Pharmaceutical compositions and methods for the prevention and treatment of lysosomal enzyme deficiency in subjects with mucopolysaccharidosis type II

Abstract

The present invention relates to the field of biotechnology, and more particularly to pharmaceutical compositions and methods for the prevention and treatment of lysosomal enzyme deficiency in a subject, which may be used in medicine. The present invention relates to an HIR-Fab-IDS compound that may be used to prevent or treat lysosomal enzyme deficiency in a subject suffering from lysosomal storage disease in the form of mucopolysaccharidosis type II (MPS II), wherein at least one dose of the HIR-Fab-IDS compound is administered to the subject in an amount of 1 to 12 mg / kg, and to a pharmaceutical composition for use in the prevention or treatment of lysosomal enzyme deficiency in a subject with the lysosomal storage disease MPS II, the composition containing the HIR-Fab-IDS compound, and a method for preventing or treating lysosomal enzyme deficiency in a subject with the lysosomal storage disease MPS II, comprising administering to the patient at least one dose of the HIR-Fab-IDS compound.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, and in particular to pharmaceutical compositions and methods for the prevention and treatment of lysosomal enzyme deficiency in a subject, said compositions and methods being usable in medicine.The present invention relates to an HIR-Fab-IDS compound for use in the prevention or treatment of lysosomal enzyme deficiency in a subject suffering from a lysosomal storage disease that is a form of mucopolysaccharidosis type II, wherein at least one dose of the HIR-Fab-IDS compound is administered to the subject in an amount of 1 to 12 mg / kg, and to a pharmaceutical composition for use in the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease that is mucopolysaccharidosis type II, said composition comprising an HIR-Fab-IDS compound, and a method for the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease that is mucopolysaccharidosis type II, comprising administering to the patient at least one dose of the HIR-Fab-IDS compound. [Background technology]

[0002] The use of enzymes in therapy has long been known from the state of the art. Modern medicine increasingly uses therapeutic enzymes in various fields of medicine due to their high activity and specificity. Currently, the following fields of enzyme therapy have emerged (Kazanskaya NF et al., 1984): 1) elimination of enzyme deficiencies to compensate for congenital or acquired dysfunctions; 2) removal of ineffective and denatured structures, cell and tissue fragments; 3) thrombolysis; 4) complex therapy of malignant neoplasms; 5) detoxification of the body.

[0003] The use of therapeutic enzymes to eliminate enzyme deficiencies to compensate for congenital or acquired deficiencies has long been practiced. Treatment of congenital enzyme deficiencies, of which over 150 have been described, is a significant challenge in replacement therapy. These genetic diseases, such as glycogen storage diseases, lipidoses, mucopolysaccharidoses, and other lysosomal storage diseases, are primarily treated by intravenous administration of recombinant analogs of the corresponding native enzymes.

[0004] Lysosomal storage diseases are a group of rare (orphan) inherited metabolic disorders caused by the absence or deficiency of lysosomal enzymes involved in the degradation of complex molecules.

[0005] Currently (Novikov PV, 2014), the following groups of lysosomal storage diseases are recognized: 1) mucopolysaccharidoses; 2) lipidoses (sphingolipidoses: GM1- and GM2-gangliosidosis, Gaucher disease, galactosialidosis, Farber granulomatosis, leukodystrophy, Niemann-Pick disease types A and B, etc.); 3) mucolipidoses; 4) glycoproteinoses (fucosidosis, sialidosis, mannosidosis, type II glycogen storage diseases: Pompe disease, Danon disease, etc.); 5) neuronal ceroid lipofuscinoses; 6) other storage diseases (Niemann-Pick disease type C, Wolman disease, cholesterol storage diseases, cystinosis, Sal's disease, pyknodysostosis, etc.).

[0006] Mucopolysaccharidoses (MPS) are a group of nine (I-IX) or 14 (including intermediate types) metabolic disorders caused by over 40 genetic disorders that result in the absence or dysfunction of many lysosomal enzymes involved in the hydrolytic degradation of glycosaminoglycans (mucopolysaccharides), oligosaccharides involved in the formation of bone, cartilage, ligaments, cornea, skin, connective tissue, and synovial fluid (Burrow TA et al, 2013).

[0007] Patients with mucopolysaccharidoses have a deficiency or absence of at least one of 11 glycosaminoglycan catabolic enzymes, which over time leads to the gradual accumulation of these carbohydrates in cells, including connective tissue, and body fluids. The resulting persistent accumulation of mucopolysaccharides leads to cell damage, tissue, and organ dysfunction, most frequently manifesting as hypertension-hydrocephalus syndrome, hepatosplenomegaly, cardiovascular insufficiency, bone and joint complications, and central nervous system dysfunction (CIS) leading to severe cognitive impairment and dementia (Table 1) (Burrow TA et al., 2013).

[0008] Table 1. Characteristics of various MPS syndromes [Table 1] TIFF2026506966000002.tif129165

[0009] Mucopolysaccharidosis type I is caused by a deficiency of the lysosomal enzyme α-L-iduronidase. This deficiency results in the accumulation of mucopolysaccharides, particularly dermatan sulfate, in tissues and organs. Excessive dermatan sulfate accumulation leads to the gradual development of numerous morphological abnormalities in tissues and organs. Mucopolysaccharidosis type I is characterized by autosomal recessive inheritance.

[0010] To date, two effective treatments for type 1 MPS have been developed: hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT).

[0011] HSCT is used exclusively to treat the severe form of MPS type 1, Hurler syndrome. HSCT corrects the deficiency of the enzyme alpha-L-iduronidase, subsequently leading to significant improvement in the patient's condition, although the severe complications of the disease may not be fully reversible. HSCT must be performed as early as possible, before the onset of severe neurological impairment. Despite the improvement in the patient's condition, HSCT is associated with a high risk of serious post-transplant complications and is a complex, multi-step, and costly procedure.

[0012] ERT is safe, well tolerated by patients, does not cause serious side effects, and leads to the degradation of non-hydrolyzable substrates. A rare possible reaction to drug administration is the formation of antibodies against the administered protein, but these are not consistent and are generally quickly stopped by standard medications. The principle of ERT is based on restoring sufficient enzyme activity levels to hydrolyze accumulated substrates and prevent further accumulation.

[0013] Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is the only mucopolysaccharidosis with X-linked recessive inheritance, unlike all other types of mucopolysaccharidoses, which are inherited in an autosomal recessive manner. MPS II is a progressive lysosomal storage pathology with heterogeneous clinical manifestations caused by deficiency of the enzyme iduronate-2-sulfatase (idursulfase). This enzyme is normally involved in the degradation of mucopolysaccharides, dermatan sulfate and heparan sulfate, by hydrolytic cleavage of O-linked sulfate groups. Therefore, in MPS II, the primary pathogenic mechanism is associated with the progressive accumulation of dermatan and heparan sulfate in lysosomes.

[0014] The most common clinical manifestations of MPS II are mental retardation, an enlarged tongue, characteristic facial skeletal abnormalities, alopecia, dental anomalies, restrictive lung disease, hepatosplenomegaly, cardiac valve abnormalities, bone and joint abnormalities, and severe short stature. Progressive neurological impairment associated with hydrocephalus and increased intracranial pressure is also common. Fatal outcomes usually occur in the teens or twenties, most frequently due to respiratory and / or cardiac failure. It is important to emphasize that the disease progresses with neurological involvement in the pathological process, including intellectual decline.

[0015] Today, there are two basic approaches to treating MPS II: ERT and supportive care (including the use of hepatoprotective, cardiovascular, and anti-inflammatory drugs, vitamins, and drugs that improve antioxidant protection). Unlike mucopolysaccharidosis type I, HSCT for the treatment of MPS II is not effective.

[0016] Elaprase® (INN: idursulfase) (Shire, USA) is a medical product equivalent to recombinant human iduronate-2-sulfatase. This enzyme is involved in the hydrolysis of the C2-sulfate ester bond of iduronic acid residues, which are part of the glycosaminoglycans (mucopolysaccharides) dermatan sulfate and heparan sulfate (Burrow TA et al., 2013). The usual dosage regimen for Elaprase® is 0.5 mg / kg body weight once a week; administration is by intravenous infusion over 3 hours (the infusion time may be gradually reduced to 1 hour).

[0017] Idursulfase contains two disulfide bonds and eight N-linked glycosylation sites occupied by complex, high-mannose oligosaccharides. The presence of M6P residues in the oligosaccharide chains enables the recombinant enzyme to bind to M6P receptors on the surface of target cells, leading to the internalization of the enzyme into the cells and lysosomes, ensuring the degradation of lysosomal mucopolysaccharides (Muenzer J., et al., Genet. Med. 2006 Aug; 8(8): 465-473).

[0018] ERT increases the lifespan of Hunter syndrome patients several-fold. However, Elaprase® also does not penetrate the blood-brain barrier, and as a result, patients receiving Elaprase® ERT die at age 20 from neurodegeneration, while patients receiving Elaprase® in their teens typically experience significant learning difficulties and require assistance with even daily activities (da Silva EM et al., 2016; Wraith JE et al., 2008).

[0019] Thus, a common drawback of all current agents used for ERT in mucopolysaccharidoses types I and II is their inability to penetrate the blood-brain barrier and reach the central nervous system, which limits the effectiveness of these agents in patients with nervous system damage associated with mucopolysaccharidoses, including mucopolysaccharidoses types I and II.

[0020] In many lysosomal storage diseases, improved internalization of enzymes into cells of specific peripheral tissues (e.g., diaphragm muscle in Pompe disease, liver and spleen in Hunter disease, and kidneys in Fabry disease) is required (Hawkes C. et al., 2004).

[0021] Therefore, it is worthwhile to develop therapeutic compounds, pharmaceutical compositions and preventative or therapeutic methods that can be used to treat lysosomal storage diseases such as MPS type II, that exhibit high activity and improved ability to be transported to the lysosomes of tissue cells of various organs, including the lysosomes of neural tissue cells, while retaining the functional properties of the corresponding therapeutic enzymes, and that allow significant improvement in the quality and duration of life of MPS type II patients. Summary of the Invention

[0022] Advantages of the invention The above-mentioned problems are successfully solved by the present invention, which relates to a compound, a pharmaceutical composition intended for the treatment of lysosomal storage diseases, containing a therapeutic enzyme and a transport element linked to each other directly or via a linker, wherein the transport element is a Fab fragment of immunoglobulin IgG specific for an epitope in the insulin receptor. The present invention is based on the unexpected discovery that a transport element, which is a Fab fragment of immunoglobulin IgG specific for an epitope in the insulin receptor, linked to a therapeutic enzyme directly or via a linker unexpectedly leads to an improved ability of the compound to transport the enzyme to lysosomes in various organs and tissues, including lysosomes in neural tissue cells, while maintaining high levels of enzymatic activity. This unexpected effect achieves an advance in the field of enzyme replacement therapy for lysosomal storage diseases, such as mucopolysaccharidosis type II, and achieves increased survival and improved quality of life for subjects suffering from lysosomal storage diseases, mucopolysaccharidosis type II, and in need of therapy.

[0023] Furthermore, it has been discovered that compounds containing a transport element that is a Fab fragment of immunoglobulin IgG specific for an epitope in the insulin receptor (human insulin receptor, or HIR) linked directly or through a linker to a therapeutic enzyme unexpectedly have increased enzymatic activity compared to therapeutic enzymes that do not contain a transport element; and administration of compounds containing a Fab fragment of immunoglobulin IgG specific for an epitope in the insulin receptor linked directly or through a linker to a therapeutic enzyme provides a greater degree of recruitment of therapeutic enzyme activity in the human brain compared to compounds containing Mab fragments.

[0024] The HIR-Fab-IDS compound was found to penetrate into the brain tissue of patients twice as well as the HIR-Mab-IDS compound.

[0025] The HIR-Fab-IDS compound was found to restore 98% of IDS enzyme function in patient brain tissue.

[0026] Doses of the compounds are indicated that are safe and effective for subjects suffering from the lysosomal storage disease, mucopolysaccharidosis type II, and in need of therapy.

[0027] More effective pharmaceutical compositions and / or administration regimens have been developed for the prevention or treatment of lysosomal enzyme deficiencies in subjects with mucopolysaccharidosis type II.

[0028] Taking into account dose escalation, more effective dosing regimens have been developed for the prevention or treatment of lysosomal enzyme deficiency in subjects with human mucopolysaccharidosis type II with a neurological component. Taking into account dose escalation, more effective dosing regimens and / or pharmaceutical compositions have been developed for the prevention or treatment of lysosomal enzyme deficiency in subjects with human mucopolysaccharidosis type II with a neurological component, which is associated with a neuropathic form in humans.

[0029] overview This Summary provides a brief description of the invention for the purpose of briefly indicating the subject matter and nature of the invention. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

[0030] A first object of the present invention is an HIR-Fab-IDS compound represented by a first amino acid sequence, SEQ ID NO: 2, and a second amino acid sequence, SEQ ID NO: 4, for the prevention or treatment of lysosomal enzyme deficiency in subjects with lysosomal storage disease, mucopolysaccharidosis type II.

[0031] Also an object of the present invention is an HIR-Fab-IDS compound represented by a first amino acid sequence, SEQ ID NO: 2, and a second amino acid sequence, SEQ ID NO: 4, for use in preventing or treating lysosomal enzyme deficiency in a subject having a lysosomal storage disease that is mucopolysaccharidosis type II, wherein the subject is administered at least one dose of 1 to 12 mg / kg of the HIR-Fab-IDS compound.

[0032] Another object of the present invention is an HIR-Fab-IDS compound represented by a first amino acid sequence, SEQ ID NO: 2, and a second amino acid sequence, SEQ ID NO: 4, for use in preventing or treating lysosomal enzyme deficiency in a subject with a lysosomal storage disease, wherein the subject is administered at least one dose of the HIR-Fab-IDS compound, and the dose is selected from the group consisting of 3 mg / kg, 6 mg / kg, and 12 mg / kg of the HIR-Fab-IDS compound.

[0033] In a particular embodiment, the subject of the present invention is an HIR-Fab-IDS compound used for the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease, wherein the lysosomal storage disease is mucopolysaccharidosis type II or mucopolysaccharidosis type II with a neurological component associated with the neuropathic form.

[0034] In a particular embodiment, the subject of the present invention is an HIR-Fab-IDS compound used for the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease that is mucopolysaccharidosis type II, wherein the subject is a human.

[0035] Another object of the present invention is a pharmaceutical composition for use in the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease that is mucopolysaccharidosis type II, comprising an HIR-Fab-IDS compound represented by a first amino acid sequence, SEQ ID NO: 2, and a second amino acid sequence, SEQ ID NO: 4, wherein the HIR-Fab-IDS compound is administered to the subject in at least one dose selected from the group consisting of 3 mg / kg, 6 mg / kg, and 12 mg / kg of the HIR-Fab-IDS compound.

[0036] In a specific embodiment, the present invention relates to a pharmaceutical composition for use in the prevention or treatment of lysosomal enzyme deficiency in a subject with a lysosomal storage disease that is mucopolysaccharidosis type II, comprising an HIR-Fab-IDS compound and further comprising sodium chloride, sodium dihydrogen phosphate dihydrate, sodium hydroxide, and polysorbate.

[0037] In a specific embodiment, the present invention relates to a pharmaceutical composition for use in preventing or treating lysosomal enzyme deficiency in a subject with a lysosomal storage disease that is mucopolysaccharidosis type II, comprising an HIR-Fab-IDS compound, further comprising 5.3 mg of the HIR-Fab-IDS compound, sodium chloride in an amount of 8.0 mg, sodium dihydrogen phosphate dihydrate in an amount of 3.12 mg, sodium hydroxide to adjust the pH to pH 6.0, polysorbate 20 in an amount of 0.2 mg, and water for injection up to 1.0 mL.

[0038] In a specific embodiment, the present invention relates to a pharmaceutical composition for use in the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease that is mucopolysaccharidosis type II, the composition comprising an HIR-Fab-IDS compound, wherein the lysosomal storage disease is mucopolysaccharidosis type II with a neurological component associated with neuropathy.

[0039] In a specific embodiment, the present invention relates to a pharmaceutical composition for use in the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease that is mucopolysaccharidosis type II, the composition comprising an HIR-Fab-IDS compound, and the subject being a human.

[0040] Another object of the present invention is a method for preventing or treating lysosomal enzyme deficiency in a subject having a lysosomal storage disease that is mucopolysaccharidosis type II, comprising administering to the patient at least one dose of an HIR-Fab-IDS compound represented by a first amino acid sequence, SEQ ID NO: 2, and a second amino acid sequence, SEQ ID NO: 4, wherein the dose is selected from the group consisting of 3 mg / kg, 6 mg / kg, and 12 mg / kg of the HIR-Fab-IDS compound.

[0041] In a specific embodiment, the present invention is a method for preventing or treating lysosomal enzyme deficiency in a subject having a lysosomal storage disease that is mucopolysaccharidosis type II, comprising administering to the patient at least one dose of an HIR-Fab-IDS compound represented by a first amino acid sequence, SEQ ID NO: 2, and a second amino acid sequence, SEQ ID NO: 4, wherein the HIR-Fab-IDS compound in a pharmaceutical composition is administered intravenously for three hours once a week.

[0042] In a specific embodiment, the present invention is a method for preventing or treating lysosomal enzyme deficiency in a subject having a lysosomal storage disease that is mucopolysaccharidosis type II, comprising administering to the patient at least one dose of an HIR-Fab-IDS compound represented by a first amino acid sequence, SEQ ID NO: 2, and a second amino acid sequence, SEQ ID NO: 4, wherein the lysosomal storage disease is mucopolysaccharidosis type II with a neurological component associated with neuropathy.

[0043] In a specific embodiment, a method for preventing or treating lysosomal enzyme deficiency in a subject having a lysosomal storage disease that is mucopolysaccharidosis type II, comprising administering to the patient at least one dose of an HIR-Fab-IDS compound represented by a first amino acid sequence of SEQ ID NO: 2 and a second amino acid sequence of SEQ ID NO: 4, wherein the subject is a human.

[0044] In other embodiments of the invention, the approaches of the present invention may also be used to treat other lysosomal storage diseases, particularly other types of mucopolysaccharidosis, such as mucopolysaccharidosis type III (Sanfilippo syndrome), including mucopolysaccharidosis types IIIA, IIIB, IIIS, and PGO; mucopolysaccharidosis type IV (Morquio syndrome), including mucopolysaccharidosis types IVA and IVB; mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome), mucopolysaccharidosis type VII (Sly syndrome), and mucopolysaccharidosis type IX. In these cases, instead of α-L-iduronidase or iduronate-2-sulfatase, another therapeutic enzyme is used to address the deficiency encountered in patients with the corresponding type of mucopolysaccharidosis: heparan sulfamidase in mucopolysaccharidosis type IIIA, N-acetylglucosaminidase in mucopolysaccharidosis type IIIB, heparan-α-glucosaminide-N-acetyltransferase in mucopolysaccharidosis type IIIC, N-acetylglucosamine-6-sulfatase in mucopolysaccharidosis type PGO, galactose-6-sulfate sulfatase in mucopolysaccharidosis type IVA, β-galactosidase in mucopolysaccharidosis type IVB, N-acetylgalactosamine-4-sulfatase in mucopolysaccharidosis type VI, β-glucuronidase in mucopolysaccharidosis type VH, and hyaluronidase in mucopolysaccharidosis type IX. The amino acid sequences of these therapeutic enzymes can be obtained using any of a number of computer programs known in the art, such as BLAST or FASTA, both of which allow offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58-7-60, the National Center for Biotechnology Information website, and the National Institutes of Health website).

[0045] In another embodiment, the approach of the present invention may also be used to treat lysosomal storage diseases other than mucopolysaccharidoses by substituting a different lysosomal enzyme, the deficiency of which is experienced by patients with the corresponding type of lysosomal storage disease, in place of α-L-iduronidase or iduronate-2-sulfatase. In particular, non-limiting embodiments of the present invention, the approach of the present invention may be used to treat disorders of amino acid metabolism, such as cystinosis; disorders of carbohydrate metabolism, such as glycogen storage diseases, particularly Pompe disease; disorders of sphingolipid metabolism and other lipid storage diseases, particularly GM2 gangliosidosis, including Sandhoff disease and Tau-Sachs disease; other gangliosidoses, particularly GMi gangliosidosis and mucolipidosis IV; and other sphingolipidoses, particularly Fabry disease, Gaucher disease, Krabbe disease, Niemann-Pick disease, Farber syndrome, and metachromatic leukodystrophy. leukodystrophy) and multiple sulfatase deficiency; neurolipofuscinosis, particularly Batten disease, Bielschowsky-Jansky disease, Kuchs disease, and Spielmeyer-Voigt disease; other lipid storage diseases, particularly Wolman disease; and glycoprotein disorders, including mucolipidosis II (Eisel's disease), mucolipidosis III (Hurler-pseudolipodystrophy), and glycoprotein degradation deficiencies, including aspartylglucosaminuria, fucosidosis, mannosidosis, and sialidosis.

[0046] In these cases, instead of iduronate-2-sulfatase, the amino acid sequence of the corresponding enzyme, the deficiency of which occurs in patients with lysosomal storage diseases, is attached to the transport element either directly or through a linker.

[0047] The most preferred embodiments of the present invention are described below, however, these preferred embodiments are provided only for the purpose of illustrating the present invention, and not for the purpose of limiting the scope of the claims.

[0048] The invention is illustrated by the following figures: [Brief explanation of the drawings]

[0049] [Figure 1] A representative autoradiogram from a male cynomolgus macaque recorded 2 hours after a single intravenous injection of [ 125 I]-HIR-Fab-IDS at a nominal dose level of 0.0020 mg / kg body weight is shown. The numbers represent the following: 1. Cerebral cortex 2. Cerebral medulla 3. Eye 4. Hypothalamus 5. Pons 6. Cerebellum 7. Medulla oblongata 8. Spinal cord 9. Myocardium 10. Blood 11. Lung 12. Liver 13. Stomach 14. Kidney 15. Small intestine 16. Large intestine 17. Muscle 18. Testis 19. Bone marrow [Figure 2] Representative autoradiograms from a male cynomolgus monkey recorded 2 hours after a single intravenous injection of [I]-HIR-Mab-IDS at a nominal dose level of 0.0015 mg / kg body weight are shown. Numbers indicate: 1. Cerebral cortex, 2. Cerebral medulla, 3. Eye, 4. Hypothalamus, 5. Pons, 6. Cerebellum, 7. Medulla oblongata, 8. Spinal cord, 9. Myocardium, 10. Blood, 11. Lung, 12. Liver, 13. Stomach, 20. Adrenal, 14. Kidney, 15. Small intestine, 21. Bladder, 19. Bone marrow. [Figure 3] Representative autoradiograms from male cynomolgus monkeys recorded 2 hours after a single intravenous injection of [I]-IDS (control) at a nominal dose level of 0.0010 mg / kg body weight are shown. Numbers indicate: 1. cerebral cortex, 2. cerebral medulla, 3. eye, 4. hypothalamus, 5. pons, 6. cerebellum, 7. medulla oblongata, 8. spinal cord, 9. myocardium, 10. blood, 11. lung, 12. liver, 13. stomach, 14. kidney, 15. small intestine, 16. large intestine, 17. muscle, 18. testis, 19. bone marrow. [Figure 4]The levels of GAGs in the urine of the animals under study are shown. Individual values ​​are indicated by Latin letters in Figure 4: A. Wild-type mice. B. Knockout mice, saline solution. C. Knockout mice, 0.3 mg / kg. D. Knockout mice, 1.0 mg / kg. E. Knockout mice, 3.0 mg / kg. [Figure 5] Liver weights in study animals are shown: I - wild-type group; II - control knockout animals; III - knockout animals, 0.3 mg / kg; IV - knockout animals, 1.0 mg / kg; V - knockout animals, 3.0 mg / kg. Individual values. [Figure 6] The levels of iduronate-2-sulfatase activity in the plasma of experimental animals are shown. I - wild-type group; II - control knockout animals; III - knockout animals, 0.3 mg / kg; IV - knockout animals, 1.0 mg / kg; V - knockout animals, 3.0 mg / kg. Individual values. [Figure 7] 1 shows the serum concentration of the HIR-Fab-IDS compound in patient 1001 determined during the dose escalation step. The NICO of the method is 50 ng / mL. [Figure 8] 1 shows the kinetics of heparan sulfate concentration in CSF when different doses of the drug are used. DETAILED DESCRIPTION OF THE INVENTION

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular genetics, nucleic acid chemistry, protein chemistry, biochemistry, organic chemistry, immunology, microbiology, genetics, etc.).

[0051] In the context of this specification, the term "compound" is understood in the broadest sense as at least one molecule that is a conjugate, fusion protein, fusion antibody, hybrid protein, fusion protein, protein construct, protein complex, etc.

[0052] A compound intended for the treatment of lysosomal storage diseases, comprising a therapeutic enzyme and a transport element linked to each other directly or through a linker, wherein the transport element is a Fab fragment of immunoglobulin IgG1 consisting of a light chain of immunoglobulin IgG1 directed against the insulin receptor (SEQ ID NO: 2) and a heavy chain with idursulfatase (SEQ ID NO: 4).

[0053] As used herein, the term "containing" means a compound that includes the listed elements, without excluding others.

[0054] As used herein, the terms "therapeutic enzyme" and "enzyme" are synonymous and refer to an enzyme for treating a disease caused by the absence, deficiency, dysfunction, etc. of that enzyme, and a subject suffering from the disease can be treated by ERT, enzyme administration, etc. In particular, the enzyme may be, but is not limited to, an enzyme for treating a disease caused by the absence, deficiency, dysfunction, etc. of a lysosomal enzyme.

[0055] Therapeutic enzymes that are part of the compounds herein include, but are not limited to, β-glucosidase, β-galactosidase, galactose-6-sulfatase, acid ceramidase, acid sphingomyelinase, galactocerebrosidase, arylsulfatase A, β-hexosaminidase A, β-hexosaminidase B, heparin-N-sulfatase, α-D-mannosidase, β-glucuronidase, N-acetylgalactosamine-6-sulfatase, lysosomal acid lipase, α-N-acetyl-D-glucosaminidase (NAGLU), glucocerebrosidase, butyrylcholinesterase, chitinase, glutamic acid decarboxylase, lipase, uricase, platelet-activating factor acetylhydrolase, neutral endopeptidase, myeloperoxidase, acetyl-CoA-glucosaminide-P-acetylase ... and any enzyme having a therapeutic effect on lysosomal storage diseases, including alpha-N-acetylgalactosaminidase, N-acetylglucosamine-6-sulfatase, galactosamine-6-sulfatase (GALN), hyaluronidase, alpha-fucosidase, beta-mannosidase, alpha-neuraminidase (sialidase), N-acetyl-glucosamine-1-phosphotransferase, mucolipin-1, alpha-N-acetyl-galactosaminidase, N-aspartyl-beta-glucosaminidase, LAMP-2 (lysosome-associated membrane protein 2), cystinosin, sialin, ceramidase, acid beta-glucosidase, galactosylceramidase, NPC1 (type C1 protein Niemann-Pick disease), cathepsin A, SUMF-1 (sulfatase modifying factor-1), lysosomal acid lipase (LIPA), and tripeptidyl peptidase 1.

[0056] In particular, therapeutic enzymes include enzymes such as agalsidase, imiglucerase, galsulfase, iduronate-2-sulfatase and α-L-iduronidase. Most preferably, the therapeutic enzyme is iduronate-2-sulfatase or α-L-iduronidase.

[0057] However, the description may also encompass, without limitation, any therapeutic enzyme, regardless of the type or origin of the enzyme.

[0058] As used herein, the terms "iduronate-2-sulfatase," as well as "idursulfase," "IDS," "IDS," and "I2S," refer to a recombinant analog of the lysosomal enzyme iduronate-2-sulfatase, an enzyme that normally hydrolyzes the O-linked sulfate groups of the mucopolysaccharides dermatan and heparan sulfate. In the present invention, the term "iduronate-2-sulfatase" may be used interchangeably with the term "idursulfase."

[0059] In the present context, the terms "HIR-Fab-IDS," as well as "rIDS-FAB-H1," "rHIR-FAB-IDS," "rIDS-FAB-HIR," and "rHIR-FAB-IDS" are synonymous and refer to a recombinant iduronate-2-sulfatase fusion protein covalently linked to a Fab fragment of a monoclonal antibody against the human insulin receptor. In the present context, "HIR-Fab-IDS" refers to, but is not limited to, iduronate-2-sulfatase with a fragment of a monoclonal antibody against the human insulin receptor. In certain (non-limiting) embodiments of the present invention, the HIR-Fab-IDS variant is represented by a first amino acid sequence selected from SEQ ID NOs: 2, 8, 9, 10, or 11, and a second amino acid sequence selected from SEQ ID NOs: 4, 5, 12, 13, 14, or 15. In certain embodiments of the present invention, the HIR-Fab-IDS is represented by a first amino acid sequence of SEQ ID NO: 2 and a second amino acid sequence of SEQ ID NO: 4.

[0060] In the present context, an "HIR-Mab-IDS" is a recombinant iduronate-2-sulfatase fusion protein covalently linked to a full-length monoclonal antibody against the human insulin receptor. In a specific (non-limiting) embodiment of the present invention, the HIR-Mab-IDS variant is according to US patent document US8834874 B2, 16.09.2014. In a specific embodiment of the present invention, the HIR-Mab-IDS is represented by the amino acid sequence of the product from the AGT-182 project of ArmaGen Technologies Inc.

[0061] As used herein, the terms "α-L-iduronidase," as well as "iduronidase," "laronidase," and "IDUA" refer to enzymes involved in the hydrolysis of glycosaminoglycans such as dermatan sulfate and heparan sulfate. As used herein, the term "α-L-iduronidase" may be used interchangeably with the term "laronidase." Iduronidase deficiency, which occurs in type I mucopolysaccharidosis (a genetically determined deficiency), leads to the gradual accumulation of glycosaminoglycans (heparan sulfate and dermatan sulfate) in the cells and tissues of the body.

[0062] "HIR-Fab-IDUA," as well as "rIDUA-FAB-HI," "rHIR-FAB-IDUA," "rIDUA-FAB-HIR," and "rHIR-FAB-IDS" are hybrid recombinant protein α-L-iduronidase covalently linked to the Fab fragment of a monoclonal antibody against the human insulin receptor. In certain (non-limiting) embodiments of the present invention, the HIR-Fab-IDUA variant is represented by a first amino acid sequence selected from SEQ ID NOs: 2, 8, 9, 10, or 11, and a second amino acid sequence selected from SEQ ID NOs: 6 and 7.

[0063] HIR-Mab-IDUA is a recombinant fusion protein of α-L-iduronidase covalently linked to a full-length monoclonal antibody against the human insulin receptor.

[0064] The therapeutic enzymes that can be included in the compounds of the present invention may be in the form of a natural form, or may be in the form of a fragment consisting of a portion of the enzyme, or an enzyme analogue having a mutation selected from the group consisting of specific amino acid substitutions, additions, deletions, modifications, and combinations thereof, provided that the enzyme has the same enzymatic activity as the natural form of the corresponding therapeutic enzyme. In a specific (non-limiting) embodiment of the present invention, an enzyme fragment having the activity of the natural form of the corresponding enzyme may be used. An enzyme analogue is, without limitation, an enzyme having different glycosylation characteristics and different degrees of glycosylation caused by expression of a known enzyme in a different host, and having different degrees of substitution of specific amino acid residues of the corresponding enzyme compared to the standard sequence, where the degree of substitution is not 100%. In a specific (non-limiting) embodiment of the present invention, analogs of α-L-iduronidase and iduronate-2-sulfatase known from patents US5932211A, 03.08.1999, US6153188A, 28.11.2006 and US6541254 B1, 01.04.2003 may be used. Those skilled in the art will understand that other fragments and analogs of the enzymes α-L-iduronidase and iduronate-2-sulfatase, which are currently known from the state of the art or which will become known in the future, may also be used, which possess the activity of the native forms of the corresponding enzymes.

[0065] Enzymes can be produced by conventional techniques known in the art, such as genetic recombination in animal cells, E. coli, yeast, insects, plants, and living animals, using a variety of expression vectors well known to those skilled in the art. The production methods are not limited to those shown, and other methods for producing enzymes known to those skilled in the art are also included. For non-limiting examples of methods for producing enzymes in cells of various organisms, as well as non-limiting examples of expression vectors, see the guidelines by Sambrook et al., "Molecular Cloning: A Laboratory Manual", CSH Press, Cold Spring Harbor, 1989; "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 2001. In a preferred embodiment, the enzyme is produced in mammalian cells. In a most preferred embodiment, the enzyme is produced in Chinese hamster ovary cells.

[0066] In certain preferred embodiments of the present invention, the enzyme may be a commercially available enzyme. Furthermore, the enzyme may comprise an amino acid sequence having at least 80%, more particularly 90%, even more particularly 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology to the above enzymes or analogs thereof, and may be obtained from a microorganism using recombinant techniques or may be purchased from a commercial source without limitation.

[0067] As used herein, the term "homology" refers to the degree of similarity to the amino acid sequence or nucleotide sequence encoding the amino acid of a wild-type protein, and includes sequences having the above-mentioned degrees of sequence similarity, expressed as percentages, with the amino acid sequence or nucleotide sequence of the present invention. Homology may be determined by comparing two given sequences with the naked eye, or by using bioinformatics algorithms that allow homology analysis by aligning the sequences in question. The homology between two given amino acid sequences may also be expressed as a percentage. Efficient automated algorithms are available in the GAP, BESTFIT, FASTA, and TFASTA software modules of the Wisconsin Genetics software package (Genetics Computer Group, Madison, WI, USA). Automated alignment algorithms in these modules include the Needleman and Wunsch, Pearson and Lipman, and Smith and Waterman sequence alignment algorithms. Other algorithms that can be used for sequence alignment and homology determination are automated in the programs FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL W. Amino acid and nucleotide sequences encoding enzymes and their analogs may be taken from well-known databases such as, but not limited to, NCBI GenBank.

[0068] In some embodiments of the invention, the compound transport element comprises a Fab fragment of immunoglobulin IgG1 consisting of a first amino acid sequence at least 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to SEQ ID NO:2.

[0069] In certain embodiments of the invention, the compound transport element comprises a Fab fragment of immunoglobulin IgG consisting of a first amino acid sequence that is at least 80% identical to SEQ ID NO:2.

[0070] In some embodiments of the invention, a compound represented by a first amino acid sequence that is at least 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to SEQ ID NO:2 and a second amino acid sequence that is at least 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to SEQ ID NO:4 is used to treat or prevent lysosomal enzyme deficiency in a subject with mucopolysaccharidosis type II.

[0071] In certain embodiments of the invention, a compound represented by a first amino acid sequence that is at least 80% identical to SEQ ID NO:2 and a second amino acid sequence that is at least identical to SEQ ID NO:4 is used to treat or prevent lysosomal enzyme deficiency in a subject with mucopolysaccharidosis type II.

[0072] The term "amino acid" refers to a group of carboxy-α-amino acids that are naturally occurring, i.e., can be encoded by nucleic acids directly or as precursors, or are not naturally occurring. Each naturally occurring amino acid is encoded by a nucleic acid consisting of three nucleotides called a codon or base triplet. Each amino acid is encoded by at least one codon.

[0073] The term "amino acid," as used herein, refers to naturally occurring α-amino acids, including the following: alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). Examples of amino acids not found in nature (non-proteinogenic amino acids) include Aad (alpha-aminoadipic acid), Abu (aminobutyric acid), Ach (alpha-aminocyclohexanecarboxylic acid), Acp (alpha-aminocyclopentanecarboxylic acid), Acpc (1-aminocyclopropane-1-carboxylic acid), Aib (alpha-aminoisobutyric acid), Aic (2-aminoindan-2-carboxylic acid;Also known as 2-2-Aic), 1-1-Aic (1-aminoindan-1-carboxylic acid), (2-aminoindan-2-carboxylic acid), allylglycine (allylglycine), alloisoleucine (allo-Ne), Asu (alpha-aminosuberic acid, 2-aminooctanedioic acid), Bip (4-phenyl-phenylalanine carboxylic acid), BnHP ((28,4K)-4-hydroxyproline), Cha (beta-cyclohexyl hydroxybenzoate), Cit (Citrulline), Cyclohexylglycine (Chg), Cyclopentylalanine, Beta-Cyclopropylalanine, Dab (1,4-Diaminobutyric Acid), Dap (1,3-Diaminopropionic Acid), p-(3,3-Diphenylalaninecarboxylic Acid), 3,3-Diphenylalanine, Di-n-Propylglycine (Dpg), 2-Furylalanine, Homocyclohexylalanine (HoCha), Homocyto Homocycloleucine (HoCit), homocycloleucine, homoleucine (HoLeu), homoarginine (HoArg), homoserine (HoSer), hydroxyproline, Lys(Ac), (1) Nal (1-naphthylalanine), (2) Nal (2-naphthylalanine), 4-MeO-Ars (1-amino-4-(4-methoxyphenyl)-cyclohexane-1-carboxylic acid), norleucine (Nle), Nva (norvaline), and hydroxyproline. Examples of suitable amines include, but are not limited to, mannose, 3-Pal (alpha-amino-3-pyridylalanine-carboxylic acid), 4-Pal (alpha-amino-4-pyridylalanine-carboxylic acid), 3,4,5,F3-Phe (3,4,5-trifluorophenylalanine), 2,3,4,5,6,F5-Phe (2,3,4,5,6-pentafluorophenylalanine), Pqa (4-oxo-6-(1-piperazinyl)-3(4H)-quinazoline-acetic acid (CAS 889958-08-1)), pyridylalanine, quinolylalanine, sarcosine (Sar), thiazolylalanine, thienylalanine, Tic (alpha-amino-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), Tic(OH), Tie (tert-butylglycine), and Tyr(Me);

[0074] The term "amino acid sequence" refers to a polypeptide having an amino acid sequence that differs to some extent from the native polypeptide of the corresponding therapeutic enzyme. Typically, an amino acid sequence variant will have at least about 70% sequence identity to the native sequence polypeptide of the corresponding therapeutic enzyme. In one embodiment, a variant has about 80% or more sequence identity to the native sequence polypeptide of the corresponding therapeutic enzyme. In one embodiment, a variant has about 90% or more sequence identity to the native sequence polypeptide of the corresponding therapeutic enzyme. In one embodiment, a variant has about 95% or more sequence identity to the native sequence polypeptide of the corresponding therapeutic enzyme. In one embodiment, a variant has about 98% or more sequence identity to the native sequence polypeptide of the corresponding therapeutic enzyme. Amino acid sequence variants have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence of the native amino acid sequence of the corresponding therapeutic enzyme. Amino acids are designated by traditional names, one-letter codes, and three-letter codes.

[0075] The term "first amino acid sequence," as used herein, refers to the amino acid sequence of immunoglobulin IgG in general, and the light chain of immunoglobulin IgG in particular. In specific (non-limiting) embodiments, the first amino acid sequence is the amino acid sequence of immunoglobulin IgG1, the amino acid sequence of the light chain of immunoglobulin IgG1, or a fragment of the amino acid sequence of the light chain of immunoglobulin IgG1, and is selected from SEQ ID NOs: 2, 8, 9, 10, or 11. In a specific embodiment, the first amino acid sequence is the amino acid sequence of the light chain of immunoglobulin IgG: SEQ ID NO: 2.

[0076] The term "second amino acid sequence" as used herein refers to the amino acid sequence of immunoglobulin IgG in general, the heavy chain of immunoglobulin IgG, a fragment of the heavy chain of immunoglobulin IgG, and in particular a fragment of the heavy chain of immunoglobulin IgG linked to an enzyme sequence directly or via a linker. In a specific (non-limiting) embodiment, the second amino acid sequence is the amino acid sequence of immunoglobulin IgG1, a heavy chain fragment amino acid sequence, SEQ ID NO: 3, a heavy chain fragment of immunoglobulin IgG1 linked to an iduronate-2-sulfatase sequence selected from SEQ ID NOs: 4, 5, 12, 13, 14, or 15.

[0077] The term "transport element," as used herein, refers to a substance capable of carrying, transporting, or delivering a therapeutic enzyme to the lysosomes of cells of various tissues. In particular, but not exclusively, the transport element will specifically interact with an epitope, antigen, receptor, or target in such a manner as to ensure effective delivery to the lysosomes of cells of neural tissue. Such an epitope, antigen, receptor, or target, in the context of this specification, may be the human insulin receptor, or a portion thereof, through which delivery of a drug, peptide, or protein (including across the BBB) is effected.

[0078] An "immunoglobulin," as used herein, is a tetrameric molecule, a "full-length antibody." In naturally occurring immunoglobulins, each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) chain and one "heavy" (about 50-70 kDa) chain. The N-terminal portion of each chain contains a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0079] In the present context, the term "antibody" is used in the broadest sense and includes, inter alia, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, provided they possess the requisite biological activity.

[0080] As used herein, "antibody fragment" includes portions of intact antibodies that retain the ability to bind to antigen. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; dimeric antibodies (diabodies); linear antibodies; single-chain antibody molecules such as single-chain Fab, scFv, and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab fragments," each with a single antigen-binding site, and a remaining "Fc fragment," the latter name reflecting their ability to crystallize readily.

[0081] A "Fab fragment," "Fab," or "FAB" contains the variable domains of the heavy and light chains, and also contains the constant domain of the light chain and the first constant domain of the heavy chain. In the present context, the transport element comprises the amino acid sequence Fab-IgG immunoglobulin fragment, where the IgG immunoglobulin is IgG1, IgG2, or IgG4. In specific (non-limiting) embodiments of the invention, the IgG immunoglobulin is IgG1. In specific embodiments, the transport element comprises the amino acid sequence of an immunoglobulin Fab fragment consisting of a first amino acid sequence selected from SEQ ID NOs: 2, 8, 9, 10, or 11, and the second amino acid sequence of SEQ ID NO: 3. In a more specific embodiment, the transport element comprises the amino acid sequence of an IgG1 immunoglobulin Fab consisting of SEQ ID NOs: 2 and 3.

[0082] In the present context, the term "linker" refers to a chemical or single-stranded peptide linker that connects the therapeutic enzyme and transport element of the compounds of the invention. The linker connects, for example, a monovalent binding element comprising the CH2-CH3 domain of an IgG to an sFab that targets the insulin receptor, i.e., the linker connects the sFab to the C-terminus of the CH3-CH2-D domain of the Ig.

[0083] In some embodiments, the linker is a chemical linker. Single-chain peptide linkers containing 1 to 20 amino acids linked by peptide bonds may be used. In certain embodiments, the amino acids are selected from the 20 naturally occurring (proteinogenic) amino acids. In other specific embodiments, the one or more amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In certain (non-limiting) embodiments, the one or more amino acids are selected from glycine, serine, and leucine.

[0084] In certain embodiments of the present invention, the linker is a single-chain peptide whose amino acid sequence consists of at least one amino acid, preferably 1 to 2 amino acids. In certain (non-limiting) embodiments of the present invention, the amino acid sequence of the linker may consist of more than 1 to 2 amino acids, for example, 3 or 15 amino acids.

[0085] Coupling of the therapeutic enzyme to the transport element may be achieved directly or using a variety of chemical linkers known in the art. In certain preferred (but non-limiting) embodiments of the present invention, coupling of the therapeutic enzyme to the transport element may be achieved using a variety of bifunctional protein coupling agents, such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 4-(1-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Those skilled in the art will also recognize that other chemical and peptide linkers known in the art, not specifically described herein, may be used for the purposes of the present invention.

[0086] In certain preferred embodiments of the present invention, the linker may be a "cleavable linker" that facilitates the release of the therapeutic enzyme after the compound has been transported into cells and tissues. For example, an acid-reactive linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker may be used (Chari R. et al., Cancer Res. 52, 1992, pp. 127-131; US ​​5,208,020, Chari Ravi J. et al., 04.05.1993). Those skilled in the art will also recognize that other cleavable linkers known in the art that facilitate the release of the therapeutic enzyme after the compound has penetrated into cells and tissues of the central nervous system may also be used for the purposes of the present invention.

[0087] The term "epitope" refers to the region of an antigen that is bound by an antigen-binding protein, including an antibody. Epitopes can be defined as structural or functional epitopes. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational epitopes, which are composed of nonlinear amino acids; in other words, conformational epitopes are composed of non-contiguous amino acids. Epitopes can include determinants that are chemically active surface groups of molecules, such as amino acids, carbohydrate chains, phosphoryl groups, or sulfonyl groups, and may also have specific three-dimensional structural characteristics and / or specific charge characteristics. In the present context, the epitope is the epitope in the insulin receptor represented by SEQ ID NO: 1, as described in the work of Zhang B.;, Roth RA (1991) Proc. Natl. Acad. Sci. USA.; 88(21): 9858-9862 and SA Prigent, K. K, Stanley, and K, Siddle. (1990) J. Biol. 1991.

[0088] The term "insulin receptor" (HIR) refers to a transmembrane glycoprotein (molecular weight approximately 320,000 Da) composed of two α subunits and two β subunits (the α subunits are located extracellularly and contain the insulin-binding domain) linked by disulfide bridges. In humans, HIR is involved in the regulation of glucose absorption and distribution, as well as the synthesis and accumulation of fats, proteins, and carbohydrates. The insulin receptor and its extracellular insulin-binding domain (ECD) are structurally and functionally well known in the art. The insulin receptor is most commonly located on the cell surface of insulin-sensitive tissues, such as connective tissue cells, skeletal muscle cells, adipose tissue cells, liver cells, etc. See, for example, Yip et al. (2003), J. Biol. Chem., 278 (30): 27329-27332; and Whittaker et al. (2005), J. Biol. Chem., 280 (22): 20932-20936. In one embodiment, the HIR herein is the human insulin receptor, comprising the amino acid sequence set forth in Kasuya et al. (Biochemistry 32 (1993) 13531-13536).

[0089] Insulin receptors are expressed almost ubiquitously, and the use of such a delivery route can significantly increase the bioavailability of the recombinant enzyme and potentially increase the efficacy of the therapy. Delivery to brain tissue is achieved by transcytosis through the capillary endothelium of the central nervous system via interaction with the human insulin receptor.

[0090] In peripheral and brain tissues expressing M6PR (Hawkes C. et al., 2004), internalization of chimeric molecules can be achieved by both antibody (HIR) and enzyme (M6PR) interactions. In this case, targeted delivery to lysosomes occurs through interaction with the M6P receptor. Internalization via the HIR (human insulin receptor) initially leads to entry into endosomes, followed by transcytosis. In addition to delivery of functional enzymes to the CNS, many lysosomal storage diseases require improved internalization by specific peripheral tissues (e.g., diaphragm muscle in Pompe disease, liver and spleen in Hunter disease, and kidneys in Fabry disease).

[0091] The term "specific" means that the molecule referred to by this term can form a complex with a specific site on another molecule. Binding can be detected by in vitro assays, such as plasmon resonance (BIAcore, GE-Healthcare, Uppsala, Sweden). The specific interaction (affinity of complex formation) of a molecule with a binding site on another molecule is determined by the value k a (rate constant for association of compounds to form a complex), k D (dissociation constant, dissociation of the complex) and K D (k D The binding or specific binding is determined by the following equation: -7 M or less, in one embodiment, about 10 -8 M ~ about 10 -13 M, in one embodiment, about 10 -9 M~10 -13 The binding affinity (K D ) means

[0092] The term "lysosome" refers to an intracellular organelle, a type of vesicle, that contains several enzymes (acid hydrolases) capable of degrading macromolecules, either from within the cell itself (e.g., when structural components of the cell are being processed) or imported from outside the cell. Inherited defects or deficiencies of lysosomal enzymes (or other lysosomal components) can result in the accumulation of undegraded metabolic products. Glycosaminoglycans (formerly called mucopolysaccharides) are common polysaccharides on cell surfaces and in extracellular matrices and structures. Enzyme deficiencies that prevent glycosaminoglycan degradation result in the accumulation of glycosaminoglycan fragments in lysosomes, which can cause widespread changes in bone, soft tissue, and the central nervous system.

[0093] The "activity" of the enzyme(s) of the present invention can be measured using any suitable test. Typically, pH and temperature determinations can be adapted to the enzyme in question. Examples of pH values ​​to be tested are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of temperatures to be tested are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 95°C. Preferred pH values ​​and temperatures are in the physiological range, e.g., pH values ​​of 4, 5, 6, 7, or 8, and temperatures of 30, 35, 37, or 40°C. For example, any test using a substrate containing a peptide bond related to the specificity of the protease in question can be used to measure protease activity.

[0094] Examples of suitable enzyme tests are included in the Experimental Section, see in particular Example 2. As used herein, the term "activity" means "enzyme activity", "specific activity", "enzyme specific activity"; "specific activity" depending on the context of the present specification.

[0095] The term "blood-brain barrier" or "BBB" refers to the physiological barrier between peripheral blood flow and the brain and spinal cord. The BBB is formed by the tight junctions in the endothelial plasma membrane of brain capillaries, which creates a tight barrier that restricts the transport of molecules into the brain, even very small molecules such as urea (60 Da). The BBB in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retinal barrier in the retina are continuous capillary barriers in the CNS, and are collectively referred to herein as the blood-brain barrier (also referred to herein as the BBB). The BBB also includes the blood-cerebrospinal fluid barrier.

[0096] The compounds of the present invention may be formulated into compositions, e.g., pharmaceutical compositions, comprising a compound or combination of compounds or moiety(ies) thereof and a pharmaceutically acceptable carrier.

[0097] A "pharmaceutical composition" refers to a mixture of one or more compounds described herein, or physiologically / pharmaceutically acceptable salts or prodrugs thereof, with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compound to the body.

[0098] Pharmaceutical compositions according to the present invention may contain one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers and / or adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.

[0099] The term "effective amount" of a compound, for example in a pharmaceutical composition, refers to an amount that is effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result (effect) in the subject undergoing treatment, with a reasonable benefit / risk balance.

[0100] The term "pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical composition, other than an active substance (compound, agent, etc.), that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In certain (non-limiting) embodiments of the present invention, a pharmaceutically acceptable carrier is co-administered with the compound.

[0101] Pharmaceutical compositions containing the compounds used according to the present invention are prepared for storage, preferably in the form of a lyophilized composition or an aqueous solution, by mixing with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th edition, edited by Osol A., 1980). Acceptable carriers, excipients, or stabilizers are non-toxic to subjects at the dosages and concentrations used, and include buffers such as phosphate, citrate, and other organic acid buffers; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and meta-cresol); low molecular weight polypeptides (containing less than about 10 residues); proteins, e.g., For example, serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as poly(vinylpyrrolidone); amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal-containing complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0102] Preferably, the pharmaceutical composition further contains sodium chloride, sodium dihydrogen phosphate dihydrate, sodium hydroxide, polysorbate.

[0103] In some embodiments of the invention, the pharmaceutical composition further contains 5.3 mg of the HIR-Fab-IDS compound, sodium chloride in an amount of 8.0 mg, sodium dihydrogen phosphate dihydrate in an amount of 3.12 mg, sodium hydroxide to adjust the pH to pH 6.0, polysorbate 20 in an amount of 0.2 mg, and water for injection to 1.0 mL.

[0104] The pharmaceutical compositions used herein may also optionally contain more than one active agent (drug) active against lysosomal storage diseases, optionally with additional activities that do not adversely affect each other. The type and effective amount of such agents will depend, for example, on the amount of therapeutic enzyme and transport element-containing compound present in the composition, as well as the clinical parameters of the subject.

[0105] The pharmaceutical compositions provided herein may be administered to a patient by any suitable route, for example, intravenously by bolus injection or continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerebrospinally, transdermally, subcutaneously, intra-articularly, sublingually, intrasynovially, orally, by inhalation, topically or externally. Intravenous administration is preferred.

[0106] In the context of this specification, the term "dose" refers to the dose used, for example, when a pharmaceutical composition provided herein is administered to a patient as an "initial dose" when first administered to the patient, or as a "therapeutic dose" when administered as a dose for subsequent administrations.

[0107] Generally, single doses in the range of about 0.3 to 30 mg / kg of patient body weight, more often 1 to 12 mg / kg, are contemplated.

[0108] Typically, treatment begins with a low dose of the HIR-Fab-IDS compound. For example, an initial dose of antibody is administered to the patient, e.g., by injection or infusion. The initial dose should contain approximately 3-12 mg / kg.

[0109] The initial dose is in the range of 3 to 12 mg / kg, for example, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, about 10 mg / kg, about 10.5 mg / kg, about 11 mg / kg, about 11.5 mg / kg, or about 12 mg / kg.

[0110] Doses for successive administrations (therapy) may be approximately equal to, or less than, or more than, the initial dose, so that the doses may vary within specified limits, and the doses administered at different times during the successive administrations may not necessarily be the same dose. For example, the dose may be gradually decreased, or different doses may be arbitrarily repeated.

[0111] Therapeutic doses range from 1 to 12 mg / kg, e.g., about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 8.5 mg / kg, about 9 mg / kg, about 9.5 mg / kg, about 10 mg / kg, about 10.5 mg / kg, about 11 mg / kg, about 11.5 mg / kg, or about 12 mg / kg.

[0112] In different instances, other therapeutic regimens may be required, for example, a therapeutic dose may correspond to 3 mg / kg, a therapeutic dose may correspond to 6 mg / kg, a therapeutic dose may correspond to 12 mg / kg.

[0113] More preferably, the therapeutic dose is selected from the group consisting of 3 mg / kg, 6 mg / kg, 12 mg / kg of the HIR-Fab-IDS compound.

[0114] In addition to expressing dose values ​​in mg / kg, dose values ​​may also be expressed as fixed doses (mg / body) and / or as doses per unit body surface area (mg / m), which correspond to the doses per unit body weight specified above. 2 ) may also be expressed as

[0115] There is no particular limit to the number of doses to be administered consecutively, and the doses may be administered, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 35, 40, 50, 60, 70, 80, 90, 100, 500, 1000, and 10,000 times.

[0116] The "inter-dosing interval" (the interval between individual doses) is the interval between the administration of the initial dose and the administration of the first dose for consecutive administrations, and the interval between the administration of the nth dose for consecutive administrations (n ​​is an integer equal to or greater than 1) and the n+1th dose for consecutive administrations. The interval between doses can be 1 day or more, e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year. The interval between doses may also be expressed in other terms, such as once daily, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, once every 14 weeks, once every 15 weeks, once every 16 weeks, once every 17 weeks, once every 18 weeks, once every 19 weeks, once every 20 weeks, once every 21 weeks, once every 22 weeks, once every 23 weeks, once every 24 weeks, once every 25 weeks, once every 26 weeks, once every 27 weeks, once every 28 weeks, once every 29 weeks, once every 30 weeks, once every 31 weeks, once every 32 weeks, once every 33 weeks, once every 34 weeks, once every 35 weeks, once every 36 weeks, once every 37 weeks, once every 38 weeks, once every 39 weeks, once every 40 weeks, once every 41 weeks, once every 42 weeks, once every 43 weeks, once every 44 weeks, once every 45 weeks, once every 46 weeks, once every 47 weeks, once every 48 weeks, once every 49 weeks, once every 50 weeks, once every 51 weeks, once every 52 weeks, once every 53 weeks, once every 54 weeks, once every 55 weeks, once every 56 weeks, once every 57 weeks, once every 58 weeks, once every 59 weeks, once every 60 weeks, once every 61 weeks, once every 62 weeks, once every 63 weeks, once every 64 weeks, once every 65 weeks, once every 66 weeks, once every 67 weeks, once every 68 The frequency may be once every 6 weeks, once every 17 weeks, once every 18 weeks, once every 19 weeks, once every 20 weeks, once every 21 weeks, once every 22 weeks, once every 23 weeks, once every 24 weeks, once every 25 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months or once every year.Additionally, the interval between doses may be expressed in other terms, such as every 1 day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 1 week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 14 weeks, every 15 weeks, every 16 weeks, every 17 weeks, every 18 weeks, 19 weeks, every 20 weeks, every 21 weeks, every 22 weeks, every 23 weeks, every 24 weeks, every 25 weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or every year.

[0117] The interval between the initial dose and the first sequential dose, and the interval between the nth sequential dose (n is an integer of 1 or more) and the (n+1)th sequential dose, may be the same; however, the intervals do not have to be the same. For example, the interval between the initial dose and the first sequential dose may be longer than the interval between the nth sequential dose and the (n+1)th sequential dose; or the interval between the initial dose and the first sequential dose may be shorter than the interval between the nth sequential dose and the (n+1)th sequential dose of the sequential administration. Furthermore, the interval between the administration of the nth dose and the (n+1)th dose of the sequential administration may be the same; however, these intervals do not necessarily have to be the same or different. For example, as the number of doses administered in the sequential administration increases, the interval between administrations may become longer or shorter.

[0118] More preferably, the interval between doses was one week (7 days).

[0119] The interval between dose administrations is also understood as the interval between administrations of the pharmaceutical composition.

[0120] The administration schedule is determined based on, for example, considerations of efficacy and safety, and further, the administration schedule is determined based on patient convenience, within the scope that does not compromise efficacy and safety.

[0121] In the context of the present invention, the term "substantially the same" means that the difference is about 20% or less, preferably that the difference is 10% or less, or more preferably that the difference is 5% or less, 4% or less, 3% or less, or 1% or less.

[0122] Administration of the dose of the claimed therapeutic compound may be carried out by any suitable route known to those skilled in the art, for example, by injection, such as intravenous, intramuscular, or subcutaneous injection. The selection of a particular administration route of the therapeutic compound will be made by those skilled in the art, and will depend, inter alia, on whether the administration is short-term or long-term. Those skilled in the art will understand that other administration routes known in the art, such as (without limitation) intrathecal, intraarterial, intraperitoneal, transdermal, inhalation, buccal, intranasal, oral, sublingual, or intranasal, can also be used to administer the dose of the claimed therapeutic compound (Felice BR, Wright TL, Boyd RB Safety Evaluation of Chronic Intrathecal Administration of Idursulfase-GG in Cynomolgus Monkeys. - Toxicol. Pathol. 2011 Aug.; 39(5):879-9, WO 2011 / 044542 A1).

[0123] In the context of this specification, a variety of dosing regimens may be contemplated, including but not limited to single or multiple administrations at different times, bolus administration, and pulsatile administration.

[0124] For treatment, an effective dose of the HIR-Fab-IDS compound ranges from 1 mg / kg body weight to 12 mg / kg body weight, preferably about 3 mg / kg body weight to 12 mg / kg body weight, administered weekly (once a week), preferably by intravenous administration, preferably over a 3-hour period.

[0125] Each vial of drug is intended for single use only and contains 15 mg of the HIR-Fab-IDS compound in 3 mL of solution. Prior to intravenous administration, the drug concentrate must be diluted with 0.9% sodium chloride solution.

[0126] The terms "approximately" and "about" refer to all values ​​expressed as numbers with one or two decimal places.

[0127] A "subject," "individual," or "patient" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys, especially apes), rabbits, and rodents (e.g., mice and rats). In some embodiments, the subject or patient is human.

[0128] In the context of this specification, the term "treatment" (and grammatical variations thereof, such as "treating" or "therapeutic process") refers to clinical intervention with the purpose of altering the natural course of disease in the individual being treated, and may be performed prophylactically or during the development of clinical pathology. Desirable therapeutic effects include (but are not limited to) preventing the onset or recurrence of disease, alleviating symptoms, reducing the direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, alleviating or palliating the disease state, and ameliorating or improving prognosis. In some embodiments, the compounds are used to slow the development of disease or slow the progression of disease.

[0129] In the context of this specification, the terms "prevention" or "prevention" include prophylactic administration, which may reduce the likelihood of developing or occurring a disease (eg, type II MPS with a neurological component).

[0130] Delivery or transport of HIR-Fab-IDS compounds to a subject (e.g., by direct administration) and practice of other methods as described herein can be used to reduce, treat, prevent, or otherwise alleviate any suitable aspect of the progression of a lysosomal storage disease (i.e., the progression of MPS II with a neurological component). Methods that reduce, prevent, or otherwise attenuate the intensity of such aspects of the progression of MPS II with a neurological component, individually or in combination, represent advantageous features.

[0131] In another aspect, a method is provided for providing a therapeutic regimen for reducing the risk of progression of MPS Type II with a neurological component in a human, reducing the risk of further progression of MPS Type II with a neurological component that has begun, and / or reducing the progression of MPS Type II with a neurological component, comprising administering to the patient one or more first-line therapies in amounts and according to a regimen sufficient to achieve a response (partial or complete response), and then administering to the patient an amount of an HIR-Fab-IDS compound, or administering the compound to the patient as a first-line therapy.

[0132] In a further aspect, there is provided a method for inducing remission of MPS type II with a neurological component in a subject, e.g., a human, comprising administering to the subject a composition containing an HIR-Fab-IDS compound to induce remission of MPS type II with a neurological component in the subject.

[0133] In yet another further aspect, there is provided a method for reducing the risk of developing MPS type II with a neurological component and reducing the time to early diagnosis of MPS type II with a neurological component, comprising administering to a patient a prophylactically effective amount of an HIR-Fab-IDS compound.

[0134] In a further embodiment, a method is provided for increasing the likelihood of survival for a reasonable period of time in an individual diagnosed with MPS Type II. In another embodiment, a method is provided for improving the quality of life of a subject with MPS Type II, comprising administering to the patient a composition in an amount effective to improve the patient's quality of life.

[0135] The term "CNS" or "central nervous system" refers to the complex of nervous tissue that controls bodily functions and includes the brain and spinal cord.

[0136] As used herein, the term "lysosomal storage disease" (LSD) refers to a rare genetic disorder in which partial or complete loss of activity of one of the lysosomal enzymes results in loss of the above-mentioned lysosomal function. In this case, ERT is required to replace the enzyme whose activity is completely or partially lost or missing. As used herein, the term "lysosomal storage disease" is used interchangeably with the term "lysosomal storage disorder" (which may also be referred to as "lysosome storage disorder"). Lysosomal storage diseases can be classified according to the enzyme defect or deficiency as follows: (i) sphingolipidoses, (ii) mucopolysaccharidoses, (iii) glycogen storage diseases, (iv) mucolipidoses; (v) oligosaccharidoses; (vi) lipidoses, (vii) lysosomal trafficking diseases, etc.

[0137] Below, lysosomal storage diseases will be described in more detail according to their classification.

[0138] As used herein, the term "sphingolipidosis" refers to a genetically determined deficiency syndrome of lysosomal enzymes that hydrolyze the carbohydrate or choline side chains of sphingolipids. These diseases are classified according to the distribution of each lipid that accumulates, and include, for example, Krabbe disease caused by galactocerebrosidase deficiency, Fabry disease caused by α-galactosidase A deficiency, Niemann-Pick disease caused by sphingomyelinase deficiency, Gaucher disease caused by glucocerebrosidase deficiency, and Tau-Sachs disease caused by hexosaminidase A deficiency. These diseases are inherited in an autosomal recessive manner, except for Fabry disease, which is an X-linked genetic disease.

[0139] As used herein, the term "mucopolysaccharidosis" (MPS) refers to a syndrome associated with a genetic deficiency of mucopolysaccharide hydrolases, caused by deficiencies of carbohydrate-chain-degrading enzymes, such as sulfatases and acetyltransferases. The primary symptom of MPS is excessive secretion of mucopolysaccharides in the urine. Currently, MPS is classified into six types: type I includes Hurler syndrome and Scheie syndrome; type II includes Hunter syndrome; type III includes Sanfilippo syndrome types A, B, C, and D; type IV includes Morquio syndrome types A and B; type VI includes Maroteaux-Lamy syndrome; and type VII includes Sly syndrome.

[0140] As used herein, the term "glycogen storage disease" (also known as "glycogen storage disease") refers to disorders of inborn errors of carbohydrate metabolism caused by glycogen accumulation, and is divided into subtypes I-VII. Subtypes of glycogen storage disease associated with lysosomal storage diseases (LSDs) are type II (Pompe disease) and IIIb (Danon disease).

[0141] A detailed description of the lysosomal storage diseases described herein and those disclosed in Table 1 is provided in: The Online Metabolic & Molecular Bases of Inherited Diseases (Scriver's OMMBID), Part 16 (David L. Valle, Stylianos Antonarakis, Andrea Ballabio, Arthur L. Beaudet, Grant A. Mitchell, McGraw-Hill Education, 2007).

[0142] Mucopolysaccharidosis type I (MPS I) is a genetic metabolic disorder caused by a deficiency in the enzyme α-L-iduronidase (IDUA), whose function is to break down the acidic mucopolysaccharides heparan sulfate and dermatan sulfate. Insufficient IDUA levels lead to the abnormal accumulation of these mucopolysaccharides in patient tissues and organs, such as the heart, liver, and central nervous system. Symptoms, including neurodegeneration and mental retardation, appear in childhood, and organ damage can lead to early death. Genetic deficiency of the carbohydrate-degrading lysosomal enzyme α-L-iduronidase causes a lysosomal storage disease known as mucopolysaccharidosis type I (MPS I). A severe form of MPS I, commonly known as Hurler syndrome, is associated with a variety of problems, including mental retardation, corneal opacity, coarse facial features, heart disease, respiratory problems, enlarged liver and spleen, hernias, and joint stiffness. Patients with Hurler syndrome usually die before the age of 10. In a moderate form known as Hurler-Scheie syndrome, mental function is usually not severely affected, but physical problems can lead to death in the teens or early twenties. Scheie syndrome is a mild form of MPS I. It is compatible with a normal lifespan, but joint stiffness, corneal clouding, and heart valve disease cause serious problems.

[0143] "Mucopolysaccharidosis type II (MPS II)" or "Hunter syndrome" is an X-linked inherited metabolic disorder caused by deficiency of the enzyme iduronate-2-sulfatase (I2S). I2S is located in lysosomes and plays a key role in the catabolism of heparan and dermatan sulfate glycosaminoglycans (GAGs). In the absence of this enzyme, these substrates accumulate in cells, gradually leading to stasis, followed by cell death and tissue destruction. Because the enzyme is widely expressed, multiple cell types, organs, and systems are affected in patients with MPS II. A characteristic clinical feature of this disease is central nervous system (CNS) degeneration, which leads to cognitive impairment (e.g., decreased IQ). Furthermore, MRI scans of patients reveal white matter lesions, perivascular space expansion in the brain parenchyma, ganglia, corpus callosum, and brainstem, atrophy, and ventricular enlargement (Wang et al., Molecular Genetics and Metabolism, 2009). The disease typically presents with organomegaly and skeletal abnormalities in the first year of life. Some patients experience progressive loss of cognitive function, and most die from disease-related complications within the first or second decade of life (Raluy-Callado M. et al. Orphanet. J. Rare Dis. 2013; 8: 101;).

[0144] As used herein, the term "neurological component" refers to a disease or disorder that affects and / or whose etiology is related to the CNS. Examples of CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, epilepsy disorder, behavioral disorder, and lysosomal storage disease. Specific examples of neurological disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, postmyelitic syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration), tauopathies (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Sträussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), Boulevard palsy, palsy), motor neuron diseases, and heterogeneous degenerative disorders of the nervous system (including but not limited to Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette's disease, Menkes kinky hair disease, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (including but not limited to Pick's disease and spinocerebellar ataxia), cancer (e.g., cancer of the central nervous system and / or brain, including brain metastases from cancer elsewhere in the body).

[0145] In the context of this specification, the term "neurological component" refers to "neuropathic" (with neurocognitive damage), "non-neuropathic" (without neurocognitive impairment), "neurocognitive effects", "neuropathic disease", "neuropathic type" and any other disorder, including peripheral ones, that reflects the nature of the therapy involving drug penetration into / through the CNS.

[0146] MPS disorders are associated with a wide range of neurocognitive effects, ranging from mild problems in attention and executive function to progressive and degenerative neuropathic disease.

[0147] The neuropathic form of MPS II presents challenges due to the variable disease course, with differing delays and timing of decline. MPS II has the greatest uncertainty regarding the neurocognitive progression of the disease. MPS II is divided into two forms: a severe neuropathic form that has a significant impact on neurocognitive function and a milder form that is thought to have little or no neurocognitive sequelae (Elsa G. Shapiro and Julie B. Eisengart, "The natural history of neurocognition in MPS disorders: a review". Molecular Genetics and Metabolism 133.1 (2021): 8-34).

[0148] In addition to neurocognitive impairment, central nervous system problems in MPS II include behavioral disturbances, communicating hydrocephalus, epilepsy, sleep apnea, and spinal cord compression. The increased risk of hydrocephalus and epilepsy in later stages of the disease may exacerbate the deterioration of neurocognitive function (S. Al Sawaf, E. Mayatepek, B. Hoffmann, Neurological findings in Hunter disease: pathology and possible therapeutic effects reviewed, J. Inherit. Metab. Dis. 31(4) (2008) 473-480; IV Schwartz, et al., A clinical study of 77 patients with mucopolysaccharidosis type II, Acta Paediatr. 96 (455) (2007) 63-70).

[0149] The compound proposed by the present invention can be used individually or in combination with other agents in therapy.For example, the compound proposed by the present invention can contain therapeutic enzyme and transport element linked by linker and be administered with at least one additional therapeutic agent.In certain embodiments of the present invention, the additional therapeutic agent is effective in treating the same neurological disorder that the compound of the present invention is used for or another neurological disorder. Examples of additional therapeutic agents include, but are not limited to, the various neurological agents described above, including cholinesterase inhibitors (e.g., donepezil, galantamine, lovastigmine, and tacrine), NMDA receptor antagonists (e.g., memantine), amyloid beta peptide aggregation inhibitors, antioxidants, gamma-secretase modulators, nerve growth factor (NGF) mimetics or NGF gene therapy agents, PPARy agonists, HMS-CoA reductase inhibitors (statins), ampakines, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulin, muscarinic receptor agonists, nicotinic receptor modulators, active or passive immunization agents against amyloid beta peptide, phosphodiesterases, serotonin receptor antagonists, and antibodies against amyloid beta peptide. Such aforementioned combination therapy includes simultaneous administration (where two or more therapeutic agents are contained in the same or different compositions) and separate administration, where administration of a compound comprising a therapeutic enzyme and a transport element linked by a linker in accordance with the present invention may occur before, simultaneously with, and / or after administration of the additional therapeutic agent(s) and / or adjuvant.

[0150] Any of the above defined terms may appear more than once, and when so appeared each term shall be defined independently of the other.

[0151] The following will describe exemplary embodiments of the present invention in detail. However, each of the descriptions and embodiments provided as examples in this document may also be true with respect to other descriptions and exemplary embodiments. Therefore, all combinations of the various factors described in this document are included in the scope of the present invention. Furthermore, the scope of the present invention is not limited to the specific descriptions provided below.

[0152] It will be clear to those skilled in the art that additions and modifications can be developed to what is disclosed in the examples presented below within the framework of the present invention without expending any inventive creativity. [Example]

[0153] The present invention will now be described in more detail with reference to the following examples. However, the examples described below are provided for illustrative purposes only and are not intended to limit the present invention.

[0154] Example 1. Preparation To prepare the compound, animal cells were cultured and purified, and an expression vector for animal cells was added thereto.

[0155] Methods for producing human antibodies are now known. For example, a human antibody of interest can be produced by immunizing transgenic animals carrying the full spectrum of human antibody genes with an antigen of interest (see International Patent Application Publications WO 93 / 12227, WO 92 / 03918, WO 94 / 02602, WO 94 / 25585, WO 96 / 34096, and WO 96 / 33735).

[0156] Genetically modified antibodies can also be produced by known methods. A specific example is a chimeric antibody, which contains the variable regions of the H and L chains of an antibody from an immunized animal and the constant regions of the H and L chains of a human antibody. Chimeric antibodies can be produced by linking DNA encoding the variable regions of an antibody from an immunized animal with DNA encoding the constant regions of a human antibody, inserting them into an expression vector, and then adding this expression vector to host cells to produce the antibody.

[0157] Humanized antibody is a modified antibody, and is often referred to as reshaped human antibody.Humanized antibody is constructed by transferring the CDR of antibody derived from immunized animal into the hypervariable region of human antibody.General genetic engineering techniques for producing such antibodies are also known (see published European patent application EP 239400; published international patent application WO 96 / 02576; Sato K. et al., Cancer Research, 53 (1993), pp. 851-856; international patent application publication WO 99 / 51743).

[0158] Construction of expression vectors The amino acid sequences of the first LC-HIR-Fab-IDS (SEQ ID NO: 1) and the second HC-HIR-Fab-IDS (SEQ ID NO: 4) were converted to nucleotide sequences to obtain HIR-Fab-IDS variants containing the MDWTWRVFCLLAVAPGAHS signal peptide. For gene synthesis with subsequent cloning into the pCLN-1 vector, the following were added to the 5' end of the sequence: a HindIII restriction site and a Kozak sequence. Two stop codons and an Xba1 restriction site were added to the 3' end of the sequence.

[0159] Nucleotide sequence optimization for Chinese hamster cell codon composition was performed using resources: http: / / gcua.schoedl.de and http: / / www.kazusa.or.jp. Synthesis of the light and heavy chains of LC-HIR-Fab-IDS and HC-HIR-Fab-IDS was performed at GenArt (USA) and transferred as part of pRA1675 and pRA1673 (containing the genes LC-HIR-Fab-IDS and HC-HIR-Fab-IDS, respectively).

[0160] The HC-HIR-Fab-IDS and LC-HIR-MAB sequences from pRA1675 and pRA1673 plasmids were cloned into the pCLN-1 expression vector at the HindIII / Xba1 sites to obtain pGNR-055-007 (pCLN-1- HC-HIR-Fab-IDS) and pGNR-055-008 (pCLN-1- LC-HIR-Fab-IDS) vectors. The resulting vectors were linearized at the BspHI / PvuI sites.

[0161] The vectors of the present invention were constructed according to molecular biology techniques well known in the art. Brown T, "Gene Cloning" (Chapman & Hall, London, GB, 1995); Watson R, et al., "Recombinant DNA", 2nd Ed. (Scientific American Books, New York, NY, US, 1992); Alberts B, et al., "Molecular Biology of the Cell" (Garland Publishing Inc., New York, NY, US, 2008); Innis M, et al., Eds., "PCR Protocols. A Guide to Methods and Applications" (Academic Press Inc., San Diego, CA, US, 1990);Erlich H, Ed., "PCR Technology. Principles and Applications for DNA Amplification" (Stockton Press, New York, NY, US, 1989);Sambrook J, et al., "Molecular Cloning. A Laboratory Manual" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, US, 1989);Bishop T, et al., "Nucleic Acid and Protein Sequence. A Practical Approach" (IRL Press, Oxford, GB, 1987);Reznikoff W, Ed., "Maximizing Gene Expression" (Butterworths Publishers, Stoneham, MA, US, 1987);Davis L, et al., "Basic Methods in Molecular Biology" (Elsevier Science Publishing Co., New York, NY, US, 1986), Schleef M, Ed., "Plasmids for Therapy and Vaccination" (Wiley-VCH Verlag GmbH, Weinheim, DE, 2001).

[0162] Obtaining monoclonal cell lines Parental cell line CHO-S. Cells were cultured in BalanCD CHO growth A medium (Invitrogen) at 37°C, 5% CO2, and 70% humidity. Stable transfection was performed on a NEON device (Invitrogen) using two linearized plasmids, pCLN-1-HC-HIR-Fab-IDS and pCLN-1-LC-HIR-Fab-IDS, according to standard protocols for CHO cells. The DNA ratio for transfection was equimolar.

[0163] After 48 hours, transfected pools were plated in minipools in 96-well plates in BalanCD CHO Growth A medium containing the selective antibiotic neomycin at 600 μg / mL. Minipools were cultured for 10 days under static conditions at 37°C, 5% CO2, and 70% humidity, followed by a series of productivity screens using ELISA. Leader minipools were cloned in semi-solid ClonaCell Flex medium (STEMCEEE) for expansion of individual colonies using 6-well plates. Plates were incubated at 37°C, 5% CO2, and 70% humidity.

[0164] During screening, the concentration of the target protein HIR-Fab-IDS secreted into the culture medium was determined by sandwich ELISA using a recombinant human insulin receptor fragment (5 μg / mL in carbonate-bicarbonate buffer, pH 9.6) instead of a primary antibody; a rat anti-iduronate-2-sulfatase antibody conjugated to horseradish peroxidase (diluted 1:10,000) was used as the secondary antibody. Color development was performed with tetramethylbenzidine solution, and the reaction was stopped with 0.5 M sulfuric acid.

[0165] Based on the results of screening in 6-well plates, 20 leader minipools with productivity ranging from 0.4 to 2.1 mg / L were selected and transferred to flasks for adaptation to suspension culture. After three passages, the 20 minipools were frozen.

[0166] Clonal selection was performed in 96-well plates using a ClonePix robot (Molecular Devices) in automated mode. The resulting clones were screened. For this purpose, a 7-day culture process was simulated in batch mode, where clones were evaluated according to the following parameters: culture viability kinetics, viable cell density kinetics, target HIR-Fab-IDS concentration, and volumetric production dependence on cumulative cell density.

[0167] Production cell culture Producer cells expressing HIR-Fab-IDS were cultured in a cyclic fed-batch mode for 12 days in a bioreactor equipped with an overhead stirrer at a temperature of 37°C and a pH of 6.9 using BalanCD CHO Growth A Nutrient Medium (Irvine Scientific) and BalanCD CHO Feed 2 Nutrient Supplement (Irvine Scientific). After the culture process, the culture medium was clarified by depth filtration and transferred for isolation.

[0168] Selection and purification Isolation and purification are carried out using standard protein isolation and purification techniques. Compounds may be isolated or purified by any method known in the art for protein isolation or purification, such as chromatography (e.g., ion exchange, high performance liquid chromatography, affinity, protein A, and size-sorting column chromatography), centrifugation, differential solubility, or any other standard protein isolation or purification technique.

[0169] Example 2. Determination of the level of enzymatic activity of a Fab fragment of an antibody against the insulin receptor linked to the amino acid sequence of iduronate-2-sulfatase The specific activity of HIR-Fab-IDS and HIR-MAB-IDS was determined by fluorimetry using 4-mutilumbelliferyl-L-iduronide-2-sulfate (4-MUS) (Moscerdam Substrates, The Netherlands) ( Voznyi YV et al., 2001 ; Toluun AA et al., 2012 ; Johnson BA et al., 2013 ; Azadeh M. et al., 2017 ). During the assay, the substrate is hydrolyzed by iduronate-2-sulfatase to produce 4-mutilumbelliferyl-L-iduronide (MUBI), which is then hydrolyzed by iduronidase (IDUA, Aldurazyme, Genzyme, USA) to 4-methylumbelliferyl (4-MU), which is fluorometrically detected using the following fluorometer settings: fluorescence excitation at a wavelength of 450 nm and fluorescence detection at a wavelength of 365 nm. A calibration curve is plotted using a standard 4-MU solution (Sigma-Aldrich, USA). During the assay, the mixture is first incubated at 37°C, pH 4.5 for 4 hours, then 12 μg of IDS is added, and the mixture is further incubated at 37°C for 24 hours. The incubation is stopped by adding 0.2 mL of 0.5 M sodium carbonate, pH 10.3. HIR-FAB-IDS exhibits specific enzyme (iduronate-2-sulfatase) activity toward the substrate 4-MU-αIdoA-2S (4-methylumbelliferyl α-L-idopyranosiduronate-2-sulfate).

[0170] Equipment and Devices 1. Thermostatic shaker PST-60 HL-4 (BioSan, Latvia). 2. Multifunction reader Spectra Max M3 (Molecular Devices, USA). 3. Multifunction reader software Soft Max Pro (Molecular Devices, USA). 4. Analytical balance ML 204 (Mettler Toledo, Switzerland). 5. Non-adsorbent surface 96-well black plate (Corning, USA, Cat. No. 3916).

[0171] reagent 1. 4-Methylumbelliferone sodium salt (Sigma-Aldrich, catalog number M1508). 2. Recombinant human α-L-iduronidase (R&D SYSTEM, Cat. No. 4119-GH). 3. 4-Methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate sodium salt, 0.5 mg / vial (USBiological, catalog number 017551). 4. Sodium acetate anhydrous (AppliChem Panreac, Catalog No. 141633.1211). 5. Acetic acid (Fluka, Cat. No. 49199). 6. Bovine serum albumin (Sigma, Cat. No. A7030). 7. Sodium carbonate (Sigma-Aldrich, Cat. No. S7795). 8. Sodium bicarbonate (Sigma-Aldrich, catalog number S6297). 9. Lead diacetate trihydrate (Aldrich, catalog number 467863). 10. Sodium phosphate dihydrate (Sigma-Aldrich, catalog number 71643). 11. Anhydrous citric acid (AppliChem Panreac, Catalog No. 141808).

[0172] Preparation of solutions Sample dilution solution, pH (5.5±0.2). Approximately 4.1 g of anhydrous sodium acetate and 0.5 g of bovine serum albumin are placed in a 1-liter beaker and dissolved in 700 mL of purified water. The pH of the solution is adjusted to (5.5±0.2) with acetic acid. The resulting solution is transferred to a 1-liter volumetric flask, the volume of the solution is adjusted to the mark with purified water, mixed, and filtered through a membrane filter with a pore diameter of 0.45 μm. Shelf life: 3 months at temperatures between 2-8°C.

[0173] Substrate dilution solution, pH (5.00 ± 0.05). Place 4.1 g of anhydrous sodium acetate and 1.9 g of lead diacetate trihydrate in a 500 mL beaker, add 450 mL of purified water, and stir until dissolved. Adjust the pH of the solution to (5.00 ± 0.05) with acetic acid (approximately 1.1 mL). Transfer the resulting solution to a 500 mL volumetric flask, bring the solution volume to the mark with purified water, stir, and filter through a 0.45 μm pore diameter membrane filter. Shelf life: 3 months at temperatures between 2-8°C.

[0174] Substrate solution (1.25 mmol / L). Add 0.83 mL of the diluted substrate solution to the vial containing the substrate (4-methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate sodium salt) and mix gently. Divide the resulting solution into 0.2 mL aliquots and freeze. Shelf life: 3 months at temperatures not exceeding -70°C.

[0175] Solution of recombinant human α-L-iduronidase. Add 400 μl of purified water to a vial containing recombinant human α-L-iduronidase (10 μg) and mix. Divide the resulting solution into 0.1 mL aliquots and freeze. Shelf life: 3 months at temperatures not exceeding -70°C.

[0176] 0.1 M citric acid solution: Place approximately 19.2 g of anhydrous citric acid in a 1 L volumetric flask, dissolve in 900 mL of purified water, bring the solution volume up to the mark with purified water, and filter through a 0.22 μm membrane filter. Shelf life: 3 months at temperatures between 15-25°C.

[0177] 0.2 M Sodium hydrogen phosphate solution. Place approximately 35.6 g of sodium hydrogen phosphate dihydrate in a 1 L volumetric flask, dissolve in 900 mL of purified water, bring the solution volume up to the mark with purified water, and filter through a 0.22 μm membrane filter. Shelf life: 3 months at temperatures between 15-25°C.

[0178] Phosphate-citrate buffer solution, pH (4.5 ± 0.1). In a 100 mL volumetric flask, mix 55.0 mL of 0.1 M citric acid solution and 45.0 mL of 0.2 M sodium hydrogen phosphate solution and filter through a 0.22 μm pore diameter membrane filter. Shelf life: 3 months at temperatures between 15-25°C.

[0179] 0.5 M sodium bicarbonate solution. Place approximately 42.0 g of sodium bicarbonate in a 1 L volumetric flask and dissolve in 900 mL of purified water. Bring the solution volume up to the mark with purified water, mix, and filter through a 0.22 μm pore diameter membrane filter. Shelf life: 6 months at temperatures between 15-25°C.

[0180] 0.5 M sodium carbonate solution. Approximately 53.0 g of sodium carbonate is placed in a 1 L volumetric flask and dissolved in 900 mL of purified water, the solution volume is brought up to the mark with purified water, mixed, and filtered through a 0.22 μm pore diameter membrane filter. Shelf life: 6 months at temperatures between 15-25°C.

[0181] Stop solution, pH (10.8±0.5). Add 900 mL of 0.5 M sodium carbonate solution and 100 mL of 0.5 M sodium bicarbonate solution to a 1 L volumetric flask, mix and filter through a 0.22 μm pore diameter membrane filter. Shelf life: 6 months at temperatures between 15-25°C.

[0182] Stock solution of 4-methylumbelliferone sodium salt (100 μmol / mL). Place approximately 1.0 g (accurately weighed) of 4-methylumbelliferone sodium salt in a 50 mL volumetric flask, add 30 mL of purified water, mix, and bring to the mark with purified water. Divide the solution into 2.0 μL aliquots. Shelf life is 6 months at temperatures not exceeding -18°C.

[0183] Working standard solution of 4-methylumbelliferone sodium salt (50 nmol / mL). An aliquot of the stock solution of 4-methylumbelliferone sodium salt is kept in a water bath for 5 minutes at 37° C. Serial dilutions are prepared according to the following scheme:

[0184] TIFF2026506966000003.tif24165 * 4-MU is 4-methylumbelliferone sodium salt.

[0185] Dilutions of the working standard solution of 4-methylumbelliferone sodium salt. Dilutions of the working standard solution of 4-methylumbelliferone sodium salt are prepared according to the following scheme:

[0186] TIFF2026506966000004.tif64165

[0187] Dilution of test sample: Based on the actual HIR-FAB-IDS content specified on the certificate, the test sample is diluted with sample diluent to a concentration of 1 mg / mL. Then, prepare serial dilutions according to the following scheme:

[0188] TIFF2026506966000005.tif59165 Keep all dilutions on ice until testing begins. In further studies, use the solutions from test tubes 5 to 7.

[0189] Conducting the analysis First enzymatic reaction 10 μl of each dilution of the test sample (two replicates) is added to the wells of the plate; the sample dilution solution is used as the comparison solution. 20 μl of substrate solution (4-methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate sodium salt) is added. The plate is covered with film and incubated in a thermostatic shaker at a temperature of (37.0±0.2)°C for 4 hours, stirring at a speed of 250 rpm. The plate is protected from light throughout the incubation period.

[0190] Second enzymatic reaction After incubation, add 20 μl of phosphate-citrate buffer solution to each well of the plate to stop the first enzymatic reaction. Then, add 10 μl of recombinant α-L-iduronidase solution to each well and mix gently. Cover the plate with film and incubate it in a thermostatic shaker at 37°C and 250 rpm for 22-26 hours. Protect the plate from light throughout the incubation period.

[0191] To stop the reaction, 200 μl of stop solution is added to each well containing the incubated mixture.

[0192] 260 μl of a dilution of the working standard solution of 4-methylumbelliferone sodium salt is added to the empty wells of the plate.

[0193] The fluorescence signal is measured in the wells of the plate at an excitation wavelength of 365 nm and a detection wavelength of 460 nm.

[0194] Evaluating the results Based on the results of measuring the fluorescent signal in the wells containing the dilutions of the working standard solution of 4-methylumbelliferone sodium salt, a graph of the linear relationship between the fluorescent signal and the concentration (nmol / mL) of 4-methylumbelliferone sodium salt is plotted. The linear function equation is used to calculate the concentration (nmol / mL) of 4-methylumbelliferone produced during the reaction in the wells containing the dilutions of the test sample and comparison solution.

[0195] The specific activity A (U / μg) for each dilution of the test sample is calculated using the following formula:

number

[0196] The final value of specific activity for the test sample is calculated as the average of the specific activities calculated for each dilution.

[0197] The results obtained to determine the enzymatic activity of the HIR-FAB-IDS and HIR-MAB-IDS variants compared to the drug, Elaprase®, are presented in Table 2.

[0198] Table 2. Evaluation of iduronate-2-sulfatase activity of HIR-FAB-IDS variants and HIR-MAB-IDS relative to unmodified iduronate-2-sulfatase enzyme (Elaprase®) [Table 2]

[0199] As can be seen from the results presented in Table 2, the HIR-FAB-IDS preparation exhibits specific enzyme (iduronate 2-sulfatase) activity toward the substrate 4-MU-αIdoA-2S (4-methylumbelliferyl α-L-idopyranosiduronate 2-sulfate) of 27.0 U / μg. At the same time, the unbound recombinant enzyme, iduronate 2-sulfatase, exhibits an activity of 14.6 U / μg. A full-length antibody against the insulin receptor coupled with the amino acid sequence of iduronate 2-sulfatase (HIR-MAB-IDS) exhibits a lower specific activity (11.0 U / μg) compared to HIR-FAB-IDS.

[0200] The performed studies showed that the iduronate-2-sulfatase in HIR-FAB-IDS retains the main functional (enzymatic) properties at the level of the free recombinant enzyme. At the same time, the specific activity of HIR-FAB-IDS is higher than that of the drugs Elaprase® and HIR-MAB-IDS (AGT-182).

[0201] Thus, increased enzymatic activity was detected for compounds containing a transport element that is a Fab fragment of immunoglobulin IgG specific for an epitope in the insulin receptor linked directly or through a linker to a therapeutic enzyme compared to a therapeutic enzyme that does not contain a transport element and a therapeutic enzyme containing a transport element represented by a MAB.

[0202] Example 3. Study of the efficacy of HIR-Fab-IDS preparations in peripheral tissues of mice of the 24744 strain: B6N.Cg-IdstmlMuen / J IDS KO In the HIR-Fab-IDS efficacy study, 60 hemizygous male iduronate-2-sulfatase knockout mice B6N.Cg-IdstmlMuen / J (JAX strain 024744) and 12 wild-type (C57BE / 6NJ) animals were used as controls. Based on their age at the time of the first dose (11 weeks), animals were randomized and treated weekly for 16 weeks with HIR-Fab-IDS at doses of 0.3, 1.0, and 3.0 mg / kg, or with saline as a control. Blood and urine samples were collected during the dosing period. The knockout mice in the control group exhibited urinary GAG levels 4.7-fold higher than those of normal animals. As shown in Figure 4, in the HIR-Fab-IDS-treated group, urinary GAG levels decreased to normal animal levels after the first dose and remained at the same level throughout the dosing period.

[0203] One hour after the final administration, the animals were anesthetized, and final blood and tissue samples were collected. Histological analysis of the tissues was performed, and iduronate-2-sulfatase activity levels and GAG accumulation in the tissues of the experimental animals were measured. Liver weights in the control knockout animals were 1.5-fold higher than those in wild-type animals. After drug administration, liver weights in the treated knockout mice returned to normal (Figure 5).

[0204] Iduronate-2-sulfatase activity levels in the plasma of knockout animals differed significantly (-70%) from those of wild-type animals. One hour after administration, all HIR-Fab-IDS-treated groups showed significantly higher values ​​of this parameter than control knockout animals (263-2408-fold) and wild-type animals (80-730-fold). Mice administered 0.3 mg / kg of the HIR-Fab-IDS compound had, on average, a 263-fold excess of iduronate-2-sulfatase activity compared to saline-treated knockout animals. The results of the analysis of iduronate-2-sulfatase activity in the plasma of experimental animals are presented in Figure 6.

[0205] Iduronate-2-sulfamidase activity levels in tissues of major organs (spleen, liver, kidney, and heart) of knockout animals not receiving drug treatment were significantly reduced (by an average of 76%) compared with normal animals. Knockout animals in all treatment groups showed significantly higher (17- to 56-fold) levels of iduronate-2-sulfatase enzyme activity 1 hour after drug administration compared with animals receiving saline, and 2.6- to 10-fold higher than that in wild-type animals. The HIR-Fab-IDS compound, when administered at low and moderate doses, did not significantly affect enzyme activity levels in brain tissue of knockout animals. At the high dose, iduronate-2-sulfatase activity increased by an average of 50% in brain tissue of knockout animals compared with placebo, but was 82% lower than that of wild-type animals.

[0206] GAG levels in tissues of major organs of knockout animals that did not receive drug were significantly (12-fold on average) higher than in normal animals. Knockout animals in all dose groups showed significantly lower (70% on average) GAG levels (-70% on average in all tissues and dose groups) compared with saline-treated animals, slightly exceeding the values ​​for this indicator in normal animals (1.0-1.5-fold in all tissues and dose groups). GAG levels in brain tissue did not differ between knockout and normal animals, nor between drug-treated knockout animals and the control group (saline-treated).

[0207] Thus, 16-week drug administration to iduronate-2-sulfatase gene knockout mice at doses of 0.3–3 mg / kg corrected the increased GAG levels in urine and tissues of major organs and normalized the increased liver weight to that of wild-type animals. One hour after dosing, animals in all three dose groups showed increased iduronate-2-sulfatase activity levels in plasma and tissues of major organs (except the brain) compared to values ​​in control knockout animals (263–2408-fold). In brain tissue from the treated knockout animals, this correction did not reach the values ​​obtained for wild-type animals. The efficacy of HIR-Fab-IDS was demonstrated at all doses studied in a mouse model of MPS II (Hunter syndrome).

[0208] Example 4. After intravenous administration to cynomolgus macaques, radiolabeled Fab fragments of antibodies against the insulin receptor linked to the amino acid sequence of iduronate-2-sulfatase [ 125 I]-HIR-Fab-IDS (SEQ ID NO: 2 and SEQ ID NO: 4), [ 125 I]-HIR-Mab-IDS, [ 125 Analysis of tissue distribution of [I]-IDS (control) Experimental animals were administered a single dose of each test molecule at a volume of 2 mL / kg body weight by intravenous bolus injection over 1–2 min (or 30 s) via the left femoral vein. The target radioactive dose level was 1 MBq / kg animal body weight for all study molecules.

[0209] Animals receiving the test substance were sedated with an intramuscular injection of ketamine hydrochloride followed by an intravenous overdose of Doretyl (pentaborbitone sodium). Two hours after administration of the test substance, the animals were humanely euthanized. During sedation, 2 mL of blood was collected from each animal's cephalic vein and centrifuged to obtain plasma. Radioactivity concentrations in the animal's plasma were measured. After death was confirmed, each animal was snap-frozen in a mixture of solid carbon dioxide in hexane. After complete freezing, the whole body was placed in a mold containing 2% (w / v) aqueous carboxymethylcellulose paste. For each animal, several longitudinal sagittal sections (nominal 30 μm) were made at at least five body levels and, if necessary, three head levels.

[0210] Sections mounted on Filmolux 610 tape (Neschan) were freeze-dried in a GVD03 benchtop freeze dryer (Girovac Ltd) and applied to a FUJI imaging plate (Type B AS-MS, Raytek Scientific Ltd). 125 I]-labeled blood standards were applied to the imaging plate.

[0211] After 7 days of development in a copper-lined lead box, the radiographic plates were processed using a FUJI FLA-5000 radiography system (Raytek Scientific Ltd). Electronic images were analyzed using a PC-based image analysis system (Seescan 2 software, LabLogic Ltd). Each autoradiogram contained a standard [ 125 I] was used to plot a calibration curve over a range of radioactivity concentrations.

[0212] The radioactivity concentration in plasma was detected by gamma measurement.

[0213] Tissue concentration data were recorded as Ig equivalents for [ 125 I]HIR-Fab-IDS, [ 125 I]HIR-Mab-IDS, and [ 125 I]-IDS (control).

[0214] Results were expressed to three significant digits, not more than two decimal places. Data tables were computer generated and individual data were rounded appropriately.

[0215] [ 125 I]HIR-Fab-IDS,[ 125 I]HIR-Mab-IDS and [ 125 [I]-IDS (control) was administered as a single intravenous dose to male cynomolgus monkeys at nominal dose levels of 0.0020 mg / kg, 0.0015 mg / kg, and 0.0010 mg / kg body weight. Because each solution contained different levels of radioactivity, the dose was varied to ensure a uniform level of 0.0015 mg / kg body weight. Two hours after each administration, animals were euthanized and frozen for whole-body autoradiography studies.

[0216] After intravenous administration, the drug was absorbed and distributed widely throughout the body tissues, and at the time of sacrifice, all tissues studied were exposed. The lower limits of quantification were 0.2, 0.03, and 0.097 ng drug equivalents / g for all animal tissue concentration measurements, respectively.

[0217] [ 125 Plasma and blood radioactivity concentrations in animals treated with [I]HIR-Fab-IDS were 2.55 and 3.11 ng eq / g, respectively (blood:plasma ratio 1.22). Quantifiable levels of radioactivity were present in several brain regions, including the medulla oblongata (1.09 ng eq / g), cerebral cortex (1.03 ng eq / g), cerebellum (0.908 ng eq / g), hypothalamus (0.869 ng eq / g), cerebellar dendritic substance (0.799 ng eq / g), pons (0.793 ng eq / g), and medulla (0.562 ng eq / g); TP ratios ranged from 0.22 to 0.43.

[0218] [ 125Radioactivity concentrations in the plasma and blood of animals treated with [I]HIR-Mab-IDS were 2.13 and 1.86 ng eq / g, respectively (blood:plasma ratio 0.87). Quantifiable levels of radioactivity were present in several brain regions of the animals. These regions included the medulla oblongata (0.803 ng eq / g), cerebellar dendritic material (0.606 ng eq / g), cerebellum (0.494 ng eq / g), cerebral cortex (0.453 ng eq / g), pons (0.438 ng eq / g), medulla (0.288 ng eq / g), and hypothalamus (0.279 ng eq / g), with TP concentration ratios ranging from 0.13 to 0.38.

[0219] [ 125 Plasma and blood concentrations in animals receiving [I]IDS were 0.910 and 0.753 ng Eq / g, respectively (blood:plasma ratio 0.83). Radioactivity was present at quantifiable levels in the whole brain (0.113 ng Eq / g), but concentrations were below the limit of quantification (0.097 ng Eq / g) in all regions examined: medulla oblongata, cerebral cortex, cerebellum, hypothalamus, cerebellar dendritic material, pons, and medulla. Concentrations in CNS tissues were significantly lower than in plasma and blood.

[0220] These results demonstrate that the HIR-Fab-IDS and HIR-Mab-IDS conjugates penetrate the blood-brain barrier better than the IDS conjugates, and that blood-borne radioactivity had little effect on the determination of CNS tissue concentrations when CNS tissues were exposed to the test substances or their labeled metabolites (Figs. 1 and 2). However, the HIR-Fab-IDS showed a broader distribution to body tissues than the HIR-Mab-IDS molecule (Table 5, Fig. 3).

[0221] It should be noted that in Example 10 of invention US8834874 B2 (ATG-182 or HIR-MAB-IDS), the penetration efficiency into human brain is evaluated at a level of 1% of the administered dose per 1000 grams of brain tissue, based on experimental data on the penetration of ATG-182 (HIR-MAB-IDS) into the brain of rhesus macaques. At this level of penetration, 20% of iduronate-2-sulfatase (IDS) activity in human brain is expected to be achieved, which is claimed to eliminate the accumulation of glycosaminoglycans in the patient's brain.

[0222] Considering the data obtained regarding penetration into the brain of cynomolgus monkeys (Table 5), it can be concluded that the compounds of the present invention penetrate into the brain of cynomolgus monkeys almost twice as well as HIR-MAB-IDS, i.e., 0.84 ng equivalents / g HIR-FAB-IDS vs. 0.43 ng equivalents / g HIR-MAB-IDS. This means that HIR-FAB-IDS penetrates into the human brain with 2% efficiency. Considering the calculations specified in Example 10 of invention US8834874 B2, it is expected that 40% of the iduronate-2-sulfatase activity in the human brain would be achieved, not taking into account the increased activity of HIR-FAB-IDS compared to HIR-MAB-IDS (AGT-182). See Table 2.

[0223] Considering the increased specific activity of HIR-FAB-IDS over HIR-MAB-IDS, shown in Example 2, Table 2, with 27 U / mcg for the actual compound and 11 U / mcg for the control, as well as better penetration into the brain: 27 / 11 = 2.4-fold higher iduronate-2-sulfatase specific activity, 0.84 / 0.43 = 1.95-fold better penetration into the brain, resulting in a 20% recovery of IDS activity in the brains of patients receiving HIR-MAB-IDS therapy, resulting in a 20% × 2.4 × 1.95 = 93.6% recovery of iduronate-2-sulfatase activity in the brains of patients with type II MPS from the IDS activity level in healthy human brains. Thus, the present invention allows for nearly complete recovery of IDS enzymatic function in the brains of patients, while the control HIR-MAB-IDS only recovers 20% (93% vs. 20%).

[0224] Thus, administration of a compound containing a transport element that is a Fab fragment of immunoglobulin IgG specific for an epitope in the insulin receptor linked directly or through a linker to a therapeutic enzyme provides a greater degree of displacement of the activity of the therapeutic enzyme in the human brain compared to a compound containing a Mab.

[0225] Table 5.[ 125 Radioactivity concentrations in cynomolgus monkey tissues after a single intravenous administration of [I]HIR-Fab-IDS [Table 5] TIFF2026506966000009.tif38165NA—Not applicable; BLQ—Concentration below the lower limit of quantification

[0226] The present specification discloses a compound containing a therapeutic enzyme that exhibits specific enzymatic activity and a transport element that has the ability to interact with insulin receptors and transport the therapeutic enzyme to tissue lysosomes (including transport to lysosomes in nervous tissue through the BBB). Thus, the compound exhibits high activity and an improved ability to be transported to lysosomes in tissue cells of various organs, including lysosomes in nervous tissue cells, suggesting the use of the present invention for enzyme replacement therapy for the treatment or prevention of subjects with lysosomal storage diseases.

[0227] Example 5. Components of pharmaceutical compositions Pharmaceutical compositions of the present invention used for therapeutic or preventive purposes may be prepared by mixing them with appropriate pharmaceutically acceptable carriers, excipients, etc., if necessary, and preparing them in the form of lyophilized preparations or solutions. Examples of suitable pharmaceutically acceptable carriers and excipients include sterile water, saline solution, stabilizers, excipients, antioxidants (e.g., ascorbic acid), buffers (e.g., phosphate, citrate, histidine buffers, or other organic acid-based buffers), preservatives, surfactants (e.g., PEG and Tween), chelating agents (e.g., EDTA), and binders. These may also contain other low-molecular-weight polypeptides, proteins such as serum albumin, gelatin, and immunoglobulins, amino acids such as glycine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, arginine, and lysine, sugars and carbohydrates such as polysaccharides and monosaccharides, and sugar alcohols such as mannitol and sorbitol. Aqueous injection solutions, such as saline, and isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, as well as suitable stabilizers, such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol and PEG), and nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer 188, and HCO-50), may be used in combination with each other. If hyaluronidase is mixed with the preparation, a large amount of liquid can be administered subcutaneously (Expert Opin. Drug Deliv., 4(4), July 2007; pp. 427-440). Furthermore, the pharmaceutical composition of the present invention may be pre-loaded into a syringe. For this purpose, the preparation may be prepared in the form of a solution according to the method described in WO2011 / 090088.

[0228] If desired, the antigen-binding molecules of the present invention may be incorporated into microcapsules (e.g., those composed of hydroxymethylcellulose, gelatin, and poly(methyl methacrylate)) or colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, e.g., Remington's Pharmaceutical Science, 16th edition, Oslo, 1980). Methods for preparing drugs in the form of sustained-release formulations are also well known, and these methods may be applied to the antigen-binding molecules of the present invention (Langer et al., J. Biomed. Mater. Res., 15, 1981, pp. 267-277; Langer, Chemtech., 12, 1982, pp. 98-105; US 3,773,919; European Patent Application Publication EP 58481; Sidman et al., Biopolymers, 22, 1983, pp. 547-556; EP 133988).

[0229] Pharmaceutical composition of the concentrate for preparing a solution for injection: Compound HIR-FAB-IDS 5.3 mg Sodium chloride 8.0 mg Sodium dihydrogen phosphate dihydrate 3.12 mg Sodium hydroxide to adjust the pH to pH 6.0 Polysorbate 20-0.2 mg Water for injection (enough to make 1.0 mL)

[0230] Example 6. Calculation and justification of starting dose for first use in humans Because the Phase I study planned single drug administration to healthy volunteers within the expected therapeutic range without titration to the maximum tolerated dose, calculation and justification of the starting dose for first use in humans was based on the NOAEL (No Observed Adverse Effect Level) and MABEL (Minimal Anticipated Biological Effect Level; the dose with the minimum expected biological effect) approach. Information on the efficacy and safety of drugs with similar mechanisms of action in humans was also taken into account.

[0231] The main results of efficacy and safety assessments in preclinical studies, reflecting the risk-benefit criteria, are presented in Table 6.

[0232] Table 6. Extrapolation to humans of doses obtained from studies of efficacy and systemic toxic effects of HIR-FAB-IDS compounds in adult animals [Table 6] *Dose extrapolation to a 70 kg human was performed without using an interspecies dose transfer factor based on body surface area.

[0233] In safety studies of the HIR-FAB-IDS compound in monkeys, doses of 3, 10, and 30 mg / kg administered weekly for 8 weeks were found to produce no adverse effects. In rats administered weekly for 8 weeks at doses of 3, 10, and 30 mg / kg, none of the doses studied produced adverse effects. Therefore, the highest nontoxic dose identified in 8-9 weekly doses in rodents and nonrodents is 30 mg / kg. The therapeutic index in preclinical studies, defined as the ratio of the highest safe dose (30 mg / kg) to the effective dose in multiple doses of the HIR-FAB-IDS compound (0.3 mg / kg), is 30 mg / kg / 0.3 mg / kg = 100.

[0234] Because the molecular weight of HIR-FAB-IDS exceeds 100 kDa, interspecies dose transfer factors based on body surface area were not used to extrapolate the doses listed in the table to humans. Therefore, the human equivalent maximum safe dose (HED NOAEL) is 30 mg / kg. In the interest of minimizing risk, a starting dose of 0.3 mg / kg should be used in clinical trials because it has been shown to cause minimal pharmacological effects in vivo and is safe.

[0235] To improve the safety of starting dose prediction in humans, calculations based on the MABEL approach were also performed. As a result of our in vitro pharmacokinetic studies, in a test to determine the activity of iduronate-2-sulfatase in fibroblasts from patients with MPS II after treatment with HIR-FAB-IDS compounds, the minimum concentration of HIR-FAB-IDS compounds with a biological effect is detected. In this test, a threshold increase (~20%) in iduronate-2-sulfatase activity is detected at 5x10 doses of HIR-FAB-IDS compounds. -9 M concentration. Considering the molecular weight of the HIR-FAB-IDS compound (105 kDa), this is 5.25 x 10 -4 This corresponds to a concentration of the HIR-FAB-IDS compound equivalent to 56L x 5.25 x 10 g / L, which may be considered the starting concentration in humans, and is achieved by adding 56L x 5.25 x 10 g / L of blood to an average human weighing 70 kg and having 56 liters (80%) of fluid in tissues. -4 g / L=294x10 -4 g of HIR-FAB-IDS compound (4.2 x 10 -4 g / kg or equivalent to 0.42 mg / kg). Thus, based on in vitro data, the MABEL approach made it possible to determine a starting effective dose of the HIR-FAB-IDS compound in humans of at least 0.42 mg / kg.

[0236] At the same time, the results of a pharmacodynamic (PD) study performed in vivo on B6N.Cg-IdstmlMuen / J mice (JAX strain no. 024744) knocked out for the iduronate-2-sulfatase gene indicate that the lowest dose of the HIR-FAB-IDS compound that elicits a pharmacodynamic effect (reducing the content of glycosaminoglycans in tissues) is 0.3 mg / kg.

[0237] Therefore, based on in vitro data, the MABEL approach determined that the starting dose of HIR-FAB-IDS in humans is at least 0.42 mg / kg, and based on in vivo data, the MABEL approach determined that the starting dose of HIR-FAB-IDS in humans is at least 0.3 mg / kg. According to existing regulations, in this case the lower of the specified values ​​must be taken as the starting dose, i.e. the starting effective dose of HIR-FAB-IDS for use in humans must be at least 0.3 mg / kg.

[0238] Therefore, the selection of the dose for the first use of the drug in humans was based on the following criteria (Table 7):

[0239] Table 7. Key data and results from preclinical studies to guide dose selection for human studies [Table 7]

[0240] Transient dose-dependent hypoglycemia was observed at a frequency of 6.4% during infusion at all dose levels and did not limit the performance of therapy.

[0241] Based on the above, the therapeutic dose in humans is expected to be in the range of 1-3 mg / kg, the lowest effective dose extrapolated to humans is 0.3 mg / kg, and drug penetration through the BBB is confirmed at doses of 0.0015-0.0020 mg / kg. The therapeutic index is 100.

[0242] The safe dose range of the drug, calculated based on preclinical safety data, reaches a maximum of 30 mg / kg of drug for adults and 10 mg / kg for children with repeated administration, which includes the expected therapeutic dose range with a safety factor of at least 10. A pharmacologically active dose of 0.3 mg / kg was adopted as the starting dose for first use in humans in the IDB-MPS-I Phase I clinical study.

[0243] The drug can only be administered intravenously as a 3-hour infusion.

[0244] Drug administration must be performed under the supervision of a physician or other medical professional experienced in treating patients with MPS II or other inherited metabolic disorders. Patients' condition will be closely monitored throughout the study, and patients will be required to remain at the research center for at least 6 hours after the first infusion of each new dose level of the HIR-FAB-IDS compound and for observation of the development of infusion reactions. Detailed information regarding drug dose, infusion duration, preparation and use of the infusion solution, and disposal of unused drug will be provided in the clinical study protocol.

[0245] Principles of dose selection for phase II-III clinical studies Based on preclinical and toxicological data, the results of the Phase I clinical study IDB-MPS-1, which showed good tolerability and a favorable safety profile of the investigational drug HIR-FAB-IDS using single doses ranging from 0.3 mg / kg to 3 mg / kg (0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, and 3 mg / kg), and taking into account the short half-life of the drug, the starting dose of the drug HIR-FAB-IDS for initial use in patients with MPS II can be determined to be <3 mg / kg.

[0246] The selection of a starting dose of 1 mg / kg also depends on the expected somatic effects of the HIR-FAB-IDS compounds, which are comparable to the therapeutic effects of Elaprase®. The HIR-FAB-IDS compounds do not differ from idursulfase in their interaction with M6PR, penetration into M6PR-presenting cells, and enzymatic activity in cell cultures from MPS II patients. Study results indicate that plasma clearance of the HIR-FAB-IDS compounds occurs primarily through interaction with the M6PR receptor, which would provide comparable exposure in M6PR cells in vivo.

[0247] HIR-FAB-IDS compounds reduce GAG ​​levels in fibroblasts from MPS II patients to the level of healthy donors at a concentration of 120 nM. Considering a blood volume of approximately 75 mL / kg in children over 3 years of age and approximately 70 mL / kg in adolescents, an effective concentration of 120 nM may be achieved by administering HIR-FAB-IDS compounds at a dose of 0.9-1.0 mg / kg.

[0248] In a 26-week toxicity study, the NOAEL was defined as 10 mg / kg / week, based on transient hypoglycemia, a predictable and expected type of effect. Hypoglycemia has been described with other fusion proteins that use the insulin receptor (HIR) to mediate drug transport across the BBB, including two HIR mAb products linked to iduronidase or idursulfase. A similar toxicity profile in preclinical studies (transient hypoglycemia at high doses up to 30 mg / kg) and individual cases of dose-related hypoglycemia in patients in the MPS II clinical trial confirm the safety of the selected starting dose of 1 mg / kg in children.

[0249] A starting dose of 1 mg / kg is also justified from an ethical point of view, as this dose avoids subtherapeutic administration and also makes it possible to reduce the already achieved effects of ERT.

[0250] Example 7. Proof of principle of penetration of HIR-Fab-IDS compounds through the BBB Proof of principle was obtained in patient 1001, a participant in the IDS-MPS-II / III clinical trial.

[0251] Patient 1001 was an 18-year-old male. The diagnosis of type II MPS (Hunter's disease) with a neurological component / neuropathy was confirmed in 2010 by a decrease in plasma iduronate-2-sulfatase level to 3.18 nM / 4 h / mL (normal: 297-705) and genotypically by the detection of a nucleotide substitution c.1327C>T in exon 09 of the IDS gene (OMIM 300823) in the hemizygous state, resulting in a translation termination p.R443. The nucleotide substitution had not been previously described, but computer analysis (Alamut Visual, version 2.10) suggested it may be pathogenic. Since 2011, the patient has been receiving enzyme replacement therapy with Elaprase® (INN iduronate-2-sulfatase).

[0252] Prior to initiation of therapy with the HIR-FAB-IDS compound, the patient's urinary glycosaminoglycan (GAG) excretion was near normal due to enzyme replacement therapy. During treatment, urinary GAG excretion rates did not change significantly, confirming comparable enzymatic activity of the drugs in the periphery. GAG concentrations in cerebrospinal fluid (CSF) were initially high: 3294.5 ng / mL heparan sulfate (HS) and 3.5 ng / mL dermatan sulfate (DS). In MPS II, GS accumulates primarily in the central nervous system (CNS), and experimental studies have shown that GS concentrations in CSF correlate with its accumulation in brain tissue (Tanaka N., Kida S., Kinoshita M., Morimoto H., Shibasaki T., Tachibana K., Yamamoto R. Evaluation of cerebrospinal fluid heparan sulfate as a biomarker of neuropathology in a murine model of mucopolysaccharidosis type II using high-sensitivity LC / MS / MS. Mol. Genet. Metab. 2018 Sep.; 125(l-2):53-58. doi: 10.1016 / j.ymgme.2018.07.013. Epub. 2018 Jul. 23. PMID: 30064964).

[0253] According to this protocol, patients received the HIR-FAB-IDS compound as a weekly intravenous infusion lasting 3 hours. After three doses, the dose was gradually increased from 1 to 3 mg / kg to assess safety and tolerability. Serum samples were collected for pharmacokinetic studies during each first administration of the drug at a new dose. Cerebrospinal fluid samples for drug content and GAG concentration were collected during screening (baseline) and 3 weeks after drug administration at doses of 2 and 3 mg / kg. CSF collection for drug concentration assessment was performed 2.5 hours after the end of the infusion.

[0254] The pharmacokinetics of the drug is nonlinear. The concentration values ​​of the HIR-FAB-IDS compound over time are presented in Table 8 and Figure 7.

[0255] Table 8. Concentration values ​​of HIR-FAB-IDS compounds in serum using different doses of the drug. [Table 8]

[0256] After drug administration at doses of 2 and 3 mg / kg 2.5 hours after the end of the infusion, drug was determined in the CSF to be at concentrations of 25.939 and 272.569 pg / mL using the 100 pg / mL NICO method (Table 9).

[0257] Table 9. Concentrations of HIR-FAB-IDS compounds in CSF and serum after drug administration at doses of 2 and 3 mg / kg. [Table 9] * LLOQ for CSF measurement, 100 pg / mL ** LLOQ for serum measurements: 50 ng / mL

[0258] A significant decrease in heparan sulfate (HS) accumulation in the CSF was also noted (Figure 8), confirming the presence of enzymatic activity of the HIR-FAB-IDS compound in central nervous system tissue and thus demonstrating the efficacy of the drug in treating patients with the neuropathic form of type II MPS.

Claims

1. 10. An HIR-Fab-IDS compound for preventing or treating lysosomal enzyme deficiency in a subject with a lysosomal storage disease, comprising a first amino acid sequence of SEQ ID NO:2 and a second amino acid sequence of SEQ ID NO:

4.

2. 2. The compound of claim 1, wherein the lysosomal storage disease is mucopolysaccharidosis type II or mucopolysaccharidosis type II with a neurological component.

3. The compound of claim 2, wherein the neurological component is indicated by a neuropathy type.

4. The compound according to any one of claims 1 to 3, wherein the subject is a human.

5. 10. The use of the compound of claim 1 for the prevention or treatment of lysosomal enzyme deficiency in a subject with a lysosomal storage disease, wherein the subject is administered at least one dose of about 1 to 12 mg / kg of the HIR-Fab-IDS compound.

6. The use according to claim 5, wherein the dose is selected from the group consisting of about 3 mg / kg, about 6 mg / kg, and about 12 mg / kg of the HIR-Fab-IDS compound.

7. The use according to any one of claims 5 to 6, wherein the lysosomal storage disease is mucopolysaccharidosis type II or mucopolysaccharidosis type II with a neurological component.

8. The use of claim 7, wherein the neurological component is indicated by a neuropathy type.

9. The use according to any one of claims 5 to 8, wherein the subject is a human.

10. A pharmaceutical composition for the prevention or treatment of lysosomal enzyme deficiency in a subject with a lysosomal storage disease, the composition comprising an HIR-Fab-IDS compound represented by a first amino acid sequence of SEQ ID NO: 2 and a second amino acid sequence of SEQ ID NO:

4.

11. 11. The pharmaceutical composition of claim 10, further comprising sodium chloride, sodium dihydrogen phosphate dihydrate, sodium hydroxide, and polysorbate.

12. 12. The pharmaceutical composition of claim 11, further comprising an HIR-Fab-IDS compound in an amount of 5.3 mg, sodium chloride in an amount of 8.0 mg, sodium phosphate dihydrate in an amount of 3.12 mg, sodium hydroxide to adjust the pH to pH 6.0, polysorbate 20 in an amount of 0.2 mg, and water for injection up to 1.0 mL.

13. The pharmaceutical composition according to any one of claims 10 to 11, wherein the lysosomal storage disease is mucopolysaccharidosis type II or mucopolysaccharidosis type II with a neurological component.

14. 14. The pharmaceutical composition of claim 13, wherein the neurological component is indicated by a neuropathy type.

15. The pharmaceutical composition according to any one of claims 10 to 14, wherein the subject is a human.

16. Use of the pharmaceutical composition described in claim 10 for the prevention or treatment of lysosomal enzyme deficiency in a subject with lysosomal storage disease, wherein the HIR-Fab-IDS compound is administered to the subject at least one dose selected from the group consisting of about 3 mg / kg, about 6 mg / kg, and about 12 mg / kg.

17. A method for preventing or treating lysosomal enzyme deficiency in a subject with a lysosomal storage disease, comprising administering to the patient at least one dose of an HIR-Fab-IDS compound represented by a first amino acid sequence of SEQ ID NO: 2 and a second amino acid sequence of SEQ ID NO: 4, wherein the dose is selected from the group consisting of about 3 mg / kg, about 6 mg / kg, and about 12 mg / kg of an HIR-Fab-IDS compound.

18. 17. The method of claim 16, wherein the HIR-Fab-IDS compound in the pharmaceutical composition is administered intravenously for 3 hours once a week.

19. 17. The method of claim 16, wherein the lysosomal storage disease is mucopolysaccharidosis type II or mucopolysaccharidosis type II with a neurological component.

20. 20. The method of claim 18, wherein the neurological component is indicated by a neuropathy type.

21. The method of any one of claims 16 to 19, wherein the subject is a human.

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