Composition for the treatment of Sanfilippo syndrome (MPSIIIA) containing heparan N-sulfatase (HNS)

Abstract

This invention relates to a pharmaceutical composition for the prevention or treatment of Sanfilippo syndrome type A (MPSIIIA) containing heparan N-sulfatase (HNS), and more specifically, to the optimal dose and frequency of heparan N-sulfatase that can effectively reduce heparan sulfate (HS) accumulation while improving the patient's cognitive function. According to this invention, it can be usefully used in enzyme replacement therapy (ERT) for the treatment of Sanfilippo syndrome type A.

Description

[[Technical Field]]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating Sanfilippo syndrome type A (MPSIIIA) comprising Heparan N-sulfatase (HNS), and more specifically to a pharmaceutical composition characterized by administering HNS at a specific dose and schedule, and to a prevention or treatment method using the same. [[Background Art]]

[0002] Lysosomal storage diseases (LSDs) are genetic metabolic diseases caused by functional defects of lysosomes. Lysosomal storage diseases are induced by lysosomal dysfunction resulting from the deficiency of single or multiple enzymes required for the metabolism of lipids, glycoproteins or mucopolysaccharides. Deficiency of lysosomal enzymes induces systemic abnormalities via lysosomal accumulation of substances such as lipids, glycoproteins or mucopolysaccharides (Nature Reviews Disease Primers. 4(1):27; Biochem. Soc. Trans. 28(2):150-4). Mucopolysaccharidosis (MPS) or mucopolysaccharidosis is a type of lysosomal storage disease, which is caused by intra-lysosomal accumulation resulting from the deficiency of lysosomal enzymes required for the degradation of glycosaminoglycans.

[0003] Sanfilippo syndrome is a type of mucopolysaccharidosis, named after Sanfilippo, an American physician who first described the disease in 1963. Sanfilippo syndrome, also known as MPSIII, is an autosomal recessive genetic disease. Clinically, it is characterized by the absence of corneal clouding, mild physical changes such as hepatosplenomegaly and skeletal system changes, but very severe and progressive central nervous system symptoms.

[0004] Sanfilippo syndrome is caused by a deficiency in four different enzymes necessary for the breakdown of polysaccharides, particularly glycosaminoglycans (GAGs). Depending on the enzyme deficiency, Sanfilippo syndrome is classified into MPSIIIA (Sanfilippo A), MPSIIIB (Sanfilippo B), MPSIIIC (Sanfilippo C), and MPSIIID (Sanfilippo D). The deficient enzymes and their gene locations (GenetIC map locus) for each type of Sanfilippo syndrome are as follows:

[0005] Type A (MPSIIIA): Heparan N-sulfatase - chromosome 17 (17q25.3) Type B (MPSIIIB): N-acetyl-α-D-glucosaminidase - chromosome 17 (17q21) Type C (MPSIIIC): Acetyl-CoA:α-glucosaminide-N-acetyltransferase - chromosome 14 Type D (MPSIIID): N-acetyl-α-D-glucosaminide-6-sulfatase-chromosome 12 (12q14)

[0006] MPSIIIA is caused by a deficiency of heparan N-sulfatase, an enzyme involved in the degradation of heparan sulfate (HS), specifically by hydrolyzing the sulfate portion attached to the amino group of the glucosamine residue of heparan sulfate. Symptoms of MPSIIIA generally appear between the ages of 2 and 6, but diagnosis can also occur after the age of 13. Patients with MPSIIIA are generally known to have significant developmental delays and a poor long-term survival rate.

[0007] Currently, there is no approved treatment for MPSIIIA, and only symptomatic treatment to alleviate symptoms is being administered. Enzyme replacement therapy (ERT), which involves administering externally manufactured heparan-N-sulfatase to MPSIIIA patients, is considered a very promising treatment for MPSIIIA.

[0008] Enzyme replacement therapy is a treatment that corrects enzyme deficiencies by administering deficient lysosomal enzymes. It is one of the primary treatment methods used for lysosomal storage disorders and has the advantage of minimizing symptoms and preventing permanent damage to the body, even with simple injection therapy. A well-known enzyme replacement therapy for enzyme storage disorders is intravenous administration (IV) of glucocerebrosidase (GCase) for Gaucher disease, which was first approved by the FDA in 1991 and is currently in use (National Gaucher Foundation. Retrieved 2017-06-08).

[0009] However, since many lysosomal storage disorders induce excessive accumulation of GAGs in the nervous system, particularly in neurons and meninges of the brain, leading to a variety of central nervous system (CNS) diseases, enzyme replacement therapy administered intravenously is ineffective in treating neurological disorders and diseases caused by lysosomal storage, especially in the brain, because the active ingredient, the lysosomal enzyme, has difficulty crossing the blood-brain barrier (BBB). As a result, the enzyme is not properly transmitted in the CNS, and therefore cannot effectively treat these conditions. Consequently, various CNS (central nervous system) transmission therapies that directly deliver drugs to the CNS to bypass the BBB are being studied.

[0010] As a form of enzyme therapy that bypasses the blood-brain barrier (BBB), various therapies have been developed to deliver drugs to the central nervous system. In particular, representative injection therapies that directly deliver proteins to the brain include intracerebral injection (IC), intracerebral injection (ICV), and intradural injection (IT).

[0011] Intradural injection (IT) and intracerebroventricular injection (ICV) have emerged as methods for delivering alternative enzymes to the central nervous system for mucopolysaccharidosis (MPS), showing significant reductions in GAG and significant improvements in neurological symptoms in various animal models of mucopolysaccharidosis (Molecular Therapy-Methods & Clinical Development, 21, 67-75). However, because the dosage of these therapies directly injected into the brain is very limited, the development of enzyme replacement therapies with appropriate effective doses and cycles is required for an effective level of therapeutic efficacy.

[0012] Against this technical backdrop, the inventors of the present invention completed the invention by confirming the optimal dosage and administration cycle that show a remarkable effect when heparan N-sulfatase (HNS) is administered to the central nervous system (CNS).

[0013] The information described above in this background section is solely for the purpose of improving understanding of the background of the present invention, and may not include information that constitutes prior art already known to those with ordinary skill in the art to which the present invention belongs. [Overview of the Initiative] [Problems that the invention aims to solve]

[0014] The object of the present invention is to provide a method for preventing or treating Sanfilippo syndrome type A (MPSIIIA) through the sustained and stable administration of heparan N-sulfatase (HNS) in an effective volume and periodic manner. [Means for solving the problem]

[0015] To achieve the above objectives, the present invention provides a pharmaceutical composition for the prevention or treatment of Sanfilippo syndrome type A (MPSIIIA) containing heparan N-sulfatase (HNS), characterized in that the heparan N-sulfatase is administered to the patient at a dose of 3 to 150 mg every 2 to 4 weeks via intracerebral injection (ICV), intracerebral injection (IC), or intradural injection (IT).

[0016] The present invention also provides a method for preventing or treating Sanfilippo syndrome type A (MPSIIIA), comprising the step of administering the above-mentioned pharmaceutical composition to a patient.

[0017] The present invention also provides the use of the above-mentioned pharmaceutical composition for the prevention or treatment of Sanfilippo syndrome type A (MPSIIIA).

[0018] The present invention also provides the use of the above-mentioned pharmaceutical composition for the manufacture of a drug for the prevention or treatment of Sanfilippo syndrome type A (MPSIIIA). [Brief explanation of the drawing]

[0019] [Figure 1]The graphs show the results of the single-dose efficacy test of GC1130A. Figure 1A shows brain HS content (All data represent as mean ± SEM. **p<0.01, ***p<0.0005, ****p<0.0001 compared with vehicle-treated MPSIIIA mICe), and Figure 1B shows CSF HS content (All data represent as mean ± SEM. **p<0.01, ***p<0.0005, ****p<0.0001 compared with vehicle-treated MPSIIIA mICe). [Figure 2] The graphs show the test results for the low-dose single-dose efficacy of GC1130A. Figure 2A shows brain HS content (All data represent as mean ± SEM. ***p<0.001, ****p<0.0001 compared with G2), and Figure 2B shows CSF HS content (All data represent as mean ± SEM. *p<0.05, **p<0.002, ****p<0.0001 compared with G2). [Figure 3] This graph shows the correlation between brain activity and CSF (Chronic Sulfate) levels after single-volume ICV administration. [Figure 4] This graph shows the HS content in brain tissue and CSF after repeated administration of GC1130A at different doses (Analyzed by one-way ANOVA Dunnett's multiple comparisons test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 VS Vehiclegroup, (mean±SEM) using GraphPad Prism version 9.4.0 software). [Figure 5] This graph shows the correlation between brain activity and CSF in patients receiving repeated ICV administration, and hypertension (HS). [Figure 6] This graph shows the results of an open-field test to evaluate mouse behavior during repeated administration of GC1130A. [Figure 7]This figure shows the results of confirming the biodistribution of GC1130A conjugated with a fluorescent dye after intravenous or intracerebroventricular administration in mice (in the graph of Figure 7B, n=4 for each time profile). [Figure 8] This is a time profile of GC1130A administered intracerebroventricularly in mouse brain, wherein error bars indicate the standard deviation of the mean (n=4). [Figure 9] This is a graph showing the results of quantitative analysis for repeatedly intracerebroventricularly administered GC1130A in mouse brain. [Figure 10] This is a graph showing the results of LAMP2 analysis after repeated intracerebroventricular administration, which is a result of quantitative IHC analysis of brain pathological changes. [Figure 11] This is a graph showing the results of CD68 analysis after repeated intracerebroventricular administration, which is a result of quantitative IHC analysis of brain pathological changes.

Mode for Carrying Out the Invention

[0020] Unless otherwise defined by other formulas, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0021] In one embodiment of the present invention, in order to confirm the effective dosage and administration interval of Heparan N-sulfatase (HNS) as enzyme replacement therapy, it was confirmed that when administered to a Sanfilippo syndrome type A (MPSIIIA) mouse model at a single dose of 15 to 60 μg per dose every 2 to 4 weeks, the therapeutic effect on Sanfilippo syndrome type A, such as HS reduction and improvement of behavioral indicators, is most prominent.

[0022] Furthermore, by comparing and analyzing allometrically scaled values ​​based on brain weight and cerebrospinal fluid, it was confirmed that the single dose for human patients when administered every 2 to 4 weeks is 3 to 150 mg, preferably 6 to 100 mg, and more preferably 6 to 60 mg.

[0023] Accordingly, in one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of Sanfilippo syndrome type A (MPSIIIA) containing heparan N-sulfatase (HNS), characterized in that the heparan N-sulfatase is administered to the patient at a dose of 3 to 150 mg every 2 to 4 weeks via intracerebral injection (ICV), intracerebral injection (IC), or intradural injection (IT).

[0024] Heparan N-sulfatase (HNS) is a lysosomal enzyme that catalyzes the hydrolysis of heparan sulfate and the sulfate group N-linked from the non-reducing glucosamine moiety of heparan (Biochem. Biophys. Res. Commun. 2001, 280, 1251-1257). Mutations in the heparan N-sulfatase gene (SGSH) are well known to induce mucopolysaccharidosis type IIIA (MPSIIIA, OMIM#252900), also known as Sanfilippo syndrome type A. Mucopolysaccharidosis type IIIA is characterized by a deficiency of heparan N-sulfatase (HNS), an enzyme involved in the lysosomal catabolism of 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 accumulate in the lysosomes of neurons and donigeliocytes, leading to severe neurological damage and subsequent abnormal symptoms.

[0025] In this specification, 'lan N-sulfatase' may be used interchangeably with N-sulfoglucosamine sulfohydrolase (SGSH) in the same sense.

[0026] In the present invention, the heparan N-sulfatase may be characterized by having a wild-type or naturally occurring amino acid sequence. For example, the heparan N-sulfatase may be characterized by being derived from a variety of organisms, and more preferably from humans, but is not limited thereto. In the present invention, the heparan N-sulfatase may be characterized by containing the amino acid sequence shown in SEQ ID NO: 1, but is not limited thereto. In the present invention, the heparan N-sulfatase may be characterized by containing a sequence having 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more homology with a wild-type or naturally occurring sequence.

[0027] JPEG2026529926000002.jpg100161

[0028] In the present invention, the heparan N-sulfatase may be characterized as a recombinantly produced recombinant enzyme. Recombinant production of heparan N-sulfatase can be easily carried out through recombinant cell production techniques for the expression of various target proteins that are known in the art.

[0029] In the present invention, the heparan N-sulfatase may also be included in the form of a fusion protein or a conjugate. In the present invention, the heparan N-sulfatase may be fused or conjugated with a moiety and / or lysosomal targeting adduct capable of binding to receptors on the surface of brain cells to facilitate cellular absorption or lysosomal targeting. Improvements to alternative enzymes such as heparan N-sulfatase are disclosed in Korean Patent Registration No. 10-2007044, etc.

[0030] In the present invention, the heparan N-sulfatase may be administered to the patient at a dose of 3 to 150 mg, preferably 5 to 120 mg, more preferably 6 to 100 mg, and most preferably 6 to 60 mg, every 2 to 4 weeks, but is not limited thereto.

[0031] Furthermore, the treatment may be characterized by administering the above-mentioned heparan N-sulfatase in a single dose of 6 to 60 mg every 2 to 4 weeks, most preferably every 2 weeks, but is not limited to this.

[0032] In the present invention, the total volume administered per single dose of the pharmaceutical composition may be 10 mL or less, preferably 9 mL or less, and more preferably 6 mL or less, but is not limited thereto.

[0033] In the present invention, the pharmaceutical composition may be characterized by containing 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, and most preferably about 12 to about 15 mg / mL, but is not limited thereto.

[0034] Furthermore, the above pharmaceutical composition may be characterized by further containing 1 to 40 mM histidine buffer.

[0035] While dosage forms containing phosphates for central nervous system transmission of heparan N-sulfatase have been reported (e.g., Registered Patent No. 10-2007044 of the Republic of Korea), research has consistently reported that the use of phosphate buffers has an unpredictable effect on the activity of heparan N-sulfatase (J. Inherit Metab. Dis. 1993;16(2):465-72; and Acta Crystallogr D biol. Crystallogr. 2014 may;70(Pt 5):1321-35). Therefore, histidine buffer can be used as a stabilizer to replace phosphates for central nervous system transmission of heparan N-sulfatase. Histidine buffer offers various advantages over existing phosphate buffers, such as significantly increased stability due to reduced protein-protein and protein-buffer interactions, and a substantial reduction in turbidity.

[0036] In the pharmaceutical composition according to the present invention, the histidine buffer may be present in a concentration of 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, but is not limited thereto. The concentration of the histidine buffer is calculated based on the concentration of histidine.

[0037] The pH of the pharmaceutical composition according to the present invention may be about 7.8 or higher, preferably about 7.8 to about 9.0, more preferably about 7.9 to about 8.9, and most preferably about 8.0 to about 8.8, but is not limited thereto.

[0038] The present invention may be characterized by further containing sugars in the above-mentioned pharmaceutical composition.

[0039] When the above pharmaceutical composition contains sugars, particularly trehalose, it exhibited remarkably superior purity (%) and potency not only when used as a liquid dosage form, but also when it was reconstituted and used in a freeze-dried dosage form.

[0040] In the present invention, the sugars may be characterized by being one or more selected from the group consisting of trehalose, sucrose, maltose, lactose, and sorbitol. In the present invention, the sugars may be characterized by being 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 specifically, the sugars may be included at a concentration of 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%.

[0041] In this invention, the % concentration of each substance means w / v% unless otherwise specified.

[0042] In the present invention, the above pharmaceutical composition may be characterized by further containing a salt.

[0043] In the present invention, the salt may be characterized by being NaCl or KCl.

[0044] In the present invention, the above salt may be characterized by being included at 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, and most preferably about 80 mM to 150 mM, but is not limited thereto.

[0045] In the present invention, the salt may be included in the pharmaceutical composition of the present invention at a concentration having an appropriate osmotic pressure for central nervous system transmission. Appropriate osmotic concentrations of drug formulations for central nervous system transmission are well known in the art.

[0046] In the present invention, the penetration 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, even more preferably about 300 mOsmol / kg or less, and most preferably about 290 mOsmol / kg or less, but is not limited thereto.

[0047] In the present invention, the osmotic concentration of the above 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, and most preferably about 280 to about 300 mOsmol / kg, but is not limited thereto.

[0048] The present invention may be characterized by further comprising the above-mentioned pharmaceutical composition or surfactant.

[0049] In the present invention, the surfactant may be characterized by being a polysorbate-based surfactant, more preferably polysorbate 20 or polysorbate 80, and most preferably polysorbate 20.

[0050] In the present invention, the surfactant may be present in a concentration of about 0.0001% to about 0.1%, preferably about 0.002% to about 0.077%, more preferably about 0.003% to about 0.05%, and most preferably about 0.004% to about 0.01%, but is not limited thereto.

[0051] However, when the pharmaceutical composition according to the present invention is formulated as a lyophilized dosage form and reconstituted for administration to a patient, the surfactant may be used in a form contained in a solution for reconstitution rather than being contained in the pharmaceutical composition and dosage form for lyophilization.

[0052] The pharmaceutical composition according to the present invention may further include suitable carriers, excipients, and diluents commonly used in pharmaceutical compositions.

[0053] In particular, pharmaceutical excipients useful for liquid protein dosage forms are well known to those skilled in the art. These may include, without limitation, body solvents or co-solvents; sugars or sugar alcohols, e.g., mannitol, sucrose, sorbitol, fructose, maltose, lactose, or dextran; buffers; preservatives, e.g., benzalkonium chloride, benzethonium chloride, tertiary ammonium salts, and chlorhexidine diacetate; carriers, e.g., poly(ethylene glycol) (PEG); antioxidants, e.g., ascorbic acid, sodium disulfite, and methionine; chelating agents, e.g., EDTA or citric acid; or biodegradable polymers, e.g., water-soluble polyesters; cryoprotectants; freeze-drying protectants; bulking agents; and stabilizers, among others. (See also Remington: "The Science and Practice of Pharmacy" 20th edition, Alfonso R Gennaro, Ed., Lippincott Williams &) Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described by Wilkins (2000), may also be included in the protein dosage forms described herein, but they should not negatively affect the preferred characteristics of the dosage form.

[0054] In the present invention, the pharmaceutical composition can be formulated into a pharmaceutical dosage form such as a liquid dosage form or a freeze-dried dosage form.

[0055] The above liquid dosage form is preferably in ampoule form or pre-filled syringe form, but is not limited thereto.

[0056] Preferably, the above-mentioned pharmaceutical composition can be formulated into a lyophilized dosage form. Lyophilized dosage forms have advantages in terms of storage and transportation, and can be manufactured by various lyophilization methods known to the industry, in addition to the methods described in the examples of the present invention.

[0057] When the composition according to the present invention is formulated in a freeze-dried form, i.e., as a dry powder, it can be reconstituted with a liquid composition for administration. In this case, the solution for reconstitution can be any general aqueous solution, sarin solution, etc., and if the composition according to the present invention does not contain a surfactant or contains an insufficient amount, the solution for reconstitution may contain a surfactant such as PS20 or PS80.

[0058] The compositions according to the present invention can be administered to the central nervous system through a variety of administration methods. They can be administered to the central nervous system by intracerebral injection (ICV), intracerebral injection (IC), or intradural injection (IT), and are most preferably characterized by administration to the central nervous system by intracerebral injection (ICV).

[0059] In this invention, the term "intracerebrovascular injection" refers to the administration of a drug by injecting it into the ventricles, which are the interconnected, empty spaces within the brain. Intracerebrovascular injection has the advantage of being able to deliver a larger amount of drug to a wider area compared to intracerebral injection. A variety of techniques for intracerebrovascular injection are known to the art, such as the Ommaya reservoir developed by Ayub Ommaya, a traditional intracerebrovascular injection device that is continuously being developed and reported, but is not limited thereto. A variety of intracerebrovascular injection devices and techniques known to the art or to be developed in the future may be used without limitation for the intracerebrovascular injection of the pharmaceutical compositions of this invention.

[0060] In this invention, the term "intracerebral injection" refers to the injection of a drug into brain tissue itself. Various techniques for intracerebral injection are publicly known in the art; for example, Mathon et al. 2015 describe an intracerebral injection method in detail.

[0061] In this invention, the term "intradural injection" refers to injection into the spinal canal. Various techniques for intradural injection are publicly known in the art, and representative methods 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.

[0062] In the present invention, when a pharmaceutical composition is administered via intraventricular injection, the subject may experience a certain amount of cerebrospinal fluid (CSF) being discharged from the ventricles before administration. This discharge of CSF can prevent an increase in intracranial pressure due to changes in CSF volume after ICV administration.

[0063] Preferably, the total volume administered when the pharmaceutical composition according to the present invention is administered intraventricularly (ICV) may be 10 mL or less, preferably 9 mL or less, and more preferably 6 mL or less, but is not limited thereto.

[0064] In the present invention, administration of the above pharmaceutical composition to the central nervous system may provide heparan N-sulfatase transmission in a variety of target tissues such as the brain, spinal cord, and periphery. In the present invention, the above target tissues include all tissues affected by the lysosomal storage disease being treated, for example, the above target tissues may be brain target tissues, spinal cord target tissues, and / or peripheral target tissues, and administration to the central nervous system may provide systemic transmission of heparan N-sulfatase.

[0065] In the present invention, administration of the above-mentioned pharmaceutical composition to the central nervous system may achieve therapeutic or clinically effective levels or activity in the various target tissues described herein. As used herein, therapeutic or clinically effective levels or activity mean levels or activity sufficient to impart a therapeutic effect in the target tissue. For example, therapeutic or clinically effective levels or activity may be enzymatic levels or activity sufficient to improve disease-related symptoms (e.g., GAG accumulation) in the target tissue.

[0066] In the present invention, administration of the above dosage form or pharmaceutical composition to the central nervous system can achieve an enzymatic level or activity such that the normal level or activity of heparan N-sulfatase in the target tissue is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In the present invention, administration of the above pharmaceutical composition to the central nervous system can achieve an enzymatic level or activity that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times higher than the control group (e.g., endogenous level or activity without treatment).

[0067] In the present invention, administration of the above pharmaceutical composition to the central nervous system may induce a decrease in GAG (e.g., heparan sulfate) storage in brain target tissues, spinal cord neurons, and / or peripheral target tissues. In the present invention, the above GAG ​​storage may be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1x, 1.5x, or 2x compared to a negative control group (e.g., GAG storage in subjects before treatment or after vehicle-monotherapy). In the present invention, administration of the above pharmaceutical composition to the central nervous system may induce reduced vacuolation in neurons. For example, it may induce a decrease of at least 20%, 40%, 50%, 60%, 80%, 90%, 1x, 1.5x, or 2x or more compared to a negative control group.

[0068] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered as a single or multiple agent.

[0069] Furthermore, the pharmaceutical composition according to the present invention is preferably administered at the fastest possible rate to improve patient convenience. As one example, the administration rate of the pharmaceutical composition according to the present invention may be about 0.1 ml / min or more, or about 0.5 ml / min or more, preferably 1 ml / min or more, more preferably about 2 ml / min or more, and most preferably about 5 ml / min or more, but is not limited thereto.

[0070] In this invention, the term "prevention" means all actions that prevent the onset of a disease or delay its progression by administering the above composition. In this invention, the term "treatment" means all actions that improve, alleviate, or completely cure the symptoms of a disease by administering the composition.

[0071] In the present invention, “patient” means a mammal that has or is at risk of having a condition or disease that can be alleviated, suppressed, or treated by administering the composition according to the present invention, preferably a human.

[0072] In other words, the present invention relates to a method for preventing or treating Sanfilippo syndrome type A (MPSIIIA), comprising the step of administering the above-mentioned pharmaceutical composition to a patient.

[0073] In another aspect, the present invention relates to the use of the above-mentioned pharmaceutical composition for the prevention or treatment of Sanfilippo syndrome type A (MPSIIIA).

[0074] In another aspect, the present invention also relates to the use of the above-mentioned pharmaceutical composition for the manufacture of a drug for the prevention or treatment of Sanfilippo syndrome type A (MPSIIIA). [Examples]

[0075] The present invention will be described in more detail below through examples. These examples are merely illustrative and it will be obvious to those ordinary in the art that the scope of the invention is not limited by these examples.

[0076] The recombinant heparan N-sulfatase (rHNS) used in the examples of the present invention was named 'GC1130A' or 'GC1130A protein'.

[0077] Example 1: High-volume single-use efficacy test of GC1130A MPSIIIA Mouse (C57BL / 6 Sgsh)mps3a The efficacy of GC1130A as a single intraventricular (ICV) injection therapy for Sanfilippo syndrome type A (MPSIIIA) was evaluated in a gene-WT, homozygous mutant, 2-week, male & female, n=12 model, with the aim of determining the dosage range and interval for repeated ICV administration efficacy studies.

[0078] The animals were classified into five different groups, as shown in Table 1 below, and the efficacy of the target drug was evaluated. [Table 1]

[0079] Both the treatment and control groups of mice were administered 1.5–15 μg / dose GC1130A (22 mg / mL, GC Biopharma), respectively. No deaths, systemic symptoms, or changes in body weight or organ weight occurred in any group after ICV administration. The drug-administered groups were divided into three dose groups. Autopsies were performed 7, 14, and 28 days after administration to collect brain and cerebrospinal fluid (CSF). These samples were stored in a cryogenic freezer until analysis began. The heparan sulfate (HS) content in the brain and cerebrospinal fluid was analyzed over time using LC-MS / MS to confirm the HS removal trend after dose-based administration (Figure 1).

[0080] We were able to confirm that HS accumulation was already present in diseased mice from day 7. In the lowest dose of 12 μg, it decreased on day 7 but showed an increasing trend from day 14, while in the intermediate dose groups of 37 μg and 110 μg, it decreased steadily until day 14 but rebounded on day 28. In other words, we confirmed that the brain and CSF were entirely dose-dependent only at day 14 after drug administration, and based on the previous results, we selected repeated doses of 15, 30, and 60 μg.

[0081] A clear downward trend in HS was observed 14 days after administration, and although the therapeutic effect of the drug weakened after 28 days, it tended to be maintained except in the low-dose group. Therefore, additional experiments were conducted with administration intervals set to every 2 weeks (2QW) or every 4 weeks (4QW).

[0082] Example 2: Low-volume single-use efficacy test of GC1130A MPSIIIA Mouse (C57BL / 6Sgsh) mps3a We aimed to evaluate the efficacy of GC1130A as a single low-dose intraventricular (ICV) injection therapy for Sanfilippo syndrome type A (MPSIIIA) in a gene-WT, homozygous mutant, 2-week, male & female (n=14) model.

[0083] The animals were classified into five different groups, as shown in Table 2 below, and the efficacy of the target drug was evaluated. [Table 2]

[0084] Both the treatment group and the control group mice were administered 1.5–15 μg / dose GC1130A (15 mg / mL, GC Biopharma), respectively. After ICV administration, there were no deaths, systemic symptoms, or changes in body weight or organ weight in any of the groups. Autopsies were performed on 14 and 28 days after administration to collect brain and cerebrospinal fluid (CSF) samples. These were stored in a cryogenic freezer until the start of analysis, and the heparan sulfate (HS) content in the brain and cerebrospinal fluid over time was analyzed by LC-MS / MS to confirm the trend of HS removal after administration according to dose (Figure 2).

[0085] Brain HS showed a statistically significant dose-dependent decrease in all treatment groups compared to the control group on days 14 and 28 (Day 14: G3 -31%, G4 -61%, G5 -80% compared to the control group; Day 28: G3 -32%, G4 -32%, G5 -67% compared to the control group). Cerebrospinal fluid HS showed a statistically significant dose-dependent decrease in all treatment groups compared to the control group on day 14, but was minimal on day 28 (Day 14: G3 -45%, G4 -62%, G5 -57% compared to the control group; Day 28: G3 -15%, G4 -23%, G5 -8% compared to the control group).

[0086] Specifically, in the GC1130A treatment group (≤1.5 μg / dose), a dose-dependent depletion effect of hematopoiesis (HS) in the brain, which was generally observed up to 28 days before autopsy, was confirmed. However, in the case of CSF, a significant HS depletion effect was observed only on day 14 in the GC1130A treatment group at a dose of 1.5 μg / dose.

[0087] In conclusion, based on the analysis of the effective concentration of GC1130A and the maximum volume (5 μL / dose) in the mouse ventricles, the effects on the brain and cerebrospinal fluid at the lowest dose of 1.5 μg / dose were confirmed.

[0088] Furthermore, strong correlations were observed across both the time-based and overall analyses, confirming a substantial correlation between brain and CSF HS levels in response to GC1130A administration (Figure 3).

[0089] Example 3: Repeated-dose efficacy test of GC1130A We aimed to evaluate the efficacy of GC1130A as an ICV injection therapy for the treatment of central nervous system symptoms in MPSIIIA mice.

[0090] Using a dosage form containing GC1130A protein (5 mM histidine, 125 mM NaCl, 1.8% Trehalose, 0.005% PS20, pH 8.11), MPSIIIA mice (C57BL / 6Sgsh) were studied. mps3a Using gene WT, homozygous mutant, 2-week, male & female models, each mouse group appeared to be as shown in Table 3 below. [Table 3]

[0091] During the autopsy, a cerebrospinal fluid (CSF) sample was collected, and the separated brain was stored in a cryogenic freezer until the HS measurement was performed.

[0092] The levels of CSF and HS in mouse brain samples were measured by LC-MS / MS, and the open field test behavioral assessment, a biochemical indicator, was performed to verify the improvement in central nervous system function. In addition, pathological analysis through MRI and immunohistochemistry was performed to confirm actual changes in brain structure (Table 4).

[0093] [Table 4]

[0094] The biochemical mediating variable, HS, was significantly reduced in both the brain and the entire CSF in the GC1130A 15-60 μg dose group, with the degree of HS suppression in the brain being greater than in the CSF. This result is thought to be due to direct injection into the ventricles. Administration every two weeks showed an even greater inhibitory effect than administration every four weeks, but HS was also effectively suppressed at every four weeks. The effect was dose-dependent, but no gender differences were observed (Figure 4).

[0095] Furthermore, a meaningful correlation was confirmed between CSF and HS content in brain tissue (Figure 5).

[0096] Through open-field behavioral tests, we confirmed improvements in various behavioral indicators in the GC1130A repeated-dosing treatment group. To confirm statistical significance, we combined data from the vehicle administration groups of WT and MPSIIIA disease mice (biweekly (Q2W) and monthly (Q4W) administration groups) and confirmed that there was no statistical difference between the two groups. We confirmed that GC1130A showed a tendency to increase in total activity compared to the control group (Figure 6).

[0097] The minimum dose expected to improve cognitive function was confirmed to be 30 μg / dose x1 / month. Even if the HS reduction in CSF is 50% or more, cognitive improvement cannot be expected if CSF HS remains at the knockout level for two weeks. For cognitive improvement, CSF HS must be maintained at 50% or less compared to the knockout level for two weeks or more (15 μg / dose x2 / month).

[0098] As a result, we were able to confirm that HNS shows an effective therapeutic effect against MPSIIIA only when administered at a mouse-based dose of 15-60 μg / dose every 2 to 4 weeks.

[0099] Example 4: Biodistribution of GC1130A conjugated with fluorescent dye after IV or ICV administration in mice. We aimed to evaluate the in vivo pharmacokinetic analysis of GC1130A in mice based on the route of administration, as well as the tissue-specific distribution profile of GC1130A over time.

[0100] After a single dose of GC1130A conjugated with a fluorescent dye at 10 mg / kg in mice via ICV, fluorescence intensity was measured using an in vivo imaging system (IVIS) at 0.083, 1, 2, 4, 8, 24, 48, 96, and 192 hours to confirm the distribution of fluorescence in different organs (Table 5). [Table 5]

[0101] When GC1130A was administered intravenously, fluorescence intensity was higher in the rest of the body compared to the brain, while when administered intravenously via intravenous catheter (ICV), fluorescence intensity in the brain was even higher compared to other organs (Figure 7A).

[0102] Figure 7B shows the average radiant efficiency of the brain-time curve after IV or ICV administration of GC1130A, confirming that the average radiant efficiency in the brain was the highest after ICV injection compared to other administration therapies.

[0103] Example 5: Pharmacokinetics of GC1130A administered via ICV to mouse brains. This study aimed to evaluate the pharmacokinetics of GC1130A in the brains of 5-week-old C57BL / 6 mice after a single ICV administration.

[0104] After administering ICV to 5-week-old C57BL / 6 mice, four mice were necropsied at 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, 168, 240, 336, 504, and 672-hour time profiles, and their brains were homogenized (Table 6). The homogenized solutions were then used to measure the concentration of GC1130A in the brain using a digital ELISA assay, and pharmacokinetic analysis was performed. [Table 6]

[0105] After single ICV administration of GC1130A, dose-dependent drug leakage was observed in the brain, and its half-life was confirmed to be approximately 7 days (Figure 8).

[0106] Example 6: Quantitative analysis of GC1130A in the brain after repeated administration of ICV After autopsy, brain tissue samples were prepared in paraffin blocks for each group, separated by sex. After immunohistochemical staining using staining equipment, the stained slides were scanned to define Regions of Interest (ROIs), and the presence or absence of expression for each marker was quantified.

[0107] Quantitative analysis of neuropathological changes after repeated administration of GC1130A via ICV confirmed drug delivery and efficacy through the central nervous system (CNS) via intracerebral administration (Figure 9).

[0108] Example 7: Quantitative IHC analysis of neuropathological changes - LAMP2 and CD68 analysis after repeated ICV administration Brain tissue samples were prepared in paraffin blocks for both males and females in each group. After immunohistochemical staining using staining equipment, the stained slides were scanned to define Regions of Interest (ROIs), and the presence or absence of expression for each marker was quantified.

[0109] We confirmed that LAMP2 and CD68, markers of inflammation and microglial cells, were increased in the MPSIIIA vehicle group and decreased in the GC1130A group (Figures 10 and 11). Furthermore, this decrease in markers was observed to be even greater in Q2W (2-week intervals) than in Q4W (4-week intervals). We also confirmed that GC1130A was detected in the same brain regions as those where LAMP2 and CD68 markers were decreased.

[0110] Example 8: Dosage analysis of GC1130A in humans Example 8-1: Selection of initial clinical dose for GC1130A in pediatric patients The pediatric dose proposed in this embodiment is based on a method validated in Hammon K, et al., Clin Transl Sci, 14, 1810-1821, 2021, and was used for human conversion with nonclinical data for ultra-rare neurodegenerative pediartic disease. Allometric scaling and PK modeling methods were developed to predict the dose from nonclinical data. These methods were compared and analyzed to select the most conservative value for the initial clinical dose.

[0111] Example 8-2: Calculation of Human Equivalent Dose (HED) based on brain weight The principle of allometric scaling is generally based on the body surface area, as it involves direct administration to body compartments. However, in the case of GC1130A, since it is administered directly to brain tissue via intracerebroventricular administration, the scaling is based on the ratio of brain weight.

[0112] Data on mouse brain weight were obtained from an in vivo efficacy repeated-dose study using the MPSIIIA mouse model. In this study, the dose groups were 15, 30, and 60 μg, administered every two weeks. All dose groups showed significant efficacy, but a clear dose-response was not observed. To calculate the brain weight ratio between humans and animals, the average mouse brain weight was 0.4662 g, obtained from the previous study. The average monkey brain weight was obtained from a 28-week safety study and was 70 g. This brain weight was consistent with reported juvenile monkey weights. Human brain weight estimates were obtained from studies examining the relationship between brain weight and body weight by age. Mean brain weights for males and females were used for each age range (Table 7).

[0113] [Table 7]

[0114] The HED was calculated by multiplying the ratio of brain weights by the dose used in the mouse efficacy test and the dose used in the monkey safety test, respectively (Table 8).

[0115] [Table 8]

[0116] When calculating the MRSD (Maximum Recommended Starting Dose), which is the maximum dose recommended in initial clinical trials, it is possible to start by determining the therapeutic index based on the MTD (maximum tolerable dose) and MED (minimum effective dose). However, due to the nature of biopharmaceuticals, no toxicity significant enough to be considered toxic has been confirmed, so in the case of this drug, the maximum dose that can be administered was selected as the NOAEL (No-observed-adverse-effect level). Considering safety factors, the criteria for the initial clinical dose were applied to 1 / 10 of the NOAEL.

[0117] In terms of selecting based on the clinical initial dose NOAEL, the Safety Factor 10, when converted from NOAEL to human dose, amounts to 23 mg.

[0118] Through a low-dose single-dose efficacy test (Example 2), the effects on the brain and cerebrospinal fluid when administered to mice at 1.5 μg / dose were confirmed, and the value converted to a human dose was 3.5 mg (1 / 10 of the human dose of 35 mg for the 15 μg group in Table 8). Through this, 23 mg can be selected as a dose that can demonstrate both safety and efficacy.

[0119] Example 8-3: Selection of initial clinical dose in children Since GC1130A is administered directly to brain tissue via intracerebroventricular injection, scaling was performed not only based on brain weight but also on cerebrospinal fluid volume. The average body weight of Cynomolgus macaque monkeys is 3.6 kg, and the average cerebrospinal fluid volume is 11.6 mL. The average body weight of a 2-year-old child is 14.0 kg, and the average cerebrospinal fluid volume is 3-4 mL / kg, approximately 42-56 mL. The ratio of cerebrospinal fluid volume between monkeys and humans was set to approximately 1:4 (William Bobadio, 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 Ned. 202Aug8;18(1):309).

[0120] In the case of GC1130A, a 28-week repeated-dose safety study in monkeys showed a NOAEL of 15 mg. Multiplying this value by 4 gives a human dose of 60 mg. Dividing this converted value by the Safety Factor 10 results in 6 mg. [Industrial applicability]

[0121] This invention relates to the optimal dose and frequency of heparan-N-sulfatase (HNS) administration, which can effectively reduce heparan sulfate (HS) accumulation while improving patient cognition, and may be usefully used in enzyme replacement therapy (ERT) for the treatment of Sanfilippo syndrome type A (MPSIIIA).

[0122] Having described in detail the specified aspects of the present invention, it will be clear to those with ordinary skill in the art that such specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for the prevention or treatment of Sanfilippo syndrome type A (MPSIIIA) containing heparan N-sulfatase (HNS), The above-mentioned heparan N-sulfatase is a pharmaceutical composition characterized by being administered to patients at a dose of 3 to 150 mg every 2 to 4 weeks via intracerebral injection (ICV), intracerebral injection (IC), or intradural injection (IT).

2. The pharmaceutical composition according to claim 1, characterized in that the heparan N-sulfatase is administered in a volume of 6 to 100 mg per single dose.

3. The pharmaceutical composition according to claim 1, characterized in that the total volume administered per single dose is 10 mL or less.

4. The pharmaceutical composition according to claim 1, characterized in that the composition further comprises 1 to 40 mM histidine buffer.

5. The pharmaceutical composition according to claim 1, characterized in that the composition further comprises sugars.

6. The pharmaceutical composition according to claim 5, characterized in that the aforementioned sugar is one or more selected from the group consisting of trehalose, sucrose, maltose, lactose, and sorbitol.

7. The pharmaceutical composition according to claim 6, characterized in that the sugars are contained in a concentration of 0.1 to 5.0 w / v%.

8. The pharmaceutical composition according to claim 1, characterized in that the composition further comprises a salt.

9. The pharmaceutical composition according to claim 8, characterized in that the salt is NaCl or KCl.

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

11. The pharmaceutical composition according to claim 1, characterized in that the composition further comprises a surfactant.

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

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

14. The pharmaceutical composition according to claim 1, characterized in that it is administered to the central nervous system via intracerebral injection (ICV).

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