Targeted medical lysosomal enzyme fusion proteins, their related formulations and uses

Abstract

This application generally relates to targeted medical lysosomal enzyme fusion proteins, their related formulations and uses, and specifically relates to a medical lysosomal enzyme fusion protein suitable for treating lysosomal storage disorders, a liquid formulation containing such fusion protein, and related methods suitable for treating lysosomal storage disorders in mammals.

Description

[0001] This application is a divisional application of Chinese patent application No. 201780025534.1, filed on February 24, 2017, entitled “Targeted Medical Lysosomal Enzyme Fusion Protein, Related Formulations and Uses thereof”. Technical Field

[0002] This application generally relates to a medical lysosomal enzyme fusion protein suitable for treating lysosomal storage diseases, a liquid formulation containing such fusion protein, and related methods for treating lysosomal storage diseases in mammals. Background Technology

[0003] Normally, mammalian lysosomal enzymes are synthesized in the cytoplasm and cross the ER, where they undergo glycosylation with N-linked high-mannose carbohydrates. In the Gorgithostomyne, high-mannose carbohydrates are modified by adding mannose-6-phosphate (M6P) to lysosomal enzymes, thereby directing these proteins to the lysosome. The M6P-modified enzymes are then delivered to the lysosome via interaction with one or both of the two M6P receptors.

[0004] The deficiency of one or more lysosomal enzymes in lysosomes can directly or indirectly lead to more than 40 types of lysosomal storage disorders (LSD). Therefore, enzyme replacement therapy for LSD is actively being explored. This therapy typically involves the uptake of LSD protein and its delivery to various lysosomes in an M6P-dependent manner. One possible approach involves purifying the LSD protein and using M6P modification to incorporate carbohydrate moieties. This modified substance may be more effectively absorbed by cells than unmodified LSD protein because it interacts with M6P receptors on the cell surface.

[0005] In the past, peptide-based targeting technologies have been developed to more effectively deliver therapeutic enzymes to lysosomes. This proprietary technology is called Glycosylation Independent Lysosomal Targeting (GILT) because it replaces the M6P with a peptide tag linked to the therapeutic enzyme, acting as a part of the protein that targets the lysosome. Detailed descriptions of GILT technology can be found in U.S. Patent Application Publications Nos. 2003-0082176, 2004-0006008, 2003-0072761, 2005-0281805, and 2005-0244400; U.S. Patent Applications Nos. 8,492,337 and 8,563,691; and International Publications WO 03 / 032913, WO 03 / 032727, WO 02 / 087510, WO 03 / 102583, WO2005 / 078077, WO 2009 / 137721, and WO 2014 / 085621, all of which are incorporated herein by reference. Summary of the Invention

[0006] This application further provides modified compositions, formulations, and methods based on GILT technology to effectively target lysosomes with medical fusion proteins. Specifically, this application provides a method and composition using a lysosome-targeting peptide to target medical lysosomal enzymes to lysosomes for the treatment of lysosomal storage diseases. This application also provides a method and composition using a lysosome-targeting peptide to target lysosomal enzymes to lysosomes, wherein the peptide has reduced or eliminated binding affinity to IGF-I receptors, and / or reduced or eliminated binding affinity to insulin receptors, and / or resists furin cleavage. This application also provides a targeted lysosomal enzyme fusion protein comprising lysosomal enzymes, IGF-II, and a spacer peptide, which can improve the manufacture and lysosomal absorption of the lysosomal enzyme fusion protein. Examples of lysosomal enzymes applicable to the medical fusion proteins incorporated in this application and diseases treated using these fusion proteins are shown in Table 1 below. In some preferred embodiments, the lysosomal enzyme is a mature human α-N-acetylglucosidase (Naglu) enzyme, and the lysosomal storage disease is mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo B Syndrome).

[0007] In one aspect, the medical fusion protein of this application comprises a functional α-N-acetylglucosidase enzyme, which has detectable enzymatic activity and has an amino acid sequence that is similar to... Figure 1The amino acid sequence (SEQ ID NO: 1) of the mature human Naglu protein shown has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. On the other hand, this application relates to a... Figure 1 The fragment shown is a mature human Naglu protein (SEQ ID NO: 1) that retains detectable Naglu enzyme activity.

[0008] On the other hand, the medically applicable fusion protein of this application contains a peptide marker having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of amino acids 8-67 of mature human IGF-II. Based on this, amino acid 8-67 of mature human IGF-II has the following amino acid sequence: LCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO: 2).

[0009] In various embodiments, the targeted therapeutic fusion protein of this application comprises a peptide tag having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence 8-67 of mature human IGF-II, wherein alanine replaces arginine at amino acid position 37. In this particularly preferred embodiment, the peptide tag has the following amino acid sequence: LCGGELVDTLQFVCGDRGFYFSRPASRVS A RSRGIVEECCFRSCDLALLETYCATPAKSE (SEQ ID NO: 3). It has been previously reported that the substitution of arginine at amino acid position 37 with alanine can disrupt at least one furin protease cleavage site (see, for example, U.S. Patent No. 8,563,691).

[0010] On the other hand, the medical fusion protein of this application includes a spacer peptide between the lysosomal enzyme and the peptide label, and links the lysosomal enzyme and the peptide label. In various embodiments, the amino acid sequence of this spacer peptide / linking peptide is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a rigid linking peptide having the following 31 amino acid sequence: GAGGGSPAPAPTPAPAPTPAPAGGGPSGAP (SEQ ID NO: 4). In various embodiments, the length of the spacer peptide is about 25-37, 26-36, 27-35, 28-34, 29-33, or 30-32 amino acids, and represents a variant of SEQ ID NO: 4, wherein 1, 2, 3, 4, 5, or 6 specific amino acids of SEQ ID NO: 4 have been substituted, added, or deleted.

[0011] On the other hand, the medical fusion protein of this application comprises (i) a functional α-N-acetylglucosidase enzyme, which has an amino acid sequence similar to that of... Figure 1 The amino acid sequence of the mature human Naglu protein shown (SEQ ID NO: 1) has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence SEQ ID NO: 3; (ii) a peptide label having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence SEQ ID NO: 3; and (iii) a spacer / linker peptide located between the enzyme and the peptide label having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of the 31-amino acid rigid linker peptide of SEQ ID NO: 4 shown herein. In one embodiment (referred to herein as BMN001), the medical fusion protein of this application comprises (i) a functionally mature human α-N-acetylglucosidase (Naglu) enzyme, which has Figure 1 The amino acid sequence shown is (SEQ ID NO: 1), (ii) a spacer peptide / linker peptide having the amino acid sequence shown in SEQ ID NO: 4, and (iii) an IGF-II peptide label having the amino acid sequence SEQ ID NO: 3. The complete amino acid sequence of the BMN001 medical fusion protein is shown in [the original text]. Figure 2 (SEQ ID NO: 5).

[0012] On the other hand, this application provides a pharmaceutical composition suitable for treating lysosomal storage diseases in mammals, wherein the composition comprises the medically applicable fusion protein of this application. In various embodiments, the pharmaceutical composition is a formulation comprising (a) a fusion protein comprising a lysosomal enzyme or a functional fragment thereof, a peptide label having at least 90% sequence identity with SEQ ID NO: 2, and a spacer peptide located between the lysosomal enzyme or its functional fragment and the peptide label, the spacer peptide having at least 90% identity with SEQ ID NO: 4; and (b) one or more components selected from the group consisting of buffers, isotonic agents, and electrolytes. The formulation of this application may be a liquid formulation, a lyophilized formulation, or a liquid formulation composed of a lyophilized formulation rehydrated with water. In various embodiments, the formulation of this application is stable.

[0013] In various embodiments, formulations or compositions of the subject matter of this application may include lysosomal enzymes comprising the amino acid sequence SEQ ID NO: 1 or functional fragments thereof. Formulations of this application may include fusion proteins comprising or constituting the amino acid sequence SEQ ID NO: 5.

[0014] The formulations of this application may contain a fusion protein containing the amino acid sequence SEQ ID NO: 5, a buffer, an isotonic agent, an electrolyte, and an anti-adsorption agent. In various embodiments, the formulations of this application contain a fusion protein containing the amino acid sequence SEQ ID NO: 5, sodium divalent phosphate heptahydrate, sodium monovalent phosphate monohydrate, sodium chloride, and trehalose. In one embodiment, the formulation of this application contains a fusion protein containing the amino acid sequence SEQ ID NO: 5 (concentration from about 25 mg / ml to about 35 mg / ml), sodium divalent phosphate heptahydrate (concentration from about 0.15 mg / ml to about 0.25 mg / ml), sodium monovalent phosphate monohydrate (concentration from about 0.03 mg / ml to about 0.05 mg / ml), sodium chloride (concentration from about 0.8 mg / ml to about 1 mg / ml), and trehalose (concentration from about 7% to about 9%), and the pH of the formulation is in the range of about 6.5 to about 7.5. In various embodiments, the formulations of this application comprise a fusion protein containing the amino acid sequence SEQ ID NO: 5 (concentration of about 30 mg / ml), sodium divalent phosphate heptahydrate (concentration of about 0.19 mg / ml), sodium monovalent phosphate monohydrate (concentration of about 0.04 mg / ml), sodium chloride (concentration of about 0.88 mg / ml), and trehalose (concentration of about 8%), and the pH of the formulation is about 7.0. These formulations may be in the form of an aqueous solution or a dry / lyophilized form.

[0015] In other embodiments, the formulation of this application comprises a fusion protein containing the amino acid sequence SEQ ID NO: 5, sodium divalent phosphate heptahydrate, sodium monovalent phosphate monohydrate, sodium chloride, trehalose, and polysorbate 20. In one embodiment, the formulation of this application comprises a fusion protein containing the amino acid sequence SEQ ID NO: 5 (concentration of about 25 mg / ml to about 35 mg / ml), sodium divalent phosphate heptahydrate (concentration of about 0.15 mg / ml to about 0.25 mg / ml), sodium monovalent phosphate monohydrate (concentration of about 0.03 mg / ml to about 0.05 mg / ml), sodium chloride (concentration of about 4.5 mg / ml to about 5.5 mg / ml), trehalose (concentration of about 3% to about 5%), and polysorbate 20 (concentration of about 0.0025% to about 0.0075%), and the pH of the formulation is in the range of about 6.5 to about 7.5. In various embodiments, the formulations of this application comprise a fusion protein containing the amino acid sequence SEQ ID NO: 5 (concentration of about 30 mg / ml), sodium divalent phosphate heptahydrate (concentration of about 0.19 mg / ml), sodium monovalent phosphate monohydrate (concentration of about 0.04 mg / ml), sodium chloride (concentration of about 5 mg / ml), trehalose (concentration of about 4%), and polysorbate 20 (concentration of about 0.005%), and the pH of the formulation is about 7.0. These formulations may be in the form of an aqueous solution or a dry / lyophilized form.

[0016] In various embodiments, the formulations of this application comprise sodium dibasic phosphate heptahydrate, sodium monobasic phosphate monohydrate, sodium chloride, potassium chloride, magnesium chloride hexahydrate, and calcium chloride dehydrate, wherein the fusion protein comprises the amino acid sequence SEQ ID NO: 5. In another embodiment, the formulation of this application comprises a fusion protein containing the amino acid sequence SEQ ID NO: 5 (concentration of about 25 mg / ml to about 35 mg / ml), sodium divalent phosphate heptahydrate (concentration of about 0.15 mg / ml to about 0.25 mg / ml), sodium monovalent phosphate monohydrate (concentration of about 0.03 mg / ml to about 0.05 mg / ml), sodium chloride (concentration of about 8 mg / ml to about 9 mg / ml), potassium chloride (concentration of about 0.15 mg / ml to about 0.3 mg / ml), magnesium chloride hexahydrate (concentration of about 0.1 mg / ml to about 0.2 mg / ml), and calcium chloride dihydrate (concentration of about 0.15 mg / ml to about 0.3 mg / ml), and the pH of the formulation is in the range of about 6.5 to about 7.5. In various embodiments, the formulations of this application comprise a fusion protein containing the amino acid sequence SEQ ID NO: 5 (concentration of about 30 mg / ml), sodium divalent phosphate heptahydrate (concentration of about 0.19 mg / ml), sodium monovalent phosphate monohydrate (concentration of about 0.04 mg / ml), sodium chloride (concentration of about 8.66 mg / ml), potassium chloride (concentration of about 0.22 mg / ml), magnesium chloride hexahydrate (concentration of about 0.16 mg / ml), and calcium chloride dihydrate (concentration of about 0.21 mg / ml), and the pH of the formulation is about 7.0. These formulations may be in the form of an aqueous solution or a dry / lyophilized form.

[0017] In another aspect, this application relates to a method for treating lysosomal storage diseases in an individual, wherein such methods include the step of administering a composition or formulation of the substance described herein. In various embodiments, this application relates to a method for treating MPS IIIB disease in an individual, wherein the method includes the step of administering a medical fusion protein having Naglu enzyme activity as described herein, or a formulation containing therein. In some embodiments, the formulation is administered intrathecally, intraventricularly, or directly to CSF ​​via lumbar puncture, and may be non-volume or isovolume. The administration of the medical formulation of this application can be continuous for about 5 minutes to about 240 minutes or longer, or about 5 minutes to about 10 minutes. In some embodiments, the formulation for treating MPS IIIB can be administered weekly for at least 24 weeks, preferably at least 48 weeks. In one embodiment, administering a medically effective amount of the present application’s medically effective fusion protein or formulation results in a reduction in the severity, intensity, or frequency of at least one symptom or feature of MPS IIIB disease, or a delay in its onset.

[0018] In another aspect, this application relates to a method for slowing the rate of decline or preventing the decline of at least one symptom of MPS IIIB in an individual suffering from MPS IIIB disease, wherein the method comprises administering to the individual a medically effective fusion protein having Naglu activity or a formulation containing therein. In various embodiments, the formulation is administered intraventricularly, wherein the intraventricular administration is isovolemic. The administration of the medically effective formulation of this application can be performed continuously for about 5 minutes to about 240 minutes or longer, or for about 5 minutes to about 10 minutes. In some embodiments, the formulation for treating MPS IIIB can be administered weekly for at least 24 weeks, preferably at least 48 weeks. These methods can result in improvement of at least one symptom of MPS IIIB disease. In various embodiments, at least one symptom of MPS IIIB disease may be selected from the group consisting of: cognitive decline, language decline, motor decline, social-emotional decline, adaptive decline, conceptual thinking decline, facial recognition decline, storytelling completion decline, hand function / dexterity decline, hearing loss, hyperactivity, aggression, or sleep disturbance.

[0019] In various aspects, the determination of the slowing or prevention of the rate of decline of at least one symptom is: (a) determining the rate of decline of the symptom before medication, and (b) determining the rate of decline of the symptom after medication; wherein comparing the rate of decline of the symptom after medication with that before medication indicates a slowing of the rate of decline. These methods may further include the steps of determining the individual's developmental quotient (DQ) before medication and determining the individual's DQ after medication, wherein a higher DQ after medication indicates a slower rate of decline. Therefore, in various embodiments, this application relates to a method for stabilizing or slowing the decline of the DQ quotient in an individual suffering from MPS IIIB disease, wherein the method includes administering a medically derived fusion protein or a formulation containing it to the individual. The developmental quotient can be determined using the BSID-III or KABC-II tools described herein and known in the relevant art.

[0020] In another aspect, this application relates to a method for slowing the rate of cognitive decline in an individual suffering from MPS IIIB, wherein the method includes administering to the individual a medically effective amount of the present application's medical fusion protein or formulation thereof. In some embodiments, the fusion protein or formulation thereof is administered via IT, ICV, or lumbar puncture, wherein the administration method may be isovolemic. Administration of the present application's medical formulation may be performed continuously for about 5 minutes to about 240 minutes or longer, or for about 5 minutes to about 10 minutes. In various different embodiments, the formulation for treating MPS IIIB may be administered weekly for at least 24 weeks, or at least 48 weeks.

[0021] In another aspect, this application relates to a method for reducing or preventing the accumulation of GAGs in one or more tissues of the CNS in an individual suffering from lysosomal storage disease, wherein the method comprises administering a medically effective amount of the medically effective fusion protein or formulation described herein. In one embodiment, the GAG ​​is heparan sulfate, and the lysosomal storage disease is MPSIIIB. As described herein, the administration method may be intraventricular, and may be isovolumetric. In various embodiments, the reduction of GAG accumulation in cellular lysosomes of one or more tissues of the CNS includes, for example: gray matter, white matter, periventricular regions, meninges, pia mater, deep tissues of the cerebral cortex, neocortex, cerebellum, caudate / putamen region, molecular layer, deep regions of the pons or medulla oblongata, midbrain, or a combination of both or more of the above.

[0022] In various embodiments, the therapeutic fusion protein is delivered to neurons, glial cells, perivascular cells, meningeal cells, and / or neurons of the spinal cord. In some embodiments, administration of the therapeutic fusion protein or a formulation containing it results in a reduction of GAG storage in one or more target brain tissues or spinal cord neurons by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1, 1.5, or 2 times or more compared to a suitable control group (e.g., GAG storage in a pre-treatment individual).

[0023] Other features, subject matter, and advantages of this application will become apparent from the following detailed description. However, it is understood that while embodiments of the application are shown in the detailed description, they are for illustrative purposes only and are not intended to be limiting. Various variations and modifications within the scope of this application will be apparent to those skilled in the art from this detailed description. Attached Figure Description

[0024] Figure 1 Present the amino acid sequence of the mature human Naglu protein (SEQ ID NO: 1).

[0025] Figure 2 Present the amino acid sequence (SEQ ID NO: 5) of the BMN001 medical fusion protein.

[0026] Figure 3 To present a set of graphs showing the heparan sulfate (HS) levels in the central nervous system (CNS) (top) and cerebrospinal fluid (CSF) (bottom) when treated with natriuretic, 12 mg BMN001, or 48 mg BMN001 in control heterozygous Naglu dogs and homozygous Naglu-resistant MPS IIIB dogs. The data confirm that BMN001 reduced HS levels in MPS IIIB dogs to levels found in these heterozygous, disease-free dogs.

[0027] Figure 4 The graph comparing the HS content of CNS and CSF shows a strong correlation (r) between the HS content in these two compartments. 2 = 0.824).

[0028] Figure 5 Western ink dot plots were performed on the canine cerebellum of wild-type dogs, untreated MPS IIIB dogs, and BMN001-treated MPS IIIB dogs using an antibody targeting the canine LAMP2 protein as a probe. The results showed that the LAMP2 content in the untreated MPS IIIB dogs was higher than that in the wild-type group, and that BMN001 treatment reduced the LAMP2 content in the treated MPS IIIB dogs to the level of the wild-type group.

[0029] Figure 6 To present a graph showing the cerebellar heparan sulfate (HS) levels in control heterozygous Naglu dogs and homozygous Naglu-resistant MPS IIIB dogs treated with natriuretic agent, 12 mg BMN001, or 48 mg BMN001. The data confirmed that BMN001 reduced HS levels in the cerebellum of MPS IIIB dogs to levels found in these heterozygous, disease-free dogs.

[0030] Figure 7 To illustrate the mean cerebellar diffusion in control group heterozygous Naglu dogs and homozygous Naglu-ineffective dogs with MPS IIIB, when treated with mordant, 12 mg BMN001, or 48 mg BMN001.

[0031] Figure 8 A set of cerebellar MRI images of wild-type dogs treated with mediator or BMN001 after treatment with mediator or BMN001.

[0032] Figure 9 This is a bar graph showing elevated heparan sulfate (HS) and MPS IIIB-specific HS non-reducing terminus (NRE) in cerebrospinal fluid (CSF) of untreated MPS IIIB patients. The dashed vertical lines represent the mean normal CSF levels of HS and NRE (0.05 mg / L and 0.0025 mg / L, respectively). This data was collected continuously for at least 24 weeks to understand the natural history of the disease. Data from early and late time points within the natural history are presented.

[0033] Figure 10 To present a set of photographs showing a reduction in HS and NRE in the CSF of two individuals who received BMN001 treatment.

[0034] Figure 11A and 11B To show the effect of trehalose and trehalose-polysorbate 20 combination on the formation of aggregate particles after being pumped through a formulation containing BMN001.

[0035] Figure 12 To show the effect of trehalose and trehalose-polysorbate 20 combination on the formation of aggregate particles after freeze-thaw pressure in a formulation containing BMN001. Artificial cerebrospinal fluid (aCSF) is BMN001 formulated without trehalose or polysorbate 20.

[0036] definition The term "lysosomal storage disorder" as used in this article refers to a group of genetic diseases caused by the absence of at least one enzyme (e.g., acid hydrolase) required in lysosomes to break down macromolecules into peptides, amino acids, monosaccharides, nucleic acids, and fatty acids. Therefore, individuals with lysosomal storage disorders accumulate substances in their lysosomes. Examples of lysosomal storage disorders are listed in Table 1.

[0037] As used herein, the term "lysosomal enzyme" refers to any enzyme that can reduce the accumulation of substances in mammalian lysosomes or rescue or alleviate one or more symptoms of lysosomal storage disorders. Lysosomal enzymes suitable for this application include wild-type or modified lysosomal enzymes, which can be manufactured using recombinant and synthetic methods or purified from natural sources. Examples of lysosomal enzymes are listed in Table 1, wherein such lysosomal enzymes can be incorporated into the therapeutic fusion proteins described herein.

[0038] As used herein, the term "human α-N-acetylglucosidase" refers to wild-type human α-N-acetylglucosidase, or a functional fragment or variant thereof, either as a precursor (i.e., containing a natural signaling peptide sequence) or processed (i.e., lacking a natural signaling peptide sequence), that can reduce glycosaminoglycan (GAG) levels in mammalian lysosomes or rescue or alleviate one or more symptoms of MPS IIIB (St. Philippian disease type B). In one embodiment, the human Naglu enzyme used herein comprises or is composed of the following: Figure 1 The amino acid sequence shown is (SEQ ID NO: 1).

[0039] The term “functional” as used in this article, in relation to lysosomal enzymes, fusion proteins containing lysosomal enzymes, or fragments thereof, refers to a polypeptide capable of being absorbed by mammalian lysosomes and possessing enzymatic activity sufficient to reduce the storage material (i.e., glycosaminoglycans (GAGs)) in mammalian lysosomes.

[0040] As used herein, the term "spacer peptide" (also known as a "linker") refers to a peptide sequence located between two protein moieties in a fusion protein. Spacer peptides are typically designed to be flexible or to be inserted between two protein moieties, such as an α-helix. The length of spacer peptides can vary, for example, 10-50, 20-40, or 25-35 amino acids. Examples of spacer peptide sequences are disclosed in more detail in this specification.

[0041] As used herein, the terms “improvement,” “enhancement,” or “reduction,” or their grammatical synonyms, refer to values ​​relative to baseline measurements (e.g., measurements of the same individual prior to initiation of the treatment described herein, or measurements of control individuals (or multiple control individuals) who did not receive the treatment described herein). A “control individual” is an individual with the same type of lysosomal storage disease (e.g., MPS IIIB (St. Philippian disease type B)) as the treated individual, and whose age is approximately the same as the treated individual (to ensure that the treatment group and control group are at comparable stages of the disease).

[0042] As used herein, the terms “mitigation”, “relief”, and their grammatical synonyms refer to the prevention, reduction, or alleviation of a disease state or symptoms, stabilization of a declining disease state or symptoms, or improvement of a disease state or symptoms for an individual. Relief includes (but does not necessarily require) complete recovery from or complete prevention of disease symptoms. In some embodiments, relief includes reducing the accumulation of substances within the lysosomes of the associated lysosomal storage disease tissue.

[0043] As used in this article, the terms “individual,” “subject,” or “patient” refer to an individual, whether human or a non-human mammal. The subject being treated (also referred to as the “patient” or “individual”) is an individual (fetus, infant, toddler, adolescent, or adult) who has lysosomal storage disease, such as MPS IIIB (St. Philippian disease type B) (i.e., juvenile, adolescent, or adult type, or severe / typical or weakened MPS IIIB (St. Philippian disease type B)) or who may develop lysosomal storage disease (e.g., MPS IIIB (St. Philippian disease type B)).

[0044] As used herein, the term "medically effective dose" or "effective dose" refers to the dosage of a target therapeutic fusion protein (or a formulation containing it) that provides therapeutic efficacy to the treated individual at a reasonable benefit / risk ratio applicable to any medical treatment. This therapeutic efficacy can be objective (i.e., measurable by certain tests or markers) or subjective (i.e., indicated or perceived by the individual). Specifically, a "medically effective dose" refers to the dosage of a therapeutic fusion protein or related pharmaceutical composition that effectively treats, alleviates, or prevents a desired disease or condition, or has detectable therapeutic or preventative efficacy (e.g., alleviating disease-related symptoms, preventing or delaying disease onset, and / or reducing the severity or frequency of disease symptoms). Therapeutic effective doses are typically administered over a course of treatment and may include multiple unit doses. For any given therapeutic fusion protein, the therapeutic effective dose (and / or the appropriate unit dose within an effective course of treatment) may vary, for example, depending on the route of administration or combination with other medications. Furthermore, the definitive medically effective dose (and / or unit dose) for any particular patient may depend on a variety of factors, including the disease being treated and its severity; the activity of the specific drug used; the specific components used; the patient's age, weight, general health condition, sex, and diet; the timing and route of administration, and / or the excretion or metabolic rate of the specific fusion protein used; the duration of treatment; and similar factors known in the medical field.

[0045] As used herein, the term "treatment" (also referred to as "manipulation" or "disposition") means any administration of a medical fusion protein or a pharmaceutical composition containing such medical fusion protein, which may partially or completely reduce, alleviate, relieve, suppress, delay the onset, reduce the severity, and / or decrease the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Such treatment may be for individuals who do not exhibit symptoms of the relevant disease, disorder, and / or condition, and / or for individuals who only exhibit early symptoms of the disease, disorder, and / or condition.

[0046] Or, in addition, such treatment may be for individuals with one or more established symptoms of a related disease, disorder, and / or condition. For example, treatment may refer to improving cardiac status (e.g., increasing end-diastolic and / or end-systolic volume, or reducing, alleviating, or preventing progressive cardiomyopathy typically seen in, for example, Pompe disease) or improving lung function (e.g., increasing crying lung capacity beyond baseline, and / or restoring oxygen saturation that has decreased during crying to normal); improving neurodevelopment and / or motor skills (e.g., improving AIMS scores); reducing the amount of glycosaminoglycans (GAGs) in tissues of individuals with the disease; or any combination of these effects. In some embodiments, the treatment includes improving GAG clearance, specifically reducing or preventing neuronal symptoms associated with MPS IIIB (St. Philippian disease type B).

[0047] A formulation containing "stable" or "well-established" proteins is one in which the protein components substantially retain their physical, functional, and / or chemical stability over storage time. Stability can be determined at a selected temperature and within a selected time period. Preferably, the formulation is stable at room temperature (approximately 30°C). o C) or about 40 o C remains stable for at least 1, 3, 6, or 12 months or longer, in approximately 2-8 weeks. o Stable at C for at least 1, 3, 6, 12, 18, 24, 30, 36, 42, or 48 months or longer, or at -30 o C, -40 o C or -60 o Stable for at least 1, 3, 6, 12, 18, 24, 30, 36, 42, or 48 months or longer. In one aspect, the degree of protein degradation or aggregation during storage is used as an indicator of protein stability. Thus, a “stable” formulation may be a non-degradable or non-aggregating formulation in which the protein component content is less than about 20%, more preferably less than about 10%, and most preferably less than about 5%, 4%, 3%, 2%, or 1%. A “stable” formulation substantially retains the same functional or medicinal characteristics, or the same physical and / or chemical integrity, as freshly prepared formulations. Various analytical techniques for determining protein stability are available from related art, and see, for example: Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993).

[0048] "Isovolition" in relation to intrathecal administration of a pharmaceutical composition to an individual's CSF means that approximately the same volume of CSF is withdrawn from the individual before administering a specified volume of pharmaceutical composition, thereby maintaining approximately the same fluid volume in the individual's CSF compartment.

[0049] The terms “about” and “approximately” are used interchangeably in this application. Any numerical values ​​used or not used in this application refer to any normal deviations that are understood by a person skilled in the art. Detailed Implementation

[0050] This application provides a composition, formulation, and method for a medical fusion protein that effectively targets lysosomes based on GILT technology. Specifically, this application provides a method and composition using a lysosome-targeting peptide to target a medical lysosomal enzyme to lysosomes for the treatment of lysosomal storage diseases. This application also provides a method and composition for targeting lysosomes with a peptide that has reduced or eliminated binding affinity to IGF-I receptors and / or reduced or eliminated binding affinity to insulin receptors, and / or resists furin cleavage, thereby targeting lysosomal enzymes to lysosomes. This application also provides a targeted lysosomal enzyme fusion protein comprising lysosomal enzymes and IGF-II, and a spacer peptide that improves the production and absorption of the lysosomal enzyme fusion protein into the lysosome. This application also provides a formulation comprising a targeted lysosomal enzyme fusion protein and its use in the treatment or prevention of lysosomal storage diseases.

[0051] Various aspects of this application are described in detail in the following sections. These sections are not intended to limit this application. Each section applies to any aspect of this application. In this specification, the word "or" means "and / or" unless otherwise stated.

[0052] Lysosomal enzymes Lysosomal enzymes suitable for inclusion in the medical fusion proteins or formulations of this application include any enzyme that can reduce the accumulation of substances in mammalian lysosomes or can rescue or alleviate the symptoms of one or more lysosomal storage disorders. Suitable lysosomal enzymes include both wild-type and modified lysosomal enzymes (and their functional fragments), and can be manufactured by recombinant or synthetic methods or purified from natural sources. Examples of lysosomal enzymes are listed in Table 1.

[0053] Table 1. Lysosomal storage diseases and related lysosomal storage diseases In some embodiments, the lysosomal enzymes included herein have an amino acid sequence that is approximately 90% to 100% identical, including 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the native or mature amino acid sequences of human enzymes as shown in Table 1, while still encoding functional proteins, i.e., capable of reducing the accumulation of substances such as GAGs in mammalian lysosomes or rescuing or alleviating one or more symptoms of lysosomal storage diseases. The sequences of the aforementioned enzymes are available to those skilled in the art and can be obtained through publicly available databases (e.g., the Center for Biotechnology Information at the US National Library of Medicine).

[0054] The "percentage of amino acid sequence identity (%)" between the major amino acid sequence and the reference amino acid sequence is defined as the percentage of identical amino acid residues obtained when the main sequence amino acid residues are sequence-aligned with the relevant reference sequence, with gaps introduced if necessary to achieve maximum sequence identity, without considering any retainable substitutions as part of the sequence identity. The alignment performed to determine the percentage of amino acid sequence identity can be done in various ways within the relevant art, using publicly available computer software (such as BLAST, ALIGN, or Megalign (DNASTAR) software). Those skilled in the art can determine the appropriate parameters used to determine the alignment, including any algorithms required to achieve maximum alignment across the full-length sequence. Preferably, WU-BLAST-2 software is used to determine amino acid sequence identity (Altschul et al., Methods in Enzymology 266, 460-480 (1996)). WU-BLAST-2 uses several search parameters, most of which are pre-defined. The adjustable parameters are set as follows: overlap interval = 1, overlap ratio = 0.125, world threshold (T) = 11. The HSP score (S) and HSP S2 parameters are dynamic values, established by the program itself based on the composition of a specific sequence; however, the minimum values ​​can be adjusted, as set above. In other embodiments, the sequence identity % between two nucleic acid or amino acid sequences is determined using the Needle (EMBOSS) or Stretcher (EMBOSS) whole sequence alignment tools from http: / / www.ebi.ac.uk / Tools / psa / , using the predefined parameters adopted therein (which are fully incorporated herein by reference).

[0055] α-N-acetylglucosidase α-N-acetylglucosidase (Naglu) is produced from a precursor molecule that has been processed into its mature form. This process typically occurs when the protein enters the endoplasmic reticulum and cleaves the 23-amino acid signal peptide. The precursor form, also known as the full-length precursor or full-length Naglu protein, typically contains 743 amino acids. Upon entry into the endoplasmic reticulum, the cleavage of the N-terminal 23 amino acids produces the processed or mature form. Therefore, it is believed that Naglu protein activity does not require the N-terminal 23 amino acids of the native full-length human Naglu protein. The amino acid sequence of the mature human Naglu protein is shown below. Figure 1 As shown in SEQ ID NO: 1. The mRNA sequence of human Naglu is described in Genbank Accession number NM_000263. In various embodiments of this application, Naglu is human Naglu, which has (amino acids 1-743) or does not have (amino acids 24-743) associated signaling sequences. In a preferred embodiment, the Naglu lysosomal enzyme incorporated into the medical fusion protein has Figure 1 The amino acid sequence shown is (SEQ ID NO: 1). In a particularly preferred embodiment, the fusion protein containing Naglu has Figure 2 The amino acid sequence shown is (SEQ ID NO: 5). In other embodiments, the fusion protein comprises a functional fragment of a mature human Naglu protein, wherein the length of the fragment is typically at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 or more amino acids.

[0056] Mucopolysaccharidosis III B (St. Philippian disease type B) One example of lysosomal storage disorder is mucopolysaccharidosis III B (MPS IIIB), also known as St. Philip's disease type B. MPS IIIB is a rare autosomal recessive genetic disorder characterized by a deficiency of the enzyme α-N-acetyl-glucosidase (Naglu). Without this enzyme, the body is unable to clear glycosaminoglycans (GAGs) (e.g., GAG heparin sulfate) and partially degraded GAG molecules, which accumulate in lysosomes in various tissues, gradually spreading and causing bodily dysfunction (Kakkis et al.). N Engl J Med. 344(3):182-8 (2001). It is known that GAGs accumulate in the lysosomes of neurons and glial cells, and less so in the external brain.

[0057] Four distinct types of MPS III have been identified, designated MPS IIIA, B, C, and D. Each type represents a deficiency of one of the four enzymes involved in the degradation of GAG heparan sulfate. All types include varying degrees of the same clinical symptoms, including coarse facial features, hepatosplenomegaly, corneal opacity, and skeletal deformities. However, the most noteworthy aspect is the severe and progressive loss of cognitive function, attributed not only to the accumulation of heparan sulfate in neurons but also to the subsequent elevation of gangliosides GM2, GM3, and GD2 due to the accumulation of primary GAG (Walkley et al.). Ann, NY Acad Sci. 845:188-99 (1998)).

[0058] The clinical features of St. Philippians type B are degeneration of the central nervous system (CNS), resulting in loss or inability to complete major developmental stages. Progressive cognitive decline eventually leads to dementia and premature death. The disease primarily affects young children, and affected individuals typically do not survive beyond late adolescence to their early twenties.

[0059] MPS III, which primarily affects young children, presents with similar symptoms. Affected infants appear outwardly normal, though some mild facial deformities may be noticeable. Other common mucopolysaccharidosis symptoms, such as joint stiffness, hirsutism, and coarse hair, typically appear only towards the end of the disease. After an initial asymptomatic period, patients usually experience developmental delays and / or behavioral problems, followed by progressive intellectual decline, leading to severe dementia and progressive motor disorders. Language development is often slow and incomplete. The disease can worsen, leading to increased behavioral disturbances, including anger, hyperactivity, destructive behavior, aggression, pica, and sleep disturbances. Because affected children possess normal muscle strength and energy, their disordered behavior is difficult to manage. In the late stages of the disease, children gradually become immobile and unresponsive, often requiring a wheelchair, and develop swallowing difficulties and seizures. Affected children typically do not survive beyond late adolescence to their early twenties.

[0060] Suitable α-N-acetylglucosidase enzymes for treating MPS IIIB (St. Philippian disease type B) include wild-type human α-N-acetylglucosidase, its functional fragments, or sequence variants, which retain the ability to be absorbed by mammalian lysosomes and are capable of hydrolyzing the α,1,4 linkages of terminal N-acetyl-D-glucosamine residues in targeted therapeutic fusion proteins that remain in linear oligosaccharides or contain wild-type human Naglu enzyme or its functional fragments. In specific embodiments, proteins comprising or composed of the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 5 may be used for the treatment of MPS IIIB.

[0061] The efficacy of the targeted therapeutic fusion protein described herein for treating MPS IIIB can be determined using relevant and known techniques, and by analyzing lysosomal and neuronal biomarkers. Initial experiments have been conducted in animals that have had Naglu removed (see Li et al.). Proc Natl Acad Sci USA 96:14505-510 (1999)). Elimination of Naglu resulted in the formation of large amounts of heparan sulfate in the liver and kidneys, and an increase in gangliosides in the brain.

[0062] Analytical methods include analyzing the activity and biodistribution of extrinsic enzymes, the decrease in GAG storage in lysosomes (especially lysosomes in brain cells), and the activation of astrocytes and microglia. The levels of various lysosomal or neuronal biomarkers may include (but are not limited to) lysosome-associated membrane protein 1 (LAMP1), glypican, gangliosides, cholesterol, mitochondrial ATP synthase subunit c (SCMAS), ubiquitin, p-GSK3b, β-amyloid, and p-tau. Survival and behavioral analyses are also performed using techniques known in the relevant fields.

[0063] In various embodiments, treatment for lysosomal storage disorders refers to reducing lysosomal storage (e.g., GAG) in various tissues. In various embodiments, treatment refers to reducing lysosomal storage in target brain tissues, spinal neurons, and / or peripheral target tissues. In some embodiments, lysosomal storage is reduced by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more compared to untreated controls. In various embodiments, lysosomal storage is reduced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more compared to controls.

[0064] In various embodiments, the treatment refers to increasing enzyme activity in various tissues. In various embodiments, the treatment refers to increasing enzyme activity in target brain tissues, spinal neurons, and / or peripheral target tissues. In various embodiments, enzyme activity is increased by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more compared to the control group. In various embodiments, enzyme activity is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more compared to the control group. In various embodiments, the enzyme activity is increased by at least about 10 nmol / hr / mg, 20 nmol / hr / mg, 40 nmol / hr / mg, 50 nmol / hr / mg, 60 nmol / hr / mg, 70 nmol / hr / mg, 80 nmol / hr / mg, 90 nmol / hr / mg, 100 nmol / hr / mg, 150 nmol / hr / mg, 200 nmol / hr / mg, 250 nmol / hr / mg, 300 nmol / hr / mg, 350 nmol / hr / mg, 400 nmol / hr / mg, 450 nmol / hr / mg, 500 nmol / hr / mg, 550 nmol / hr / mg, 600 nmol / hr / mg, or more. In various embodiments, the lysosomal enzyme is Naglu.

[0065] Enzyme replacement therapy Enzyme replacement therapy (ERT) is a medical strategy that corrects enzyme deficiencies by infusing the missing enzyme into the patient's bloodstream or other body tissues. As the tissue is infused into the bloodstream, the enzyme is absorbed by the cells and transported to lysosomes, where it eliminates substances accumulated in the lysosomes due to enzyme deficiency. Effective lysosomal enzyme replacement therapy requires the delivery of the therapeutic enzyme to the appropriate lysosomes within the cells of the tissue with the storage deficiency. Commonly used lysosomal enzyme replacement therapies utilize naturally occurring carbohydrate delivery attached to proteins to bind to specific receptors on the surface of target cells. One such receptor, the cation-independent M6P receptor (CI-MPR), is particularly suitable for targeted replacement of lysosomal enzymes because CI-MPRs are present on the surface of most cell types.

[0066] The terms “cation-independent mannose-6-phosphate receptor (CI-MPR),” “M6P / IGF-II receptor,” “CI-MPR / IGF-II receptor,” “IGF-II receptor,” or “IGF2 receptor,” or their abbreviations, may be used interchangeably herein to refer to cellular receptors that can bind to both M6P and IGF-II simultaneously.

[0067] Glycosylation-independent lysosomal targeting A glycosylation-independent lysosomal targeting (GILT) technology has been developed to enable medical enzymes to target lysosomes. Specifically, GILT technology uses peptide labeling instead of M6P binding to CI-MPR for lysosomal targeting. Typically, GILT labels are proteins, peptides, or other moieties that can bind to CI-MPR in a mannose-6-phosphate-independent manner. The advantage of this technology is that it mimics normal biological mechanisms for the absorption of lysosomal enzymes while still maintaining a mannose-6-phosphate-independent approach.

[0068] The preferred GILT marker is derived from human insulin-like growth factor II (IGF-II). Human IGF-II is a high-affinity ligand for CI-MPR, also known as the IGF-II receptor. Therapeutic enzymes labeled with GILT bind to the M6P / IGF-II receptor, allowing the protein to target lysosomes via endocytosis. This method offers several advantages over methods involving glycosylation, including simplicity and cost-effectiveness, because once the protein is isolated, no further modification is required.

[0069] Detailed descriptions of GILT technology and the GILT marking can be found in U.S. Patent Application Publications Nos. 2003-0082176, 2004-0006008, 2003-0072761, 2005-0281805, 2005-0244400, U.S. Patent Applications Nos. 8,492,337 and 8,563,691, and International Publications WO 03 / 032913, WO 03 / 032727, WO 02 / 087510, WO 03 / 102583, WO2005 / 078077, WO 2009 / 137721 and WO 2014 / 085621, all of which are incorporated herein by reference.

[0070] In various embodiments, the GILT label is a furin-resistant GILT label, the amino acid sequence of which is shown in SEQ ID NO: 3 herein (see, for example, U.S. Patent No. 8,563,691).

[0071] Binding affinity to insulin receptor Many IGF-II mutant proteins (including furin-resistant IGF-II mutant proteins) have reduced or eliminated binding affinity to the insulin receptor. Therefore, in some embodiments, the peptide markers suitable for this application have reduced or eliminated binding affinity to the insulin receptor relative to the binding affinity of natural human IGF-II. In some embodiments, peptide markers suitable for this application with reduced or eliminated binding affinity to the insulin receptor include peptide markers with a binding affinity to the insulin receptor that is more than 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times lower than that of wild-type mature human IGF-II. Binding affinity to the insulin receptor can be determined using various in vitro and in vivo assays known in relevant techniques. 。

[0072] Dosing of medical proteins and formulations According to this application, the medical fusion protein is typically administered alone to an individual, or as a composition or pharmaceutical containing the medical protein (e.g., for manufacturing a medicine to treat a disease), as described herein. The composition may be formulated into a pharmaceutical composition using one or more physiologically acceptable carriers or excipients. The carrier and composition may be sterile. The formulation should be appropriate for the administration method.

[0073] Suitable pharmaceutically acceptable carriers include (but are not limited to): water, saline solutions (e.g., NaCl), physiological saline, buffered physiological saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (e.g., lactose, amylose, or starch), sugars (e.g., mannitol, sucrose, or others), dextrose, magnesium stearate, talc, sialic acid, viscous paraffin, perfume oils, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, etc., and combinations thereof. Pharmaceutical preparations may, if necessary, be mixed with excipients (e.g., lubricants, preservatives, stabilizers, humectants, emulsifiers, salts affecting osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances, and the like) that do not impair the reaction with or interfere with the activity of the active compound.

[0074] The composition or pharmaceutical may also contain small amounts of humectants, emulsifiers, or pH buffers, if necessary. The composition may be in the form of a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained-release formulation, or powder. The composition may also be formulated into suppositories using conventional binders and carriers (e.g., triglycerides). Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0075] Typically, the ingredients are supplied separately or mixed together in unit dosage forms, such as dry lyophilized powders or anhydrous concentrates, contained in airtight containers, such as ampoules or pouches indicating the dosage of the active ingredient. If the composition is administered by infusion, it can be delivered using infusion bottles containing sterile pharmaceutical-grade water, physiological saline, or dextran / water. If the composition is administered by injection, ampoules of sterile water or physiological saline for injection can be provided to allow for pre-mixing of the ingredients before administration.

[0076] Medical proteins can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include those that form with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those that form with free carboxyl groups, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0077] Medical proteins (or compositions or medicines containing medical proteins) are administered via any suitable route. In various embodiments, medical proteins are administered intravenously. In other embodiments, medical proteins are administered directly to target tissues (e.g., the heart or muscle (e.g., intramuscularly) or the nervous system (e.g., direct injection into the brain, CNS, CSF; ventricles; intrathecal). In various embodiments, medical proteins are administered intrathecally. Methods of intrathecal administration of medical fusion proteins are known in related art (see, for example, U.S. Patent Nos. 7,442,372 and 9,044,473). Alternatively, medical proteins (or compositions or medicines containing medical proteins) may be administered non-enteral, transdermal, or transmucosal (e.g., orally or nasally). More than one route may be used concurrently if necessary; for example, medical proteins may be administered intravenously and intrathecally. Intravenous and intrathecal administration do not necessarily have to be simultaneous, but can be sequential.

[0078] Medical proteins (or compositions or medicines containing medical proteins) may be administered alone or in combination with other agents, such as antihistamines (e.g., diphenhydramine) or immunosuppressants or other immunotherapeutic agents that counteract antibodies against anti-GILT zymosomal enzymes. The term "in combination" means that the agent is administered before, approximately simultaneously with, or after the medical protein (or composition or medicine containing the medical protein). For example, the agent may be mixed into a composition containing the medical protein so that it is administered simultaneously with the medical protein; or the agent may be administered without mixing (e.g., by "piggybacking" the agent into an intravenous line while also administering the medical protein, or vice versa). In another example, the agent may be administered separately (e.g., without mixing) but within a short timeframe (e.g., within 24 hours) of administering the medical protein.

[0079] A medically effective protein (or a component or medicine containing a medically effective protein) is a dose that, when administered at regular intervals, is sufficient to treat a disease (e.g., alleviate disease-related symptoms, prevent or delay disease onset, and / or reduce the severity or frequency of disease symptoms as described above). This medically effective dose for treating a disease will depend on the nature and extent of the disease effect and may be determined by standard clinical techniques. Furthermore, in vitro or in vivo analytical methods, employing methods known in relevant techniques, may be used as needed to assist in determining the optimal dose range. The precise dose used will also depend on the route of administration and the severity of the disease, and should be determined based on the operator's judgment and the individual patient's circumstances. The effective dose can be obtained by extrapolation from dose-response curves from in vitro or animal model testing systems. In various embodiments, the medically effective dose may be, for example: about 0.1-1 mg / kg, about 1-5 mg / kg, about 2.5-20 mg / kg, about 5-20 mg / kg, about 20-50 mg / kg, or about 20-100 mg / kg. The effective dose may vary over time (e.g., increase or decrease) depending on individual needs. For example, the dose may be increased during periods of physical pain or stress, or if symptoms worsen.

[0080] The therapeutically effective dose of a therapeutic protein (or a component or medicine containing a therapeutic protein) is determined by regular intervals of administration, depending on the nature and extent of the disease effect and its ongoing status.

[0081] The term "interval" used in this article refers to the regular administration of the therapeutically effective dose (as opposed to a single dose). This interval can be determined by standard clinical techniques. In some embodiments, the therapeutic protein is administered every two months, every month, twice a month, every three weeks, every two weeks, weekly, twice a week, three times a week, or daily. The dosing interval for an individual is not necessarily fixed but can vary over time, depending on the individual's needs. For example, the interval between doses can be shortened during periods of physical pain or stress, or if symptoms worsen.

[0082] The terms “every two months” as used in this article mean medication once every two months (i.e., medication once every two months); “every month” means medication once every month; “every three weeks” means medication once every three weeks (i.e., medication once every three weeks); “every two weeks” means medication once every two weeks (i.e., medication once every two weeks); “every week” means medication once a week; and “every day” means medication once a day.

[0083] This application further relates to a pharmaceutical composition which contains the medically applicable protein described herein in a container (e.g., vial, bottle, intravenous administration bag, syringe, etc.) and is accompanied by a label containing instructions such as: administer the composition for the treatment of mucopolysaccharidosis type IIIB (St. Philippian disease type B) using the methods described herein.

[0084] In some embodiments, this application relates to a formulation comprising lysosomal enzymes, targeted medical lysosomal enzyme fusion proteins, or functional fragments thereof. In some embodiments, such formulations are liquid formulations, preferably liquid formulations suitable for intrathecal administration. In other embodiments, the formulation may be a lyophilized formulation or a liquid formulation reconstituted from a lyophilized formulation.

[0085] In various embodiments, the formulations of this application comprise lysosomal enzymes, targeted medical lysosomal enzyme fusion proteins, or functional fragments thereof, at concentrations ranging from about 0.1 mg / ml to about 300 mg / ml, or about 1 mg / ml to about 75 mg / ml, or about 5 mg / ml to about 50 mg / ml, or about 10 mg / ml to about 40 mg / ml, or about 20 mg / ml to about 40 mg / ml, or about 25 mg / ml to about 35 mg / ml. In some embodiments, the concentration or maximum concentration of the lysosomal enzymes, fusion proteins, or fragments thereof may be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 mg / ml or higher. In various embodiments, the formulations of this application comprise lysosomal enzymes, targeted medical lysosomal enzyme fusion proteins, or functional fragments thereof at a concentration of approximately 30 mg / ml. In various embodiments, the lysosomal enzymes, targeted medical lysosomal enzyme fusion proteins, or functional fragments thereof are combined with... Figure 2 The amino acid sequence shown (SEQ ID NO: 5) has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity. More preferably, it is a lysosomal enzyme, a targeted medical lysosomal enzyme fusion protein, or a functional fragment thereof. Figure 2 The amino acid sequence shown (SEQ ID NO: 5) is BMN001.

[0086] In another embodiment, the formulation of this application includes one or more buffers suitable for maintaining the pH of the formulation within a desired range. In a preferred embodiment, the pH of the liquid formulation of this application is in the range of about 5.0 to about 9.0, or about 5.5 to about 8.5, or about 6.0 to about 8.0, or about 6.5 to about 7.5, or about 6.8 to about 7.2. More preferably, the pH of the liquid formulation of this application is about 7.0. Various buffers and their use in protein-containing formulations are known to those skilled in the art, and non-limiting examples of buffers that can be used in the liquid formulation of this application include, for example, sodium acetate, citrate monohydrate, sodium citrate dihydrate, sodium monobasic phosphate monohydrate, and sodium dibasic phosphate heptahydrate, and the like.

[0087] When used in formulations of this application, the preferred concentration range of sodium monohydrate monobasic phosphate is about 0.005 mg / ml to about 0.1 mg / ml, or about 0.01 mg / ml to about 0.1 mg / ml, or about 0.02 mg / ml to about 0.08 mg / ml, or about 0.03 mg / ml to about 0.05 mg / ml. In a particularly preferred embodiment, the concentration of sodium monohydrate monobasic phosphate is about 0.04 mg / ml.

[0088] When used in formulations of this application, the preferred concentration range of sodium dibasic phosphate heptahydrate is about 0.005 mg / ml to about 0.5 mg / ml, or about 0.01 mg / ml to about 0.5 mg / ml, or about 0.05 mg / ml to about 0.4 mg / ml, or about 0.1 mg / ml to about 0.4 mg / ml, or about 0.15 mg / ml to about 0.25 mg / ml. In a particularly preferred embodiment, the concentration of sodium dibasic phosphate heptahydrate is about 0.19 mg / ml.

[0089] In another embodiment, the formulation of this application comprises one or more isotonic agents suitable for maintaining the desired isotonicity and imparting more preferably compatibility when the formulation is administered to an individual (specifically, intrathecal administration). Various isotonic agents and their use in protein-containing formulations are known to those skilled in the art, and unlimited examples of isotonic agents that can be used in the liquid formulations of this application include, for example, sodium chloride, trehalose, mannitol, dextrose, glucose, glycerol, sorbitol, xylitol, ethanol, and the like. In specific embodiments, the use of trehalose ranges from about 3% (w / v) to about 10% (w / v), or about 3% (w / v) to about 5% (w / v), or about 7% (w / v) to about 9% (w / v). In a preferred embodiment, trehalose is used at about 8% (w / v). In yet another preferred embodiment, trehalose is used at about 4% (w / v).

[0090] In various embodiments, the formulation includes an anti-adsorption agent (e.g., reducing the adsorption of protein components onto glass or plastic and decreasing the formation of aggregates and polymers). The anti-adsorption agent includes (but is not limited to): benzyl alcohol, polysorbate 20, and polysorbate 80. In some embodiments, the concentration of the anti-adsorption agent is about 0.001% to about 0.5%, or about 0.01% to about 0.5%, or about 0.1% to about 1%, or about 0.5% to about 1%, or about 0.5% to about 1.5%, or about 0.5% to about 2%, or about 1% to about 2%. In some embodiments, the anti-adsorption agent is polysorbate 20 in the range of about 0.004% to about 0.006%. In a preferred embodiment, polysorbate 20 is used at 0.005%.

[0091] When used in formulations of this application, the preferred concentration range of sodium chloride is from about 0.5 mg / ml to about 20 mg / ml, or from about 2 mg / ml to about 15 mg / ml, or from about 5 mg / ml to about 10 mg / ml, or from about 7 mg / ml to about 10 mg / ml, or from about 8 mg / ml to about 9 mg / ml. In a preferred embodiment, the concentration of sodium chloride is about 0.88 mg / ml. In another preferred embodiment, the concentration of sodium chloride is about 5 mg / ml.

[0092] In another embodiment, the formulations of this application comprise one or more electrolyte agents suitable for maintaining the levels of key electrolytes (groups) in an individual's cerebrospinal fluid (CSF) or mimicking the natural composition of human CSF. Various electrolyte agents and their use in protein-containing formulations are well-known to those skilled in the art, and unlimited examples of electrolyte agents that can be used in the liquid formulations of this application include, for example, potassium chloride, magnesium chloride, magnesium chloride hexahydrate, calcium chloride, calcium chloride dihydrate, and the like.

[0093] When used in formulations of this application, the preferred concentration range of potassium chloride is about 0.01 mg / ml to about 1 mg / ml, or about 0.1 mg / ml to about 0.5 mg / ml, or about 0.2 mg / ml to about 0.8 mg / ml, or about 0.15 mg / ml to about 0.4 mg / ml, or about 0.15 mg / ml to about 0.3 mg / ml. In a preferred embodiment, the concentration of potassium chloride is about 0.22 mg / ml.

[0094] When used in formulations of this application, the preferred concentration range of magnesium chloride hexahydrate is about 0.01 mg / ml to about 1 mg / ml, or about 0.1 mg / ml to about 0.8 mg / ml, or about 0.1 mg / ml to about 0.5 mg / ml, or about 0.1 mg / ml to about 0.3 mg / ml, or about 0.1 mg / ml to about 0.2 mg / ml. In a preferred embodiment, the concentration of magnesium chloride hexahydrate is about 0.16 mg / ml.

[0095] When used in formulations of this application, the preferred concentration range of calcium chloride dihydrate is about 0.01 mg / ml to about 1 mg / ml, or about 0.1 mg / ml to about 0.8 mg / ml, or about 0.1 mg / ml to about 0.5 mg / ml, or about 0.15 mg / ml to about 0.4 mg / ml, or about 0.15 mg / ml to about 0.3 mg / ml. In a preferred embodiment, the concentration of calcium chloride dihydrate is about 0.21 mg / ml.

[0096] In a preferred embodiment, the formulation of this application is a liquid formulation comprising a BMN001-targeted medical fusion protein, one or more buffers, one or more isotonic agents, and one or more electrolytes. More preferably, the liquid formulation comprises BMN001, sodium dibasic phosphate heptahydrate, sodium monobasic phosphate monohydrate, sodium chloride, and trehalose. In one embodiment, the liquid formulation comprises BMN001 (concentration of about 25 mg / ml to about 35 mg / ml), sodium dibasic phosphate heptahydrate (concentration of about 0.15 to about 0.25 mg / ml), sodium monobasic phosphate monohydrate (concentration of about 0.02 mg / ml to about 0.06 mg / ml), sodium chloride (concentration of about 0.8 mg / ml to about 1 mg / ml), and trehalose (about 7% (w / v) to about 9% (w / v)). In a preferred embodiment, the liquid formulation comprises BMN001 (concentration of about 30 mg / ml), sodium divalent phosphate heptahydrate (concentration of about 0.19 mg / ml), sodium monovalent phosphate monohydrate (concentration of about 0.04 mg / ml), sodium chloride (concentration of about 0.88 mg / ml), and trehalose (about 8% (w / v)). Preferably, the liquid formulation has a pH of about 7.0.

[0097] In another preferred embodiment, the formulation of this application is a liquid formulation comprising BMN001 targeted medical fusion protein, one or more buffers, one or more isotonic agents, and one or more electrolytes. More preferably, the liquid formulation comprises BMN001, sodium dibasic phosphate heptahydrate, sodium monobasic phosphate monohydrate, sodium chloride, trehalose, and polysorbate 20. In one embodiment, the liquid formulation comprises BMN001 (concentration of about 25 mg / ml to about 35 mg / ml), sodium dibasic phosphate heptahydrate (concentration of about 0.15 to about 0.25 mg / ml), sodium monobasic phosphate monohydrate (concentration of about 0.02 mg / ml to about 0.06 mg / ml), sodium chloride (concentration of about 4.5 mg / ml to about 5.5 mg / ml), trehalose (about 3% (w / v) to about 5% (w / v)), and polysorbate 20 (about 0.004% to about 0.006%). In a preferred embodiment, the liquid formulation comprises BMN001 (concentration of about 30 mg / ml), sodium divalent phosphate heptahydrate (concentration of about 0.19 mg / ml), sodium monovalent phosphate monohydrate (concentration of about 0.04 mg / ml), sodium chloride (concentration of about 5 mg / ml), trehalose (about 4% (w / v)), and polysorbate 20 (about 0.005%). Preferably, the liquid formulation has a pH of about 7.0.

[0098] In various embodiments, the formulation may include preservatives. Preservatives include (but are not limited to) m-cresol and benzyl alcohol. In some embodiments, the concentration of the preservative is about 0.4% ± 0.2%, or about 1% ± 0.5%, or about 1.5% ± 0.5%, or about 2.0% ± 0.5%. In some embodiments of this application, the formulation does not contain preservatives.

[0099] In various embodiments, the formulation includes a stabilizer. Unlimited examples of stabilizers include glycerin, glycerol, thioglycerol, methionine, and ascorbic acid and its salts. In some embodiments, when the stabilizer is thioglycerol or ascorbic acid or its salts, the concentration of the stabilizer is from about 0.1% to about 1%. In other embodiments, when the stabilizer is methionine, the concentration of the stabilizer is from about 0.01% to about 0.5%, or from about 0.01% to about 0.2%. In yet another embodiment, when the stabilizer is glycerol, the concentration of the stabilizer is from about 5% to about 100% (net).

[0100] In various embodiments, the composition includes an antioxidant. Examples of antioxidants include (but are not limited to) methionine and ascorbic acid. In some embodiments, the molar ratio of the antioxidant to the protein is about 0.1:1 to about 15:1, or about 1:1 to about 15:1, or about 0.5:1 to about 10:1, or about 1:1 to about 10:1, or about 3:1 to about 10:1.

[0101] Pharmaceutically acceptable salts that can be used in formulations include (but are not limited to): mineral salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate), organic acid salts (e.g., acetate, propionate, malonate, benzoate, methanesulfonate, toluenesulfonate), and ammonia salts (e.g., isopropylamine, trimethylamine, dicyclohexylamine, diethanolamine). For a detailed discussion of pharmaceutically acceptable salts, see Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, (Easton, Pennsylvania (1990)).

[0102] Other examples of formulation additives and compositions suitable for intrathecal or ICV delivery are described in WO2013 / 096899, which is incorporated herein by reference.

[0103] The formulations in this application are stable and can be stored for extended periods without unacceptable changes in quality, potency, or purity. In one aspect, the formulation is stable at about 5°C (e.g., 2°C to 8°C) for at least 1 month, for example, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or longer. In another aspect, the formulation is stable at a temperature below or equal to about -20°C for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer. In yet another aspect, the formulation is stable at a temperature below or equal to about -40°C for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer. In yet another aspect, the formulation is stable at a temperature below or equal to about -60°C for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer.

[0104] Suitable formulations for this application include liquids, lyophilized products, or rehydrated lyophilized formulations. In various aspects, the formulations of this application are contained within a container, which may include single-dosage-form formulations. Container examples include, for example, ampoules, vials, tubing, reservoirs, and pre-filled syringes.

[0105] Intrathecal administration of pharmaceutically acceptable formulations In various embodiments, enzymes, enzyme-fused proteins, or formulations containing them are directly introduced into an individual's central nervous system, for example, into the individual's cerebrospinal fluid. In some aspects of this application, enzymes are introduced intrathecally, for example, into the lumbar region or the greater ventricle, or intraventricularly into the ventricular space. Methods of intrathecal or intraventricular administration of lysosomal enzymes or fusion proteins containing functional lysosomal enzymes are described in U.S. Patent Nos. 7,442,372, 9,044,473, and 9,089,566, the entire contents of which are incorporated herein by reference.

[0106] As those skilled in the art will understand, devices for intrathecal administration of medical components may be employed. For example, therapies using the Ommaya reservoir, commonly used for intrathecal drug administration in meningeal carcinoma (Ommaya et al.), are available. Lancet2: 983-84 (1963)). More specifically, in this method, a ventricular catheter is inserted through a small opening formed in the anterior horn and connected to an Ommaya reservoir placed under the scalp. The reservoir is inserted subcutaneously into a sheath to deliver the specific enzyme to be replaced, which has been injected into the reservoir. Other devices for intrathecal administration of medical components to an individual are described in U.S. Patent No. 6,217,552, the contents of which are incorporated herein by reference. Alternatively, the component may be administered intrathecally, for example, as a single injection or as a series of injections. It should be understood that dosing therapy may be administered as a single dose or as multiple doses.

[0107] The term "intrathecal administration" as used in this article refers to a plan that involves the direct delivery of a pharmaceutical ingredient into the cerebrospinal fluid of an individual, with delivery techniques including injection through a skull burr hole into the lateral ventricle (i.e., within the brain) or cisterns, or lumbar puncture, or similar techniques (described in Lazorthes et al.). Advances in Drug Delivery Systems And Applications in Neurosurgery , 143-192 (1991) and Ommaya et al., Cancer Drug Delivery 1:169-179 (1984), the contents of which are incorporated herein by reference. The term “lumbar region” is planned to include the area between the third and fourth lumbar vertebrae (lower back), and further to include the L2-S1 region of the spine.

[0108] The term "cistern" refers to a space surrounding and below the cerebellum accessible through an opening between the skull and the top of the spine. The term "ventricle" refers to a lumen extending from the brain into the central canal of the spinal column. The pharmaceutical composition according to this application can be administered to any of these sites by direct injection or by infusion pump. For injection, the composition can be prepared as a liquid solution, preferably in a physiologically compatible buffer solution, such as Hank's solution, Ringer's solution, or phosphate buffer. Furthermore, the enzyme can be prepared in a solid form and then dissolved or suspended immediately before use. Lyophilized forms are also included. Enzyme injection can be, for example, by bolus injection or continuous infusion (e.g., using an infusion pump).

[0109] In various embodiments of this application, the enzyme is administered to the individual's brain via lateral ventricle injection. This injection method can be, for example, by drilling a hole in the individual's skull. In another embodiment, the enzyme, fused with proteins and / or other pharmaceutical formulations, is delivered into the individual's ventricle via surgical insertion of a shunt. For example, it can be injected into the lateral ventricle, which is a larger area, or even into the third and fourth lateral ventricles.

[0110] In various embodiments, the pharmaceutical composition used in this application is administered via injection to the cisterna magna or spinal cord of an individual. In another embodiment of the method of this application, a pharmaceutically acceptable formulation provides sustained delivery; for example, the "sustaining release" of the enzyme or other pharmaceutical composition used in this application to an individual is a sustained release for at least 1, 2, 3, 4 weeks or longer after administration of the pharmaceutically acceptable formulation to the individual.

[0111] In various embodiments, the medical fusion protein is delivered to one or more surface or superficial tissues of the brain or spine. For example, in various embodiments, the medical fusion protein is delivered to one or more surface or superficial tissues of the brain or spine. In some embodiments, the target surface or superficial tissue of the brain or spine is located within 4 mm of the brain surface. In some embodiments, the target surface or superficial tissue of the brain is selected from: pia mater tissue, cerebral cortical bands, hippocampus, perivascular space, blood vessels within the VR space, hippocampus, hypothalamus portion on the lower surface of the brain, optic nerve and optic tract, olfactory bulb and projections, and combinations thereof.

[0112] In some embodiments, the medically fusion protein is delivered to one or more deep tissues of the brain or spine. In some embodiments, the target one or more deep tissues of the brain or spine are located less than 4 mm (or deeper) from the surface of the brain (e.g., 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm). In some embodiments, the target deep tissues of the brain include the bands of the cerebral cortex. In some embodiments, the target deep tissues of the brain include one or more of the following: the diencephalon (e.g., hypothalamus, thalamus, anterior thalamus, hypothalamus, etc.), the hindbrain, the lentiform nucleus, the basal ganglia, the caudate nucleus, the putamen, the amygdala, the globus pallidus, and combinations thereof.

[0113] In various embodiments, the target surface or superficial tissue of the spine includes pia mater and / or white matter fiber bundles. In various embodiments, the target deep tissue of the spine includes spinal gray matter and / or tubular cells. In some embodiments, the medically relevant fusion protein is a neuron that transmits signals to the spine.

[0114] In various embodiments, the therapeutic fusion protein is delivered to one or more tissues of the cerebellum. In some embodiments, one or more target tissues of the cerebellum are selected from the group consisting of: tissues of the molecular layer, tissues of the Purkinje cell layer, tissues of the granular cell layer, cerebellar peduncle, and combinations thereof. In some embodiments, the therapeutic agent (e.g., an enzyme) is delivered to one or more deep tissues of the cerebellum, including (but not limited to): tissues of the Purkinje cell layer, tissues of the granular cell layer, deep cerebellar white matter tissue (e.g., deep relative to the granular cell layer), and deep cerebellar nucleus tissue.

[0115] In various embodiments, the therapeutic fusion protein is delivered to one or more tissues of the brainstem. In some embodiments, one or more target tissues of the brainstem include brainstem white matter and / or brainstem nuclei.

[0116] In various embodiments, the medical fusion protein is delivered to a variety of different brain tissues, including (but not limited to): gray matter, white matter, periventricular regions, pia mater, meninges, neocortex, cerebellum, deep tissues of the cerebral cortex, molecular layers, caudate nucleus / putamen, midbrain, deep regions of the pons or medulla oblongata, and combinations thereof.

[0117] In various embodiments, the therapeutic fusion protein is delivered to various cells in the brain, including (but not limited to): neurons, glial cells, perivascular cells, and / or meningeal cells. In some embodiments, the therapeutic protein is delivered to oligodendrocytes in the deep white matter.

[0118] In certain preferred embodiments, in order to treat MPS IIIB disease in human individuals, slow the rate of decline of at least one symptom of MPS IIIB in human individuals (including cognitive decline), or reduce or prevent GAG accumulation in one or more tissues of the CNS in individuals with MPS IIIB disease, a medical fusion protein of about 30 mg, 100 mg, or 300 mg, or about 30 mg-300 mg, 30 mg-200 mg, or 30 mg-100 mg is administered once weekly in ICV (isovolute) for at least about 24 weeks, preferably 48 weeks.

[0119] The reagent kit used in this application method The formulations used in the methods of this application may be provided as kits, which may further include instructions for use. Such kits will contain lysosomal enzymes or fusion proteins described herein, comprising enzymes for treating lysosomal storage disorders and portions targeting lysosomes, typically in a dose and formulation suitable for administration to the host. In various embodiments, the kit may include one or more devices for delivering the enzyme into the sheath.

[0120] In addition to the medical components, the kit of this application may also include instructions for intrathecal administration of the medical components of this application. In some embodiments, the kit of this application may include catheters (groups), pumps (groups), or other devices for intrathecal administration of enzyme replacement therapy (which are pre-loaded with the medical components of this application). Specifically included are, for example, catheters pre-loaded with pharmaceutically acceptable formulations containing 0.001-0.01 mg, 0.01-0.1 mg, 0.1-1.0 mg, 1.0-10 mg, 10-100 mg, or more of a medically acceptable fusion protein containing lysosomal enzymes and partial bodies of lysosomes targeting lysosomes (e.g., Naglu and IGF-II peptide labeling). Examples of catheters may include single-use catheters that are disposable after use.

[0121] In some embodiments, the kit of this application may include one or more of the following components: extension cord (e.g., Smiths Medical PN:536040), in-line filter (e.g., Smiths Medical PN:FS116), needle implantation port (e.g., Smiths Medical PN:21-2737-24), syringe (e.g., Becton Dickinson PN:309604), or needle (e.g., Becton Dickinson PN:305196).

[0122] Treatment methods for St. Philippian disease type B Intrathecal administration of Naglu enzymes or their fusion proteins (including BMN001) to a patient's CSF (e.g., ICV or lumbar spine) may be used to prevent or treat one or more symptoms or adverse effects of MPS IIIB disease in humans. Therefore, intrathecal administration of a medically effective amount of Naglu enzymes or their fusion proteins (including BMN001) is expected to improve one or more symptoms or adverse effects of MPS IIIB disease, slow or reduce the progression of one or more symptoms or adverse effects of MPS IIIB disease, or stabilize the decline of one or more symptoms or adverse effects of MPS IIIB disease. Known symptoms or adverse effects of MPS IIIB disease in humans include, for example, one or more of the following detectable declines: cognitive function, language function, motor function, socio-emotional function, adaptive function, conceptual thinking ability, facial recognition, storytelling completeness, reasoning ability, and hand function / dexterity.

[0123] To quantify the therapeutic efficacy of the administered enzyme or its fusion protein, a Development Quotient (DQ) score can be calculated using any known and routinely used neurocognitive testing method. In one embodiment, the DQ score is a cognitive function DQ score.

[0124] In one embodiment of this application, the DQ score of a human individual can be obtained through the Bayley Scales of Infant Development, Version 3 (BSID-III). Bayley Scales of Infant and Toddler Development (Bayley-III). Technical Manual. Third Edition. San Antonio: Psychological Corp., 2006 (the contents of which are incorporated herein by reference). BSID-III is a tool for assessing developmental function in children aged 1 to 42 months across five domains (cognitive, linguistic, motor, social-emotional, and adaptive). In some implementations, the social-emotional and adaptive domains of the BSID-III test are not used. In one implementation, the DQ score is determined only in the cognitive domain.

[0125] In some embodiments, the cognitive domains of BSID-III may be the primary focus of this test. The Cognitive Strength Scale is individually assessed and recorded by qualified testers, who record the development of key skills such as processing speed, problem-solving, and play. Importantly, this cognitive assessment method does not require verbal responses from the individual; therefore, this test is particularly suitable for assessing cognitive function in individuals with expressive language problems, such as those with MPSIIIB disorder. Raw data from one domain can be converted into scale scores, and then into composite scores covering multiple domains. Age-equivalent scores and DQs can also be generated from average raw scores correlated with different ages.

[0126] In some embodiments, the language and motor domains of BSID-III may also be used. The language domain consists of two subtests (receptive communication and expressive communication), and the motor domain consists of two subtests (fine motor skills and gross motor skills).

[0127] In another embodiment of this application, the Kaufman Assessment Battery for Children, Version 2 (KABC-II) (Kaufman et al.) can be used. Kaufman Assessment Battery for ChildrenThe Kaufman Nonverbal Index (KABC-II) is a clinical instrument (psychological diagnostic tool) for assessing cognitive development. Similar to the BSID, the KABC can be used to generate age-appropriate scores and thus the DQ. Because many test components are not verbal, it is particularly suitable for assessing the functioning of children with difficulties in both hearing and verbal communication (both conditions associated with MPS IIIB). Furthermore, the test has been translated into many different languages ​​worldwide. Subtests, including the Kaufman Nonverbal Index, include: conceptual thinking, facial recognition, storytelling completeness, trigonometry, reasoning, and hand-eye coordination. In addition to the nonverbal index subtests, knowledge set subtests (riddles, expressive vocabulary, and verbal knowledge) can also be administered to verbal individuals.

[0128] In some embodiments, an algorithm is used to determine whether to use the BSID-III or KABC-II test to determine the DQ quotient, as described in Delaney et al. JIMD Rep. 13:129-137 (2014).

[0129] Before, during, or after treatment with medical enzymes or their fusion proteins, an individual's DQ score is determined using results from the BSID-III tool (or its cognitive subtest) or the KABC-II nonverbal index. More specifically, using the aforementioned BSID-III or KABC-II tool, an individual's performance on the tool is assigned to an "equivalent age level" (in months). The DQ score is then calculated by dividing the equivalent age level by the individual's actual age (in months) and multiplying by 100. For example, an MPS IIIB individual with an actual age of approximately 60 months, whose assigned "equivalent age level" based on their performance on the tool is 48 months, has a DQ calculated as follows: (48 divided by 60) x 100 = 80. On the other hand, a functionally intact 60-month-old individual with an assigned "equivalent age level" of 60 months has a DQ of (60 divided by 60) x 100 = 100. When cognitive, language, motor, socio-emotional, adaptive, conceptual thinking, facial recognition, storytelling completeness, reasoning ability, and hand function / dexterity decline over time in human MPS IIIB patients, the DQ score of untreated MPS IIIB individuals is expected to decrease over time. This application aims to mitigate, stabilize, or improve the observed DQ decline over time by administering the therapeutic protein described herein. The benefits of administering the therapeutic fusion protein of this application or formulations containing it can be assessed by comparing the DQ score measured before and after treatment.

[0130] The following examples will be used further and more explicitly in this application. However, the examples are for illustrative purposes only and are not intended to be limiting.

[0131] Example 1 - Compound Development Lysosomal enzyme fusion proteins containing GILT-tagged and spacer peptides (including mature human α-N-acetylglucosidase [Naglu] fusion proteins) have been disclosed in U.S. Patent Applications Nos. 2003-0082176, 2004-0006008, 2003-0072761, 2004-0005309, 2005-0281805, 2005-0244400, U.S. Patent Applications Nos. 8,492,337 and 8,563,691, and International Publications WO 03 / 032913, WO 03 / 032727, WO 02 / 087510, WO 03 / 102583, WO 2005 / 078077, WO 2009 / 137721 and WO The contents disclosed herein are all incorporated herein by reference, as of 2014 / 085621.

[0132] In a particularly preferred embodiment (referred to herein as BMN001), a Naglu / IGF-II fusion protein was prepared comprising a functional, mature human Naglu enzyme linked via a rigid linker to a furin-resistant IFG-II peptide (composed of amino acids 8-67 of mature human IGF-II, with arginine replacing amino acid position 37 by alanine) and formulated for in vivo safety and efficacy studies. The peptide linker for BMN001 has the amino acid sequence shown in SEQ ID NO: 4. The complete amino acid sequence of the medically viable fusion protein BMN001 is shown below. Figure 2 (SEQ ID NO: 5).

[0133] To determine the excipients and formulation conditions suitable for intrathecal clinical use of BMN001 liquid formulations, various experiments were conducted. First, the aggregation tendency of BMN001 in liquid formulations at different pH values ​​was tested. BMN001 was added to liquid formulations containing citrate buffer at pH 5.0 or 6.5, or to artificial human CSF liquid formulations at pH 6.0, 6.5, 7.0, and 8.0, and static light scattering analysis was performed to determine the aggregation tendency of BMN001 at different pH values ​​and at gradually increasing temperatures. These analyses confirmed that BMN001 aggregates more readily at lower pH values ​​(pH 5.0 to 6.0) than at higher, more neutral pH values ​​(approximately pH 7.0) when heated. Furthermore, the aggregation tendency of BMN001 liquid formulations at 25°C was also measured. o SEC analysis at different pH levels confirmed that the percentage of aggregated polyBMN001 was significantly higher at pH levels below 6.5 than at pH levels between approximately 6.5 and 7.5, which is less acidic and more neutral. Finally, SEC analysis of the BMN001 liquid formulation after 10 freeze / thaw cycles at different pH levels confirmed that the percentage of aggregated polyBMN001 was significantly higher at pH levels below 6.5 than at pH levels between approximately 7.0, which is less acidic and more neutral. These data combined suggest that a pH range of approximately 6.5 to 7.5, preferably approximately 7.0, is more favorable for the clinical application of the BMN001 liquid formulation.

[0134] Secondly, liquid formulations containing different concentrations of BMN001 were tested to determine the effect of fusion protein concentration on the formation of aggregates / polymers during five freeze / thaw cycles. In these experiments, liquid formulations containing 1, 5, 15, or 24 mg / ml BMN001 and at pH values ​​of 5.0, 6.0, 7.0, and 8.0 were prepared. After five freeze / thaw cycles, the percentage of aggregates / polymers relative to monomers was more likely to be higher in formulations with lower protein concentrations than in formulations with higher fusion protein concentrations. Furthermore, aggregates / polymers formed more frequently at higher acidity pH levels than at neutral pH levels. The addition of 2% trehalose prevented fusion protein aggregation compared to the same formulation without trehalose. These results indicate that liquid formulations containing at least approximately 24 mg / ml BMN001 fusion protein are superior to formulations with lower fusion protein concentrations. These remarkable results confirm that liquid formulations with higher protein concentrations (i.e., approximately 30 mg / ml) exhibit a lower relative percentage of agglomerate / polymer formation, providing significant benefits for intrathecal administration to humans, where such administration methods are highly sensitive to the total volume of fluid administered. Therefore, formulations of this application (including those containing BMN001) with protein concentrations of at least 24 mg / ml, including those with protein concentrations of approximately 30 mg / ml, are well-suited for intrathecal administration to human individuals.

[0135] Secondly, the ability of different excipients for formulation to prevent the formation of aggregates induced by agitation or freezing / thawing of BMN001 can be screened, as determined by static light scattering. A liquid formulation containing equal amounts of BMN001 was prepared, comprising (i) 180 mM N-acetylglucosamine, (ii) 222 mM glucose, (iii) 234 mM sucrose, (iv) 212 mM trehalose, (v) 220 mM sorbitol, (vi) 200 mM glutamic acid, (vii) 200 mM glutamylamine, (viii) 200 mM arginine, (ix) 200 mM histidine, (x) 200 mM glycine, (xi) 0.1% w / v polysorbate 20, or (xii) 0.1% w / v poloxamer 188. These analytical results confirm that the addition of one or more amino acids easily leads to instability in liquid formulations, as evidenced by an increase in agglomerates induced by agitation or freezing / thawing. Conversely, the addition of one or more sugars / polyols easily reduces the relative amount of agglomerates / polymers induced by agitation or freezing / thawing.

[0136] Based on the above formulation development process and other experiments not described herein, the final BMN001 liquid formulation was developed for further development for human clinical use as follows. Example 3 below illustrates the BMN001 clinical formulation used in human clinical trials, which consists of the following components: (i) 30 mg / ml BMN001 fusion protein, (ii) 0.19 mg / ml sodium divalent phosphate heptahydrate, (iii) 0.04 mg / ml sodium monovalent phosphate monohydrate, (iv) 8.66 mg / ml sodium chloride, (v) 0.22 mg / ml potassium chloride, (vi) 0.16 mg / ml magnesium chloride hexahydrate, and (vi) 0.21 mg / ml calcium chloride dihydrate. The final pH of this BMN001 clinical formulation was 7.0. BMN001 clinical compound can be packaged in clear borosilicate glass vials, sealed with fluoropolymer-coated bromobutyl rubber stoppers, and capped with aluminum seals. It should be stored at approximately -40°C. o Freeze at C until thawed and used.

[0137] Example 2 – BMN001 for the treatment of St. Philippian disease type B (preclinical trial) If non-clinical trials are conducted as described above and similar medical benefits are demonstrated, there is no significant risk of BMN001-related toxicity in human patients with MPS IIIB disease.

[0138] The non-clinical trial design described in this article is intended to support long-term ICV infusion of BMN001 for the treatment of human patients with MPS IIIB. A single-dose trial has been conducted in normal animals (cynomolgus monkeys) and four repeated-dose trials in normal and disease-prone models of MPS IIIB (Naglu-knockout [KO] mice, WT and NAGLU-nullified dogs (Ellinwood et al.). J. Inherit. Metab. Dis. 26(5):489-504 (2003)) and cynomolgus monkeys) analyzed the main pharmacodynamics (PD), cardiovascular (CV) and CNS safety pharmacology, PK, CNS distribution and toxicity characteristics of BMN001 administered via the ICV route. These species were selected based on high NAGLU amino acid sequence homology, CI-MPR performance and amino acid sequence identity. The animal models of the disease also exhibit some key features of human MPS IIIB disease, including the accumulation of lysosomal stores, neuronal death, functional decline, and a shortened lifespan with similar relative disease progression time. They also exhibit certain neurological symptoms, including tremor and ataxia, which can be used to functionally explore the underlying CNS pathology and may be used to track the response to treatment. Therefore, these models provide valuable insights into pathologically mitigating disease progression by utilizing clinically interpretable endpoints.

[0139] The primary drug efficacy (PD) analysis of BMN001 was performed using IGF2 receptor binding assays, MPS IIIB human fibroblasts, and two available MPS IIIB animal models (NAGLU knockout (KO) mice and juvenile NAGLU-nullified dogs), confirming the reliable pharmaceutical activity of BMN001. These studies used a batch of BMN001 IGF2 receptor binding assays, and the results yielded a calculated mean IC50. 50 The concentration was 0.28 nM. The in vitro cell uptake and half-life of BMN001 were determined in MPS IIIB human fibroblasts. The lysosomal K+ of BMN001... 吸收 The concentration of lysosomes absorbed into the lysosomes at half the maximum rate is defined as 3.7–6.4 nM (average 5.3 nM), and the lysosomal half-life is approximately 9.5 days.

[0140] In the MPS IIIB mouse disease model, administration of BMN001 via ICV reversed the pathology of the disease. Repeated-dose studies in these mice showed consistent distribution of BMN001 in the CNS. Primary PD analysis confirmed the efficacy of BMN001 at both biochemical and histological endpoints. Administration of BMN001 via ICV to Naglu-KO mice reduced the accumulation of lysosomal storage material (i.e., GAG / heparan sulfate accumulation) while improving histological and immunohistochemical indices of lysosomal function. More specifically, when assessed 24 hours after the last dose of BMN001, the treatment results showed a significant increase in Naglu enzyme activity, while reducing β-hexamethylenetetramine activity and the levels of total heparan sulfate and LAMP-2. Naglu activity was detectable in brain tissue, not only in the cerebral cortex, hippocampus, dentate gyrus, and thalamus, but also in distal geographic areas, including the amygdala, olfactory cortex, and hypothalamus. It was also found that the levels of CD68, SCMAS, β-amyloid, p-Tau, P-GSK3β, and phosphatidylinositol proteoglycan-5 were significantly decreased. The levels of heparan sulfate, Naglu-specific NRE, and β-hexamethylenetetramine activity continued to decrease at 7, 14, and 28 days after the last administration.

[0141] In NAGLU-ineffective dogs, the pharmacodynamic efficacy of BMN001 was observed 6 months after ICV administration, including reducing lysosomal storage material in cerebrospinal fluid (CSF) and maintaining motor function. Other pharmacodynamic endpoints (including cognitive function and delay in disease progression) were analyzed as follows. Six independent groups, each with four immunized dogs that had developed tolerance, aged 4 to 18 months, received ICV infusions every two weeks, as follows: Group 1 (Normal NAGLU+ dogs) – ICV mediator only (10 ml / kg); Group 2 (Normal NAGLU+ dogs) - BMN001 (12 mg / kg); Group 3 (normal NAGLU+ dogs) - BMN001 (12 mg / kg, gradually increasing to 48 mg / kg at the 3rd dose); Group 4 (NAGLU - Ineffective Dogs) – ICV mediator only (10 ml / kg); Group 5 (NAGLU-ineffective dogs) - BMN001 (12 mg / kg); Group 6 (NAGLU-ineffective dogs) - BMN001 (12 mg / kg, gradually increasing to 48 mg / kg at dose 3).

[0142] In dogs in groups 1, 4, 5, and 6, the levels of heparan sulfate (HS) in CNS tissue, CSF, and cerebellar tissue were measured. Figure 3 As shown, BMN001, following a dose-dependent pattern, reduced the HS content in the CNS tissue and CSF of MPS IIIB dogs, reducing it to wild-type levels at a dose of 48 mg / kg. The HS content in the CNS and CSF of each group of dogs was analyzed and compared. Figure 4 As shown, there is a strong correlation between the HS content in these two brain compartments, confirming that BMN001 has the ability to reduce HS that is evenly distributed in the brain.

[0143] The effect of BMN001 treatment on LAMP2 levels was also investigated. Cerebellar tissue homogenate samples were obtained from wild-type untreated MPSIIIB dogs and MPSIIIB dogs treated with BMN001 for electrophoresis (18 µg protein / line). The samples were spotted under non-reducing conditions and then detected using an unlabeled AC17 probe and a secondary anti-mouse IgG antibody conjugated to HRP. The specks included lysate representing LAMP2 or lysate from control CHO-K1 cells as a control group (5 µg protein / line). To analyze the total protein content loaded on each line, the cerebellar homogenate specks were excised, and β-actin was detected using a probe. Figure 5 As shown, untreated dogs with MPSIIIB had higher LAMP2 levels than wild-type dogs. However, treatment with BMN001 reduced LAMP2 levels in MPSIIIB-affected dogs to levels observed in wild-type dogs.

[0144] The effects of BMN001 on cerebellar atrophy were also investigated. Figure 6 As shown, BMN001 caused a significant reduction in cerebellar white matter at both doses. Furthermore, diffusion tensor magnetic resonance imaging (DTI) determined that BMN001 reduced the degree of cerebellar white matter degeneration at both doses. Figure 7 BMN001 also clearly reduced cerebellar atrophy in MRI images of wild-type and BMN001-treated MPS IIIB dogs. Figure 8 ).

[0145] In another trial, healthy juvenile cynomolgus monkeys weighing approximately 1-2 kg were randomly assigned to the following five dosage groups: Group 1 – ICV mediator only (5 min, 2.5 ml isovolemic ICV, 0.5 ml / min); Group 2 - 30 mg BMN001 (5 minutes, 2.5 ml isovolemic ICV, 0.5 ml / min); Group 3 - 73 mg BMN001 (5 minutes, 2.5 ml isovolemic ICV, 0.5 ml / min); Group 4 - 73 mg BMN001 (240 minutes, 2.5 ml non-isovolute ICV, 0.88 ml / hr); and Group 5 - 200 mg / kg BMN001 (5 minutes, non-isovolume IV, 3 ml / min).

[0146] Forty-eight hours after administration, animals were euthanized, and designated CNS tissues were collected. Superficial and deep tissue samples relative to the ventricles were collected from seven brain regions and three spinal cord regions for biodistribution analysis. These analyses confirmed that the ICV delivery pathway dominates CNS enzymatic replacement to achieve a superior biodistribution in the CNS compared to IV administration, and that BMN001 can be safely and rapidly administered after the removal of an equal volume of CSF, with good in vivo tolerability. Finally, similar and widespread BMN001 distribution was observed in both superficial and deep CNS tissues from both rapid (approximately 5 minutes) equal volume administration and slow (approximately 240 minutes) non-equal volume administration.

[0147] In single-dose and weekly repeated-dose toxicity studies in monkeys, cardiovascular, respiratory, and CNS safety pharmacological parameters were analyzed. In bi-weekly repeated-dose studies in WT and NAGLU-ineffective dogs, CNS and cardiovascular safety pharmacological parameters were analyzed. No adverse effects related to the CNS, cardiovascular, or respiratory systems were observed in these studies. Following ICV administration, no CNS or systemic organ toxicity associated with BMN001 was observed due to exaggerated pharmacological phenomena (e.g., rapid clearance of accumulated lysosomal stores). Furthermore, no systemic toxicity, including hypoglycemia, was observed after repeated IV administration of BMN001.

[0148] In human clinical trials, the dosage equivalent to that of humans is calculated based on brain mass proportions. The human brain reaches approximately 75% of adult mass at age 2 and 100% of adult mass at age 5. If the adult brain mass is 1400 g, and the brains of MPS IIIB patients gradually atrophy, the estimated average mass of the patient population in the project is 1000 g. Using the average macaque brain mass of 100 g, the proportionality factor is 10. Therefore, based on current non-clinical project analysis, when pediatric patients in the project receive 4-hour infusions or equivalent boluses weekly, at doses up to 730 mg (calculated based on brain weight proportions), the safety and efficacy profile of BMN001 supports long-term ICV administration of BMN001.

[0149] Example 3 – BMN001 for the treatment of St. Philippians type B (human clinical trial) This is the first-in-human, multicenter, multinational, open-label, escalation-dose Phase 1 / 2 trial conducted in human patients diagnosed with MPS IIIB. BMN001 was prepared as described in Example 1, administered weekly via ICV infusion, and individuals' neurocognitive function, behavior, sleep, quality of life (including individual and family / caregiver), angiographic features, and biochemical markers of disease burden were assessed. The primary objective of this trial was to analyze the safety and tolerability of BMN001 administered via ICV reservoir and catheter to individuals with MPS IIIB, and to analyze the impact of BMN001 on cognitive function in human patients with MPS IIIB using the applicable development quotient (DQ). To analyze the effect of this treatment on cognitive function, data from the human individuals treated in this trial were compared with data from previous observational trials (i.e., “natural history trials”) of the same group of individuals with progressive MPS IIIB symptoms.

[0150] The current Phase 1 / 2 human clinical trial consists of two parts. Part 1, the escalation period, involves three human individuals (who have not previously participated in natural history trials) receiving at least four weekly doses of BMN001 at up to three escalation doses (30 mg, 100 mg, and 300 mg) until the maximum tolerated dose (MTTD) is established. Part 2, the steady-state period, involves up to 30 human individuals who have previously participated in natural history trials receiving weekly BMN001 treatment starting at the MTTD for 48 weeks. The three individuals from Part 1 are also moved to Part 2 for baseline analysis, and then continue weekly treatment for 48 weeks at the MTTD established in Part 1.

[0151] This infusion regimen involves eliminating an equal volume of 10 ml CSF, followed by continuous infusion of a total volume of 10 ml of BMN001 over approximately 5 to 10 minutes via ICV. This trial selected a rapid infusion rate to address the specific needs of the MPS IIIB patient population; specifically, these patients often have severe behavioral problems, and prolonged infusion times could pose logistical challenges. Eliminating an equal volume of CSF before rapidly delivering a large volume (e.g., 10–12 ml) of medication within the ventricles is routine in pediatric oncology. However, longer ICV deliveries are also possible. Therefore, a dose of BMN001 (which may or may not be equal volume) can be administered via ICV over at least 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 150, 180, 210, or 240 minutes, or longer.

[0152] The trials conducted at the baseline period in Part 1 and at the baseline period in Part 2 and at weeks 12, 24, 36, and 48 included the Vineland Adaptive Behavior Scales, Version 2 (VABS-II), the Bayley Scales of Infant Development, Version 3 (BSID-III), or the Kaufman Assessment Battery for Children, Version 2 (KABC-II), and the Sanfilippo Behavior Rating Scale (SBRS). Other trial procedures conducted at the baseline visits in Parts 1 and 2, and at weeks 24 and 48 in Part 2, included the Infant Toddler Quality of Life questionnaire (ITQOL) or the Child Health Questionnaire Parent Form (CHQ-PF50), the Children's Sleep Habits Questionnaire (CSHQ), the Parenting Stress Index, the PEDIATRIC QUALITY OF LIFE INVENTORY™ (PEDSQL™) Family Impact Module, MRI of the brain and abdomen (under anesthesia), and brainstem auditory evoked response (BAER) assessment.

[0153] Three individuals received at least eight doses of 30 mg QW BMN001 and at least three doses of 100 mg QW. Part of this trial involved continuous follow-up of individuals for at least 24 weeks prior to treatment initiation to understand the natural progression of the disease. Baseline levels of heparan sulfate (HS) and MPS IIIB-specific HS non-reduced terminus (NRE) in each patient's cerebrospinal fluid (CSF) were measured before and after treatment. Figure 9 As shown, the three individuals (A, B, and C) had significantly higher HS and NRE levels than the disease-free (normal) control group before treatment. However, treatment with BMN001 significantly and persistently induced a decrease in HS and NRE in individuals A and B. Figure 10 (Individual C participated in this trial later, and data is still unavailable.) BMN001 was well tolerated, and no serious treatment-related side effects were observed.

[0154] These results confirm that BMN001 can be safely administered into the ventricular space using the equal-volume bolus method, and that this treatment method can achieve a significant pharmacodynamic response in the CNS of MPS IIIB patients.

[0155] Example 4 – Reconstituted BMN001 It has been found that physical stress in formulations containing BMN001 can cause the formation of aggregates and / or polymers of the active fusion protein. Aggregates and / or polymers are undesirable in pharmaceutical products because they can reduce effective drug concentrations, potentially clog in-line filters during dosing, and may trigger unwanted immune responses in the drug product. Therefore, additional procedures are needed to identify formulations that resist aggregation and / or polymer formation under physical stress. Thus, the effects of various excipients on aggregate / polymer formation were screened (see Table 2). Formulations containing these excipients were subjected to physical stress (recirculation pump pressure), and aggregate formation was determined using differential scanning calorimetry (DSC) and static light scattering (SLS).

[0156] Table 2: Tests on the reduction of agglomerates by excipients As shown in Table 2, N-acetylglucosamine, glucose, sucrose, trehalose, sorbitol, polysorbate 20, and poloxamer 188 were found to reduce aggregate / polymer formation. Trehalose and polysorbate 20 were selected as the primary candidate excipients for the next step.

[0157] Formulas containing various concentrations of trehalose and / or polysorbate 20 are formed in a matrix containing sodium dibasic phosphate heptahydrate, sodium monobasic phosphate monohydrate, and sodium chloride. Their isostatic reduction was then tested by recirculation pump pressure, through four pump pressure cycles, and freeze / thaw cycles (-40). o C / 25 o The ability of each formulation to produce aggregates / polymers (C, 20 cycles) was also tested. Additionally, the ability of each formulation to produce aggregates / polymers was tested at an accelerating temperature of 40°C. o C and 25 o Stability at C was assessed under each condition, measured by the number of visible and sub-visible particles, solution turbidity (OD550), particle size uniformity, and percentage of polymers. For example... Figure 11A and 11B As shown, trehalose reduces particle formation in a dose-dependent manner. Furthermore, the combination of trehalose and polysorbate 20 exhibits the strongest effect in reducing particle formation. Similar results were observed after each of the four pumping passes, with the combination of trehalose and polysorbate 20 resulting in the greatest reduction in particle number (Tables 3 and 4).

[0158] Table 3: Number of particles per milliliter after each pump pressure stage, trehalose or polysorbate 20 * PS20 is polysorbate 20.

[0159] Table 4: Number of particles per milliliter after each pump pressure stage, trehalose and polysorbate 20 *PS20 is polysorbate 20.

[0160] After discovering an additive or synergistic effect of the combination of trehalose and polysorbate 20 in inhibiting particle formation due to pump pressure, many trehalose / polysorbate 20 combinations were tested. As shown in Table 5, increasing the amount of trehalose or polysorbate 20 in the combination reduced particle formation, with the largest reduction observed in the highest trehalose / polysorbate 20 combination (8% trehalose and 0.005% polysorbate 20). The combination of trehalose and polysorbate 20 also reduced agglomerates formed after 20 freeze / thaw cycles more effectively than trehalose or polysorbate 20 alone. Figure 12 As summarized in Table 6, compared to the original formulation, the excipient trehalose and trehalose combined with polysorbate 20 effectively reduced the formation of BMN001 aggregates and polymers.

[0161] Table 5: Effect of different trehalose / polysorbate 20 combinations on particle formation (number of particles per milliliter). * PS20 is polysorbate 20.

[0162] Table 6: Summary of the effect of excipients on reducing aggregates and polymers (reduction %) Based on these excipient tests, two formulations were further identified for clinical trials. One formulation comprises BMN001 (approximately 30 mg / ml), sodium divalent phosphate heptahydrate (approximately 0.19 mg / ml), sodium monovalent phosphate monohydrate (approximately 0.04 mg / ml), sodium chloride (approximately 0.88 mg / ml), and trehalose (approximately 8% (w / v)), with a pH of approximately 7.0. The other formulation comprises BMN001 (approximately 30 mg / ml), sodium divalent phosphate heptahydrate (approximately 0.19 mg / ml), sodium monovalent phosphate monohydrate (approximately 0.04 mg / ml), sodium chloride (approximately 5 mg / ml), trehalose (approximately 4% (w / v)), and polysorbate 20 (approximately 0.005%), with a pH of approximately 7.0.

[0163] equivalent Those skilled in the art will understand, or through routine experimentation, many equivalents to the specific embodiments of this application described herein. The scope of this application is not limited to the foregoing description, but is as indicated by the appended claims. The articles “a,” “an,” and “the” used in this specification and claims, unless otherwise specified, should be understood to include their plural forms. A claim or description including “or” between one or more numbers in a group shall be considered applicable if one, more than one, or all members of that group are present, applied to, or related to the specified product or process, unless otherwise stated in the text. This application includes embodiments in which only one member of the group is present, applied to, or related to the specified product or process. This application also includes embodiments in which one, more than one, or all members of the group are present, applied to, or related to the specified product or process. Furthermore, it is understood that this application encompasses variations, combinations, and arrangements of one or more limitations, elements, clauses, descriptive terms, etc., derived from one or more claims when referenced to another claim under the same underlying claim (or any other related claim), unless otherwise indicated or unless it can be demonstrated by a person skilled in the art that such contradictions or inconsistencies may occur. If, for example, elements are listed in a Markush group or similar format, it should be understood that each subgroup of such elements is also claimed, and any element (group) can be removed from that group. It should be understood that generally, if this application, or an aspect thereof, refers to the inclusion of specific elements, features, etc., certain embodiments of this application or the composition or essential components of an aspect thereof are such elements, features, etc. For simplicity, these embodiments are not explicitly described in every example herein. It should also be understood that any embodiment of this application, such as any embodiment appearing in the prior art, is expressly excluded from the claims, regardless of whether specific exclusions are stated in this specification.

[0164] It should also be understood that, unless otherwise stated to the contrary, when any method claimed herein includes more than one operation, the order of operations is not necessarily limited to the order described in the method, but this application includes embodiments in which such order is limited. Furthermore, if a composition is specified in the claims, this application includes methods of using the composition and methods of manufacturing the composition.

[0165] All publications and patent documents extracted in this application are incorporated herein by reference in their entirety, as if they were incorporated herein by reference individually.

Claims

1. A formulation comprising: (a) a fusion protein comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 5, wherein the concentration of the fusion protein is from about 30 mg / ml to about 35 mg / ml; and (b) a mixture of sodium divalent phosphate heptahydrate at a concentration of about 0.15 mg / ml to about 0.25 mg / ml, sodium monovalent phosphate monohydrate at a concentration of about 0.03 mg / ml to about 0.05 mg / ml, sodium chloride at a concentration of about 8 mg / ml to about 9 mg / ml, potassium chloride at a concentration of about 0.15 mg / ml to about 0.3 mg / ml, magnesium chloride hexahydrate at a concentration of about 0.1 mg / ml to about 0.2 mg / ml, and calcium chloride dihydrate at a concentration of about 0.15 mg / ml to about 0.3 mg / ml, wherein the pH of the mixture is in the range of about 6.5 to about 7.

5.

2. The formulation as described in claim 1 is aqueous.

3. The formulation of claim 1, wherein the fusion protein comprises the amino acid sequence SEQ ID NO:

5.

4. The formulation of claim 1, wherein the fusion protein is composed of the amino acid sequence SEQ ID NO:

5.

5. The formulation of claim 1, wherein the fusion protein comprises the amino acid sequence SEQ ID NO: 5 at a concentration of about 30 mg / ml, sodium divalent phosphate heptahydrate at a concentration of about 0.19 mg / ml, sodium monovalent phosphate monohydrate at a concentration of about 0.04 mg / ml, sodium chloride at a concentration of about 8.66 mg / ml, potassium chloride at a concentration of about 0.22 mg / ml, magnesium chloride hexahydrate at a concentration of about 0.16 mg / ml, and calcium chloride dihydrate at a concentration of about 0.21 mg / ml, and the pH of the formulation is about 7.

0.

6. The formulation as described in claim 1, wherein it is a freeze-dried powder.

7. The formulation as described in claim 1, which is suitable for intrathecal administration to a human individual.

8. A container comprising the formulation as claimed in claim 1.

9. The container as described in claim 8, wherein it is a glass vial.

10. Use of a medically effective amount of the formulation as described in any one of claims 1 to 7 for the preparation of a medicament for treating MPS IIIB disease in an individual suffering from MPS IIIB disease.

11. The use as described in claim 10, wherein the medically effective amount comprises at least about 30 mg / ml of the fusion protein.

12. The use as described in claim 11, wherein the formulation is the formulation as described in claim 5.

13. The use as described in claim 10, wherein the drug is an intrathecally administered drug.

14. The use as described in claim 10, wherein the drug is administered intraventricularly.

15. The use as described in claim 14, wherein the intraventricular drug delivery is of equal volume.

16. The use as described in claim 14, wherein the intraventricular drug delivery is intended to be performed continuously for about 5 minutes to about 240 minutes.

17. The use as described in claim 14, wherein the intraventricular drug delivery is intended to be performed continuously for about 5 minutes to about 10 minutes.

18. The use as described in claim 14, wherein the medication is administered weekly.

19. The use as described in claim 18, wherein the drug is administered weekly for at least 24 consecutive weeks.

20. The use as described in claim 18, wherein the drug is administered weekly for at least 48 consecutive weeks.

21. The use as described in claim 11, wherein the fusion protein comprises the amino acid sequence SEQ ID NO:

5.

22. Use of a medically effective amount of the formulation as described in any one of claims 1 to 7 for the preparation of a medicament for individuals suffering from MPS IIIB disease to slow the rate of decline of at least one symptom of MPS IIIB disease.

23. The use as described in claim 22, wherein the formulation is the formulation as described in claim 5.

24. The use as described in claim 22 or 23, wherein the drug is administered intraventricularly.

25. The use as described in claim 24, wherein the intraventricular drug delivery is of equal volume.

26. The use as described in claim 24, wherein the intraventricular drug delivery is intended to be performed continuously for about 5 minutes to about 240 minutes.

27. The use as described in claim 24, wherein the intraventricular drug delivery is intended to be performed continuously for about 5 minutes to about 10 minutes.

28. The use as described in claim 24, wherein the medication is administered weekly.

29. The use as described in claim 28, wherein the drug is administered weekly for at least 24 consecutive weeks.

30. The use as described in claim 28, wherein the drug is administered weekly for at least 48 consecutive weeks.

31. The use as described in claim 22 or 23, wherein the drug is a drug for improving at least one symptom of the individual’s MPS IIIB disease.

32. The use as described in claim 22 or 23, wherein the at least one symptom is selected from the group consisting of: cognitive decline, language decline, motor decline, social-emotional decline, adaptive decline, conceptual thinking decline, facial recognition decline, storytelling completion decline, hand function / dexterity decline, hearing loss, hyperactivity, aggression, and sleep disorders.

33. The use as described in claim 22 or 23, wherein the slowing of the rate of decline of the at least one symptom can be determined by the following: (a) Determine the rate of symptom deterioration before drug administration, and (b) Determine the rate of symptom deterioration after drug administration; If the rate of symptom decline after medication is lower than the rate of symptom decline before medication, it indicates that the rate of decline has slowed down.

34. The use as described in any one of claims 22 or 23, wherein the individual’s development quotient (DQ) before drug administration can be determined, and the individual’s DQ after drug administration can be determined, wherein when the individual’s DQ after drug administration is higher than the DQ before drug administration, it indicates that the rate of decline has slowed down.

35. The use as described in claim 34, wherein the developmental quotient is determined using the Berry Infant Development Scale, 3rd Edition (BSID-III) or the Kaufman Intelligence Scale for Children, 2nd Edition (KABC-II).

36. The use of a medically effective amount of the formulation as described in any one of claims 1 to 7 in the preparation of a medicament for individuals suffering from MPSIIIB disease to slow the rate of cognitive decline.

37. The use as described in claim 36, wherein the formulation is the formulation as described in claim 5.

38. The use as described in claim 36 or 37, wherein the drug is administered intraventricularly.

39. The use as described in claim 38, wherein the intraventricular drug delivery is of equal volume.

40. The use as described in claim 38, wherein the intraventricular drug delivery is intended to be performed continuously for about 5 minutes to about 240 minutes.

41. The use as described in claim 38, wherein the intraventricular drug delivery is intended to be performed continuously for about 5 minutes to about 10 minutes.

42. The use as described in claim 38, wherein the medication is administered weekly.

43. The use as described in claim 42, wherein the drug is administered weekly for at least 24 consecutive weeks.

44. The use as described in claim 42, wherein the drug is administered weekly for at least 48 consecutive weeks.

45. The use as described in claim 36 or 37, wherein the drug is a drug for improving the individual's cognitive function.

46. ​​The use as described in claim 36 or 37, wherein the slowing of the rate of cognitive decline in the individual can be determined by the following: (a) Measuring the rate of cognitive decline before drug administration, and (b) Determine the rate of cognitive decline after drug administration; When the rate of cognitive decline after drug administration is lower than the rate of cognitive decline before drug administration, it indicates that the rate of decline has slowed down.

47. The use as described in any one of claims 36 or 37, wherein the individual’s development quotient (DQ) before drug administration can be determined, and the individual’s DQ after drug administration can be determined, wherein when the individual’s DQ after drug administration is higher than the DQ before drug administration, it indicates that the rate of decline has slowed down.

48. Use of a medically effective amount of the formulation as described in any one of claims 1 to 7 for the preparation of a medicament to reduce or prevent the accumulation of glycosaminoglycans (GAGs) in one or more tissues of the CNS of an individual suffering from lysosomal storage disease.

49. The use as described in claim 48, wherein the GAG ​​is heparan sulfate and the lysosomal storage disease is MPSIIIB.

50. The use as described in claim 49, wherein the formulation is the formulation as described in claim 5.

51. The use as described in any one of claims 48 to 50, wherein the drug is formulated for intraventricular administration.

52. The use as described in claim 51, wherein the intraventricular drug delivery is of equal volume.

53. The use as described in claim 51, wherein the intraventricular drug delivery is intended to be performed continuously for about 5 minutes to about 240 minutes.

54. The use as described in claim 51, wherein the intraventricular drug delivery is intended to be performed continuously for about 5 minutes to about 10 minutes.

55. The use as described in claim 51, wherein the medication is administered weekly.

56. The use as described in claim 55, wherein the drug is administered weekly for at least 24 consecutive weeks.

57. The use as described in claim 55, wherein the drug is administered weekly for at least 48 consecutive weeks.

58. The use as described in any one of claims 48 to 50, wherein the drug is a drug for improving at least one symptom of MPS IIIB disease in the individual.

59. The use as described in any one of claims 48 to 50, wherein the tissues of the CNS are selected from the group consisting of: gray matter, white matter, periventricular regions, meninges, pia mater, deep tissues of the cerebral cortex, neocortex, cerebellum, caudate nucleus / putamen region, molecular layer, deep regions of the pons or medulla oblongata, midbrain, and spinal cord neurons.

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