WO2016020426A1 - Compositions for the therapy of mucopolysaccharidoses - Google Patents

Abstract

Compositions for the treatment of mucopolysaccharidoses (MPS) including the hepatocyte growth factor (HGF) or its variants as active ingredients are described. HGF and its variants are able to reduce the content of mucopolysaccharides in the organism of patients affected by the disease, preventing the onset of the symptoms coupled with the disease, thus improving the quality and life span of patients.

Description

COMPOSITIONS FOR THE THERAPY OF

MUCOPOLYSACCHARIDOSES

This invention refers to compositions to be used for the treatment of lysosomal storage diseases (LSD), and in particular for the treatment of patients affected by mucopolysaccharidoses (MPS).

STATE OF ART

The lysosomal storage diseases (LSD), due to congenital metabolic disorders, include about 50 different inherited diseases. They show an incidence of 1/7000 newborn and are caused by the deficiency of lysosomal enzymes or transporters with the consequent intra-lysosomal accumulation of undegraded metabolites. Common LSD features include bone disorders, organomegaly, and degeneration of central and peripheral nervous system. Although these diseases have been described for the first time since 1980, and more than 100 years of studies on the genetic and molecular basis of LSD have been passed, there are still open issues on the pathways that link intra-lysosomal accumulation to the degeneration of cellular mechanisms and to the clinical phenotypes related to the different type of LSD (Klein AD, Futerman AH. Pediatr Endocrinol Rev. 2013 Nov; 1 1 Suppl 1 :59-63).

For some LSD, treatments aimed to stop the disease progression or delay the pathologic process and increase the life expectancy of patients are available or under development. To date, different methods and compositions for the treatment of lysosomal diseases have been reported [AU2012267588, US2014017212, US2013331309, PL399467, PL399359, WO2014022841, EP2666476, JP5301719, JP201301 1621, WO2012166653, US2012288489, US8609088, EP2700717, US2012183502 , WO2012094600, US201 1237538, CA2731346, US2010266571, US2010221235, US2010184803, US2010183577, US2010173979, IL193282, PT1083899, US2010068183, EP2333074, WO20091 14729, US2009016998, US5798366, US2001031741, US2004204379, US6537785, US2006045874, US5433946, US2004023218, US7090836, WO03068255, WO2007091 159]. However, these methods and compositions demonstrated limited efficacy. For example, the enzyme replacement therapy, which is the most common, is unable to correct all defects coupled with these diseases, especially the disorders of central nervous system, due to the inability of recombinant enzymes to cross the blood-brain barrier.

The LSD include the mucopolysaccharidoses (MPS), inherited rare diseases, chronic and progressive, caused by the deficiency of lysosomal enzymes. MPS include a group of 1 1 diseases, each one characterized by mutations in the DNA sequence coding for an enzyme involved in the degradation of mucopolysaccharidoses (glycosaminoglycans, GAG). This causes the gradual accumulation of GAG in the tissues and organs of affected patients. The MPS are classified on the basis of the causative gene and of the storage products which are the following: heparan and dermatan sulfate for MPS I; heparan and dermatan sulfate for MPS II; heparan sulfate for MPS III; cheratan sulfate and chondroitin 6 sulfate for MPS IV; dermatan sulfate for MPS VI; heparan sulfate, dermatan sulfate and chondroitin 6 sulfate for MPS VII; hyaluronic acid for MPS IX. The intra-lysosomal storage of GAG, along with other pathogenetic mechanisms, results in different clinical consequences with a wide range of phenotypic variability (Atul Mehta and Brian Winchester. Lysosomal Storage Disorders: A Practical Guide, First Edition 2012). The MPS typical symptoms include organomegaly, mutiplex dysostosis, and mental and developmental retardation (Lampe C, et al. Rheum Dis Clin North Am. 2013 May; 39(2):431-55).

An early diagnosis is essential for MPS patients, and only few therapeutic choices are currently available, which show variable and limited efficacy (Hollak CEM, Wijburg FA. J Inherit Metab Dis. 2013). The first therapeutic approach involves the enzyme replacement therapy (ERT) which is based on the administration of a recombinant enzyme replacing the deficient lysosomal enzyme. The second one involves the substrate reduction therapy (S T) by using molecules which prevent the synthesis of storage products. Another approach involves the hematopoietic stem cell transplantation (HSCT). Finally, it has been proposed the use of gene therapy which, however, is still under investigation. Most of these therapies are described in US2013302308, WO2012177778, US8623910, US2012190642, US201 1008810, EP2081023, KR100762945, KR20040084881, US8105788, US2009092996, WO02055064, RU2196988, RU2083205, JP20081021 14, JP4965999, JP2003265196. Such therapeutic approach show many limits. In particular, the enzyme replacement therapy which is the most common results to be unable to correct all defects coupled with the disease, especially the disorders of central nervous system, due to the inability of recombinant enzymes to cross the blood-brain barrier. The stem cell treatment has also shown poor efficacy, but above all this treatment is extremely hazardous due to the unknown fate of stem cells injected into the patient, including the possibility that these cells acquire a tumoral phenotype. The gene therapy is not currently used in clinical practice because of the high immunogenicity of vectors and the risk of viral genome integration in the treated patients. Due to the limits of these therapeutic strategies, research is still in progress focused to a better understand of the MPS physiopathology and the development of new therapeutic strategies for MPS. Thus, to date there no exist suitable therapies to reduce or remove the acute symptoms of patients affected by MPS.

The hepatocyte growth factor (HGF), also known as scatter factor (SC) plays a critical role in the development of organs such as the liver, through the activation of the tyrosine kinase receptor c-MET (Schmidt, C. et al. (1995). Nature 373: 699-702.; Bottaro, D. P. et al (1991) Science 251 : 802-804.). It has been also shown that HGF regulates the migration of muscle cells (Bladt et al. (1995) Nature 376: 768-71.) and motoneurons (Caton et al. (2000). Development 127: 1751-66.; Ebens et al (1996) Neuron 17: 1 157-72.). HGF chemical structure can be divided in six domains: an N-terminal domain (N), four kringle domains (K), and a serine -protease domain which is catalytically inactive (Donate, L. E. et al. (1994) Protein Sci. 3: 2378-2394). The primary transcript of HGF may undergo to alternative splicing processes which give rise to two distinct products: NKl fragment which includes the N-terminal domain and the first kringle domain, and NK2 which includes the N-terminal domain and the first two kringle domains (Cioce, V., et al (1996) J. Biol. Chem. 271 : 131 10-131 15.; Miyazawa, K. et al (1991). Eur. J. Biochem. 197: 15-22).

The growth factor HGF acts locally in the tissues in a paracrine way and, once secreted, from mesenchymal cells, is captured by extracellular matrix. The GAG such as heparin sulfate bind HGF with nanomolar affinity. The elevated isoelectric point and the high affinity of HGF for the extracellular matrix components reveal the behavior of recombinant HGF in solution. In low ionic strength or physiological buffers, HGF is not structured, tends to form aggregates as demonstrated by analytical ultracentrifugation experiments, and cannot be eluted from a size- exclusion chromatographic column. However, the protein acts as a monomer and it is stable in buffer containing at least 0.5 M NaCl. By contrast, the NKl fragment is much more stable in solution than HGF. The protein NKl has its high affinity binding site for heparan sulfate in the N-terminal domain and in the first kringle domain. Recently, NKl high affinity for dermatan sulfate has also been demonstrated (Deakin et al JBC 284, (10) 631 1-63212009). Furthermore, it has also been demonstrated that NKl is able to cross the blood-brain barrier (Weihong Pan et al, 2006, Experimental Neurology 201 : 99-104).

HGF variants are described in the literature (Lietha D, et al, (2001) EMBO J 20:5543-5555; Rituparna SR et al. 2010 PNAS 107(31): 13608-13613). They have been developed in order to obtain new compounds able to enhance c-MET receptor activation and stimulate regenerative processes, or to obtain new antitumoral drugs able to deactivate c-MET. These variants include NKl, 1K1 e 1K2, which have the ability to activate c-Met, whereas NK4 variant deactivates this receptor.

DESCRIPTION OF THE INVENTION

We found that the hepatocyte growth factor (HGF) and its variants are able to reduce glycosaminoglycan (GAG) content in different organs of patients affected by mucopolysaccharidoses (MPS).

Therefore, this invention has as object compositions to be used for the treatment of clinical symptoms coupled to MPS and caused by GAG accumulation in the different organs. The invention allows to restore the functionality of damaged organs and tissues, thus improving the quality and life span of MPS affected patients.

The compositions of the invention are especially efficient in the treatment of

MPS I, II, III (A, B, C, D), VI, VII types, coupled with the accumulation of heparan sulfate and/or dermatan sulfate, and all their subtypes (Neufeld EF, Muenzer J. The mucopolysaccharidoses. In: Scriver C , Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. 8th ed. New York: McGraw- Hill; 2001, 3421 - 3452).

In particular, we found that HGF and its variants reduce GAG accumulation in in vitro experiments, and the progression of the pathology typical of MPS in in vivo experiments. Most surprisingly, we found that the therapeutic efficacy of HGF and its variants is independent from their scatter activity which is known in literature. In fact, fibroblasts do not express the HGF high affinity c-MET receptor, thus indicating that the reduction of [3H]-glucosamine incorporation into fibroblasts from MPS IIIB affected patients treated with NK1 is not ascribable to c-MET receptor activation. Furthermore, the therapeutic efficacy of HGF and its variants is not strictly related to their ability to bind heparan sulfate and dermatan sulfate. In fact, for example, fibronectin, a membrane protein which has high affinity for heparan sulfate, not only is unable to reduce GAG content in the fibroblasts from MPS IIIB affected patients, but has the opposite effect significantly increasing GAG accumulation. It is preferred the use of HGF variants NK1, 1K1, 1K2 which, tested in vitro at low doses (up to 10^"8M) on fibroblasts from MPS patients, greatly reduced [3H]-glucosamine incorporation as compared to untreated pathologic samples.

In particular, as described in the following examples, the in vivo treatment of knockout mice affected by MPS with 10 mg of one of the HGF variants NK1 , 1K1 , 1K2 per kg of body weight, once a week starting from the second month of age until the eighth month, reduced GAG accumulation in the liver, brain and urine of treated mice as compared to untreated mice, reduced craniofacial and neurological dysfunctions which conversely were present in the untreated mice, and increased the survival of affected mice.

The HGF variants NK1, 1K1, 1K2 do not show toxicity in vivo at the dose administered after six months of treatment.

The compositions of the invention can be administered through injection, i.e. subcutaneous or parenteral, intravenous, intraarterial, intraperitoneal, or intrathecal route.

Preferably, the pharmaceutical composition is formulated in "lactated ringer's solution/1% BSA".

The administration can be also performed per os, through continuous infusion or multiple injections, or intranasal route.

A composition of this invention will generally be formulated in unit dosage forms for injections (solution, suspension, emulsion). The injectable formulations include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, liquid polyethylene glycol and similar), mixtures of solvents or vegetable oils.

In addition, various additives may be added that enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. In many cases, it may be appropriate to include isotonic agents, for example sugars, sodium chloride and similar. The prolonged absorption of the injectable pharmaceutical form can be achieved by the use of agents which delay the absorption, for example, aluminum monostearate and gelatin.

The amount of the composition to be administered will vary depending on the patient to be treated. Preferably, the daily dose is between 1 and 40 mg/ kg per day of HGF or its variants. However, the precise determination of the effective dose will depend on individual factors for each patient, such as weight, height, age, severity of hepatic, cardiac and/or neurologic dysfunctions and/or dysfunctions of other organs of the patient, etc.

Examples of compositions of the invention include: a) liquid preparations such as suspensions, syrups or elixirs for oral, nasal, anal, vaginal, intragastric, mucosal administration (i.e., peritongue, alveolar, gingival, olfactory or respiratory mucosa); b) sterile suspensions or emulsions for parenteral, subcutaneous, intradermal, intramuscular administration or intravenous injection. Such compositions may comprise a suitable carrier, diluent or excipient such as sterile water, physiological saline solution, glucose or similar. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling agents to improve the viscosity, additives, preservatives, flavoring agents, dyes and similar, depending on the route of administration and the desired preparation, as reported for example in "Remington's Pharmaceutical Sciences Handbook", 17th edition, 1985.

The compositions of the invention are conveniently in liquid form, for example, isotonic aqueous solutions, suspensions, emulsions or viscous compositions which may be buffered to a selected pH.

The viscous compositions may be in the form of gels, lotions, ointments, creams and similar (for example, for transdermal administration) and will typically contain a sufficient amount of a thickening agent so that the viscosity is about 2500-6500 cps, although it is also possible to use more viscous compositions, up to 10,000 cps. The viscous compositions preferably have a viscosity of 2500-5000 cps.

The viscous compositions may be formulated in a "range" of viscosity that provides longer contact times with the mucosa, such as the lining of the stomach or nasal mucosa. The compositions can be isotonic, i.e., they may have the same osmotic pressure of blood and or the tear fluid. The desired isotonicity of the compositions of the invention can be accomplished using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.

The viscosity of the compositions can be maintained at the selected level using pharmaceutically acceptable thickening agents such as xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, carbomer, and similar. The compositions of the invention are prepared by mixing the ingredients by using known procedures.

DEFINITIONS

As used herein, the term "variant" may include amino acid substitutions, insertions, deletions, alternative splicing variants, or fragments of the native protein. The term "variant" with respect to a polypeptide refers to an amino acid sequence that is altered by one or more amino acids compared to a reference sequence. The variant may have "conservative" changes, in which a substituted amino acid has similar chemical or structural characteristics, for example, the replacement of leucine with isoleucine. Alternatively, a variant can have "non-conservative" variations, for example, the replacement of a glycine with a tryptophan. Analogous minor variations may include the deletion of an amino acid or an insertion or both. The indication for the determination of amino acid residues that can be substituted, inserted or deleted without eliminating the biological activity of the protein can be obtained using known bioinformatic programs, for example, DNASTA software. The pharmaceutical compositions of the invention may be used as therapeutic agents. As used herein, the terms "treatment" and "therapy" include the curative effects, the effects of reduction and prophylactic effects.

HGF is defined as the protein with the following amino acid sequence (SEQ ID 1):

MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAK TTLIKIDPALKIKTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCL WFPFNSMSSGVKKEFGHEFDLYENKDYIRNCIIGKGRSYKGTVSITKSGIKC QPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYE VCDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTPHRHKFLPER YPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIKTCADNTMNDTD VPLETTECIQGQGEGYRGTVNTIWNGIPCQRWDSQYPHEHDMTPENFKCK DLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGK NYMGNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYCRNPDD DAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNLDHPVISCAKTKQLRVV NGIPTRTNIGWMVSLRYRNKHICGGSLIKESWVLTARQCFPSRDLKDYEA WLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVS TIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQH HRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGR GCAIPNRPGIFVRVAYYAKWIHKIILTYKVPQS .

NK1 is the HGF variant with the following amino acid sequence:

YVEGQRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCADRC TRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKD YIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQEN YCRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVE (SEQ ID 2).

1K1 is the HGF variant with the following amino acid sequence:

YAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRC TRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKD YI NCIIGEGESYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSY GKDLQENY CRNPRGEEGGPWCFTSNPEVRYEVCDIPQCSEVE (SEQ ID 3).

1K2 is the HGF variant with the following amino acid sequence:

YAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRC TRNKGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKD YIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGEDLQENY CRNPEGEEGGPWCFTSNPEVRYEVCDIPQCSEVE (SEQ ID 4).

The invention is further described by the following examples.

EXAMPLE 1: Production and purification of NKl .

The production of NKl can be performed by one of the methods known from the literature (Chirgadze et al. Nat. Struct. Biol, 6 (1999), 72-79.).

The expression vector pPIK9K, inducible by methanol, containing the cDNA coding for NKl was used to transform the GS115 strain of Pichia pastoris yeast. The transformed clones were selected for the best expression of NKl and grown at 30°C under stirring for 72 hours in BMMY medium supplemented with 0.5% methanol every day for the induction of NKl expression. The recombinant protein NKl expressed and secreted into the culture medium was purified by affinity chromatography with a column of heparin (GE Healthcare). The peak corresponding to NKl was collected and the fractions containing the recombinant protein were analyzed by SDS-PAGE. The appropriate fractions were further purified by molecular exclusion chromatography G200. Finally, the purified protein was concentrated up to about 3 mg/ml, dialyzed into 20 mM phosphate buffer, pH 7.4, and stored at -20°C. The purity of recombinant NKl (> 95%) was evaluated preliminarily by SDS-PAGE electrophoresis, and subsequently measured by capillary electrophoresis. The total amount of NKl purified from 1 liter of culture medium containing methanol after 72 hours is about 20 mg evaluated with the Lowry method (Lowry OH, et al, J Biol Chem. November 1951 ; 193 (1): 265- 75.).

The described procedure allows to obtain large quantities of recombinant NK1 with a high degree of purity.

EXAMPLE 2: Reduction of GAG content in MPS IIIB cells.

Fibroblasts from MPS IIIB patients and control individuals were cultured and incubated for 48 hours with 7uCi/ml [3H]-glucosamine (Perkin Elmer, Waltham, MA, USA) in medium containing 2% FBS and 10^"7M of NK1 described in example 1. Before harvesting, the cells were washed with DPBS, suspended in water and lysed using cycles of freezing and thawing. An aliquot of the extract was used for the determination of protein concentration by the method of Lowry . The lipids were extracted by addition of chloroform, methanol and water (4:8:3, v/v/v). After 10 minutes of incubation at room temperature, the extracts were recovered by centrifugation at 13000 rpm for 10 min, washed with acetone, dried and subjected to proteolysis over night at 65°C with 1 mg/ml of papain (Sigma) in 100 mM sodium acetate buffer containing 5 mM EDTA and 5 mM cysteine (pH 5.5). The incorporation of [3H]-glucosamine was measured by liquid scintillation counting, and normalized with protein concentration.

Relevant effect of NK1, obtained as described in example 1, were observed on the rate of incorporation of [3H]-glucosamine in MPS IIIB fibroblasts at a concentration of 10^"7 M after 48 hours. The incorporation is reduced by 40% compared to untreated cells (Table 1).

TABLE 1: Reduction of [3H]-glucosamine content in MPS IIIB fibroblasts after treatment with 10^"7M NK1 for 48 hours.

Sample [3H] -glucosamine incorporation

CTR NT 100%

CTR + NK1 10^"7M 100%

MPS IIIB NT 100%

MPS IIIB NK1 10 ⁷M 60%

Moreover, MPS IIIB fibroblasts were incubated with different concentrations (from 10^"8M to 10^"6M) of NK1 for 48 hours and the incorporation of [3H]-glucosamine was measured. MPS IIIB fibroblasts incorporated less [3H]-glucosamine with increasing concentration of NKl until the optimal dose of 10^"6M (Table 2).

TABLE 2: Decrease of the content of [3H] -glucosamine in the GAG of MPS IIIB fibroblasts after 48 hours of treatment with concentrations of NKl from 10-8M to 10-6M.

MPS MPS MPS MPS

MPS IIIB IIIB IIIB IIIB

IIIB NKl NKl NKl NKl

NT 5* 10^"8M 10^"7M 5* 10^"7M 1 * 10^"6M

[3H] -glucosamine

100% 77% 60% 54% 48% incorporation

At these concentrations no interference with growth and cell viability was observed, nor changes in cell morphology.

The incorporation of [3H] -glucosamine was measured for periods ranging from 24 to 48 hours at the concentration of 10^"6 M of NKl . In this experiment, the effect of NKl on the incorporation of [3H]-glucosamine in GAG was already detectable after 24 hours and increased over time from 20% reduction of incorporation at 24 hours up to 55% reduction after 72 hours (Table 3).

TABLE 3 : Decrease of the content of [3H]-glucosamine in the GAG of MPS

IIIB fibroblasts after treatment with NKl 10^"6M for 24, 36, 48 hours.

Sample NKl [3H]- [3H]- [3H]- glucosamine glucosamine glucosamine incorporation incorporation incorporation

Tempo 24 ore 36 ore 48 ore

MPS IIIB NT 100% 100% 100%

MPS IIIB 10 ⁶M 70% 60% 45%

These experiments show that NKl at the concentration of 10^"6M after 48 hours reduces by 55% the incorporation of [3H]-glucosamine in the GAG of cultured fibroblasts from MPS IIIB patients as compared with untreated cells, showing a significant effect already at 24 and 36 hours of exposure.

EXAMPLE 3: NK1 reduces the content of GAG in MPS IIIB cells independently from c-MET receptor activation.

To demonstrate that the effectiveness of NK1 in decreasing the content of GAG is independent from the activation of the c-MET receptor, an experiment of Western blotting using a specific antibody toward the receptor c-MET was carried out. The tested samples included protein extracts from a human prostate epithelial cell line (PNT1A) expressing the c-MET receptor as a positive control, from MPS IIIB fibroblasts used in the previous experiments, and immortalized fibroblast cells (NIH3T3) commonly used in laboratory. As shown in the Figure, the cells from MPS IIIB patients do not express the c-MET receptor: lane 1 , protein extracts from MPS IIIB fibroblasts; lane 2, protein extracts from human prostate epithelial cells PNT1 A; lane 3, protein extracts from immortalized fibroblasts (NIH3T3).

This result highlights the properties of NK1 to reduce the content of GAG in the cells of affected patients, regardless of its already known property to activate its specific receptor c-MET.

EXAMPLE 4: Fibronectin does not reduce the content of GAG in MPS IIIB cells.

Fibronectin, a membrane protein with high affinity for heparan sulfate, was chosen as a negative control of the therapy for the reduction of GAG content in the fibroblasts of MPS IIIB patients.

The MPS IIIB fibroblasts were grown and incubated for 48 hours with 7uCi/ml [3H]-glucosamine (Perkin Elmer, Waltham, MA, USA) in medium containing 2% FBS and 10^"6M fibronectin. Before harvesting, the cells were washed with DPBS, suspended in water and lysed using cycles of freezing and thawing. An aliquot of the extract was used for the determination of protein concentration by the method of Lowry. The lipids were extracted by addition of chloroform, methanol and water (4:8:3, v/v/v). After 10 minutes of incubation at room temperature, the extracts were recovered by centrifugation at 13000 rpm for 10 minutes, washed with acetone, dried and subjected to proteolysis over night at 65 °C with 1 mg/ml papain (Sigma) in 100 mM sodium acetate buffer containing 5 mM EDTA and 5 mM cysteine (pH 5.5). The incorporation of [3H]-glucosamine was measured by liquid scintillation counting, and normalized with protein concentration.

The results on the effects of fibronectin at a concentration of 10^"6M on the rate of incorporation of [3H]-glucosamine in MPS IIIB fibroblasts after 48 hours show that fibronectin promoted an increased accumulation of [3H]-glucosamine by 200% (Table 4).

TABLE 4: Increase of the content of [3H] -glucosamine in the GAG of MPS IIIB fibroblasts after treatment with 10-6M fibronectin for 48 hours.

Sample [3H] -glucosamine incorporation

MPS IIIB NT 100%

MPS IIIB Fibronectin 10 ⁶M 200% These results demonstrate the absolutely non- obviousness of the invention since not all proteins (such as fibronectin) with high affinity for the substrate of accumulation of the disease appear to be effective for the treatment of the disease.

EXAMPLE 5: NK1 reduces GAG content also in other MPS types: MPS I, II, IIIA e VI.

To demonstrate the effectiveness of therapy for the treatment of MPS that accumulate heparan and dermatan sulfate, we tested NK1 on fibroblasts from patients affected by MPS I, II, IIIA, and VI. Fibroblasts were cultured and incubated for 48 hours with 7DCi/ml [3H]-glucosamine (Perkin Elmer, Waltham, MA, USA) in medium containing 2% FBS and 10^"6M NK1. Before harvesting, the cells were washed with DPBS, suspended in water and lysed using cycles of freezing and thawing. An aliquot of the extract was used for the determination of protein concentration by the method of Lowry. The lipids were extracted by addition of chloroform, methanol and water (4:8:3, v/v/v). After 10 minutes of incubation at room temperature, the extracts were recovered by centrifugation at 13000 rpm for 10 minutes, washed with acetone, dried and subjected to proteolysis over night at 65 °C with 1 mg/ml papain (Sigma) in 100 mM sodium acetate buffer containing 5 mM EDTA and 5 mM cysteine (pH 5.5). The incorporation of [3H]-glucosamine was measured by liquid scintillation counting, and normalized with protein concentration.

Significant effects of NK1 were observed on the rate of [3H]-glucosamine incorporation in fibroblasts from MPS I, II, IIIA and VI patients, at a concentration of 10^"6 M, after 48 in all tested lines in accordance with the values reported in Table 5.

TABLE 5: Decrease of [3H]-glucosamine content in the GAG of MPS I, II, IIIA and VI fibroblasts after treatment with 10^"6M NK1 for 48 hours compared to untreated controls which did not show any decrease of [3H]-glucosamine incorporation.

Sample NK1 concentration [3H] -glucosamine incorporation

MPS I 10^"6M 55%

MPS II 10^"6M 58%

MPS IIIA 10^"6M 55%

MPS VI 10^"6M 56%

These results indicate the specificity of the treatment for all MPS which accumulate heparan and dermatan sulfate.

EXAMPLE 6: NK1 prevents the occurrence of the disease phenotype in the mouse model of MPS IIIB

A mouse model of MPS IIIB (Li HH, et al, 1999 96 (25): 14505-10), missing of the gene NAGLU, N-acetylglucosaminidase, coding for the lysosomal enzyme required for the gradual degradation of the heparan sulfate, was used to evaluate the therapeutic efficacy in vivo.

These mice, called NAGLU knockout mice, are healthy and fertile when young up to the second month of age, and survive for 8-12 months. They are completely deficient in the protein N-acetylglucosaminidase and show a massive accumulation of heparan sulfate in the liver and kidney as well as other minor changes in the activity of several other lysosomal enzymes present in the liver and brain.

Although most of the vacuoles contain granular material of accumulation of glycosaminoglycans, large pleomorphic inclusions are visible in some neurons and pericytes in the brain (Li HH, et al, 1999 96 (25): 14505-10). A hypoactive anomalous behavior occurs at 4-5 months of age. The general phenotype of these mice is similar enough to that of patients with MPS IIIB to make these mice a good model to study its pathophysiology and to develop new therapies.

NAGLU knock-out mice were treated for 6 months with intraperitoneal injections of 10 mg/kg (w/w) of NKl, once a week starting from the second month of age until the eighth month of age. Treated mice show no phenotype of the disease. In fact, the treated animals are perfectly able to freely move throughout all the space at their disposal, eat and drink like a normal mouse, have a fur color and quality completely normal, and does not show the typical craniofacial phenotype of the disease.

The analysis of GAG accumulation in the urine after two months of treatment showed a significant reduction of 70% of urinary GAG in mice under treatment compared to untreated mice. Moreover, the same analysis carried out on liver and brain samples of treated mice showed a complete reduction of GAG accumulation after 4 months of treatment compared to untreated mice.

These results show that NKl treatment prevents the occurrence of malformations in the murine model of MPS IIIB, and therefore is an effective therapy for this disease.

Claims

1. Compositions comprising hepatocyte growth factor or variants thereof for use in the treatment of mucopolysaccharidoses.

2. Compositions according to claim 1 comprising the variants NK1, 1K1, 1K2 of hepatocyte growth factor.

3. Compositions according to claim 1 or 2 for use in the treatment of mucopolysaccharidoses of type I, II, III (A, B, C, D), VI, VII and of sub-types thereof.

4. Compositions according to one or more of claims 1 to 3 in form suitable for the administration by injective route.

5. Compositions according to claim 4 in form suitable for the subcutaneous, parenteral, intravenous, intra-arterial, intramuscular, intraperitoneal or intrathecal administration.

6. Compositions according to claim 5 in which the carrier is a lactate ringer/1% BSA solution.

7. Hepatocyte growth factor or variants thereof for use in the treatment of mucopolysaccharidoses.

8. Hepatocyte growth factor or variants thereof for the use according to claim 7 in the treatment of mucopolysaccharidoses of type I, II, III (A, B, C, D), VI, VII and of sub-types thereof.

9. Variants of hepatocyte growth factor for the use according to claim 7 or 8 selected from NK1, 1K1 , 1K2.