WO2012137991A1

WO2012137991A1 - Pharmaceutical composition for the prevention or treatment of mucolipidosis ii, comprising a lysosomal enzyme as an active ingredient

Info

Publication number: WO2012137991A1
Application number: PCT/KR2011/002317
Authority: WO
Inventors: 이성열; 박성익
Original assignee: 주식회사 메디진바이오
Priority date: 2011-04-04
Filing date: 2011-04-04
Publication date: 2012-10-11
Also published as: KR20140048105A; KR101619815B1

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of mucolipidosis II, comprising a lysosomal enzyme complex. The composition improves the clinical findings of mucolipidosis II, and thus can be used to advantage in the prevention or treatment of mucolipidosis II.

Description

Specification

Of Mucofatosis II Containing Lysosomal Enzyme as an Active Ingredient

Pharmaceutical composition for prophylaxis or treatment Field of the invention The present invention relates to a pharmaceutical composition for preventing or treating mucodiosis I I comprising a lysosomal enzyme as an active ingredient. Background Art of the Invention Mucolipidosis I l Onucol ipidosis I I; Mycolipidosis I I; Mycolipidosis I I) is also called I cell disease and is a metabolic disease inherited by autosomal recessive. Characteristic symptoms are 1) unique facial features, 2) skeletal abnormalities, and 3) mental retardation. Symptoms of Mucofatosis I I are similar to Hurler Syndrome, but appear much more seriously. Symptoms associated with the disease are usually pronounced in infancy, with various lesions on the skull and face and slow growth.

This disease belongs to a group known as lysosomal storage disease. Lysosomes (lysosomes) are substances in the cytoplasm and contain various enzymes and play an important role in the intracellular digestion of extracellular subtypes by phagocytosis as well as metabolism of intracellular components. Lack of these enzymes results in the accumulation of mucoids in tissue cells.

Mucofatosis II is caused by a mutation in the GNPTA gene located on chromosome 4 (4q21-q23). GNPTA gene mutations result in a deficiency of an enzyme called UDP—N-acetylglucosamine-1-phosphortransferase, which is involved in the synthesis of mannose-6-phosphate. Lack of this enzyme causes cells with lysosomal enzyme levels to It falls within and rises in blood and body fluids. Symptoms of Mucofatosis II are caused by a lack of lysosomal enzymes, which result in the accumulation of certain fatty substances and carbohydrate complexes in the tissues and cells of the body.

Prenatal diagnosis of Mucofatosis II can be diagnosed as a decrease in the activity of UDP-N-acetylglucosamine-1-phosphortransferase on prenatal amniotic fluid and chorionic membrane tests. And special laboratory test findings. In addition, the activity of UDP-N-acetylglucosamine-1-phosphorase enzyme in leukocytes or fibroblasts can be measured. Levels of lysosomal enzymes are particularly elevated in serum and decreased in fibroblasts. To date, the ultimate treatment for mucofatosis II is limited and only a few have undergone bone marrow transplantation. At present, supportive therapy, such as nutrition, treatment of recurrent respiratory tract infections, etc. is best, and no specific treatment has been developed. Therefore, there is an urgent need for a new treatment for mucofatosis II. Therefore, the inventors of the present invention tried to develop mucofatosis II treatment, and confirmed that the lysosomal enzyme obtained from the human placenta is excellent in mucofatosis Π treatment effect and completed the present invention. SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of new Mucofatosis I I.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating Mucofatosis II, comprising a lysosomal enzyme as an active ingredient. Since the pharmaceutical composition comprising the lysosomal enzyme according to the present invention improves the clinical findings of mucofatosis II, it can be usefully used for the prevention or treatment of mucofatosis II. 1 is a schematic diagram of a targeting vector for the production of GNPTA knockout mice. In the figure, the black square bars represent exons and the other parts represent introns. 2 is a detailed view for the construction of a targeting vector for the production of GNPTA knockout mice.

3 shows the results of Southern blotting of embryonic stem cell clones identified as having the GNPTA knockout gene. In the figure, WT represents the wild type GNPTA gene and K0 represents the GNPTA knockout gene.

4 is a result of measuring the body weight from the birth to 24 weeks of age of mucofatosis I I mice (homo 1 and 2) and normal mice (control 1 to 4).

FIG. 5 is a photograph comparing normal mice and mucofatosis II mice at 5 weeks of age. The upper mouse is Mucofatosis II mouse and the lower mouse is normal mouse.

6A and 6B show the results of bone characteristics using normal-energy X-ray absorpt iometry (DXA) in normal mice and mucofatosis I I model mice, respectively.

Figure 7 shows the results of measuring the amount of lysosomal enzymes in the serum according to the culture time in normal mice (wi ld-type) and mucofatosis II mice (mutant).

8 is a graph showing the body weight change according to the administration of the lysosomal enzyme of the present invention in the Mucofatosis II mouse model. DETAILED DESCRIPTION OF THE INVENTION Hereinafter, terms used in the present invention are defined.

The term "lysosomal enzyme" as used herein refers to a lysosomal enzyme obtained through lysosomal fractionation and purification from normal tissue isolated from a mammal, preferably a human. The "lysosomal enzyme" used in the present invention is a complex lysosomal enzyme including two or more enzymes among about 50 known lysosomal enzymes, except for a single lysosomal enzyme. That is, the "lysosomal enzyme" used in the present invention may be used interchangeably with the "complex lysosomal enzyme", the "lysosomal enzyme complex" or the "lysosomal rich fract ion". Lysosomal enzymes of the present invention can be obtained in any tissue in vivo, including placenta, liver and the like.

The term "GNPTA gene" as used herein refers to

It refers to a gene encoding N-acetylglucosamine-1-phosphor transferase, which gene is well known to those skilled in the art. In this regard, the term "GNPTA knockout gene" as used herein means that a part of the gene involved in the synthesis of GNPTA is deleted and is not normally expressed. In particular, the present invention refers to a gene characterized in that the exon 20 part of the axon 12 in the GNPTA gene is deleted. "GNPTA Knockout Gene", "Modified GNPTA Gene" GNPTA Mutation Gene "nests can be used interchangeably.

"Mucofatosis II mouse" as used herein refers to a mouse model showing symptoms of mucofatosis II. The mouse model is used to search for substances that have an effect on Mucofatosis II. The most important symptom of mucopyridosis II is a condition in which weight is not greatly increased. Therefore, the prophylactic or therapeutic effect of mucofatosis II can be confirmed by the rapid weight gain of the mouse model. . The present invention provides a pharmaceutical composition for preventing or treating mucofatosis II, comprising a lysosomal enzyme as an active ingredient.

The lysosomal enzyme used as an active ingredient in the pharmaceutical composition according to the present invention can be separated from the human placenta through conventional fractionation and purification procedures known in the art. In one embodiment of the invention, the lysosomal enzyme is isolated from the human placenta, but can be obtained in the same way from mammals in addition to humans, and obtained from other tissues in vivo, including the liver, which is known to contain lysosomal enzymes in addition to the placenta You may.

Lysosomal enzymes used in pharmaceutical compositions according to the invention are described in C. ALQUIER et al. , Biochem. J. (1985) 232, 529-537. Specifically, the method may include the following steps:

1) suspending human placenta in TS complete solution after passing through a sieve;

2) recovering the pellet by centrifuging the suspension twice at a rate of 300-10,000 X g and resuspending in TS buffer;

3) homogenizing the resuspension using a homogenizer, and centrifuging it at a rate of 300-1, 000 xg to recover the supernatant;

4) recovering the supernatant by centrifuging the supernatant at a rate of 2,000-6,000 xg, and centrifuging it at a rate of 10, 000-40, 000 X g to recover the pellet;

5) suspending the pellet in TS complete layer, and adding 30% percol to mix; and

6) centrifuging the mixture twice at a rate of 50, 000-70, 000 X g to recover the lysosomal enzyme layer, and washing it to obtain a lysosomal enzyme. The lysosomal enzyme comprises two or more of the 50 known lysosomal enzymes. Preferably, the lysosomal enzymes of the present invention include arylsulfatase A, arylsulfatase B, alpha-galactosidase, beta-galactosidase, beta-glucosidase, nucleososaminidase A, and the like. can do. Arylsulfatase A, arylsulfatase B, alpha-galactosidase, beta-galactosidase and nucleosamidase A are each present in an amount of 0.01 to 1.0 X 10 ^"3 units per mg of lysosomal enzyme of the present invention. It may be included as, beta-glucosidase may be included in an amount of 5 to 20 X 10 ^"3 units, but is not limited to the above content. The effect of lysosomal enzyme isolated according to the present invention on mucolipidosis can be confirmed through in vivo experiments using Mucolipidosis II mouse model. The mucofatosis Π mouse model may be a knock-out mouse obtained by removing a part of the exon of the GNPTA gene of a wild-type mouse. In experiments with the Mucofatosis II mouse model, the lysosomal enzyme according to the present invention has an effect of improving mucofatosis. In particular, it prevents weight gain failure, the most important indicator of mucofatosis II, and shows a steady weight gain effect. The compositions of the present invention can be prepared in pharmaceutical formulations according to conventional methods. In the preparation of the formulation, it is preferred that the lysosomal enzyme is mixed or diluted with the carrier or enclosed in a carrier in the form of a container. When the carrier is used as a diluent, it may be a solid, semi-solid or liquid substance which acts as a carrier, excipient or medium for the antibody. Thus, the formulation may be in the form of tablets, pills, powders, sasei, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectables, sterile powders and the like. Examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, solbi, manni, stag silicate salose, methyl salose, microcrystalline salose, polyvinylpyridone, water, methyl Hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The formulation may further comprise a layering agent, an antifoaming agent, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like. The composition of the present invention It can be formulated using methods well known in the art to provide rapid, sustained or delayed release after administration to a mammal. Accordingly, the present invention provides the use of lysosomal enzymes for the manufacture of a medicament for the prevention or treatment of mucofatosis II. Also provided is a method of preventing or treating mucofatosis Π in a mammal, comprising administering a lysosomal enzyme to a mammal in need of prevention or treatment of mucofatosis II.

The composition of the present invention can prevent or treat mucofatosis I I by administering to a mammal including a human. At this time, the dosage of the composition depends on the subject to be treated, the severity of the disease or condition, the rate of administration and the judgment of the prescribing physician. As an active ingredient, the lysosomal enzyme is administered via a parenteral route in mammals in an amount of 0.001 to 10 mg / kg (weight) per day, preferably 0.005 to 1 mg / kg (body weight), once a day or divided into mammals. Can be. In some cases, smaller dosages may be more suitable than the above-mentioned ranges, and more may be used without causing adverse side effects, and higher dosages may be dispensed in small portions over a day. May be The route of administration of the pharmaceutical composition of the present invention is not particularly limited, but may be preferably administered intravenously. Hereinafter, the present invention will be described in detail with reference to Examples. However, this is merely to illustrate the present invention, and thus the scope of the present invention is not limited thereto. Example 1 Isolation of Lysosomal Enzymes

Human placenta sections (GCJBP, South Korea) were passed through a metal sieve (pore size 0.3 mm 3) and then suspended in 1 L TS buffer (10 mM-Tris / HCl, 0.25 M sucrose, pH 7.4). The tissue suspension was centrifuged at 770 Xg for 20 minutes to remove supernatant (SO). It was removed and the precipitate was recovered. The recovered precipitate was resuspended with TS complete layer solution, washed and centrifuged at 770Xg for 20 minutes to recover the precipitate. The obtained precipitate was resuspended in 50 mL of TS complete solution, which was named 'open follicle fraction'.

An open vesicles fraction obtained in step 1, a glass / Teflon Potter-was used to obtain a homogenate (P1) by using a Elbe hem (gl _ass / T _e fl ₀ n Potter Elvehjera) homogeneous, which was separated 20 minutes and centrifuged at 800Xg supernatant ( S2) and pellets (P2) were recovered, respectively. The pellet (P2) was resuspended in 40 mL TS complete solution, re-homogenized and centrifuged under the same conditions. The supernatant obtained in the above process was recovered and centrifuged at 4,000 × g for 20 minutes to obtain a supernatant (S3) and pellets (P3). The supernatant (S3) was centrifuged at 26,000Xg for 20 minutes to recover the pellet (P4) and the supernatant (S4). The resulting pellets (P4) were resuspended in 15 mL of TS complete solution.

Nine volumes of percoll (Pharmacia) were added to one volume of 2.5M sucrose / 10 mM Tris-HCl to make i so-osmotic solution and then 30% (v / v) with TS complete solution. And 30 mL of this 30% percol were mixed with 1.2 mL of pellet (P4) suspension. The mixture was centrifuged at 60,000 × g for 45 minutes to recover the portion that sank below. The recovered product was diluted with 3 times TS complete solution and centrifuged at 26,000Xg for 20 minutes to remove percol. The washing process was repeated twice to finally obtain a lysosomal enzyme pellet (L). The pellet was resuspended in TS complete solution and stored at -20 ^° C. Example 2: Analysis of Lysosomal Enzymes Representative components of the lysosomal enzyme obtained in Example 1 were analyzed according to the conventional analytical method as follows. Arylsulfatase A and B were measured spectrophotometrically to measure the enzyme activity by measuring the sulfate released when P-nitrocatechol sulfate was degraded by arylsulfatase A or B. Alpha-galactosidase was measured by measuring the NuF released when MuF-galactoside was hydrolyzed by alpha-galactosidase using fluorescence photometry (f luorometry). Similarly, beta-galactosidase

The enzyme activity was measured by measuring the MuF released when MuF-beta-galactopyranoside was hydrolyzed by beta-galactosidase. Beta-glucosidase measured enzyme activity by measuring MuF released when MuF-beta-glucoside was hydrolyzed by beta-glucosidase. Nucleosaminidase A measured the enzyme activity by measuring the MuF released when MuF-glucosamine was hydrolyzed by nucleosaminedase A.

The analysis results are shown in Table 1.

Table 1

As shown in the table, the lysosomal enzyme assay showed the highest amount of beta-glucosidase, and arylsulfatase A, arylsulfatase B, alpha-galactosidase and nucleosamynidase A. Was found to be present in trace amounts. Repeated experiments showed that arylsulfatase A, arylsulfatase B, alpha-galactosidase, beta-galactosidase, and nucleosaminidase A were each 0.01 to 1.0 X 10 ^"3 units per mg of lysosomal enzyme. Present in an amount, beta-glucosidase has been shown to be present in an amount of 5 to 20 X 10 &^quot; Example 3 Preparation of Mucofatosis II Mouse Model A mouse model showing Mucofatosis II for verifying the efficacy of the lysosomal enzyme isolated by Example 1 was prepared according to the following method.

<3-1> Preparation of Clones Containing GNPTA Knockout Gene A homologous recombinat ion method was used to remove the GNPTA knockout (K0) gene from which axons of the GNTPA gene causing mucofatosis II were removed. Clones were prepared.

Specifically, after obtaining genomic DNA from embryonic stem cells (JBI) of 129 / SvJ mouse J1, about 10.1 kb of Not l− corresponding to the left arm of the GNPTA gene of genome 1) ^ Sal l fragments were amplified by PCR using primer sets of SEQ ID NO: 1 and SEQ ID NO: 2. In addition, the 5.8 kb Sal l-Nhel fragment corresponding to the right arm of the GNPTA gene from the genomic DNA was amplified by PCR using primer sets of SEQ ID NO: 3 and SEQ ID NO: 4 (see FIGS. 1 and 2). ). The basic structure of the GNPTA gene shown in FIGS. 1 and 2 is described in Stepahn Tiede et al. , Nature Medicine, Vol. 11, No. 10, October 2005.

Using the left arm and the right arm obtained above, a targeting vector for deleting axon 20 (about 8.5 kb) from exon 12 of the GNPTA gene was prepared as shown in FIG. 1. Consisting of left arm (10105 bp), MCI promoter and neomycin resistance gene (MCl-neo; -1300 bp), a positive selection marker, and right arm (-5800 bp) Targeting vectors were prepared by ligation of the gene cassette with pOsdupdel vector (Gene Targeting Laboratory). The targeting vector does not express normal GNPTA genes because homologous recombinat ions occur in the complementary fragments, resulting in deletion of the GNTPA gene exon 12 through exon 20, resulting in the loss of a portion of the gene (see FIGS. 1 and 2).

The targeting vector was linearized by cleavage with Not I, and then cultured by electroporation into J1 embryonic 21 cells of 129 / SvJ mice. After the incubation, 336 clones that were resistant to G418 and ganciclovir were selected.

<3-2> Screening of Clones by Southern Blotting Among the 336 clones obtained in <3-1>, the clones containing the GNPTA gene from which axon 12 to axon 20 were deleted were screened by Southern blotting as follows. It was. Mouse genomic DNA was isolated from each clone, treated with Hindi I I and Sai l, followed by Southern blotting to homologous recombination to select clones with knockout genes (about 8.0 kb). As a probe for Southern blotting, a 310 bp sequence present in the exon 23 region of the GNTPA gene was used (see FIG. 1), and the probe was obtained by PCR using primer sets of SEQ ID NOs: 5 and 6.

Southern blotting confirmed that 4 of the 336 clones had a modified GNPTA gene of 8.0-kb (FIG. 3).

<3-3> Preparation of Mucofatosis II Mouse Model The four clones screened in Example <3-2> were injected into the 3.5-day pc blastocysts of C57BL6 mice 9-9: L0 cells. Specifically, 3.5 days after male and female mating The females were regenerated by cervical dislocation and the uterus was extracted and 1 ml syringe was used to perfusion 1 ml of injection solution containing 20 mM HEPES, 10% fetal bovine serum, 0.1 mM 2-mercaptoethanol and DMEM. Embryos were isolated from uterine tissue using microglass tubes under a dissecting microscope, and the isolated embryonic embryos were transferred onto 35 mL Petri dishes and the embryonic stem cell clones selected above were introduced into blastocysts of the blastocysts. The clone-implanted blastocysts were transplanted into gestational surrogate mouse uterus to induce the development of chimeric mice, a type of heterotypic hybrids formed from embryonic stem cell clones and C57BL6 mice. As described above, the embryonic stem cell-injected blastocyst was transplanted into the surrogate mouse uterus and cultured for about 19 days, thereby transforming the embryonic stem cell-derived transformed cells and the mouse blastocyst-derived cells to GNPTA +/- genotype. Genie produced chimeric mice. Thereafter, the chimeric mice obtained above were crossed with C57BL6 mice six times or more, thereby preparing GNPTA + / + and GNPTA-/-mice in the F1 step, and finally preparing GNPTA-/-nick out mice in which all GNPTA genes were knocked out. It was. Example 4 Analysis of Mucofatosis Pathology in Mucofatosis II Mouse Model Mucofatosis pathology was analyzed while mucofatosis II mice prepared according to Example 3 and normal mice were bred under the same conditions.

<4-1> Weight Comparison

Body weights of Mucofatosis II mice and normal mice were measured weekly, and the results are shown in FIG. 4. As shown in FIG. 4, normal mice (controls 1 to 4) gain weight over time, whereas mucofatosis II mouse models (homo 1 and 2) of the present invention show that growth is delayed compared to normal mice. appear. Body weight is the most important characteristic that represents Mucofatosis II, the subject with Mucofatosis II does not increase the body weight at all compared to the normal control group based on this can determine whether Mucofatosis II. 4 of the above The results show that the GNPTA knockout mice of the present invention are mouse models representing Mucofatosis II.

On the other hand, looking at Figure 5 showing the appearance of the mouse after 5 weeks, it was found that the size of the upper Mucofatosis I I mouse is significantly smaller than the normal mouse at the bottom. In addition, even after 12 weeks, the GNPTA knockout mice of the present invention exhibited mucofatosis I I symptoms such as small size, short mouth and unique coarse face compared to normal mice.

The above results demonstrate that the right mucofatosis II mouse model was prepared by Example 3 of the present invention.

<4-2> Bone Formation Comparison by DXA

Bone characteristics were compared in 10-week-old Mucofatosis II mice and normal mice using DXACDual-energy X-ray absorpt iometry. 6A shows the DXA results of normal mice, and FIG. 6B shows the DXA results of the mucodilipidemic II mice prepared in the present invention.

As shown in FIG. 6A, in the case of normal mice, BMD of 0.0624 g / cm ² , BMC of 0.623 g, and area (area) of 9.98 cm ² were observed. BMP} 0.0527g / cm ² , BMC 0.443 g, area 8.40 cm ² . From the above results, it can be seen that the mucofatosis II mouse model prepared in the present invention has shorter bones and problems in bone formation than normal mice, and bone mineral density (BMD) is also reduced compared to normal mice.

This demonstrates that the correct Mucofatosis II mouse model was prepared by Example 3.

<4-3> Biochemical Analysis

The amount of lysosomal enzyme in serum was measured in normal mice and mucofatosis II mice prepared in the present invention. The measurement results are shown in FIG. 7. Degree As shown in Fig. 7, mucofatosis II mice (mutant) were measured in serum compared to normal mice (wi ld-type)

N-acetylglucosaminidase,

Beta—galactosidase,

Beta-glucuronidase and nucleosaminidase

^ hexosaminidase A) was significantly increased and beta-glucosidase independent of mannose-6-phosphate was significantly reduced. This was the same as the result measured in the serum of human Mucofatosis I I patients. The results demonstrate that the right mucofatosis I I mouse model was prepared by Example 3. Example 5: Mucofatosis Pathology Analysis of Mucofatosis II Mouse Model Following Lysosomal Enzyme Treatment Mucofatosis II prepared in Example 3 to confirm the effect of lysosomal enzyme on mucofatosis II The mouse model was examined for weight change. Weight loss is the most important indicator of Mucofatosis II. Since patients with Mucofatosis II rarely gain weight despite a normal diet, a substance that rapidly increases weight by administration is effective for Mucofatosis II. It can be determined that there is.

The Mucofatosis II mouse model prepared in Example 3 was divided into 10 experimental groups and 10 control groups, the experimental group was administered with the lysosomal enzyme of the present invention prepared in Example 1, and the control group was administered normal saline. Test substances were administered intravenously in the tail vein for a total of 4 weeks, twice a week, 2 mg per kg body weight of the mouse.

After 4 weeks of administration, the change in body weight over time was examined. The weight measurement results are shown in FIG. 8. As shown in Figure 8, in the case of the control group was not much change in weight, in the group treated with the lysosomal enzyme according to the present invention steadily And it appeared to increase weight rapidly. The results show that the lysosomal enzyme according to the present invention has a prophylactic and therapeutic effect against Mucofatosis II.

Claims

Claims: Claim 1. A pharmaceutical composition for the prevention or treatment of Mucofatosis II, comprising a lysosomal enzyme as an active ingredient. 2. The pharmaceutical composition for preventing or treating mucofatosis II according to claim 1, wherein the lysosomal enzyme is derived from human placenta. 3. The lysosomal enzyme of claim 1, wherein the lysosomal enzyme is arylsulfatase A, arylsulfatase B, alpha-galactosidase, betacogalactosidase, beta-glucosidase and nucleosamidase A. Characterized in that the pharmaceutical composition for the prevention or treatment of mucofatosis II. 4. The method according to claim 1, wherein the lysosomal enzyme is 0.01 to mg of arylsulfatase A, arylsulfatase B, alpha-galactosidase, beta-galactosidase and nucleosaminidase A, respectively. A pharmaceutical composition for preventing or treating mucofatosis II, comprising beta -glucosidase in an amount of 1.0 X 10 ^"3 units, and comprising beta -glucosidase in an amount of 5 to 20 X 10 ^{" 3} units. 5. The method of claim 1, wherein said lysosomal enzyme is

1) passing the human placenta through a sieve and suspending it in TS complete solution (101111 ^ 1 3 / strip, 0.25 M sucrose, pH 7.4);

2) recovering the pellet by centrifuging the suspension twice at a rate of 300˜1,000 × g and resuspending in TS complete layer;一 _

3) homogenizing the resuspension using a homogenizer, and centrifuging it at a rate of 300 to l, 000Xg to recover the supernatant; 4) recovering the supernatant by centrifuging the supernatant at a rate of 2,000-6, 000 xg, and centrifuging it at a rate of 10,000-40, 000 X g to recover the pellet;

5) suspending the pellet in TS complete layer, and adding by adding 30% percol l; And

6) centrifuging the mixture twice at a rate of 50, 000—70,000 xg to recover the lysosomal enzyme layer, and washing it to obtain a lysosomal enzyme.

A pharmaceutical composition for the prevention or treatment of mucofatosis I I, which is obtained by a method comprising a. 6. Use of a lysosomal enzyme for the manufacture of a medicament for the prophylaxis or treatment of mucofatosis I I. 7. A method for preventing or treating mucofatosis II in a mammal, comprising administering a lysosomal enzyme to a mammal in need of prevention or treatment of mucofatosis II. 8. The method according to claim 7, wherein the lysosomal enzyme is administered intravenously.