about 15 mg / mL, but is not limited thereto.

[0047] The pharmaceutical composition may further comprise 1 to 40 mM histidine buffer.

[0048] Although formulations for the central nervous system delivery of heparan N-sulfatase, including phosphate, have been reported (e.g., Korean Patent No. 10-2007044), studies have consistently reported negative effects of phosphate-buffered saline (PBS) on the activity of heparan N-sulfatase (J. Inherit Metab. Dis. 1993;16(2):465-72; and ActaCrystallogr D Biol. Crystallogr. 2014 May;70(Pt 5):1321-35). Therefore, histidine buffer can be used as a stabilizer instead of phosphate for the central nervous system delivery of heparan N-sulfatase. Histidine buffer offers several advantages over conventional phosphate buffers, including significantly increased stability due to reduced protein-protein and protein-buffered saline interactions, and a significant reduction in turbidity.

[0049] In the pharmaceutical composition according to the invention, the histidine buffer may be, but is not limited to, 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 of the histidine buffer described above is a concentration calculated based on the histidine concentration.

[0050] The pH of the pharmaceutical composition according to the invention may be, but is not limited to, about 7.8 or higher, preferably about 7.8 to about 9.0, more preferably about 7.9 to about 8.9, and most preferably about 8.0 to about 8.8.

[0051] In this invention, the pharmaceutical composition may further include sugars.

[0052] The inclusion of sugars, particularly trehalose, in pharmaceutical compositions results in very high purity (%) and titers, not only when the composition is used as is in liquid form, but also when it is formulated and reconstituted into lyophilized formulations.

[0053] In this invention, the sugar may be at least one selected from the group consisting of trehalose, sucrose, maltose, lactose, and sorbitol. In this invention, the concentration of the included sugar may be about 0.1% or more, about 0.5% or more, about 1.0% or more, or about 1.3% or more, more specifically, 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%.

[0054] In this invention, unless otherwise stated, the % concentration of each substance refers to w / v.

[0055] In this invention, the pharmaceutical composition may further include a salt.

[0056] In this invention, the salt may be NaCl or KCl. The concentration of the salt included in this invention may be from about 30 mM to about 500 mM, preferably from about 50 mM to about 400 mM, more preferably from about 60 mM to about 200 mM, and most preferably from about 80 mM to 150 mM, but is not limited thereto.

[0057] In this invention, the concentration of the included salt can be appropriately osmotic for the central nervous system delivery of the pharmaceutical composition of this invention. Suitable osmotic concentrations for pharmaceutical formulations used for central nervous system delivery are known in the art.

[0058] In this invention, the permeation concentration of the pharmaceutical composition can be, for example, about 400 mOsmol / kg or less, preferably about 350 mOsmol / kg or less, more preferably about 330 mOsmol / kg or less, even more preferably about 300 mOsmol / kg or less, and most preferably about 290 mOsmol / kg or less, but is not limited thereto. In this invention, the permeation concentration of the pharmaceutical formulation can be, for example, about 200 to about 400 mOsmol / kg, preferably about 220 to about 360 mOsmol / kg, more preferably about 250 to about 330 mOsmol / kg, and most preferably about 280 to about 300 mOsmol / kg, but is not limited thereto.

[0059] In this invention, the pharmaceutical composition may further include a surfactant.

[0060] In this invention, the surfactant may be a polysorbate-based surfactant, more preferably polysorbate 20 or polysorbate 80, and most preferably polysorbate 20.

[0061] In this invention, the concentration of the surfactant may be from about 0.0001% to about 0.1%, preferably from about 0.002% to about 0.07%, more preferably from about 0.003% to about 0.05%, and most preferably from about 0.004% to about 0.01%, but is not limited thereto.

[0062] However, when the pharmaceutical composition according to the 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 used for reconstitution, rather than included in the pharmaceutical composition and dosage form used for lyophilization.

[0063] The pharmaceutical compositions according to the invention may further include suitable carriers, excipients, and diluents conventionally used in pharmaceutical compositions.

[0064] In particular, pharmaceutical excipients used 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, benzyl chloride, tertiary ammonium salts, and chlorhexidine diacetate; carriers such as polyethylene 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 polyesters; cryoprotectants; lyophilization protectants; fillers; and stabilizers, and the 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 desired properties of the formulation.

[0065] In this invention, the pharmaceutical composition can be formulated into a pharmaceutical dosage form, such as a liquid dosage form or a lyophilized dosage form.

[0066] Liquid formulations are preferably, but not exclusively, in the form of ampoules or pre-filled syringes.

[0067] Preferably, the pharmaceutical composition can be formulated into a lyophilized preparation. Lyophilized preparations have advantages in terms of storage and transportation, and can also be prepared by various freeze-drying methods known in the art, in addition to those methods described in the embodiments of the present invention.

[0068] If the composition according to the invention is formulated into a lyophilized form, i.e., a dry powder, it can be reconstituted into a liquid composition for administration. Non-limiting examples of solutions for reconstitution may include ordinary aqueous solutions, saline solutions, etc., and if the composition according to the invention does not contain a surfactant or contains an insufficient amount of a surfactant, the solution for reconstitution may contain surfactants such as PS20 or PS80.

[0069] The compositions according to the invention can be administered to the central nervous system via various methods of administration. They can be administered to the central nervous system via intraventricular injection (ICV), intracerebral injection (IC), or intrathecal injection (IT), with intraventricular injection (ICV) being the most preferred method.

[0070] As used herein, intraventricular injection refers to the administration of a drug by injecting it into the ventricles of the brain, which are interconnected hollow spaces within the brain. Compared to intracerebral injection, intraventricular injection has the advantage of delivering a larger volume of drug over a larger area. Various techniques for intraventricular injection are known in the art, such as, but not limited to, the Ommaya reservoir developed by Ayub Ommaya as a conventional intraventricular injection device, and various techniques for intraventricular injection are continuously being developed and reported. Various other intraventricular injection devices and techniques known in the art or developed thereafter can be used, without limitation, for intraventricular injection of the pharmaceutical compositions of the present invention.

[0071] As used herein, intracerebral injection refers to the injection of drugs into the brain tissue itself. Various intracerebral injection techniques are known in the art, such as those described in detail by Mathon et al. 2015.

[0072] 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, for example, 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, which serve as representative methods.

[0073] In this invention, when the pharmaceutical composition is administered via intraventricular injection, a certain amount of cerebrospinal fluid (CSF) can be drained from the ventricles before administration. This drainage of CSF prevents an increase in intracranial pressure due to changes in CSF volume after intraventricular administration.

[0074] Preferably, the total administration volume of the pharmaceutical composition according to the invention for intraventricular (ICV) administration may be 10 mL or less, preferably 9 mL or less, more preferably 6 mL or less, but is not limited thereto.

[0075] In this invention, administration of the pharmaceutical composition to the central nervous system can deliver heparin N-sulfatase to various target tissues, such as the brain, spinal cord, and peripheral tissues. In this invention, target tissues include any tissue affected by the lysosomal storage disease to be treated; for example, target tissues can be brain target tissues, spinal cord target tissues, and / or peripheral target tissues, and administration to the central nervous system can provide systemic delivery of heparin N-sulfatase.

[0076] In this invention, administration of the pharmaceutical composition to the central nervous system can achieve therapeutic or clinically effective levels or activities in the various target tissues described herein. As used herein, therapeutic or clinically effective levels or activities refer to levels or activities sufficient to impart a therapeutic effect in the target tissue. For example, a therapeutic or clinically effective level or activity may be an enzyme level or activity sufficient to improve disease-related symptoms (e.g., GAG accumulation) in the target tissue.

[0077] In this invention, administration of the formulation or pharmaceutical composition to the central nervous system can achieve enzyme levels or activities of 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 target tissues. In this invention, administration of the pharmaceutical composition to the central nervous system can achieve enzyme levels or activities that are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than those of a control group (e.g., untreated endogenous levels or activities).

[0078] In this invention, administration of the pharmaceutical composition to the central nervous system can result in a reduction of GAG (e.g., heparan sulfate) storage in brain target tissues, spinal cord neurons, and / or peripheral target tissues. In this invention, GAG storage can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, or 2-fold compared to a negative control group (e.g., GAG storage in subjects before treatment or after administration of the carrier alone). In this invention, administration of the pharmaceutical composition to the central nervous system can result in a reduction of vacuolation 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.

[0079] The pharmaceutical compositions according to the invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in single or multiple doses.

[0080] Furthermore, for patient comfort, the pharmaceutical composition according to the invention is preferably administered at a preferably rapid rate of administration. As an example, the administration rate of the pharmaceutical composition according to the invention may be, but is not limited to, about 0.1 ml / min or higher, or about 0.5 ml / min or higher, preferably about 1 ml / min or higher, more preferably about 2 ml / min or higher, and most preferably about 5 ml / min or higher.

[0081] As used herein, the term "prevention" means any action taken to prevent the onset of disease or to delay the progression of disease by administering a composition. Furthermore, as used herein, the term "treatment" means any action taken to improve or alleviate or cure disease symptoms by administering a composition.

[0082] As used herein, "patient" means a mammal, preferably a human, that suffers from or is at risk of suffering from a symptom or disease that can be relieved, suppressed or cured by administration of the composition according to the invention.

[0083] In another aspect, the present invention relates to a method for preventing or treating Santa Felipe syndrome type A (MPS IIIA), the method comprising administering the pharmaceutical composition to a patient.

[0084] In another aspect, the present invention relates to the use of the pharmaceutical composition for the prevention or treatment of San Phillips syndrome type A (MPS IIIA).

[0085] In another aspect, the present invention relates to the use of the pharmaceutical composition in the preparation of a medicament for the prevention or treatment of San Phillips syndrome type A (MPS IIIA).

[0086] The present invention will now be described in more detail with reference to the following embodiments. These embodiments are merely illustrative and it will be apparent to those skilled in the art that the scope of the invention should not be construed as limited to these embodiments.

[0087] The recombinant heparin N-sulfatase (rHNS) used in the embodiments of the present invention is named “GC1130A” or “GC1130A protein”.

[0088] Example 1: Efficacy study of high-dose single-dose administration of GC1130A

[0089] The aim of this study was to develop [the technology / initiative] in MPS IIIA mice (C57BL / 6 Sgsh). mps3a The efficacy of GC1130A as a single intraventricular (ICV) injection for the treatment of San Philippo syndrome type 2 (MPS IIIA) was evaluated in a model of WT gene homozygous mutant, 2 weeks, male & female, n=12, and the dose range and interval were established for repeated ICV dosing efficacy studies.

[0090] As shown in Table 1 below, the animals were divided into five different groups to evaluate the efficacy of the targeted drug.

[0091] [Table 1]

[0092]

[0093]

[0094] Mice in the treatment and control groups received 12 to 110 μg / dose of GC1130A (22 mg / mL, GCBiophana) and a carrier, respectively, and there were no deaths, systemic symptoms, or changes in body weight or organ weight in either group after ICV administration. Animals were randomly assigned to three dosage groups and necropsy was performed at 7, 14, and 28 days post-administration. Brain and cerebrospinal fluid (CSF) were collected and stored at low temperatures until analysis. Changes in heparan sulfate (HS) content in the brain and CSF over time were analyzed by LC-MS / MS to determine the trend of HS clearance after each administration. Figure 1 ).

[0095] It can be seen that HS had accumulated in the diseased mice by 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, a dose-dependent effect was found in both the brain and CSF only on day 14 after administration, and repeated doses of 15, 30, and 60 μg were selected based on the above results.

[0096] After 14 days of treatment, HS showed a significant downward trend, and after 28 days, except for the low-dose group, the therapeutic effect of the drug weakened but remained unchanged. Therefore, further experiments were conducted at dosing intervals of every two weeks (2QW) or every four weeks (4QW).

[0097] Example 2: Efficacy study of low-dose single-dose administration of GC1130A

[0098] The aim of this study was to develop MPS IIIA mouse model (C57BL / 6 Sgsh) mps3a The efficacy of GC1130A as a single low-dose intraventricular (ICV) injection for the treatment of Santa Felipe syndrome type A (MPS IIIA) was evaluated in a WT gene homozygous mutant, 2-week-old males & females, n=14.

[0099] As shown in Table 2 below, the animals were divided into five different groups to evaluate the efficacy of the targeted drug.

[0100] [Table 2]

[0101]

[0102] Mice in the treatment and control groups received 1.5 to 15 μg / dose of GC1130A (15 mg / mL, GCBiophama) and a carrier, respectively, and there were no deaths, systemic symptoms, or changes in body weight or organ weight in either group after ICV administration. Autopsies were performed on the treatment group at days 14 and 28 post-administration, and brain and cerebrospinal fluid (CSF) were collected and stored at low temperatures until analysis. Changes in heparan sulfate (HS) levels in the brain and CSF over time were analyzed by LC-MS / MS to determine the trend of HS clearance after specific dose administration. Figure 2 ).

[0103] On days 14 and 28, compared with the control group, HS in the brain showed a statistically significant dose-dependent decrease in all treatment groups (day 14: -31% for G3, -61% for G4, and -80% for G5 compared with the control group; day 28: -32% for G3, -32% for G4, and -67% for G5 compared with the control group). On day 14, HS in cerebrospinal fluid showed a statistically significant dose-dependent decrease in all treatment groups compared with the control group, but this decrease was not significant on day 28 (day 14: -45% for G3, -62% for G4, and -57% for G5 compared with the control group; day 28: -15% for G3, -23% for G4, and -8% for G5 compared with the control group).

[0104] In other words, the GC1130A treatment group (≥1.5 μg / dose) was shown to have dose-dependent HS depletion in the brain, which was usually observed up to 28 days post-mortem, while in CSF, significant HS depletion was only observed on day 14 in the GC1130A treatment group with 1.5 μg / dose.

[0105] In summary, based on the effective concentration of GC1130A and the analysis of the maximum volume in the mouse ventricles (5 μL / dose), the effects on the brain and cerebrospinal fluid were determined at the lowest dose of 1.5 μg / dose.

[0106] Furthermore, strong correlations in hourly and overall analyses confirmed a significant correlation between HS levels in the brain and CSF in response to GC1130A administration. Figure 3 ).

[0107] Example 3: Repeated-dose efficacy study of GC1130A

[0108] The aim of this study was to evaluate the efficacy of GC1130A with ICV injection for the treatment of central nervous system symptoms in MPS IIIA mice.

[0109] A formulation containing GC1130A protein (5 mM histidine, 125 mM NaCl, 1.8% trehalose, 0.005% PS20, pH 8.11) was used, along with MPS IIIA mice (C57BL / 6 Sgsh). mps3a The WT gene model (homozygous mutant, 2 weeks old, male & female) was used, and each mouse group is shown in Table 3 below.

[0110] [Table 3]

[0111]

[0112]

[0113] Cerebrospinal fluid (CSF) samples were collected during the autopsy, and the isolated brain was stored in a cryogenic freezer until HS measurements were performed.

[0114] The levels of hemoglobin (HS) in mouse CSF and brain samples were measured by LC-MS / MS, and biochemical markers, namely open field test behavioral assessment, were performed to verify the improvement in central nervous system function. In addition, immunohistochemistry was used for MRI analysis and pathological analysis to confirm the changes in actual brain structure (Table 4).

[0115] [Table 4]

[0116]

[0117] In the GC1130A 15-60 μg treatment groups, the biochemical parameter HS in both the brain and CSF was significantly reduced, with greater HS inhibition in the brain than in the CSF. Direct injection into the ventricles of the brain is likely the reason for these results. A 2-week interval administration produced a greater inhibitory effect than a 4-week interval administration, but HS was also effectively inhibited at the 4-week interval, and no dose-dependent or sex-specific differences were observed. Figure 4 ).

[0118] Furthermore, a significant positive correlation was found between CSF and HS levels in brain tissue. Figure 5 ).

[0119] Open field behavior experiments confirmed that all behavioral parameters were improved in the treatment group receiving repeated GC1130A. To ensure statistical significance, data from the vector-treated groups in WT and MPSIIIA mice were calculated by summing the treatment groups administered every two weeks (Q2W) and once a month (Q4W), and no statistically significant differences were found between the two groups. Compared with the control group, the total activity level of GC1130A showed a trend of increasing activity (Figure 6).

[0120] The minimum dose at which expected cognitive improvement was found was 30 μg / dose x 1 month. Even if HS in the CSF is reduced by 50% or more, cognitive improvement cannot be expected if the CSF HS remains below the level of carrier therapy for 2 weeks. For cognitive improvement, CSF HS must be maintained at 50% or less of carrier therapy for at least 2 weeks (15 μg / dose x 2 months).

[0121] As a result, HNS was found to be effective against MPS IIIA in mice when administered at a dose of 15-60 μg / dose every 2 to 4 weeks.

[0122] Example 4: Biodistribution of GC1130A conjugated with fluorescent dye after IV or ICV administration in mice

[0123] The aim of this study was to analyze the pharmacokinetics of GC1130A in mice according to the route of administration and to assess the distribution profile of GC1130A in different tissues over time.

[0124] Following administration of a single dose of 10 mg / kg of GC1130A conjugated with a fluorescent dye to mice via IV or ICV, fluorescence intensity was imaged at time curves of 0.083, 1, 2, 4, 8, 24, 48, 96, and 192 hours using an in vivo imaging system (IVIS) to determine organ-specific distribution (Table 5).

[0125] [Table 5]

[0126]

[0127] When GC1130A was administered via IV, higher fluorescence intensity was observed in the rest of the body compared to the brain, while when administered via ICV, higher fluorescence intensity was observed in the brain compared to other organs. Figure 7 A).

[0128] Figure 7 B shows the mean radiation efficiency-time curves of the brain after IV or ICV administration of GC1130A. The mean radiation efficiency in the brain was the highest after ICV injection compared to other administration regimens.

[0129] Example 5: Pharmacokinetics of GC1130A administered via ICV in the mouse brain

[0130] This study aimed to evaluate the pharmacokinetics of GC1130A in the brain of 5-week-old C57BL / 6 mice after a single ICV administration.

[0131] Following ICV administration to 5-week-old C57BL / 6 mice, four mice underwent necropsy, and the brains were homogenized for each of the following time curves: 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 using a digital ELISA assay, and pharmacokinetic analyses were performed.

[0132] [Table 6]

[0133]

[0134] Dose-dependent drug exposure was observed in the brain following a single ICV administration of GC1130A, with a half-life of approximately 7 days. Figure 8 ).

[0135] Example 6: Quantitative analysis of GC1130A in the brain based on repeated ICV administration

[0136] Following autopsies, brain tissue from one male and one female in each group was embedded in paraffin. After immunostaining using a staining device, the stained slides were scanned to establish regions of interest (ROIs), and the expression of each biomarker was quantified.

[0137] Quantitative analysis of brain pathological changes was performed after repeated ICV administration of GC1130A to confirm the drug delivery and efficacy via the CNS through brain administration. Figure 9 ).

[0138] Example 7: Quantitative IHC analysis of brain pathological changes after repeated ICV administration - LAMP2 and CD68 analysis

[0139] Following autopsies, brain tissue from one male and one female in each group was embedded in paraffin. After immunostaining using a staining device, the stained slides were scanned to establish regions of interest (ROIs), and the expression of each biomarker was quantified.

[0140] The study found that markers of inflammation and microglia, namely LAMP2 and CD68, were increased in the MPS IIIA vector group but decreased in the GC1130A group. Figure 10 and Figure 11 Furthermore, these markers were observed to decrease more at Q2W (2-week interval) than at Q4W (4-week interval). GC1130A was also detected in the same brain regions where LAMP2 and CD68 markers were reduced.

[0141] Example 8: Dosage analysis of GC1130A in humans

[0142] Example 8-1: Determination of the starting dose of GC1130A in pediatric clinical practice

[0143] The pediatric dosage recommended in this embodiment is based on the method validated in Hammon K, et al, Clin Transl Sci, 14, 1810-1821, 2021, and was used to translate non-clinical data into human applications for ultra-rare pediatric neurodegenerative diseases. Allometric growth scaling and PK modeling methods were developed to predict the dosage from non-clinical data. These methods were compared and analyzed to select the most conservative value as the clinical starting dose.

[0144] Example 8-2: Calculation of Human Equivalent Dose (HED) Based on Brain Weight

[0145] The principle of the allometric growth scale is based on direct administration to body parts and is usually based on body surface area, but in the case of GC1130A, it is based on a scale of brain weight because it is administered directly to brain tissue via intraventricular administration.

[0146] Data on mouse brain weight were obtained from repeated-dose in vivo efficacy studies in MPSIIIA mouse models. Treatment groups in this study received 15, 30, and 60 μg, administered every 2 weeks. All treatment groups showed significant efficacy, but no significant dose-response was observed. To calculate the human-to-animal brain weight ratio, the mean mouse brain weight obtained from previous studies was 0.4662 g. The mean monkey brain weight, obtained from a 28-week safety study, was 70 g. This brain weight is consistent with reported brain weights in juvenile monkeys. Estimates of human brain weight were obtained from a paper investigating the relationship between brain weight and body weight with age. Brain weights for males and females were used as averages for each age category (Table 7).

[0147] [Table 7]

[0148] The weight ratio of human brain to animal brain

[0149] The brain weight ratio was multiplied by the dose used in the mouse efficacy study and the dose used in the monkey safety study to obtain the HED (Table 8).

[0150] [Table 8]

[0151]

[0152]

[0153] Human ICV Dosage Based on Brain Weight

[0154] Therapeutic indices based on MTD (Maximum Tolerated Dose) and MED (Minimum Effective Dose) can be used to calculate MRSD (Maximum Recommended Starting Dose), which is the maximum dose recommended in the initial clinical trial. However, due to the nature of biopharmaceuticals, toxicity has not yet been determined; therefore, the maximum dosable dose is selected as the NOAEL (No Observed Adverse Effect Level) for the drug. Considering safety factors, 1 / 10 NOAEL is used as the standard for the clinical starting dose.

[0155] Since the selection was based on the clinical starting dose NOAEL, NOAEL was converted to a human dose and divided by a safety factor of 10, which equals 23 mg.

[0156] 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 g / dose, which translates 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 indicates that 23 mg is a safe and effective dose.

[0157] Example 8-3: Selection of clinical starting dose in pediatric populations

[0158] Because GC1130A is administered intraventricularly to directly enter brain tissue, it is scaled not only by brain weight but also by cerebrospinal fluid volume. Cynomolgus macaques have an average weight of 3.6 kg and an average cerebrospinal fluid volume of 11.6 mL. Two-year-old children have an average weight of 14.0 kg and an average cerebrospinal fluid volume of 3 to 4 mL / kg, approximately 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).

[0159] For GC1130A, the NOAEL in a 28-week repeated-dose monkey safety study was 15 mg, which, multiplied by four, yields a human dose of 60 mg. Dividing this converted value by a safety factor of 10 gives 6 mg.

[0160] Industrial applicability

[0161] This invention relates to the optimal dosage and cycle of heparin-N-sulfatase (HNS), which effectively reduces the accumulation of heparin sulfate (HS), improves patient cognition, and can be used as an enzyme replacement therapy (ERT) for the treatment of San Phillips syndrome type A (MPS IIIA).

[0162] Although certain aspects of the invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the invention. Therefore, the essential scope of the invention will be defined by the appended claims and their equivalents.

[0163] Sequence List Free Text

[0164] Electronic documents are attached.

Claims

1. A pharmaceutical composition for the prevention or treatment of San Phillips syndrome type A (MPS IIIA), comprising heparin N-sulfatase (HNS), in, The heparin N-sulfatase is administered to patients at doses of 3 to 150 mg per dose, at intervals of 2 to 4 weeks, via intraventricular injection (ICV), intracerebral injection (IC), or intrathecal injection (IT).

2. The pharmaceutical composition according to claim 1, wherein, The heparin 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 volume of each dose is 10 mL or less.

4. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition further comprises 1 to 40 mM histidine buffer.

5. The pharmaceutical composition according to claim 1, wherein, The pharmaceutical composition further includes sugars.

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

7. The pharmaceutical composition according to claim 6, wherein, The concentration of the sugars included is from 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 concentration of the salts included is from 30 mM to 500 mM.

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

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

13. The pharmaceutical composition according to claim 12, wherein, The concentration of the surfactant included is from 0.0001 to 0.1 w / v.

14. The pharmaceutical composition according to claim 1, wherein, It is administered to the central nervous system via intraventricular injection (ICV).

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