WO2023012092A1 - A combined preparation for the treatment of pompe disease - Google Patents

Abstract

The present invention relates to a combined preparation comprising an acid alpha-glucosidase (GAA) enzyme and at least one allosteric chaperone of the acid alpha-glucosidase enzyme selected from the group consisting of L-carnitine, D-carnitine, acetyl-D-carnitine, vitamin B1, vitamin B6, and any combination thereof. The combined preparation of the invention is effective for the treatment of Pompe disease.

Description

A combined preparation for the treatment of Pompe disease

The present invention concerns the field of therapeutic treatment of lysosomal storage disorders, particularly Pompe disease.

Glycogen storage disease type 2, or Pompe disease (PD, OMIM 232300) is an inborn metabolic disorder caused by the functional deficiency of the acid lysosomal alphaglucosidase (GAA, acid maltase, E.C.3.2.1.20), the enzyme hydrolyzing alpha- 1,4 and alpha- 1,6-glucosidic bonds in glycogen and belonging to family GH31 of the carbohydrate active enzyme (CAZy) classification (www.cazy.org; {Lombard, 2014 #1589}). GAA deficiency results in glycogen accumulation in lysosomes and in secondary cellular damage, with mechanisms not fully understood (Parenti G, et al. “A chaperone enhances blood a- glucosidase activity in Pompe disease patients treated with enzyme replacement therapy”; Mol Ther. 2014 Nov;22(l l):2004-12. doi: 10.1038/mt.2014.138). In PD, muscles are particularly vulnerable to glycogen storage, and disease manifestations are predominantly related to the involvement of cardiac and skeletal muscles. However, central nervous system involvement is emerging as part of the clinical spectrum in infantile-onset patients.

It is assumed that to obtain positive therapeutic effects it is enough that the enzymatic activity of GAA is rescued at about 10% of the wild type, meaning that relatively small increase in activity can mitigate the clinical course. Therapeutic strategies include the supply of wild type enzyme, such as enzyme replacement therapy (ERT), gene therapy, or small-molecule drugs able to adjust cellular networks controlling protein synthesis, folding, trafficking, aggregation, and degradation, thus facilitating the escape of mutated proteins from the endoplasmic reticulum- associated degradation (ERAD) machinery (Kohler L, et al.; “Pompe Disease: From Basic Science to Therapy”. Neuro therapeutics (2018) 15:928-942 https://doi.org/10.1007/sl3311-018-0655-y; Mu TW, et al, “Chemical and biological approaches synergize to ameliorate protein-folding diseases”. Cell. 2008 Sep 5; 134(5):769- 81. doi: 10.1016/j.cell.2008.06.037). Since early 2006, enzyme replacement therapy (ERT) with recombinant human alphaglucosidase has been approved and is currently considered the standard of care for the treatment of PD, improving survival by stabilizing the disease course.

However, there are significant limitations in connection with enzyme replacement therapy in that, despite treatment, some patients experience little clinical benefit or show signs of disease progression. Several factors concur in limiting therapeutic success of ERT, including the age at start of treatment, the immunological status of patients, the insufficient targeting of the enzyme to skeletal muscle, the possible instability at neutral pH of the recombinant enzyme during the transit to lysosomes, the relative deficiency of the cation-independent mannose-6-phosphate receptor, required for enzyme uptake, in muscle cells, and the build up of the autophagic compartment observed in myocytes (Porto C, et al.; “Pharmacological Enhancement of a-Glucosidase by the Allosteric Chaperone N-acetyl cysteine”. Molecular Therapy vol. 20 no. 12, 2201-2211 dec. 2012).

In recent years, other therapeutic approaches have been proposed and evaluated to target lysosomal storage diseases. Among them, pharmacological chaperone therapy (PCT) represents a particularly promising strategy. This approach, which has been designed for the treatment of protein misfolding diseases (PMD), exploit small-molecule ligands that may bind directly to the defective enzymes, templating the protein folding in the most stable conformation(s) and preventing their recognition and disposal by the ERAD machinery (Parenti G, et al. “A chaperone enhances blood a-glucosidase activity in Pompe disease patients treated with enzyme replacement therapy”; Mol Ther. 2014 Nov;22(l l):2004-12. doi: 10.1038/mt.2014.138).

Most pharmacological chaperones (PC) proposed or used for the treatment of lysosomal storage diseases (LSD) are reversible competitive inhibitors of the target enzymes. Compared to ERT, small-molecule chaperones have important advantages in terms of biodistribution, oral availability, and reduced impact on patients’ quality of life. Recent studies have shown that 1-deoxynojirimycin (DNJ, 1), N-butyl-deoxynojirimycin (NB-DNJ) and 1-deoxygalactonojirimycin (DGJ, 2) may also potentiate the effects of the enzymes used for ERT in Pompe (Porto C, et al., “The pharmacological chaperone N- butyldeoxynojirimycin enhances enzyme replacement therapy in Pompe disease fibroblasts”, Mol Ther. 2009 Jun;17(6):964-71) and Fabry diseases, respectively (Porto C, et al., “Synergy between the pharmacological chaperone 1-deoxygalactonojirimycin and the human recombinant alpha-galactosidase A in cultured fibroblasts from patients with Fabry disease”; J Inherit Metab Dis. 2011 Dec 21).

However, the majority of PCs so far identified for the treatment of LSD are active-site directed competitive inhibitors which interfere with the activity of the targeted enzymes (Parenti G, et al. “A chaperone enhances blood a-glucosidase activity in Pompe disease patients treated with enzyme replacement therapy”; Mol Ther. 2014 Nov;22(l l):2004-12. doi: 10.1038/mt.2014.138). Enzyme inhibition is therefore a major concern on the clinical use of pharmacological chaperones.

The paradox that an inhibitor can increase the enzymatic activity is explained by the fact that therapeutic levels can be reached at sub-inhibitory intracellular concentrations and that the high concentrations of the natural substrate accumulated in the lysosome or the acidic conditions within the organelle may displace the PC inhibitor from the active site.

Alternative pharmacological chaperone therapies have been proposed in the art.

WO2013/182652 describes the therapeutic use of N-acetyl cysteine (NAC) and related compounds N-acetyl serine (NAS) and N-acetyl glycine (NAG) as allosteric non-inhibitory chaperones for lysosomal acid alpha-glucosidase (GAA). As shown in WO2013/182652, these chaperones do not interact with the GAA catalytic domain, and consequently are not competitive inhibitors of the enzyme. Despite this clear advantage, clinical translation of NAC and related compounds has several potential drawbacks. First, only a limited number of GAA gene mutations appeared to be responsive to these compounds in fibroblast cells from Pompe disease patients indicating drug’s effectiveness only in a restricted number of patients. In addition, exceedingly high doses of NAC are required in vivo for the chaperone effect to take place thereby making this compound unsuitable for long-term therapies (Porto C. et al., Mol Ther. 2012 Dec;20(12):2201-l l). In view of the foregoing, the need for novel therapeutic approach for the treatment of Pompe disease is felt in the art.

It is therefore an object of the present invention to provide an effective therapeutic approach for the treatment of Pompe disease, particularly aiming at reducing the severity of this disease.

It is another object of the present invention to provide a therapeutic approach which enables to achieve a long-lasting clinical response, thereby allowing for an efficient and safe therapeutic intervention in both infantile and late-onset forms of Pompe disease.

These and other objects are met by the present inventors, who conducted a drug discovery study by assaying various compounds, including amino acid derivatives, sugars and vitamins, and surprisingly found that the carnitine compounds L-camitine, D-carnitine, and acetyl-D-carnitine as well as the water-soluble vitamins vitamin Bl, vitamin B6 and vitamin C, all exert a stabilizing effect on the lysosomal enzyme acid alpha-glucosidase (GAA). In particular, these compounds acted beneficially in cell-free assays by preventing the loss of enzyme activity at neutral pH and increasing the thermal stability of GAA in a concentration dependent manner. Moreover, as shown in Figures 3 and 11, the carnitine and vitamin B compounds, when assayed in combination with active site-directed pharmaceutical chaperones, show a clear additive effect on GAA stability demonstrating that these compounds all act as allosteric chaperones not binding to the active site of the enzyme.

Based on these findings, the inventors have carried out further experimental studies in order to exploit the identified GAA allosteric chaperones as a possible therapeutic strategy to potentiate enzyme replacement therapy for Pompe disease. Surprisingly, when the acid alpha-glucosidase enzyme was administered to fibroblasts from patients with Pompe disease in combination with at least one of the compounds as above indicated, either at the same time, separately or in sequential order, a dramatic increase was observed in the lysosomal trafficking, the maturation, and the intracellular activity of the administered enzyme (Figures 13-15). Besides the effect on the externally supplied GAA enzyme, the use of the allosteric chaperones as above defined advantageously resulted also in significantly enhanced levels of residual activity of the endogenous GAA enzyme present in Pompe patients’ fibroblasts. As further validation, the present inventors observed that enzyme replacement therapy in a mouse model of Pompe disease resulted in a significant increase of the therapeutic enzyme levels in key disease-relevant tissues when administered in combination with the carnitine compound (Figure 22).

Therefore, an aspect of the present invention is a combined preparation comprising an acid alpha-glucosidase (GAA) enzyme and at least one allosteric chaperone of the acid alphaglucosidase enzyme, for simultaneous, separate or sequential use in the therapeutic treatment of Pompe disease in a patient, wherein the at least one allosteric chaperone of the acid alphaglucosidase enzyme is selected from the group consisting of L-camitine, D-carnitine, acetyl- D-carnitine, vitamin Bl, vitamin B6, and any combination thereof.

Other features and advantages of the combined preparation according to the invention are defined in the appended claims which form an integral part of the description.

Carnitine is an essential nutrient for the transport of long-chain fatty acids into the mitochondrial matrix. This molecule (beta-hydroxy-gamma-trimethylaminobutyric acid) is a quaternary ammonium compound biosynthesized from the amino acid lysine and methionine, and exists as one of two stereoisomers: D-camitine and L-carnitine. Both are biologically active, but only L-carnitine naturally occurs in animal. Accordingly, the generic term carnitine is usually used as referring to L-carnitine.

L-carnitine has proved therapeutically beneficial for the treatment of several cardiovascular diseases, such as acute and chronic myocardial ischemia, angina pectoris, heart failure and cardiac arrhythmias, as well as for the treatment of patients with chronic uremia on hemodialysis, or to combat muscle asthenia and muscle cramps.

Derivatives of L-carnitine are also available, such as acetyl-L-carnitine and propionyl-L- camitine. US 4,343,816 describes the use of acyl L-camitine for the therapeutic treatment of peripheral artery disorders, such as Raynaud's disease and acrocyanosis.

US 4,346,107 discloses therapeutic methods involving the administration of acetyl L- camitine to patients with altered brain metabolism, associated, for example, with senile or pre-senile dementia and Alzheimer's disease.

US 4,194,006 describes the use of acetyl D, L-camitine in the therapeutic treatment of myocardial ischemia and arrhythmia at a therapeutic dose of 50 mg/kg.

US 5,432,199 discloses that acetyl-D-camitine and pharmacologically acceptable salts thereof are particularly effective in the therapeutic treatment of glaucoma.

The studies described in Huang HP et al. (Hum Mol Genet. 2011 Dec 15;20(24):4851-64) show that L-camitine treatment of Pompe disease-induced pluripotent stem cells improves mitochondrial dysfunction.

Vitamin Bl (VitBl) (thiamine, 3 - [(4-amino-2-metholpyrimidin-5-yl) methyl] -5- (2- hydroxyethyl)-4-methyl-l,3-thiazol-3-io) plays a critical role in carbohydrate metabolism and is a coenzyme involved in the metabolism of pymvate and other alpha-keto acids to produce energy via the Krebs cycle. It is widely distributed in foods and primarily absorbed in the small intestine by both passive diffusion and active transport. However only small amounts of this vitamin are stored in the liver, so a daily intake of thiamin-rich foods is needed.

As a drug, vitamin Bl is used to treat thiamine (Beriberi) and niacin deficiency states, Korsakoff alcoholic psychosis, Wernicke-Korsakoff syndrome, delirium and peripheral neuritis (Lewis and Hotchkiss).

Vitamin B6 (VitB6), in its biologically active pyridoxal 5'-phosphate form, is involved in various reactions of amino acid and glycogen metabolism, in the synthesis of nucleic acids, hemogloblin, sphingomyelin and other sphingolipids, and in the synthesis of neurotransmitters such as serotonin, dopamine, norepinephrine and gamma-aminobutyric acid (GABA) (Wilson MP, et al. “Disorders affecting vitamin B 6 metabolism”. Inherit Metab Dis. 2019 Jul;42(4):629-646. doi: 10.1002/jimd.12060. Epub 2019 Mar 20.). VitB6 is used in medicine for the treatment of vitamin B6 deficiency, for the treatment of nausea and vomiting in pregnancy, and as a food supplement.

Vitamin C (VitC) (ascorbic acid, (2~{R})-2-[(l~{S})-l,2-dihydroxyethyl]-3,4-dihydroxy- 2~{H}-furan-5-one) is a powerful reducing and antioxidant agent which acts against bacterial infections and has roles in detoxifying reactions and collagen formation. Vitamin C is used in the treatment of scurvy, clinical syndrome that results from Vitamin C deficiency. The average vitamin C requirement for adults is defined as between 70 and 150 mg per day.

As used herein, the term “chaperone” refers to a molecule capable of facilitating protein folding, which acts by mediating folding of de novo synthesized proteins, or by assisting refolding of misfolded proteins.

As used herein, the term “allosteric” refers to the ability of a molecule to modulate the activity of a protein, such as e.g. an enzyme, by binding to a site topographically distinct from the site, called the active site, in which the catalytic activity characterizing the enzyme is carried out.

As outlined above, the inventors have surprisingly found that in enzyme replacement therapy (ERT) the administration of at least one allosteric chaperone selected from the group consisting of L-carnitine, D-camitine, acetyl-D-carnitine, vitamin Bl, vitamin B6, and any combination thereof, improves considerably the effectiveness of the administered replacement acid alpha-glucosidase without any inhibitory effect on the activity of this enzyme and independently of mutations affecting individual patients. Moreover, the combined therapy of the invention also provides for enhancing the stability of a mutant, endogenous GAA protein that is deficient due to defective folding. Stability and, hence, activity of the endogenous protein is enhanced concurrently with the increased stability of the administered replacement GAA enzyme that corresponds to the mutant protein. Within the context of the present description, the expression “protein stability” means the resistance to denaturing conditions (heat, pH) that allows it to be active. Protein stability is usually measured by testing protein unfolding and/or inactivation by denaturants.

The combination therapy of the present invention has been found to be particularly effective in increasing the stability of the replacement acid alpha-glucosidase (GAA) enzyme at a pH value comprised between 7.0 and 7.5, i.e. a pH which is no longer optimal for the function of the lysosomal enzyme.

An ideal chaperone should indeed be able to protect the enzymes from degradation without interfering with its activity, be largely bioavailable in tissues and organs, reach therapeutic levels in cellular compartments where its action is required, show high specificity for the target enzyme with negligible effects on other enzymes, and have a good safety profile.

Advantageously, the aforementioned compounds acting as allosteric chaperones in the combined preparation for use according to the invention are all already approved as pharmaceutical drugs and/or nutraceuticals for human therapy.

Accordingly, the combined preparation for use according to the invention might be promptly included in clinical protocols for the treatment of Pompe disease without the need of long and expensive clinical trials, which are even more challenging in the case of rare diseases because of the small sample size.

As a further advantage, the toxicity of the allosteric chaperones compounds for use according to the invention is reported to be low even at doses higher than those used in the studies conducted by the present inventors. In fact, for the treatment of Beriberi, vitamin Bl is usually administered intramuscularly at a dose of 10- 20 mg three times daily or, generally, it is used as a dietary supplement (for adults, a 50-100 mg tablet per day) (Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline Institute of Medicine (US) Standing Committee on the Scientific Evaluation of Dietary Reference Intakes and its Panel on Folate, Other B Vitamins, and Choline. Washington (DC): National Academies Press (US); 1998). As known in the art, vitamin B6 is used for the treatment of vitamin B6 deficiency and for the prophylaxis of isoniazid-induced peripheral neuropathy. Additionally, this compound is used in combination with doxylamine (such as the commercially available product Diclectin) for the treatment of nausea and vomiting in pregnancy, and as a food supplement (in adults, 100 mg/day) (Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline Institute of Medicine (US) Standing Committee on the Scientific Evaluation of Dietary Reference Intakes and its Panel on Folate, Other B Vitamins, and Choline. Washington (DC): National Academies Press (US); 1998). In patients with scurvy, provision of 300 mg to 1 g of vitamin C is used once a day intravenously. In addition, up to 6 g of vitamin C may be administered parenterally in adult subjects without evidence of toxicity (Abdullah M, Attia FN. SourceS tatPearls 2018 Oct 27). U-carnitine, with doses of 3 grams daily as oral supplement, is used to treat patients affected by congestive heart failure, end-stage renal disease, hyperthyroidism, male infertility, myocarditis, polycystic ovary syndrome, and toxic side effects caused by the drug valproic acid. Instead, intravenous infusion of 60 mg/kg of U-carnitine are used for patients suffering of angina pectoris (Pepine C.J., Welsch M.A. (1995) “Therapeutic potential of L-camitine in patients with angina pectoris”. In: De Jong J.W., Ferrari R. (eds) The Carnitine System. Developments in Cardiovascular Medicine, vol 162. Springer, Dordrecht. https://doi.org/10.1007/978-94-011-0275-9_16).

In the combined preparation for use according to the invention, the acid alpha-glucosidase (GAA) enzyme may be a recombinant protein, wherein the term "recombinant", as used herein, refers to a polypeptide produced using genetic engineering approaches at any stage of the production process, for example by fusing a nucleic acid encoding the polypeptide to a strong promoter for overexpression in cells or tissues or by engineering the sequence of the polypeptide itself. The person skilled in the art is familiar with methods for engineering nucleic acids and encoded polypeptides (for example, described in Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989), Molecular Cloning, CSH or in Brown T. A. (1986), Gene Cloning - an introduction, Chapman & Hall) and for producing and purifying native or recombinant polypeptides (for example Handbooks "Strategies for Protein Purification", "Antibody Purification", published by GE Healthcare Life Sciences , and in Burgess, R. R., Deutscher, M. P. (2009): Guide to Protein Purification ).

Alternatively, the acid alpha-glucosidase (GAA) enzyme in the combined preparation for use according to the invention may be purified from a variety of tissues, such as e.g. liver, muscle and placenta, using any of a variety of conventional methods including liquid chromatography such as normal or reversed phase, affinity chromatography, size exclusion chromatography, immobilized metal chelate chromatography and gel electrophoresis. The selection of the most appropriate enzyme purification method is within the reach of those skilled in the art.

In a preferred embodiment of the invention, the acid alpha-glucosidase (GAA) enzyme is a recombinant human acid alpha-glucosidase (rhGAA).

The present invention relates to therapeutic treatment of Pompe disease. Within the context of the present description, the expression “Pompe disease” is intended to encompass all the various clinical presentations of this disease, including patients with infantile, juvenile, and late-onset forms.

According to the invention, it is envisaged that any possible combination of the allosteric chaperones in the combined preparation as above defined is encompassed within the present invention.

A preferred combined preparation for use according to the invention comprises a recombinant acid alpha-glucosidase (GAA) enzyme and the GAA allosteric chaperone L- camitine.

Another preferred combined preparation for use according to the invention comprises a recombinant acid alpha-glucosidase (GAA) enzyme and the GAA allosteric chaperone L- camitine in combination with D-camitine or a racemic mixture of L- and D- carnitine. In still another preferred embodiment, the combined preparation for use according to the invention comprises a recombinant acid alpha-glucosidase (GAA) enzyme and one of vitamin Bl and vitamin B6, or any combination thereof.

The combined preparation of the invention may be administered alone or in combination with one or more active-site directed molecular chaperones. Exemplary active-site directed molecular chaperones include, but are not limited to, 1-deoxynojirimycin (DNJ) and N- butyl-deoxynojirimycin (NB-DNJ).

The administration of the constituents of the combined preparations of the present invention can be made simultaneously, separately or sequentially in any order. Namely, the present invention intends to embrace administration of an acid alpha-glucosidase (GAA) enzyme and at least one allosteric chaperone of the acid alpha-glucosidase as above-defined in a sequential manner in a regimen that will provide beneficial effects of the drug combination, and intends as well to embrace co-administration of these agents in a substantially simultaneous manner, such as in a single dosage device having a fixed ratio of these compounds or in multiple, separate dosage devices for each compound, where the separate dosage devices can be taken together contemporaneously, or taken within a period of time sufficient to receive a beneficial effect from both of the constituent compounds of the combination.

The exact dose of the combined administration of an acid alpha-glucosidase (GAA) enzyme and of the at least one allosteric chaperone of GAA according to the invention may vary depending on the disease stage as well as on the specific components of the combined preparation, i.e. the allosteric chaperone compound and the type of GAA enzyme, and on the patient’s characteristics (e.g. sex, age, weight, etc.).

For replacement therapy, the acid alpha-glucosidase (GAA) enzyme is generally administered by infusion every week or every other week, preferably in an amount ranging from 20 mg and 40 mg/kg of body weight. According to the combined administration of the invention, the at least one allosteric chaperone of GAA may be administered on the same day as the GAA enzyme or, alternatively, it may be administered for a period of time ranging from 3 to 6 consecutive days, for example 3, 4, 5, 6 days, said period of time including the enzyme administration day. A preferred combined therapeutic regimen consists of three consecutive administration days of the allosteric chaperone, i.e. from the day before ERT administration to the day after enzyme administration.

In the above embodiment, the at least one allosteric chaperone of GAA may be administered once daily, or multiple times per day, for example three times per day, depending upon the condition of the patient. Preferably, the daily dose of single or multiple administrations is in the range of 100 to 250 mg/kg of body weight.

In another embodiment of the combined administration according to the invention, the course of therapy preferably contemplates a continuous daily therapy of the at least one allosteric chaperone of GAA, preferably over one or more years. In the continuous treatment regimens, the daily dosage of the at least one allosteric chaperone of GAA according to the invention is preferably comprised between 100 mg and 200 mg per kilogram of body weight.

Preferably, in the above regimen, the daily dosage of the at least one allosteric chaperone of GAA according to the invention is comprised between 20 mg and 40 mg/kg of body weight/infusion.

In a preferred embodiment, the therapeutic treatment of the invention comprises administering to a patient a dose of a recombinant human acid alpha-glucosidase (rhGAA) comprised between 20 mg and 40 mg/kg of body weight/infusion every other week and a dose of L-camitine comprised between 100 and 250 mg/kg of body weight/die.

In another preferred embodiment according to the invention, the therapeutic treatment of the invention comprises administering to a patient a dose of a recombinant human acid alphaglucosidase (rhGAA) comprised between 20 and 40 mg/kg of body weight/infusion and a dose of vitamin C comprised between 1 and 2 g/die on the same day of enzyme infusion or on the following days.

In the combined therapy according to the invention the GAA enzyme and the at least one allosteric chaperone of said enzyme can be administered to a patient in any acceptable manner that is medically acceptable including the enteral (oral or gastro-enteral, rectal, sublingual, buccal) or parenteral (intravenous, intraarterial, transcutaneous, intramuscular, intradermal, subcutaneous, intraperitoneal) routes.

Preferably, in the combined preparation for use according to the invention the acid alphaglucosidase (GAA) enzyme is formulated for parenteral administration, more preferably for intravenous administration by infusion or injection.

As mentioned, the acid alpha-glucosidase (GAA) enzyme and the at least one allosteric chaperone as above defined, may also be effectively administered in the form of a pharmaceutical composition, i.e. of a physical mixture of the two compounds.

Accordingly, a second aspect of the present invention is a pharmaceutical composition for use in the therapeutic treatment of Pompe disease in a patient, comprising an acid alphaglucosidase (GAA), at least one allosteric chaperone of the acid alpha-glucosidase, and pharmaceutically acceptable vehicles, excipients and/or diluents, wherein the at least one allosteric chaperone of the acid alpha-glucosidase is selected from the group consisting of L-carnitine, D-carnitine, acetyl-D-camitine, vitamin Bl, vitamin B6, and any combination thereof.

According to the invention, any combination of the allosteric chaperones of the GAA enzyme are contemplated in the pharmaceutical composition. Particularly preferred embodiments are as above defined with reference to the combined preparation for use according to the invention. The pharmaceutical compositions of the present invention can be formulated using pharmaceutically acceptable vehicles, excipients and/or diluents well known in the art in dosages suitable for oral or parenteral, such as intravenous, administration.

The term "pharmaceutically acceptable" refers to compounds which may be administered to mammals without undue toxicity at concentrations consistent with effective activity of the active ingredient.

Formulations of the pharmaceutical composition according to the invention suitable for parenteral administration, include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.

For the purpose of oral therapeutic administration, the pharmaceutical composition according to the invention can be used in the form of tablets, troches, capsules, e.g., gelatin capsules, syrups, slurries, or suspensions.

The selection of suitable vehicles, excipients and/or diluents is carried out depending on the desired form of administration and this selection is within the skills of those of ordinary skill in the art.

The amount of the compounds contained in the pharmaceutical composition for use according to the invention may vary quite widely depending upon many factors such as e. g. the administration route and the vehicle.

A preferred pharmaceutical composition according to the invention comprises the acid alpha-glucosidase enzyme (GAA) at a concentration comprised within the range of from 0.05% to 1% w/v on the total weight of the composition and/or the at least one allosteric chaperone of the acid alpha-glucosidase enzyme at a concentration comprised within the range of from 0.5% to 5% w/v on the total weight of the composition. In a particularly preferred embodiment according to the invention, the concentration of the acid alpha-glucosidase enzyme (GAA) in the composition is of 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, or 1.0% w/v on the total weight of the composition.

In another particularly preferred embodiment according to the invention, the concentration of the at least one allosteric chaperone of the acid alpha-glucosidase enzyme in the composition is of 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5.0% w/v on the total weight of the composition.

As an example, the pharmaceutical composition for use according to the invention may contain 0.05% w/v of an acid alpha-glucosidase (GAA) enzyme and 1.0% of L-camitine w/v on the total weight of the composition.

The selection of the dose of active principles and the dosage regimen also fall within the skills of those of ordinary skill in the art, and their selection depends on several factors, such as for example the age of the patient and the degree of progression of the disease.

The following experimental section is provided purely by way of illustration and is not intended to limit the scope of the invention as defined in the appended claims. In the following experimental section reference is made to the appended drawings, wherein:

Figure 1 shows a comparison of the effect of L-carnitine on the stability of rhGAA. (a) Effect of L-CAR on the rhGAA stability: L-CAR at various concentrations was incubated with rhGAA and the enzymatic activity was measured after 5 h of incubation at pH 7.4. (b) Effect of L-CAR on the structural stability of rhGAA: L-CAR was incubated with rhGAA at ten concentrations (from 2 to 20 mM). Changes in the fluorescence of SYPRO Orange were monitored by DSF as a function of temperature at pH 7.4. (c) Summary of the Tms measured by DSF: Tms were calculated according to Niesen FH, et al., Nat Protoc. 2007;2(9):2212- 21. The standard deviations for each melting temperature were calculated from three replicates, (d) Determination of the KD rhGAA-L-CAR by DSF. Thermal scans were performed in triplicate and melting temperatures were calculated according to Niesen FH, et al., Nat Protoc. 2007;2(9):2212-21. For the determination of the dissociation constant (KD) of L-CAR experimental data were best fitted according to a simple cooperative model equation as reported in Vivoli M., et al, J Vis Exp 51809 (2014);

Figure 2 shows the effect of a racemic mixture of D/L-CAR on the structural stability of rhGAA. (a) DSF analysis. L-CAR and D-CAR were incubated with rhGAA either alone (10 and 20 mM) or in combination (at 5 mM or 10 mM each). Changes in the fluorescence of SYPRO Orange were monitored by DSF as a function of temperature at pH 7.4. (b) Summary of the Tm measured by DSF;

Figure 3 shows a comparison of the effect of allosteric and non- allosteric chaperones on the stability of rhGAA. (a) Analysis of the synergistic effect of L-CAR and NAC. L-CAR was incubated with rhGAA either alone (10 or 20 mM) or in combination with NAC, at 10 mM each, (b) Analysis of the synergistic effect of L-CAR and DNJ. L-CAR was incubated with rhGAA either alone (10 or 20 mM) or in combination with DNJ (10 and 0.1 mM, respectively). For both experiments, changes in the fluorescence of SYPRO Orange were monitored by DSF as a function of temperature at pH 7.4;

Figure 4 illustrates the effect of L-CAR and D-CAR on rhGAA (a) Time course of the stabilizing effect of L- and D-CAR on the activity of rhGAA. The stability of rhGAA activity was measured in the absence and in the presence of L-CAR (10 or 20 mM) and D- CAR for 48 h. (b) Effect of L-CAR on rhGAA activity. The specific activity of rhGAA was measured in the absence and in the presence of L-CAR at various concentrations;

Figure 5 shows a comparison of the effect of D-CAR and A-D-CAR on the stability of rhGAA (a) Effect on the rhGAA stability. The specific activity of rhGAA was measured in the absence and in the presence of D- and A-D-CAR at various concentrations (0.1-10 mM). (b) Effect on rhGAA activity: D- and A-D-CAR at various concentrations (0.1-10 mM) were incubated with rhGAA and the enzymatic activity was measured after 5 h of incubation at pH 7.4. (c) Effect of D-CAR on the stability of the rhGAA activity. rhGAA was incubated alone or with D-CAR (2-10 mM) in sodium phosphate buffer pH 7.4 at 37 °C. After 5h, the residual alpha-glucosidase activity was measured with the standard assay, (d) Effect of D-CAR on the structural stability of rhGAA: D-CAR was incubated with rhGAA at 5 concentrations (from 2 to 10 mM). Changes in the fluorescence of SYPRO Orange were monitored by DSF as a function of temperature at pH 7.4. (e) Summary of the Tms measured by DSF: Tm values were calculated according to Niesen FH, et al., Nat Protoc. 2007;2(9):2212-21. The standard deviations for each melting temperature were calculated from three replicates;

Figure 6 shows the effect of L-CAR on rhGAA stability in the medium. PD fibroblasts were incubated in Dulbecco’s modified Eagle’s medium (DMEM) in the presence (black) or in the absence of L-CAR 10 mM. GAA activity decreased over time, with significant differences between rhGAA in combination with L-CAR and rhGAA alone already detectable after 2 hrs;

Figure 7 illustrates the effects of vitamin Bl (VitBl), vitamin B6 (VitB6), vitamin C (VitC), carbocisteine, saccharose and trehalose on the structural stability of rhGAA. The compounds (10 mM) were incubated in 25 mM sodium phosphate buffer, pH 7.4, and 150 mM NaCl. Scans were performed at l°C/min in the range 25-95°C with rhGAA. Changes in the fluorescence of SYPRO Orange were monitored by Differential Scanning Fluorimetry (DSF) as a function of temperature at pH 7.4. (a) The fluorescence of SYPRO orange was normalized to the maximum fluorescence value for each scan so that relative fluorescence was calculated, (b) Melting temperatures (Tm) were calculated according to Niesen FH, et al., Nat Protoc. 2007;2(9):2212-21;

Figure 8 shows a comparison of the effect of VitBl, VitB6 and VitC on rhGAA activity. Effect on enzyme activity, measured under standard conditions, normalized for the amount of rhGAA used (specific activity - U/mg) in the absence and presence of increasing concentrations of chaperones. VitB 1, VitB6 e VitC at four concentrations (1, 5, 10 e 20 mM);

Figure 9 shows the effect of VitBl(10 mM and 20 mM), VitB6 (5 mM e 10 mM) and VitC (10 mM e 20 mM) on the stability of the specific activity of rhGAA. RhGAA was incubated in the presence and absence of vitamins, and at regular time intervals (0 to 360 minutes) activity was assayed. Activity is expressed as the percentage of residual activity considering the specific activity of non-incubated rhGAA as 100%;

Figure 10 shows the measurement of the dissociation constant (KD) of VitBl, VitB6 and VitC. Dissociation constants (KD) of the different molecules were measured by rhGAA thermal stability scans according to Vivoli M., et al, J Vis Exp 51809, 2014. DSF scans were performed as described above, in the range 0-30 mM for each chaperone. Melting temperature (Tm) values were plotted as a function of ligand concentration. KD values were calculated by applying the cooperative binding model equation as reported in Vivoli M., et al, 2014, using GraphPAD Prism software (GraphPad Software, San Diego, CA, USA);

Figure 11 shows the effect of (A) vitamin B 1 and (B) vitamin C in combination with non- allosteric chaperones on the structural stability of rhGAA. The structural stability of rhGAA was evaluated in the presence of VitBl (A) and VitC (B) alone (10 mM) and with DNJ (0.1 mM DNJ). Fluorescence changes of SYPRO-orange dye were followed by temperature-dependent DSF at pH 7.4. RhGAA was incubated in the presence and absence of vitamins, and activity was assayed at regular time intervals (0 to 360 minutes). Activity is expressed as the percentage of residual activity considering the specific activity of nonincubated rhGAA as 100%;

Figure 12 illustrates the effect of L-CAR in PD fibroblasts, (a) Effect of L-CAR on the residual activity of mutated GAA in fibroblasts. Fibroblasts derived from three PD patients were incubated in the presence and in the absence of 2 and 10 mM L-CAR before being harvested and used for GAA assay;

Figure 13 illustrates the synergy between L-CAR and rhGAA in PD fibroblasts, (a) Setting the conditions for evaluation of synergy between L-CAR and rhGAA. Different treatment protocols were evaluated: (i) pre-incubation of cells with L-CAR for 24 hours, followed by co-incubation of L-CAR and rhGAA for additional 24 hrs; (ii) co-incubation of L-CAR and rhGAA for 24 hrs. (b) Setting the optimal L-CAR concentrations for evaluation of synergy between L-CAR and rhGAA. Fibroblasts were incubated with rhGAA and different L-CAR concentrations (1 to 20 mM). GAA activity enhancements were observed at 5, 10 and 20 mM L-CAR concentrations with the highest and statistically most significant enhancements at 10 and 20 mM. (c) Effect of L-CAR on rhGAA processing in PD fibroblasts. Cells were incubated for 24 hours with rhGAA alone or with rhGAA in combination with 10 mM L-CAR. In the cells treated with the combination of rhGAA and L-CAR the amount of the 70-76 kDa mature GAA active peptides was dramatically improved, as indicated by a quantitative analysis by western blot. Glyceraldehyde 3- phosphate dehydrogenase (GAPDH) is the loading control, (d) GAA activities measured in PD fibroblasts. The increase of GAA activity confirms the enhancing effect of L-CAR;

Figure 14 shows the kinetics of GAA enhancements at different time-points in PD fibroblasts treated with rhGAA or rhGAA in combination with 10 mM L-CAR. (a) GAA activity increased progressively over time and an enhancing effect of co-incubation with L- CAR was already detectable at 2 hours and became progressively more pronounced up to 24 hours (Figure 14, left). The amounts and the processing of rhGAA, analyzed by western blot, also improved over time (b);

Figure 15 shows the effects of rhGAA and L-CAR co-dosing on lysosomal trafficking of the recombinant enzyme. The cells were incubated under the conditions selected in the previous experiments, and co-localization of rhGAA with Lamp2 was analyzed by confocal immune-fluorescence microscopy. In all three cells lines the colocalization was improved (A). This result was confirmed by a quantitative analysis of total GAA signal (B) and of GAA signal co-localized with Lamp2 (C). Overexposed Images: brightness +40%; contrast -20%;

Figure 16 shows the comparison of the effect of VitBl (A), VitB6 (B), Acetyl-L- camitine (C) on rhGAA stability: VitB / VitB6 / A-L-CAR at various concentrations were incubated with rhGAA and the enzymatic activity was measured after 5 hours of incubation at pH 7.4; (D) Effect of A-L-CAR on rhGAA activity;

Figure 17 shows the effect of A-L-CAR on the structural stability of rhGAA: (A) A- L-CAR was incubated with rhGAA at three concentrations (from 5 to 20 mM). Changes in SYPRO Orange fluorescence were monitored by DSF as a function of temperature at pH 7.4; (B) Summary of Tm values measured by DSF: Tm was calculated according to Niesen FH, et al., Nat Protoc. 2007; 2 (9): 2212-21. Standard deviations for each melting temperature were calculated from measurements performed in triplicate; (C) Determination of the dissociation constant (KD) of A-L-CAR with rhGAA by DSF. A-L-CAR was incubated with rhGAA at ten concentrations (from 1 to 20 mM). Thermal denaturation curves were performed in triplicate and the melting temperatures were calculated according to Niesen FH, et al., Nat Protoc. 2007; 2 (9): 2212-21. For the determination of the KD of A- L-CAR, the experimental data were adapted to the equation of the cooperative model reported by Vivoli M., et al, J Vis Exp 51809 (2014);

Figure 18 shows the comparison of the effect of allosteric and non-allosteric chaperones on the stability of rhGAA. (A) Analysis of the synergistic effect of A-L-CAR and NAC. A-L-CAR was incubated with rhGAA alone (10 or 20 mM) or in combination with NAC, at 10 mM concentration each; (B) Analysis of the synergistic effect of A-L-CAR and DNJ. A-L-CAR was incubated with rhGAA alone (10 or 20 mM) or in combination with DNJ (10 and 0.1 mM, respectively). (C) Analysis of the synergistic effect of Vitamin B6 and DNJ. VitB6 was incubated with rhGAA alone (10 or 20 mM) or in combination with DNJ (10 and 0.1 mM, respectively). Changes in SYPRO Orange fluorescence were monitored by DSF as a function of temperature at pH 7.4;

Figure 19 shows the comparison of the effect of allosteric chaperones on the stability of rhGAA. Analysis of the synergistic effect of (A) VitB 1 and NAC and (B) VitB6 and NAC. VitB l/VitB6 were incubated with rhGAA alone (10 or 20 mM) or in combination with NAC, at 10 mM concentration each. For both experiments, changes in SYPRO Orange fluorescence were monitored by DSF as a function of temperature at pH 7.4;

Figure 20 shows the combination of L-CAR with A-L-CAR (A), VitBl (B) and VitB6 (C) on the stability of rhGAA. L-CAR was incubated with rhGAA alone (10 or 20 mM) or in combination with A-L-CAR / VitBl / VitB6, at 10 mM concentration each; changes in SYPRO Orange fluorescence were monitored by DSF as a function of temperature at pH 7.4;

Figure 21 shows the combination of A-L-CAR with (A) VitBl and (B) VitB6 on the stability of rhGAA. A-L-CAR was incubated with rhGAA alone (10 or 20 mM) or in combination with VitBl / VitB6, at 10 mM concentration each; changes in SYPRO Orange fluorescence were monitored by DSF as a function of temperature at pH 7.4; (C) VitBl and VitB6 combination on rhGAA stability. VitB 1 and VitB6 were incubated with rhGAA alone (10 or 20 mM) or in combination with VitB 1 / VitB6, at 10 mM concentration each; changes in SYPRO Orange fluorescence were monitored by DSF as a function of temperature at pH 7.4;

Figure 22 shows the results of in vivo studies in GAA^-/“ mice evidencing the enhancement of ERT with rhGAA in some target tissues from the Pompe disease mouse model. (Top) Schematic diagram of the experimental design (Bottom) Graphs showing GAA activity in heart, diaphragm, gastrocnemius and quadriceps tissues of GAA^-/“ animals receiving ERT alone or ERT in combination with carnitine.

Declaration under Art 170 bis of the Italian Industrial Property Code

The present invention has been attained in accordance with the provisions established by Article 170-bis, paragraphs 2, 3 and 4, of the Italian Industrial Property Code (Legislative Decree No. 30 of February 10, 2005, as amended up to Legislative Decree No. 131 of August 13, 2010).

MATERIALS AND METHODS

Fibroblast cultures

Fibroblasts from Pompe disease (PD) patients were derived from skin biopsies after obtaining the informed consent of patients. Normal age-matched control fibroblasts were available in the laboratory of the Department of Pediatrics, Federico II University of Naples. All cell lines were grown at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (Invitrogen, Grand Island, NY) and 20% fetal bovine serum (Sigma- Aldrich, St Louis, MO), supplemented with 2 mM/L glutamine, 100 U/ml penicillin and 100 pg/ml streptomycin.

Reagents rhGAA (alpha-glucosidase, Myozyme), was from Genzyme Co, Cambridge, MA, USA. As source of enzyme, authors used the residual amounts of the reconstituted recombinant enzyme prepared for the treatment of PD patients at the Department of Traslational Medical Sciences of the University of Naples, ‘Federico II’. D-CAR, A-D-CAR were from Sigma- tau; L-CAR, DNJ, 4-nitrofenil-a-glucopiranoside (4NP-Glc) (N1377), vitamin C (VitC) (A0278), and carbocisteine (C0470000) were from Sigma- Aldrich. Vitamin Bl (VitBl) (FT28200) and vitamin B6 (VitB6) (FP27320) were from Carbosynth.

Thermal stability of rhGAA

Thermal stability scans of rhGAA were performed as described in Porto C, et al., Mol Ther. 2012 Dec;20(12):2201-l 1. Briefly, 0.9 pM of enzyme were incubated in the absence and in the presence of L-CAR, D-CAR A-D-CAR, A-L-CAR, NAC, VitBl, VitB6, VitC, carbocisteine, saccharose, trehalose and DNJ at the indicated concentrations with SYPRO Orange dye, 25 mM sodium phosphate buffer, pH 7.4, and 150 mM NaCl. Thermal stability scans were performed at l°C/min in the range 25-95°C in a Real Time LightCycler (Bio- Rad). SYPRO Orange fluorescence was normalized to maximum fluorescence value within each scan to obtain relative fluorescence. Melting temperatures were calculated according to Niesen FH, et al., Nat Protoc. 2007;2(9):2212-21.

The dissociation constant were measured by thermal stability scans of rhGAA as described in Roig-Zamboni V, et al., Nat Comm. 2017 Oct 24;8(1): 1111. DSF scans were performed as described above, in the range 0-40 mM chaperone. The melting temperature values were plotted as function of ligand concentration. The experimental data were best fitted according to a simple cooperative model equation as reported in Vivoli M., et al, “Determination of protein- ligand interactions using differential scanning fluorimetry”. J Vis Exp 51809 (2014), by using the software GraphPAD Prism (GraphPad Software, San Diego, CA, USA).

Enzyme characterization

The standard activity assay of rhGAA was performed in 200 pL by using 0.2 pM at 37°C in 100 mM sodium acetate pH 4.0 and 20 mM 4NP-Glc. The reaction was started by adding the enzyme. After suitable incubation time (1-2 min) the reaction was blocked by adding 800 pL of 1 M sodium carbonate pH 10.2. Absorbance was measured at 420 nm at room temperature, the extinction coefficient to calculate enzymatic units was 17.2 mM-1 cm-1. One enzymatic unit is defined as the amount of enzyme catalyzing the conversion of 1 pmol substrate into product in 1 min, under the indicated conditions.

The effect of pH on the rhGAA stability was measured by preparing reaction mixtures containing 6.8 pM of enzyme in the presence of 50 mM sodium phosphate, pH 7.4. After incubations at 37°C, aliquots were withdrawn at the times indicated and the residual alphaglucosidase activity was measured with the standard assay. To test the effect on the pH stability of rhGAA of chemical chaperons and of the other molecules, experiments were performed as described above by adding to the reaction mixtures the amounts of the different compounds indicated in the text.

Incubation of fibroblasts with rhGAA and GAA assay To study the rhGAA uptake and correction of GA A activity in PD fibroblasts, the cells were incubated with 50 p M rhGAA for 24 hours, in the absence or in the presence of 10 mM L- CAR. Untreated cells or were used for comparison. After the incubation, the cells were harvested by trypsinization and disrupted by 5 cycles of freezing and thawing.

GAA activity was assayed by using the fluorogenic substrate 4-methylumbelliferyl-a-D- glucopyranoside (4MU) (Sigma- Aldrich) according to a published procedure (Porto C, et al., Mol Ther. 2009 Jun;17(6):964-71). Briefly, 25 pg of cell homogenates were incubated with the fluorogenic substrate (2 mM) in 0.2 M acetate buffer, pH 4.0, for 60 minutes in incubation mixtures of 100 pl. The reaction was stopped by adding 1 mL of glycinecarbonate buffer, 0.5 M, pH 10.7. Fluorescence was read at 365 nm (excitation) and 450 nm (emission) on a Promega GloMax Multidetection system fluorometer. Protein concentration in cell homogenates was measured by the Lowry assay.

Immunofluorescence analysis and confocal microscopy

For immunofluorescence studies, cells (human fibroblasts) grown on coverslips were fixed using methanol (5 minutes at -20°C to study the colocalization GAA-LAMP2), permeabilized using 1% PBS (phosphate buffered saline) - Triton 0,1% and blocked with 0.05% saponin, 1% BSA diluted in 1% PBS at room temperature for 1 h. The cells were incubated with the primary antibodies anti-GAA rabbit polyclonal antibody (PRIMM) and anti-LAMP2 mouse monoclonal antibody (Santa Cruz Biotechnology) overnight at 4°C diluted in blocking solution, washed with 1% PBS and then incubated with appropriate autofluorescent secondary antibodies (anti-rabbit or anti-mouse antibodies conjugated to Alexa Fluor 488 or 596) and DAPI (4',6-diamidino-2-phenylindole, Invitrogen) in 0.05% saponin, 3% BSA, 1% PBS. Samples were then washed, mounted with Mowiol (Sigma) and examined with a Zeiss LSM700 confocal microscope. Colocalization and quantitative analysis were performed with Fiji (ImageJ) software.

Study of L-carnitine in vivo Knock-out mice for Pompe disease were purchased from the Charles River laboratories (Wilmington, MA) and kept at the Animal Facility of Tigem (Pozzuoli, Italy).

The in vivo studies were conducted in accordance with EU directives 86/609, regarding the protection of animals used for experimental purposes.

Each procedure on the mice was conducted ensuring minimal discomfort, stress, and pain for the animals.

The efficacy of L-camitine was tested in 4-month GAA knock out mice, treated with a single injection of 40 mg/kg rhGAA alone or in combination with 250 mg/kg L-carnitine for 5 days.

Mice treated with rhGAA alone (n = 3) received a single retrorbital injection of rh-GAA (40 mg/kg).

Mice treated with rhGAA in combination with L-carnitine (n = 5) received L-carnitine (250 mg/kg per day) via gavage for 5 days. On the third day, they received a single retrorbital injection of rhGAA (40 mg/kg).

The animals were all sacrificed by perfusion on day 5, 48h after the injection of rhGAA, and the heart, diaphragm, gastrocnemius, quadriceps and liver organs were removed for enzyme assays.

The tissues were mechanically homogenized by Tissue Lyser (27 oscillations for 3 minutes, twice) in water and subjected to cycles of freezing and thawing in liquid nitrogen and centrifuged at 13000 rpm for 15 minutes at 4 °C.

The activity of GAA was evaluated using the fluorogenic substrate 4-methylumbelliferil-a- D-glucopyranoside (4MU) (Sigma- Aldrich) according to the published procedure (Porto C, et al., Mol Ther. 2009 Jun; 17 (6): 964-71). Briefly, 50 pg of homogenates were incubated with the fluorogenic substrate (2 mM) in 0.2 M acetate buffer, pH 4.0, for 60 minutes in 100 pl incubation mixes. The reaction was stopped by adding 200 pl of glycine-carbonate buffer, 0.5 M, pH 10.7. Fluorescence was analysed at 365 nm (excitation) and 450 nm (emission) with a Promega GloMax system fluorometer in tissue lysates and measured by the BCA assay.

RESULTS

Example 1: Carnitine compounds and vitamin Bl, B6 and C improve rhGAA stability in vitro

In the course of the discovery study, the present inventors analyzed the effects of numerous compounds on the pH stability of the acid alpha-glucosidase (GAA) enzyme as already performed in previous studies on lysosomal enzymes (Lieberman RL, et al., Nat Chem Biol. 2007 Feb;3(2): 101-7; Shen JS, et al., Biochem Biophys Res Commun. 2008 May 16;369(4): 1071-5; Porto C, et al., Mol Ther. 2012 Dec;20(12):2201-l l).

Among the tested compounds, L-camitine, D-carnitine, acetyl-D-carnitine, vitamin Bl, vitamin B6, vitamin C, trehalose, saccharose and carbocisteine were assayed. In particular, the inventors analyzed rhGAA stability incubating the enzyme at different pHs and assaying the residual activity on 100 mM 4-nitrophenyl-a-D-glucopyranoside (4NP-Glc) in 100 mM sodium acetate buffer, pH 4.0 in which rhGAA is stable for up to 24 hours. Instead, at pHs, either acidic (pH 3.0) or neutral (pH 7.0), lower and higher, respectively, when compared to the lysosomal compartment, the enzyme halved its activity in about 5 hrs (Porto C, et al., Mol Ther. 2012 Dec;20(12):2201-l l).

L-CAR, already at the concentration of 10 mM, rescued the activity of rhGAA on 4NP-Glc after 5h of incubation at pH 7.4 (Figure la). The stabilizing effect on the rhGAA activity was maintained even after 48 h of incubation in the presence of 20 mM L-CAR (Figure 4a). No effect on the specific activity of rhGAA was observed when L-CAR was included at any concentration in the alpha-glucosidase assay, indicating that it did not interact with the active site of the enzyme (Figure 4). Surprisingly, L-CAR increased in dose-dependent manner also the structural stability of rhGAA as analysed by Differential Scanning Fluorimetry (DSF) (Figure lb). The variations of the melting temperature (ATm) increased by 2.4±0.1°C at every 2 mM increment of L- CAR concentration (Figure 1c).

The dissociation constant of L-CAR for rhGAA was measured by DSF according to Vivoli M., et al, “Determination of protein- ligand interactions using differential scanning fluorimetry”. J Vis Exp 51809 (2014) (Figure Id). L-CAR showed a KD similar to that of the allosteric chaperone NAC (9.16 ± 1.02 mM and 11.57 ± 0.74 mM, respectively) (Roig- Zamboni V, et al., Nat Comm. 2017 Oct 24;8( 1): 1111). As for molecules that do not bind to the rhGAA active site, these values are higher than the typical Ki of 3.4 pM exhibited by active-site directed molecular chaperones such as DNJ inhibitor (Porto C, et al., Mol Ther. 2012 Dec;20(12):2201-l l).

The chaperone effect on rhGAA of vitamin Bl (VitBl), vitamin B6 (VitB6), vitamin C (VitC), carbocisteine, saccharose, and trehalose molecules was tested by thermal stability analysis (Figure 7) at pH 7.4. VitBl, VitB6, VitC at 10 mM concentration increased the thermal stability of rhGAA analyzed by DSF (Figure 7). The measured melting temperature (Tm) of rhGAA was 54.5+0.1 °C, 53.9±0.9°C, 54.0±0.4°C, respectively, for VitBl, VitB6, and VitC vs 49.8±0.2°C in the absence of chaperones. In contrast, carbocisteine destabilized the enzyme by reducing the melting temperature to 29.8+0.3 °C. Saccharose and trehalose showed no effect on rhGAA (Tm of 50.3+0.5 °C and 50.5+0.2 °C, respectively).

The present inventors did not observe any significant effect on the specific activity of rhGAA when the enzyme was assayed in the presence of VitB l, VitB6 and VitC at pH 4.0 and temperature of 37°C, except for an increase in activity in the presence of 20 mM VitB6, suggesting that these compounds do not interact with the active site of the enzyme (Figure 8). Instead, as already known in the art, DNJ inhibited the activity of the enzyme, already at the concentration of 1 pM leading to almost total inhibition at 0.1 mM.

As reported in Porto C, et al., Mol Ther. 2012 Dec;20(12):2201-l l, rhGAA is stable up to

24 hr at pH 5.0, while at neutral pH (i.e. pH 7.0, present in non-lysosomal cellular compartments), the enzyme is unstable and rapidly loses its activity, with about 50% residual activity after 4 hr, and is almost completely inactive (less than 10% residual activity) after 16 hr. In contrast, when incubated in the presence of 10 and 20 mM VitBl/VitB6/VitC at pH 7.4, rhGAA retained over 50% of its activity for 1 hour and 2 hours, respectively (Figure 9).

The dissociation constants of VitBl, VitB6, and VitC were measured by DSF (Figure 10) using compound concentrations ranging between 0 and 30 mM for each chaperone. VitB 1 and VitC showed a KD of 11.14+0.90 mM and 10.28+0.98 mM, respectively. Differently, VitB6 showed a lower KD than the other chaperones, which amounted to 5.59+0.43 mM.

Example 2: Effect on rhGAA stability by combined action of allosteric and active-site directed PCs

The present inventors observed similar stabilizing effects with the related compounds D- CAR and A-D-CAR (7 and 8, respectively). Both compounds rescued the activity of rhGAA on 4NP-Glc after 5h of incubation at pH 7.4 (Figure 5a). Again, no effect on the specific activity of rhGAA at 0.1-10 mM concentrations was observed (Figure 5b), indicating that D-CAR and A-D-CAR also did not interact with the active site of the enzyme. Compared to the L-isomer, D-CAR showed a complete rescue of rhGAA activity already at 10 mM concentration vs 20 mM of L-CAR (Figure 5c), maintaining the stabilizing effect even after 24 h of incubation (Figure 4a). DSF analysis showed that D-CAR increased the structural stability of rhGAA in a dose-dependent manner (Figure 5d) and that the ATm increased every 2 mM increment of D-CAR concentration (Figure 5e).

Nutraceutical preparations of carnitine are often racemic mixtures of the L- and D- enantiomers; thus, the stabilizing effect on rhGAA of equimolar amounts of D- and L-CAR was analyzed. As shown in Figure 2, when rhGAA was incubated with 10 mM total concentration of the two enantiomers (resulting from L-CAR 5 mM + D-CAR 5 mM), the ATms of 9.4+0.8 °C corresponds to the sum of the ATms measured when the enzyme was incubated with either L- or D-CAR at 5 mM concentration (ATms of 4.3±0.2°C and 4.9+0.1 °C, respectively). A similar additive effect was observed when the concentration of each enantiomer was increased to 10 mM D- and L-CAR (Figure 2b).

The combined effect on rhGAA by L-CAR and other active-site directed or allosteric chaperones, is shown in Figure 3. At a concentration of 10 mM, L-CAR increased the Tm of rhGAA by 9.0 + 0.3 (Tm 58.6±0.2°C vs 49.6+0.1°C of rhGAA alone) a value similar to that obtained with NAC at the same concentration (9.6 + 0.2°C), but slightly lower than that of the active-site directed pharmacological chaperone DNJ (1) at a concentration of 0.1 mM (+12.1 + 0.3°C) (Figure 3a and 3b). To understand the mechanism of stabilization towards rhGAA the inventors combined these molecules in DSF experiments. L-CAR was mixed at 10 mM concentration in equimolar ratios with NAC (Figure 3a) or with 0.1 mM DNJ (Figure 3b). The stabilizing effect of L-CAR in the presence of 10 mM equimolar amounts of NAC (20 mM total) was identical to the effect observed when each of the allosteric PCs was used individually at 20 mM concentration (Figure 3a). The effect of L-CAR and NAC was nonadditive with ATm of 14.3 ± 0.2°C, 14.3 ± 0.13°C, and 14.4 ± 0.2°C with L-CAR, NAC, and L-CAR+NAC, respectively (Figure 3a).

The ATms obtained with either 10 mM L-CAR combined with 0.1 mM of the non-allosteric chaperone DNJ, were exactly additive with ATm of +9.0 ± 0.3°C, +12.1 ± 0.3°C, and +23.2 + 0.2°C with L-CAR, DNJ, and L-CAR+DNJ, respectively, confirming that these PCs interact with different sites of rhGAA (Figure 3b).

The present inventors further investigated whether the stabilizing effect of VitB 1 and VitC is increased in the presence of known non-allosteric chaperones, by mixing these compounds at 10 mM concentration with 0.1 mM DNJ (Figures 11A and 11B). VitBl/VitC in combination with 0.1 mM DNJ showed an additive effect on the ATms thus obtained (Figures 11 A and 1 IB), and these data indicate that also VitB 1/VitC bind rhGAA at different sites than DNJ.

Example 3: Effect of L-CAR in PD fibroblasts Based on the results as above described, the present inventors conducted a study to evaluate the effect of L-CAR on mutant GAA activity in cultured fibroblasts from three PD patients carrying different mutations and with early-onset phenotypes (Table 1).

Table 1

Fibroblasts were incubated in the presence of 0.1 to 10 mM L-CAR for 24 hours and the results were compared to those obtained in untreated cells. A surprising enhancing chaperone effect was observed on endogenous residual activity in patient cells homozygous for the p.L552P mutation (Figure 12). Significant increments in activity were observed in a range of L-CAR concentrations between 1 and lOmM, with a 2.8-fold increase at 2 mM.

Based on these surprising findings, the present inventors tested whether the allosteric PC L- CAR is able to enhance also the efficacy of the recombinant enzymes used for ERT in PD disease. The experiments were conducted on the patient cell lines as indicated above. As a first step, the inventors studied the optimal conditions to evaluate this effect. More specifically, a study was conducted by comparing a protocol based on pre-incubation of cells with L-CAR for 24 hours, followed by co-incubation of L-CAR and rhGAA for additional 24 hrs, with a protocol based on co-incubation of L-CAR and rhGAA for 24 hrs (Figure 13a). The results of both protocols were compared with those obtained in cells treated with rhGAA alone. The second treatment protocol gave the best results and was selected to evaluate the optimal L-CAR concentration for rhGAA enhancement.

With the co-dosing of rhGAA and L-CAR (1 to 20 mM) GAA activity enhancements were observed at 5, 10 and 20 mM L-CAR concentrations (Figure 13b). The highest and statistically most significant enhancements were obtained at 10 and 20 mM. Higher L-CAR concentrations (up to 50 mM) were toxic for fibroblasts (not shown). Accordingly, a concentration of 10 mM was selected for further experiments, as this concentration appeared to combine efficacy and safety for cells.

Subsequently, the inventors studied the effect of L-CAR on rhGAA processing in PD1 and PD2 fibroblasts. For enzyme replacement therapy rhGAA is provided by the manufacturer as a 110 kDa precursor. Once internalized by cells through the mannose-6-phosphate receptor and the endocytic pathways, the enzyme is converted into an intermediate of 95 kDa and the active molecular isoforms of 76 and 70 kDa.

Cells were incubated for 24 hours with rhGAA alone or with rhGAA in combination with 10 mM L-CAR. In the cells treated with the combination of rhGAA and L-CAR the amount of the 70-76 kDa mature GAA active peptides was dramatically improved (Figure 13c). The corresponding GAA activities measured in PD1 and PD2 cells (Figure 13d) confirmed the enhancing effect of L-CAR and were in line with those observed in previous experiments.

Moreover, the kinetics of GAA enhancements was studied at different time-points in PD fibroblasts treated with rhGAA or rhGAA in combination with 10 mM L-CAR. GAA activity increased progressively over time and an enhancing effect of co-incubation with L- CAR was already detectable at 2 hours and became progressively more pronounced up to 24 hours (Figure 14a). The amounts and the processing of rhGAA, analyzed by western blot, also improved over time (Figure 14b).

The inventors investigated also the effects of rhGAA and L-CAR co-dosing on lysosomal trafficking of the recombinant enzyme. The cells were incubated under the conditions selected in the previous experiments, and co-localization of rhGAA with Lamp2 was analyzed by confocal immune-fluorescence microscopy. In all three cells lines the colocalization was improved (Figure 15A). This result was confirmed by a quantitative analysis of total GAA signal (Figure 15B) and of GAA signal co-localized with Lamp2 (Figure 15C) performed by ImageJ Software.

Example 4: Compound A-L-CAR, Vitamin Bl and Vitamin B6 and their formulations improve the stability of rhGAA in vitro The present inventors have observed similar stabilizing effects with the VitBl, VitB6 and A-L-CAR compounds. The compounds preserved the rhGAA activity on 4NP-Glc after 5 hours of incubation at pH 7.4 (Figure 5a, b and c, respectively). Again, no effect of A-L- CAR was observed on the specific activity of rhGAA at 5-20 mM concentrations (Figure 5d), indicating that A-L-CAR too did not interact with the active site of the enzyme. DSF analysis showed that A-L-CAR increased the structural stability of rhGAA in a dosedependent manner (Figure 17a) and that ATm increased every 5mM increase in A-L-CAR concentration (Figure 17b). The dissociation constant of A-L-CAR was measured by DSF (Figure 17c) using compound concentrations ranging from 0 to 30 mM. A-L-CAR showed a KD value of 10.56 ± 1.44 mM, which is similar to that of L-CAR (Figure Id).

The combined effect on rhGAA by A-L-CAR and VitB6 with active-site or allosteric chaperones is shown in Figure 18. At a concentration of 10 mM, A-L-CAR increased the rhGAA Tm by 7.2 ± 0.4 °C (Tm 54.0 ± 0.2 °C vs 46.8 ± 0.2 °C of rhGAA alone) a value slightly lower than that obtained with NAC at the same concentration (9.6 ± 0.2 °C), and to that of the pharmacological chaperone directed to the active site DNJ (1) at the concentration of 0.1 mM (12.1 ± 0.3 °C) (Figure 3a and 3b).

To understand the stabilization mechanism produced on rhGAA, the inventors combined these molecules in DSF experiments. A-L-CAR was mixed at a concentration of 10 mM in equimolar ratios with NAC (Figure 18a) or with DNJ 0.1 mM (Figure 18b). The stabilizing effect of A-L-CAR in the presence of equimolar amounts of 10 mM of NAC (20 mM total) was identical to the effect observed when each of the allosteric PCs was used individually at a concentration of 20 mM (Figure 18a). The effect of A-L-CAR and NAC was non-additive with ATm of 16.7 ± 0.4 °C, 14.3 ± 0.13 °C and 16.2 ± 0.3 °C, with A-L-CAR, NAC and A- L-CAR + NAC, respectively (Figure 18a). The ATms obtained with A-L-CAR 10 mM combined with 0.1 mM of the non- allosteric chaperon DNJ, were higher than the sum of the ATm of the single chaperones, respectively by 7.2 ± 0.3 °C, 12.1 ± 0.3 °C and 23.5 ± 0.2 °C for A-L -CAR, DNJ and A-L-CAR + DNJ, confirming that these PCs interact with different rhGAA sites (Figure 18b). Furthermore, the inventors evaluated the combined effect of VitB6 with DNJ (Figure 18c). The ATms obtained with VitB6 at 10 mM concentration combined with 0.1 mM of the non- allosteric DNJ chaperone, were higher than the sum of the ATm of the single chaperones, respectively by 8.2 ± 0.3 °C, 12.1 ± 0.3 °C and 23.1 ± 0.3 °C for VitB6, DNJ and VitB6 + DNJ, confirming that these PCs too, interact with different rhGAA sites (Figure 18c).

The present inventors have further investigated whether the stabilizing effect of VitB 1 and VitB6 increases in the presence of known allosteric chaperones, by mixing these compounds at a concentration of 10 mM with 10 mM NAC (Figures 19A and 19B). As shown for A-L- CAR, VitBl / VitB6 in combination with 10 mM NAC did not show an additive effect on the ATms obtained (Figures 19A and 19B).

The present inventors have further investigated whether the stabilizing effect of L-CAR, A- L-CAR, VitBl and VitB6 increases in the presence of a mixture of these compounds at a concentration of 10 mM each (Figures 20A, 20B, 20C, 21A, 21B, 21C). As observed with NAC, the combinations of L-CAR / A-L-CAR (Figure 20A), L-CAR / VitBl (Figure 20B), L-CAR / VitB6 (Figure 20C), A-L-CAR / VitBl (Figure 21A), A-L-CAR / VitB6 (Figure 2 IB), VitBl / VitB6 (Figure 21C) did not show an additive effect on the ATms if compared to the single compounds at 20 mM.

Example 5: Studies in an animal model of Pompe disease

To further validate the therapeutic approach of the invention, the present inventors performed dedicated experiments in a Gaa-/- mouse model of Pompe disease.

As shown in Figure 22, the combined administration of ERT and L-carnitine to the animals with Pompe disease led to improved therapeutic effects in certain target tissues, thus proving a synergistic effect of the combined preparation of the invention. In particular the diaphragm is a critical tissue, as it is involved in respiratory function in Pompe disease patients. The correction and stabilization of respiratory function is one of the most challenging aspects in treating the disease and the increased levels of GAA enzyme observed in the diaphragm in the animals is an important signal that the combined therapy of the invention leads to the correction of the respiratory defect. The results observed in the quadriceps are also of high relevance, as achieving increased enzyme levels in skeletal muscles is a major therapeutic challenge.

Claims

34 CLAIMS

1. A combined preparation comprising an acid alpha-glucosidase (GAA) enzyme and at least one allosteric chaperone of the acid alpha-glucosidase enzyme, for simultaneous, separate or sequential use in the therapeutic treatment of Pompe disease in a patient, wherein the at least one allosteric chaperone of the acid alpha-glucosidase enzyme is selected from the group consisting of L-camitine, D-carnitine, acetyl-D-carnitine, vitamin Bl, vitamin B6, and any combination thereof.

2. The combined preparation for use according to claim 1, wherein the acid alphaglucosidase enzyme is a recombinant human acid alpha- glucosidase (rhGAA).

3. The combined preparation for use according to claim 1 or 2, wherein the administration of the at least one allosteric chaperone of the acid alpha-glucosidase enzyme increases the stability of said acid alpha-glucosidase (GAA) enzyme at a pH value comprised between 7.0 and 7.5.

4. The combined preparation for use according to any of claims 1 to 3, wherein the acid alpha-glucosidase (GAA) enzyme is in a form suitable for parenteral administration.

5. The combined preparation for use according to claim 4, wherein the acid alphaglucosidase (GAA) enzyme is in a form suitable for administration by intravenous infusion or by injection.

6. The combined preparation for use according to any of claims 1 to 5, wherein the at least one allosteric chaperone of the acid alpha-glucosidase enzyme is in a form suitable for enteral or parenteral administration.

7. A pharmaceutical composition for use in the therapeutic treatment of Pompe disease in a patient, comprising an acid alpha-glucosidase (GAA) enzyme, at least one allosteric chaperone of the acid alpha-glucosidase enzyme, and pharmaceutically acceptable vehicles, excipients and/or diluents, wherein the at least one allosteric chaperone of the acid alpha- 35 glucosidase enzyme is selected from the group consisting of L-camitine, D-carnitine, acetyl- D-carnitine, vitamin Bl, vitamin B6, and any combination thereof.

8. The pharmaceutical composition for use according to claim 7, wherein the acid alpha- glucosidase enzyme is a recombinant human acid alpha- glucosidase (rhGAA).

9. The pharmaceutical composition for use according to claim 7 or 8, which is in a form suitable for enteral or parenteral administration.

10. The pharmaceutical composition for use according to any of claims 7 to 9, comprising the acid alpha-glucosidase enzyme (GAA) at a concentration comprised within the range of from 0.05% to 1% w/v on the total volume of the composition and/or the at least one allosteric chaperone of the acid alpha-glucosidase enzyme at a concentration comprised within the range of from 0.5% to 5% w/v on the total volume of the composition.