Methods and compositions for treating hunt syndrome

Abstract

This application relates to methods and compositions for treating Hunter syndrome. In particular, the present invention provides compositions and methods for effectively treating Hunter syndrome by delivering idurosulfatase-β (human recombinant iduronate-2-sulfatase protein) to the CNS. The compositions and methods provided by the present invention effectively alleviate symptoms not only in the brain and spinal cord but also in peripheral tissues, including the heart, liver, spleen, lungs, and kidneys.

Description

[0001] This application is a divisional application of Chinese patent application filed on December 21, 2016, with application number "201680076842.2" and invention title "Method and Composition for Treating Hunter Syndrome". The original application was the Chinese national phase application of international application PCT / KR2016 / 015060. Technical Field

[0002] This invention relates to improved methods and compositions for treating Hunter syndrome. Background Technology

[0003] Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is an X-linked recessive lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase, which is responsible for degrading mucopolysaccharides[1]. The lack of iduronate-2-sulfatase leads to the accumulation of undegraded glycosaminoglycans (GAGs) in cells and causes progressive multi-organ damage[2]. Among the various types of GAGs, dermatan sulfate (DS) and heparan sulfate (HS) are the main GAGs that accumulate in MPS II[2].

[0004] The clinical phenotype of MPS II is divided into attenuated and severe forms. Patients with the attenuated form exhibit physical manifestations, including a coarse face, hepatosplenomegaly, multiple osteodystrophys, and joint stiffness without neurological involvement, while patients with the severe form, in addition to physical symptoms, have neurological damage and progressive neurodegeneration. Insufficient levels of endogenous IDS lead to the pathological accumulation of heparan sulfate and dermatan sulfate in organs such as the heart, liver, and central nervous system (CNS). Symptoms including neurodegeneration and mental retardation appear during childhood; and early death can occur due to organ damage in the brain.

[0005] Enzyme replacement therapy (ERT) involves the systemic administration of proteins and / or enzymes of natural or recombinant origin to the subject. Approved therapies are typically administered intravenously and are generally effective in treating physical symptoms of underlying enzyme deficiency.

[0006] Treatment of diseases with CNS etiology is particularly challenging because intravenously administered proteins and / or enzymes have limited distribution to the cells and tissues of the central nervous system (CNS), as they cannot adequately cross the blood-brain barrier (BBB).

[0007] The blood-brain barrier (BBB) ​​is a structural system composed of endothelial cells that protects the central nervous system (CNS) from harmful substances in the bloodstream, such as bacteria, macromolecules (e.g., proteins) and other hydrophilic molecules, by restricting their spread across the BBB and into the underlying cerebrospinal fluid (CSF) and CNS.

[0008] Many people believe that the barriers to diffusion on the brain surface and the lack of effective and convenient delivery methods are too great obstacles to achieving adequate therapeutic effects in the brain for any disease.

[0009] Hunter syndrome affects the nervous system and therefore presents unique challenges in treating these conditions with conventional therapies. Large amounts of glycosaminoglycans (GAGs) often accumulate in the neurons and meninges of affected individuals, leading to various forms of CNS symptoms.

[0010] Therefore, there remains a great need to effectively deliver therapeutic agents to the brain. More specifically, there is a great need to more effectively deliver active agents to the central nervous system to treat lysosomal storage disorders (such as Hunter syndrome).

[0011] To overcome BBB, direct drug delivery via intrathecal or intraventricular injection of recombinant enzymes has shown promising results in several types of MPS animal models [3-7]. In addition, phase I / II clinical trials have reported that intrathecal (IT) injection of idursulfase (IDS) reduced GAG concentrations in cerebrospinal fluid (CSF) by approximately 80-90% in children with severe forms of MPS II. However, numerous adverse events have been reported in clinical trials, most of which are related to malfunctions of the IT drug delivery device [8].

[0012] Although the cellular mechanisms of neurodegeneration in severe forms of MPS II are not fully understood, several recent studies have demonstrated a correlation between disaccharides derived from HS and intellectual disability in large MPS patient populations [9, 10]. Furthermore, animal studies have shown that HS may contribute to neurological disorders associated with MPS III mouse models by activating integrin-based focal adhesions in astrocytes or neural stem cells [11, 12]. Akiyama et al.

[13] reported that “pathological GAG” measured by the Sensi-Pro non-reducing end HS assay

[14] had higher sensitivity and specificity than total GAG in MPS II mouse brain tissue

[13] . Based on these data, the amount of accumulated HS could be a more sensitive biomarker for representing the neuropathological status and neurological function of MPS II compared to the amount of total GAG. However, measuring brain tissue HS is not feasible in a clinical setting. Therefore, there is a need to discover available clinical biomarkers representing brain tissue HS. If the concentration of HS in CSF is correlated with the amount of brain tissue GAG ​​(especially HS), we hypothesize that it could be one of the clinical biomarkers.

[0013] A single ICV (intracerebroventricular) injection of IDS-β according to the present invention was well tolerated, and it resulted in a significant reduction in HS and GAG in the brain and other body tissues. We also found a significant positive correlation between brain HS and brain GAG in CSF HS content, suggesting that CSF HS concentration may be a usable biomarker for representing the brain pathology in MPS patients with CNS involvement.

[0014] This invention provides an efficient method for the direct delivery of therapeutic agents to the central nervous system (CNS). The invention is based on the discovery that idurosulfatase-β (IDS-β) (a recombinant human iduronate-2-sulfatase protein developed as an effective alternative enzyme for Hunter syndrome) can be directly introduced into the ventricle of a subject via intraventricular (ICV) administration, allowing the enzyme to efficiently and extensively diffuse across multiple surfaces and penetrate multiple regions throughout the brain, including deep brain regions. The inventors have demonstrated that such protein delivery can be achieved using simple saline-based formulations without causing significant adverse effects (e.g., severe immune responses) in the subject. Therefore, this invention provides a highly efficient, clinically desirable, and patient-friendly method for the direct CNS delivery of therapeutic agents for Hunter syndrome. Summary of the Invention

[0015] Technical issues

[0016] For the treatment of Hunter syndrome, there remains a great need for efficient delivery of therapeutic agents to the brain. Intrathecal (IT) injection or administration of therapeutic proteins to cerebrospinal fluid (CSF) has recently been attempted to treat patients with Hunter syndrome, but a number of adverse events have been reported in clinical trials [8]. Although most adverse events are related to failure of the IT drug delivery device, other methods, such as intraventricular (ICV) administration, will be needed to treat patients with Hunter syndrome. ICV administration would be an effective method for delivering therapeutic agents directly to the patient's ventricles, and there are currently no approved or developing products for the treatment of Hunter syndrome by ICV administration.

[0017] Technical solution

[0018] 1. A method for treating Hunter syndrome, comprising the step of administering (ICV administration) a therapeutically effective dose of an ICV preparation to the ventricle of a subject requiring treatment, said ICV preparation comprising idosasulfatase-β (IDS-β) protein at a concentration of about 0.1 mg / ml to about 60 mg / ml, sodium chloride at a concentration of about 150 mM, polysorbate 20 at a concentration of about 0.05 mg / ml, and a pH of about 6.

[0019] 2. The method of technical solution 1, wherein the ICV preparation comprises idosasulfatase-β (IDS-β) protein at a concentration of about 15 mg / ml, sodium chloride at a concentration of about 150 mM, polysorbate 20 at a concentration of about 0.05 mg / ml, and a pH of about 6.

[0020] 3. The method of technical solution 1, wherein the effective therapeutic dose is about 1 mg to about 30 mg.

[0021] 4. The method described in technical solution 1, wherein the effective therapeutic dose is approximately 10 mg.

[0022] 5. The method described in technical solution 1, wherein the ICV application is performed every three weeks.

[0023] 6. The method of the technical solution 1, wherein the ICV application is performed once a month.

[0024] 7. The method of the first technical solution, wherein the ICV is administered via an intraventricular catheter system comprising a reservoir and a catheter connected to the reservoir.

[0025] 8. The method of technical solution 7 further includes the following steps: surgically implanting the intraventricular catheter system, wherein the reservoir is placed between the scalp and brain of the subject to be treated, and the end of the catheter is placed in the ventricle of the subject, such that the internal space of the reservoir is connected to the internal space of the ventricle through the internal space of the catheter, so that cerebrospinal fluid flows from the ventricle into the reservoir to fill the reservoir; 0.1 to 5 ml of cerebrospinal fluid is removed from the reservoir at a flow rate of 0.1 to 60 ml / min; 0.1 to 5 ml of the ICV preparation is injected into the reservoir at a flow rate of 0.1 to 60 ml / min; and the ICV preparation flows from the reservoir through the catheter into the ventricle.

[0026] 9. The method of claim 1, wherein the ICV administration is combined with at least one other form of enzyme replacement therapy for Hunter syndrome.

[0027] 10. The method of claim 9, wherein the additional form of enzyme replacement therapy for Hunter syndrome is selected from intravenous administration and subcutaneous administration.

[0028] 11. The method of the technical solution 10, wherein the ICV administration is performed monthly and the intravenous administration is performed weekly.

[0029] 12. The method of the technical solution 10, wherein the ICV administration is performed every three weeks and the intravenous administration is performed weekly.

[0030] 13. The method of the technical solution 10, wherein the ICV application is performed monthly and the subcutaneous application is performed weekly.

[0031] 14. The method of the technical solution 10, wherein the ICV application is performed every three weeks and the subcutaneous application is performed weekly.

[0032] 15. The method of the technical solution 10, wherein the ICV application is performed once a month and the subcutaneous application is performed twice a week.

[0033] 16. The method of the technical solution 10, wherein the ICV application is performed once every three weeks and the subcutaneous application is performed twice a week.

[0034] 17. The method of the aforementioned technical solution 10, wherein the ICV administration is performed once a month, and the intravenous administration and the subcutaneous administration are alternated at a weekly interval.

[0035] 18. The method of the technical solution 10, wherein the ICV administration is performed every three weeks, and the intravenous administration and the subcutaneous administration are alternated at a one-week interval.

[0036] 19. A formulation for intraventricular administration for the treatment of Hunter syndrome, comprising idosasulfatase-β (IDS-β) protein at a concentration of about 0.1 mg / ml to about 60 mg / ml, sodium chloride at a concentration of about 150 mM, polysorbate 20 at a concentration of about 0.05 mg / ml, and a pH of about 6.

[0037] Beneficial effects

[0038] According to the present invention, intraventricular administration of IDS-β reduces the levels of heparan sulfate (HS) and glycosaminoglycans (GAG) in the brain and cerebrospinal fluid (CSF) of MPS II mice.

[0039] According to the present invention, intraventricular administration of IDS-β reduces the levels of heparan sulfate (HS) and glycosaminoglycans (GAG) in somatic (peripheral) tissues (including heart, lung, liver, spleen and kidney) in MPS II mice.

[0040] According to the present invention, the trend of GAG concentration in the brain can be predicted by the level of heparan sulfate in the CSF, which allows for a safer and easier diagnosis of the severity of brain GAG accumulation. Attached Figure Description

[0041] Figure 1 The levels of GAG in the brain tissue of IDS KO mice following a single ICV injection of IDS-β are shown.

[0042] Figure 2 The levels of HS in CSF and brain tissue of IDS KO mice after a single ICV injection of IDS-β are shown.

[0043] Figure 3 The correlation between HS levels in CSF and HS levels in brain tissue was shown in IDS KO mice following a single ICV injection of IDS-β.

[0044] Figure 4 The levels of GAG in the somatic tissues of IDS KO mice following a single ICV injection of IDS-β are shown.

[0045] Figure 5 The brain tissue harvested at different time points after injection and visualized with trypan blue is shown.

[0046] Figure 6The levels of GAG in the heart tissue of IDS KO mice following a single ICV injection of IDS-β are shown.

[0047] Figure 7 The levels of GAG in the lung tissue of IDS KO mice after a single ICV injection of IDS-β are shown.

[0048] Figure 8 The levels of GAG in the liver tissue of IDS KO mice following a single ICV injection of IDS-β are shown.

[0049] Figure 9 The levels of GAG in the spleen tissue of IDS KO mice following a single ICV injection of IDS-β are shown.

[0050] Figure 10 The levels of GAG in the kidney tissue of IDS KO mice following a single ICV injection of IDS-β are shown. Detailed Implementation

[0051] As described in detail below, the inventors have successfully developed a stable formulation for the effective intraventricular (ICV) administration of idosasulfatase-β (IDS-β) protein.

[0052] In several embodiments, the present invention includes a stable formulation for direct intraventricular (ICV) administration comprising idosasulfatase-β (IDS-β) protein, a salt, and a polysorbate surfactant. In some embodiments, the IDS-β protein is present in the ICV formulation at a concentration of about 0.1 to 60 mg / ml (e.g., 0.1 to 60 mg / ml, 0.1 to 30 mg / ml, 0.3 to 30 mg / ml, 0.2 to 20 mg / ml, 0.2 to 6 mg / ml, 0.6 to 6 mg / ml, 5 to 60 mg / ml, or 10 to 60 mg / ml). In some embodiments, the IDS-β protein is present in the ICV formulation at a concentration selected from 0.1 mg / ml, 0.2 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 1 mg / ml, 2 mg / ml, 5 mg / ml, 6 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 55 mg / ml, or 60 mg / ml, or higher.

[0053] In several embodiments, the present invention includes stable formulations of any of the embodiments described herein. In some embodiments, IDS-β comprises a protein having the amino acid sequence of SEQ ID NO: 1. In some embodiments, IDS-β further comprises a protein having the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 1 is a recombinant human iduronate-2-sulfatase protein. SEQ ID NO: 2 is a recombinant human iduronate-2-sulfatase protein in which cysteine ​​at position 59 is replaced by formyl-glycine (G*).

[0054] In some embodiments, IDS-β comprises about 35% (mol percentage) or less of the protein having SEQ ID NO: 1 and about 65% (mol percentage) or more of the protein having SEQ ID NO: 2. In some embodiments, IDS-β comprises about 20 to 35% (mol percentage) of the protein having SEQ ID NO: 1 and about 65 to 80% (mol percentage) of the protein having SEQ ID NO: 2.

[0055] In some embodiments, IDS-β comprises a protein having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NO: 1. In some embodiments, IDS-β comprises a protein having an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NO: 2.

[0056] In some embodiments, the stabilizing agent of any of the embodiments described herein comprises a salt. In some embodiments, the salt is sodium chloride (NaCl). In some embodiments, NaCl is present at a concentration of about 0 to 300 mM (e.g., 0 to 250 mM, 0 to 200 mM, 0 to 150 mM, 50 to 250 mM, or 100 to 200 mM). In some embodiments, NaCl is present at a concentration of about 125 to 175 mM. In some embodiments, NaCl is present at a concentration of about 150 mM.

[0057] In several embodiments, the present invention includes a stable formulation of any of the embodiments described herein, wherein the polysorbate surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof. In some embodiments, the polysorbate surfactant is polysorbate 20 (Tween 20). In some embodiments, polysorbate 20 is present at a concentration of about 0 to 0.02% (0 to 0.2 mg / ml). In some embodiments, polysorbate 20 is present at a concentration of about 0.005% (0.05 mg / ml).

[0058] In several embodiments, the present invention includes a stable formulation of any of the embodiments described herein, wherein said formulation further comprises a buffer. In some embodiments, the buffer is selected from phosphates, acetates, histidine, succinates, Tris, and combinations thereof. In some embodiments, the buffer is a phosphate. In some embodiments, the phosphate is present at a concentration not greater than 50 mM (e.g., not greater than 45 mM, 40 mM, 35 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM, 0.25 mM, or 0.12 mM). In some embodiments, the phosphate is present at a concentration not greater than 20 mM. In several aspects, the present invention includes stable formulations of any embodiment described herein, wherein the pH of said formulation is about 3 to 8 (e.g., about 4 to 7.5, 5 to 8, 5 to 7.5, 5 to 6.5, 5 to 7.0, 5.5 to 8.0, 5.5 to 7.7, 5.5 to 6.5, 6 to 7.5, or 6 to 7.0). In some embodiments, the pH of said formulation is about 5.5 to 6.5 (e.g., 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5). In some embodiments, said formulation is about 6.0.

[0059] In several embodiments, the present invention includes a stable formulation of any embodiment described herein, wherein said formulation is a liquid formulation. In several embodiments, the present invention includes a stable formulation of any embodiment described herein, wherein said formulation is formulated as a lyophilized powder.

[0060] In some embodiments, the present invention includes a stable formulation for ICV administration comprising IDS-β protein at a concentration of about 0.1 to 60 mg / ml, NaCl at a concentration of about 150 mM, polysorbate 20 at a concentration of about 0.005% (0.05 mg / ml), and a pH of about 6.0. In some embodiments, the concentration of IDS-β protein is about 0.1 mg / ml, 0.2 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 1 mg / ml, 2 mg / ml, 5 mg / ml, 6 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 55 mg / ml, or 60 mg / ml.

[0061] In several aspects, the present invention includes containers comprising a single-dose form of a stable formulation as described in several embodiments herein. In some embodiments, the container is selected from ampoules, vials, bottles, cartridges, reservoirs, lyo-jects, or pre-filled syringes. In some embodiments, the container is a pre-filled syringe. In some embodiments, the pre-filled syringe is selected from borosilicate glass syringes with a baked silicone coating, borosilicate glass syringes with sprayed silicone, or plastic resin syringes without silicone. In some embodiments, the stable formulation is present in a volume of less than about 50 mL (e.g., less than about 45 ml, 40 ml, 35 ml, 30 ml, 25 ml, 20 ml, 15 ml, 10 ml, 5 ml, 4 ml, 3 ml, 2.5 ml, 2.0 ml, 1.5 ml, 1.0 ml, or 0.5 ml). In some embodiments, the stable formulation is present in a volume of about 6.0 ml. In some embodiments, the stable formulation is present in a volume of about 3.0 ml. In some embodiments, 2.0 ml of the stabilizing agent is contained in a 6.0 ml vial. In some embodiments, 1.5 ml of the stabilizing agent is contained in a 5.0 ml vial. In some embodiments, 1.0 ml of the stabilizing agent is contained in a 3.0 ml vial.

[0062] In several aspects, the present invention includes a method for treating Hunter syndrome, comprising the step of administering a formulation according to any embodiment described herein into the ventricles of a subject requiring treatment.

[0063] In some embodiments, the present invention includes a method for treating Hunter syndrome, comprising the step of administering a formulation to the ventricle of a subject requiring treatment, the formulation comprising IDS-β protein at a concentration of about 0.1 to 60 mg / ml, sodium chloride at a concentration of about 150 mM, polysorbate 20 at a concentration of about 0.005% (0.05 mg / ml), and a pH of about 6.

[0064] In some embodiments, the subject requiring treatment has an implanted intraventricular catheter system for ICV administration, comprising a reservoir and a catheter, such as the Ommaya reservoir. In some embodiments, ICV administration is performed by infusing the aforementioned ICV formulation into the reservoir at a flow rate of 0.1 to 60 ml / min. In some embodiments, prior to ICV administration, the subject's cerebrospinal fluid (CSF) is removed from the reservoir at a flow rate of 0.1 to 60 ml / min to prevent a net increase in the subject's CSF volume after ICV administration, thus preventing an increase in intracranial pressure. In some embodiments, the formulation injected into the reservoir is propelled through the catheter into the subject's ventricles by gently pressing and releasing the reservoir.

[0065] In some embodiments, ICV administration to subjects does not result in significant adverse effects (e.g., severe immune responses). In some embodiments, ICV administration to subjects does not result in significant adaptive T cell-mediated immune responses.

[0066] In some embodiments, ICV administration of the formulation results in the delivery of IDS-β protein to various target tissues in the brain, spinal cord, and peripheral organs. In some embodiments, ICV administration of the formulation results in the delivery of IDS-β protein to brain target tissues. In some embodiments, brain target tissues include neurons in gray matter and / or white matter. In some embodiments, IDS-β protein is delivered to neurons, glial cells, perivascular cells, and / or meningeal cells. In some embodiments, IDS-β protein is also delivered to neurons in the spinal cord.

[0067] In some embodiments, ICV administration of the formulation also results in systemic delivery of the IDS-β protein to peripheral target tissues. In some embodiments, peripheral target tissues are selected from, but are not limited to, the heart, liver, spleen, lungs, and / or kidneys.

[0068] In some embodiments, ICV administration of the formulation results in lysosomal localization of cells in target brain tissue, spinal cord neurons, and / or peripheral target tissue. In some embodiments, ICV administration of the formulation results in a reduction in GAG accumulation in target brain tissue, spinal cord neurons, and / or peripheral target tissue. In some embodiments, GAG accumulation is 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 (e.g., GAG accumulation in a subject before treatment or after administration of the load-only agent). In some embodiments, ICV administration of the formulation results in reduced vacuolation formation in neurons (e.g., a reduction of at least 20%, 40%, 50%, 60%, 80%, 90%, 1-fold, 1.5-fold, or 2-fold compared to a negative control). In some embodiments, the neurons comprise Purkinje cells.

[0069] In some embodiments, ICV administration of the formulation results in increased IDS-β enzyme activity in brain target tissues, spinal cord neurons, and / or peripheral target tissues. In some embodiments, IDS-β enzyme activity is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times compared to negative controls (e.g., endogenous enzyme activity in subjects before treatment or after administration of the load-only agent). In some implementations, the enhanced IDS-β enzyme activity is at least about 10 nmol / hr / mg, 20 nmol / hr / mg, 40 nmol / hr / mg, 50 nmol / hr / mg, 60 nmol / hr / mg, 70 nmol / hr / mg, 80 nmol / hr / mg, 90 nmol / hr / mg, 100 nmol / hr / mg, 150 nmol / hr / mg, 200 nmol / hr / mg, 250 nmol / hr / mg, 300 nmol / hr / mg, 350 nmol / hr / mg, 400 nmol / hr / mg, 450 nmol / hr / mg, 500 nmol / hr / mg, 550 nmol / hr / mg, or 600 nmol / hr / mg.

[0070] In some embodiments, administration of the formulation to the ICV results in a reduction in the intensity, severity, or frequency of at least one symptom or feature of Hunter syndrome, or a delay in its onset. In some embodiments, at least one symptom or feature of Hunter syndrome is cognitive impairment; white matter lesions; dilated perivascular spaces in the brain parenchyma, ganglia, corpus callosum, and / or brainstem; atrophy; and / or ventricular enlargement.

[0071] In some embodiments, ICV administration occurs every two weeks. In some embodiments, ICV administration occurs every three weeks. In some embodiments, ICV administration occurs monthly. In some embodiments, ICV administration occurs every two months. In some embodiments, administration is continuous, for example, via a continuous infusion pump. In some embodiments, ICV administration is used in conjunction with intravenous (IV) administration. In some embodiments, IV administration occurs weekly. In some embodiments, IV administration occurs every two weeks. In some embodiments, IV administration occurs monthly. In some embodiments, IV administration occurs every two months.

[0072] In some implementations, IV and ICV administrations are performed on the same day. In some implementations, IV and ICV administrations are not performed within a certain time frame of each other (e.g., not within at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, or at least 1 week). In some implementations, IV and ICV administrations are performed on an alternating schedule, such as weekly, every other week, twice a month, or monthly alternating administrations. In some implementations, ICV administration replaces IV administration in an administration schedule (e.g., in a schedule of weekly, every other week, twice a month, or monthly IV administrations), where every three, four, or five administrations in that schedule can be replaced by ICV administrations that replace IV administrations.

[0073] In some implementations, IV and ICV administration are performed sequentially, for example, first IV administration (e.g., weekly, every other week, every three weeks, twice a month, or monthly administration for two weeks, one month, two months, three months, four months, five months, six months, one year, or longer), followed by ICV administration (e.g., weekly, every other week, every three weeks, twice a month, or monthly administration for more than two weeks, one month, two months, three months, four months, five months, six months, one year, or longer). In some implementations, ICV administration is performed first (e.g., weekly, every other week, every three weeks, twice a month, monthly, every two months, every three months for two weeks, one month, two months, three months, four months, five months, six months, one year, or longer), followed by IV administration (e.g., weekly, every other week, every three weeks, twice a month, or monthly administration for more than two weeks, one month, two months, three months, four months, five months, six months, one year, or longer).

[0074] In some implementations, ICV is used in the absence of IV administration.

[0075] In some implementations, ICV is administered in the absence of concurrent immunosuppressive therapy.

[0076] Example

[0077] The present invention will be further described in detail below with reference to embodiments. It will be apparent to those skilled in the art that these embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0078] Example 1

[0079] 1-1: Overview

[0080] This study was conducted to investigate the pharmacological effects and dose-response relationship of a single intraventricular (ICV) injection of idosasulfatase-β (IDS-β) in MPS II mice. Furthermore, we measured the concentration of HS in the CSF and investigated the correlation between CSF HS and brain tissue HS and GAG in MPS II mice.

[0081] Three doses of ICV IDS-β were administered (3 μg, 10 μg, and 30 μg), and tissue GAG ​​(brain, heart, lung, liver, spleen, and kidney) was measured at 7, 14, and 28 days post-injection. HS was measured in mouse CSF and brain using LC / MS-MS. Total GAG in the brain and other somatic tissues was significantly reduced in all IDS-β-treated groups. The significant reduction in the 30 μg injection group was maintained for 28 days. We also showed that HS content was reduced in both CSF and brain tissue in all IDS-β-treated groups. Furthermore, we showed that HS concentration in CSF was significantly correlated with brain HS and brain tissue GAG.

[0082] A single ICV injection of the IDS-β according to the invention was well tolerated, and it produced a significant reduction in HS and GAG in the brain and other body tissues. We also found a significant positive correlation between brain HS and brain GAG in CSF HS content, suggesting that CSF HS concentration may be a usable biomarker for representing the brain pathology in MPS II patients with CNS involvement.

[0083] 1-2: Method

[0084] animal

[0085] We used previously reported IDS knockout (KO) mice. In short, the Ids gene is deleted from exon 2 through exon 3

[15] . IDS KO mice were bred from the C57BL / 6.129S background strain and had a null mutation in the Ids gene. Wild-type (WT) control mice were bred from the C57BL / B6.129S strain. The genotypes of all mice were confirmed by polymerase chain reaction of DNA obtained by tail snip. This study was approved by the Institutional Animal Care and Use Committee (Approval No. 20140925005) and conducted in accordance with the animal welfare policy of the Samsung Biomedical Research Institute in Seoul, South Korea.

[0086] Research Design

[0087] Six-week-old animals were randomly assigned to five groups (n=12 per group) using stratified randomization. IDS KO mice were assigned to four groups: IDS KO mice with ICV injection and IDS KO mice injected with three different doses (3 μg, 10 μg, and 30 μg) of IDS-β (Green Cross Corp., Yongin, Korea). Four animals in each group were sacrificed every 7, 14, and 28 days following ICV injection. GAG concentrations in various tissues (brain, heart, lung, liver, spleen, and kidney) were analyzed, and HS concentrations from CSF and brain were measured at 7, 14, and 28 days post-injection.

[0088] Preparation of IDS-β for ICV injection

[0089] The carrier solution was a 150 mM sodium chloride solution at 0.05 mg / mL Tween 20 (Merck Millipore, Darmstadt, Germany). The concentrated IDS-β (50 mg / mL) drug solution was diluted with the carrier to obtain concentrations of 0.6 mg / mL, 2 mg / mL, and 6 mg / mL.

[0090] ICV injection

[0091] Mice were given a single ICV injection at 6 weeks of age. Each drug solution or carrier was administered to mice in a total ICV volume of 5 μL. On the day of administration, mice were anesthetized by inhalation of isoflurane (Hana Pharm., Korea) and placed in a stereotactic apparatus. After a small incision, the skull was exposed and cleaned. ICV injection was performed according to a modified method previously reported [16, 17]. Using the following coordinates (Benchmark, Neurolab), IDS-β or the carrier was injected into the right ventricle with a 31-gauge needle at a flow rate of 10 mL / min controlled by an infusion pump (Harvard Apparatus, Holliston, MA, USA): 0.58 mm caudal to the anterior fontanelle, 1.25 mm lateral to the sagittal suture, and 1.77 mm deep. Ruptured vessels or facial swelling at the injection site were monitored. The needle was then removed 15 seconds after stopping the plunger movement to prevent backflow. The incision was closed with a wound clip, and the mouse was placed on an isothermal mat at 37°C and observed post-surgery until recovery. The entire procedure took 10 to 15 minutes per animal. To demonstrate successful ICV injection technique, an ICV dye solution was injected. The brain was collected and visualized at different time points after injection. Proper injection into one of the ventricles allowed trypan blue (0.05%) to distribute on the injection side of the brain approximately 10 to 15 minutes after injection. Extensive distribution of trypan blue in the cerebral hemispheres was visible approximately 1 hour after injection. Inaccurate injection can be distinguished by the lack of blue in the cerebral hemispheres. Figure 5 ).

[0092] CSF and organization collection

[0093] Mice were euthanized at 7, 14, and 28 days post-injection by injection of an overdose of Alfaxan solution (15 mg / kg). CSF was collected from the cisterna magna via borosilicate glass (OD: 10 mm, ID: 0.75 mm) and frozen for HS concentration measurements. Blood in mouse brain tissue was cleansed with phosphate-buffered saline (PBS) via transcardiac perfusion for 15-20 minutes. Brain tissue was collected and frozen on dry ice. The samples were then homogenized and aliquoted into four portions (half for GAG measurements and half for HS measurements). Other somatic tissues (heart, lung, liver, spleen, and kidney) were also collected and homogenized in PBS.

[0094] Measuring total GAG concentration in tissues

[0095] Homogenized tissue samples were shaken overnight at 4°C and centrifuged at 12,000×g for 15 minutes, followed by collection of the supernatant. Total GAG levels were measured using an sGAG assay kit (Kamiya Biochemicals, Japan). First, 50 μL of the homogenized sample was incubated at room temperature (RT) with 50 μL of 8 mol / L guanidine-HCl for 15 minutes. Then, at RT, 50 μL of STA solution (0.3% H2SO4, 0.75% Triton X-100) was added for 15 minutes, followed by the addition of alcian blue solution for 15 minutes. The sample was then centrifuged at 12,000 rpm for 15 minutes and rinsed with DMSO solution (40% DMSO, 0.05 mol / L MgCl2). Finally, 500 μL of Gu-prop solution (4 mol / L guanidine-HCl, 33% 1-propanol, 0.25% Triton X-100) was added to the precipitate and the mixture was completely dissolved. Alternatively, the absorbance was read at 600 nm in an X-Mark (Bio-Rad, Hercules, CA). The GAG ​​concentration was normalized to the protein concentration, which was measured using a BCA protein assay kit (ThermoFisher, Waltham, MA). The GAG ​​concentration was expressed as g GAG / mg protein, calculated using a standard curve of the GAG ​​substrate chondroitin sulfate-6. Data for each sample are averages of repeated measurements.

[0096] Measuring HS in CSF and brain tissue

[0097] HS levels in mouse CSF and brain tissue samples were determined using LC-MS / MS. Stock solutions of each calibration standard (STD) at 5 mg / mL were prepared by dissolving sodium HS or DS in water. The STD stock solutions were diluted with appropriate volumes of water to prepare STDs of 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10, and 20 μg / mL, and quality control (QC) samples of 0.2, 2, and 15 μg / mL. Additionally, the 5 mg / mL STD stock solutions were deuterated to prepare HS-d6 and DS-d6 as internal standards (IS). 20 μL of STD solution was added to a glass tube and evaporated under nitrogen. The residue was methanolyzed by mixing with 200 μL of methanol-d4-acetyl chloride (400:64, v / v) and heating at 65 °C for 90 min. After methanolyzation, the solvent was evaporated under nitrogen. The residue was reconstituted in 1 mL of water, diluted with water-methanol-formic acid (950:50:1, v / v / v: buffer A), and the prepared solution was used as an IS stock solution. Mouse CSF samples were centrifuged at 2100×g for 5 min at 4 °C, and the supernatant was diluted with an equal volume of PBS. Mouse brain tissue was homogenized with 0.01 mol / L sodium hydroxide (50 or 100 times the brain weight by volume). The homogenate was incubated at room temperature for 24 h, and 20 μL of the homogenate was added to 180 μL of water-chloroform (4:5, v / v). After mixing, the sample was centrifuged at 10000×g for 5 min at 4 °C, and the supernatant was diluted with an equal volume of PBS. These samples prepared from CSF and brain were used as test samples for LC-MS / MS analysis. 4 μL of test samples from CSF (20 μL from brain), STD, or QC were evaporated in tubes under nitrogen. Add 50 μL of 3M HCl-MeOH and 5 μL of 2,2-dimethoxypropane to the residue, sonicate for 3 min, heat at 65 °C for 90 min, and evaporate. Reconstitute the residue with 200 μL of IS stock solution, sonicate for 3 min, transfer to a centrifuge filter, centrifuge at 10000×g for 3 min at 4 °C, and analyze the resulting filtrate. Inject 5 μL of each sample into an API5000 (AB / MDS Sciex) triple quadrupole mass spectrometer equipped with an ACQUITY UPLC system (Waters). Separate the test samples and IS using an ACQUITY UPLC HSS T3 column (100 A 1.8 μm, 2.1 mm × 100 mm) heated at 40 °C. The initial mobile phase consisted of 100:0 (v / v) buffer A: buffer B [water-methanol-formic acid (500:500:1, v / v / v)], and gradient elution was performed at a flow rate of 0.4 mL / min.Elution was performed with a linear gradient, where buffer B was increased from 0% to 45% between 0.5 and 4 minutes, then increased to 60% at 4.01 minutes, held at 60% for 1 minute, decreased to 0% at 5.01 minutes, and held at 0% for 1 minute. Mass spectrometry was performed with electrospray ionization as the selected ion method and positive ion polarity. Nitrogen was used as the curtain gas (40 psi), and air was used as both the nebulizer gas (50 psi) and the heater gas (40 psi). Ion monitoring conditions were limited to an ion spray voltage of 4.5 kV and a turbine probe temperature of 600 °C. These settings for declustering potential, inlet potential, and collision energy were 110 V, 8 V, and 22 eV, respectively. Data were obtained using the following multiple reaction monitoring (MRM): for HS mass-to-charge ratio (m / z) 384→162, for DS m / z 426→236, for HS-d6 m / z 390→162, and for DS-d6 m / z 432→162. Peak areas, STD curves, and measured concentrations were calculated using Analyst ver. 1.5.1 (AB Scix).

[0098] Statistical analysis

[0099] Statistical analyses were performed using GraphPad Prism 6. The Mann-Whitney U test was used to compare differences between drug-treated and load-treated groups in KO mice. A p-value less than 0.05 was considered statistically significant. Data are presented as mean and SEM. To determine the relationships between CSF HS and brain HS, and between CSF HS and brain GAG, we evaluated 73 samples of mouse CSF and brain tissue and analyzed the data using Spearman's rho and linear regression. Intraclass correlation coefficients (ICC) and 95% confidence intervals were calculated.

[0100] 1-3: Results

[0101] weight

[0102] During the study, there were no significant changes in body weight in any of the experimental groups. The body weight of mice in the ICV ERT groups was not significantly different from that in the control groups (WT and untreated IDSKO mice). No abnormal clinical signs were observed in any of the ERT groups during the study.

[0103] A single ICV injection of IDS-β reduced GAG in the brain tissue of IDS KO mice.

[0104] Compared with WT mice, KO mice in the load-injection group had significantly higher levels of total GAG in their brain tissue. Figure 1 Seven days after administration, total GAG in the brain tissue of all IDS-β treatment groups was significantly reduced compared to that in disease control mice. Figure 1 However, GAG re-accumulation was observed in the 3 μg and 10 μg injection groups 14 days post-injection. At 28 days post-injection, a significant GAG reduction remained in the 30 μg injection group, although total GAG levels slightly increased again compared to days 7 and 14. Figure 1 ).

[0105] A single ICV injection of IDS-β reduced CSF and HS in brain tissue of IDS KO mice.

[0106] Compared with WT control mice, HS levels were significantly increased in CSF and brain tissue of IDS KO mice. Figure 2 Seven days after ICV injection, HS levels in CSF and brain tissue were significantly reduced in all three IDS-β treatment groups. This significant reduction in HS levels in brain tissue was maintained throughout the entire 28 days. Figure 2 At 14 and 28 days post-ICV injection, HS levels in CSF remained decreased. However, statistical significance was only observed in the 30 μg IDS-β treatment group at 28 days post-ICV injection. Figure 2 ).

[0107] The HS content in CSF is positively correlated with HS and GAG in brain tissue.

[0108] A significant positive correlation was found between the HS content in CSF and the HS concentration in brain tissue of mouse samples (r=0.785, P<0.0001). Figure 3 A). Furthermore, the HS content in CSF was also significantly positively correlated with the GAG ​​concentration in brain tissue (r=0.703, P<0.0001). Figure 3 B).

[0109] A single ICV injection of IDS-β reduced GAG in the somatic tissues of IDS-KO mice.

[0110] We measured total GAG concentrations following a single ICV injection in both brain and other somatic tissues (heart, lung, liver, spleen, and kidney). GAG accumulation was observed in all analyzed tissues from IDS KO mice with load-bearing injections, compared to WT mice. Figure 4 ). 28 days after ICV injection, administration of 30 μg IDS-β maintained a significant reduction in total GAG in all examined tissues. Figure 4 ). Figure 6-10 The figure shows the GAG ​​concentrations in body tissues 7 and 14 days after ICV injection.

[0111] 1-4: Discussion

[0112] MPS II is the most common type of MPS in Asia, and approximately 70% of MPS II patients have the severe form [18,19]. Therefore, correcting the neuropathological condition is one of the most important and challenging issues in the treatment of MPS II patients. Intrathecal or intraventricular injection of recombinases has been proposed as a strategy for delivering therapeutic drugs to the brain. In this study, we performed single ICV injections of three different doses of IDS-β in 6-week-old IDS KO mice to evaluate dose-response relationships and the time course of pharmacological action.

[0113] Total GAG was significantly reduced in brain tissue in all IDS-β treatment groups, and the significant GAG reduction in the 30 μg injection group was maintained for 28 days. Figure 1 A significant decrease in CSF HS concentration was consistently observed in the 30 μg treatment group 28 days after ICV injection. Figure 2 Therefore, we propose that intraventricular injection of 30 μg IDS-β every 4 weeks effectively reduces and maintains accumulated brain GAGs in MPSII mice. Furthermore, these results can serve as a basis for determining the dosage and injection frequency of ICV-injected recombinant enzymes in clinical applications, although the differences in brain size and metabolic rate between mice and humans should be considered. However, to further elucidate the efficacy of ICV enzyme administration in improving CNS pathology in MPSII, we need to expand the study with repeated injections and conduct functional assessments including behavioral tests and histological analyses of the brain.

[0114] In various types of MPS, CNS involvement is present in the severe form of MPS I (Hurler disease), and the severe forms of MPS II, MPS III, and MPS VII. In contrast, patients with MPS IV, MPS VI, attenuated type MPS I (Scheie syndrome), and attenuated type MPS II do not have cognitive impairment [2]. HS is one of the major accumulated GAGs in MPS I, II, III, and VII

[10] . Some reports have suggested that accumulated HS in brain tissue is a cause of the neurological manifestations of MPS [9-12]. Furthermore, HS concentration has been shown to be a more sensitive and specific biomarker in the brain tissue of MPS II mice [13, 20]. However, direct measurement of the amount of HS in brain tissue is not possible in a clinical setting. Therefore, we analyzed HS concentration in CSF and attempted to find a correlation between CSF and HS levels in brain tissue. We showed that, compared with WT mice, HS content was significantly increased in both CSF and brain tissue of IDS KO mice, and decreased in all IDS-β treatment groups. Figure 2 Furthermore, this is the first study to demonstrate a significant correlation between HS concentration in CSF and HS and GAG in brain tissue. Figure 3 Therefore, we propose that HS content in CSF could be one of the potential biomarkers for hypothetical brain tissue HS levels or GAG. However, to demonstrate that HS content in CSF truly represents CNS pathology, further studies including brain tissue pathology are needed, as the CNS pathology of MPS II is caused not only by the amount of GAG accumulation but also by secondary substrate accumulation, inflammation, and degenerative changes in the CNS [12, 21, 22]. If the correlation is demonstrated, CSF HS content could be a usable parameter for assessing CNS pathology in potential future clinical trials in MPS II patients with CNS involvement.

[0115] Furthermore, we show that IDS-β administration via ICV significantly reduces GAG accumulation in somatic tissues (liver, spleen, kidney, heart, and lungs) and brain tissue in a dose-dependent manner, although the effect varies across tissues. Figure 4 and Figures 6 to 10The degree of GAG reduction was observed to be dose-dependent, and we noted that administration of 30 μg IDS-β significantly reduced and maintained accumulated GAG in somatic tissues for up to 28 days, as was the case in brain tissue. Overall, these data demonstrate the physiological transport of therapeutic proteins from CSF to systemic organs following ICV administration, suggesting a clinically feasible route for delivering therapeutic enzymes to both the brain and systemic organs. Furthermore, CSF can act as an intermediate reservoir for the injected enzymes, from which some amounts gradually transfer to the systemic circulation after ICV injection. Although the mechanism of enzyme delivery from CSF to systemic circulation is not fully understood, it has been proposed that CSF containing iduronate-2-sulfatase may be connected to the systemic venous circulation via the subarachnoid space [6,23-25].

[0116] In summary, a single ICV injection of IDS-β was well tolerated and resulted in significant reductions in HS and GAG in the brain tissue of IDS-β KO mice, as well as significant reductions in GAG in somatic tissues. Furthermore, the effect was maintained for 28 days after ICV injection, especially at a dose of 30 μg. Moreover, since CSF HS concentration is positively correlated with brain tissue HS and GAG, CSF HS concentration could be a usable biomarker for indicating neuropathological conditions.

[0117] 1-5: Sequence List

[0118] SEQ ID NO:1

[0119] Length: 525

[0120] Type: PRT

[0121]

[0122] SEQ ID NO:2

[0123] Length: 525

[0124] Type: PRT

[0125]

[0126]

[0127] (The amino acid "G*" at position 59 of SEQ ID NO: 2 represents formyl-glycine.)

[0128] 1-6: References

[0129]

[0130]

[0131]

Claims

1. A method for treating Hunter syndrome, comprising the step of administering (ICV administration) a therapeutically effective dose of an ICV preparation to the ventricle of a subject requiring treatment, said ICV preparation comprising idosasulfatase-β (IDS-β) protein at a concentration of about 0.1 mg / ml to about 60 mg / ml, sodium chloride at a concentration of about 150 mM, polysorbate 20 at a concentration of about 0.05 mg / ml, and a pH of about 6.

2. The method of claim 1, wherein the ICV formulation comprises idosasulfatase-β (IDS-β) protein at a concentration of about 15 mg / ml, sodium chloride at a concentration of about 150 mM, polysorbate 20 at a concentration of about 0.05 mg / ml, and a pH of about 6.

3. The method of claim 1, wherein the therapeutically effective dose is about 1 mg to about 30 mg.

4. The method of claim 1, wherein the therapeutically effective dose is about 10 mg.

5. The method of claim 1, wherein the ICV application is performed every three weeks.

6. The method of claim 1, wherein the ICV application is performed monthly.

7. The method of claim 1, wherein the ICV application is performed via an intraventricular catheter system comprising a reservoir and a catheter connected to the reservoir.

8. The method of claim 7, further comprising the step of: The intraventricular catheter system is surgically implanted, wherein the reservoir is placed between the scalp and brain of the patient requiring treatment, and the end of the catheter is placed within the ventricle of the patient, such that the internal space of the reservoir is connected to the internal space of the ventricle through the internal space of the catheter, allowing cerebrospinal fluid to flow from the ventricle into the reservoir to fill it. 0.1 to 5 ml of cerebrospinal fluid was withdrawn from the reservoir at a flow rate of 0.1 to 60 ml / min; Inject 0.1 to 5 ml of the ICV formulation into the reservoir at a flow rate of 0.1 to 60 ml / min; as well as The ICV formulation is allowed to flow from the reservoir through the catheter into the ventricle.

9. The method of claim 1, wherein the ICV administration is combined with at least one other form of enzyme replacement therapy for Hunter syndrome.

10. The method of claim 9, wherein the additional form of enzyme replacement therapy for Hunter syndrome is selected from intravenous and subcutaneous administration.

11. The method of claim 10, wherein the ICV administration is performed monthly and the intravenous administration is performed weekly.

12. The method of claim 10, wherein the ICV administration is performed every three weeks and the intravenous administration is performed weekly.

13. The method of claim 10, wherein the ICV application is performed monthly and the subcutaneous application is performed weekly.

14. The method of claim 10, wherein the ICV administration is performed every three weeks and the subcutaneous administration is performed weekly.

15. The method of claim 10, wherein the ICV administration is performed monthly and the subcutaneous administration is performed twice weekly.

16. The method of claim 10, wherein the ICV administration is performed every three weeks and the subcutaneous administration is performed twice a week.

17. The method of claim 10, wherein the ICV administration is performed monthly, and the intravenous administration and the subcutaneous administration are alternated at weekly intervals.

18. The method of claim 10, wherein the ICV administration is performed every three weeks, and the intravenous administration and the subcutaneous administration are alternated at a one-week interval.

19. A formulation for intraventricular administration for the treatment of Hunter syndrome, comprising idosasulfatase-β (IDS-β) protein at a concentration of about 0.1 mg / ml to about 60 mg / ml, sodium chloride at a concentration of about 150 mM, polysorbate 20 at a concentration of about 0.05 mg / ml, and a pH of about 6.

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