RU2793184C2 - TREATMENT SCHEME FOR TREATMENT OF FABRY'S DISEASE, USING STABILIZED α-GALACTOSIDASE - Google Patents

Abstract

FIELD: medicine; therapy.

SUBSTANCE: invention relates to the field of medicine, namely to therapy; it is intended for the treatment of Fabry’s disease. For the treatment of Fabry’s disease, a single dosage form is used, containing a human vegetative recombinant α-galactosidase crosslinked using bis-NHS-PEG45. Monomers of the specified human recombinant α-galactosidase are covalently bound to each other by means of a poly(alkylene) glycol binding fragment containing at least 20 alkylene groups. The specified single dosage form is composed in the form of liquid for intravenous injection, and injection is performed at a dose of 2 mg/kg at intervals of 4 weeks.

EFFECT: use of the invention is safe, and it provides significant improvement of patient’s disease parameters in terms of reduction in accumulation of globotriaosylceramide, pain parameters, and gastrointestinal tract, stabilization of kidney and heart functions in clinical conditions.

17 cl, 4 dwg, 3 tbl, 7 ex

Description

Field and prior art

The present invention, in some embodiments, relates to α-galactosidase homodimeric protein structures, and in particular, but not exclusively, to effective treatment regimens for the treatment of Fabry's disease using enzyme replacement therapy with stabilized covalently linked α-galactosidase homodimeric protein structures.

The lysosomal enzyme α-galactosidase-A (α-GAL or α-Gal A; EC 3.2.1.22) catalyzes the removal of galactose from oligosaccharides, glycoproteins and glycolipids during the catabolism of macromolecules. Deficiency of lysosomal enzymes leads to accumulation of their substrates in various tissues, a condition known as lysosomal storage diseases. In humans, the absence of functional α-galactosidase-A leads to the accumulation in tissues of glycolipids containing terminal α-galactose residues (primarily globotriaosylceramide, which is also called "ceramidetrihexoside", "STN" or "Gb ₃ "), leading to Fabry disease. Fabry disease is an X-linked recessive disorder first described in 1898 that is characterized by chronic pain, cloudy eyes, liver and kidney damage, skin lesions, vascular deterioration, and/or heart failure. Recombinant human α-galactosidase-A has the ability to provide and replace reduced enzymatic activity in patients, and enzyme replacement therapy (ERT) using α-GAL was approved in the United States and European countries in 2003 for the treatment of Fabry disease. α-GAL became the second recombinant protein approved for the treatment of lysosomal storage disease after β-glucosidase for the treatment of Gaucher's disease.

Endogenous and recombinant α-GAL enzymes catalyze the hydrolysis of terminal galactosylated glycolipids in the lysosomes of cells in organs such as skin, kidneys, heart, etc. This natural environment is characterized by an acidic pH of only 4.5. Therefore, lysosomal enzymes, including α-GAL, are designed to exhibit their maximum activity at such low pH levels.

Current treatments for ERT Fabry disease are based on recombinant mammalian-derived α-GAL, which is considered to have limited clinical efficacy and currently does not offer a satisfactory clinical solution for patients with Fabry disease.

X-ray diffraction analysis shows that human α-GAL is a homodimeric glycoprotein, with each monomer consisting of two domains: a (β/α) ₈ domain containing an active site, and a C-terminal domain containing eight antiparallel β-chains on two sheets in β sandwich [Garman & Garboczi, J Mol Biol 2004, 337:319-335].

Both structural (X-ray diffraction crystallography) and biochemical (kinetic) data indicate the cooperativity of active sites between the monomeric units of the homodimeric structure, highlighting the importance of dimerization for the enzymatic activity and stability of α-GAL therapeutic compositions.

In the present patentee's international patent publication WO 2009/024977, which is incorporated herein by reference as if it were set forth herein in its entirety, saccharide-biomolecule conjugates covalently linked together via a non-hydrophobic linker are described, as well as medical applications using such conjugates.

The present patentee's International Patent Publication WO 2011/061736, which is incorporated herein by reference as if it were set forth herein in its entirety, describes procedures that use α-galactosidase that exhibits lysosomal activity at pH levels higher than pH level of lysosomes.

In the international patent publication WO 2011/107990 of the present patent owner, which is incorporated herein by reference, as if it were fully set forth herein, a covalently linked multimeric protein structure containing at least two α-galactosidase monomers, having a stable catalytic α-galactosidase activity under physiological conditions and improved pharmacodynamics, and proposals for its therapeutic use.

In the present patentee's international patent publication WO 2012/098537, which is incorporated herein by reference as if it were set forth herein in its entirety, nucleic acid constructs for recombinant expression of catalytically active α-galactosidase in plants are described and suggestions for their therapeutic use.

Additional prior art includes Bendele et al. [Toxicological Sciences 1998, 42:152-157], US Patent Nos. 5256804, 5580757 and 5766897, International Patent Publication PCT/NL2007/050684 (published as WO 2008/075957), US Patent Publication US 20160184409, Treco, et al. and Seely & Richey [J Chromatography A 2001, 908:235-241].

The essence of the invention

According to an aspect of some embodiments of the present invention, there is provided a method of treating Fabry disease in a human subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of recombinant human α-galactosidase, wherein the therapeutically effective amount of recombinant human α-galactosidase is 0.2-2, 0 mg/kg, which allows the treatment of Fabry disease in the subject, and the introduction is carried out at intervals of more than two weeks, and moreover, the monomers of recombinant human α-galactosidase are covalently linked to each other through a linking fragment of 20-600 atoms in length.

According to an aspect of some embodiments of the present invention, there is provided a method of treating Fabry disease in a human subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of recombinant human α-galactosidase, wherein the therapeutically effective amount of recombinant human α-galactosidase is 0.2-2, 0 mg/kg, which allows the treatment of Fabry disease in the subject, and the introduction is carried out at intervals of more than two weeks - every 4 weeks, and moreover, the monomers of recombinant human α-galactosidase are covalently linked to each other through a linking fragment of 20-600 atoms in length.

According to an aspect of some embodiments of the present invention, the recombinant human α-galactosidase is a plant recombinant human α-galactosidase.

According to an aspect of some embodiments of the present invention, plant recombinant human α-galactosidase is cross-linked with bis-NHS-PEG ₄₅ .

According to an aspect of some embodiments of the present invention, the intervals are three weeks or four weeks.

According to an aspect of some embodiments of the present invention, the intervals are 17 days - 8 weeks.

According to an aspect of some embodiments of the present invention, the intervals are 17 days - 6 weeks.

According to an aspect of some embodiments of the present invention, the intervals are 17 days - 5 weeks.

According to an aspect of some embodiments of the present invention, the intervals are 3 weeks - 6 weeks.

According to an aspect of some embodiments of the present invention, the intervals are 3 weeks - 5 weeks.

According to an aspect of some embodiments of the present invention, the intervals are 3 weeks - 4 weeks.

According to an aspect of some embodiments of the present invention, the intervals are 4 weeks - 6 weeks.

According to an aspect of some embodiments of the present invention, the intervals are 4 weeks - 5 weeks.

According to an aspect of some embodiments of the present invention, the administration is intravenous administration.

According to an aspect of some embodiments of the present invention, administration is at a dose of 1.0 mg/kg.

According to an aspect of some embodiments of the present invention, administration is at a dose of 2.0 mg/kg.

According to an aspect of some embodiments of the present invention, the administration is carried out once every three weeks.

According to an aspect of some embodiments of the present invention, the administration is carried out once every four weeks.

According to an aspect of some embodiments of the present invention, the administration is carried out once every five weeks.

According to an aspect of some embodiments of the present invention, the administration is carried out once every six weeks.

According to an aspect of some embodiments of the present invention, a therapeutically effective amount of recombinant human α-galactosidase reduces Gb3 and/or lyso-Gb3 in a subject.

According to an aspect of some embodiments of the present invention, a therapeutically effective amount of recombinant human α-galactosidase maintains stability or attenuates deterioration in cardiac parameters in a subject.

According to an aspect of some embodiments of the present invention, the cardiac parameters are LVM (left ventricular myocardial mass) or LVMI (left ventricular myocardial mass index) measured by MRI compared to pre-treatment values.

According to an aspect of some embodiments of the present invention, a therapeutically effective amount of recombinant human α-galactosidase maintains a stable reduction in plasma Gb3 and/or lyso-Gb3 concentrations in a subject.

According to an aspect of some embodiments of the present invention, a therapeutically effective amount of recombinant human α-galactosidase maintains a stable reduction in urinary Gb3 concentrations in a subject.

According to an aspect of some embodiments of the present invention, a therapeutically effective amount of recombinant human α-galactosidase ameliorates Fabry disease-associated deterioration in renal function in a subject.

According to an aspect of some embodiments of the present invention, a therapeutically effective amount of recombinant human α-galactosidase maintains the stability of kidney function in a subject.

According to an aspect of some embodiments of the present invention, a therapeutically effective amount of recombinant human α-galactosidase maintains stability or attenuates deterioration in at least one parameter of the gastrointestinal tract in a subject.

According to an aspect of some embodiments of the present invention, the gastrointestinal tract parameter is abdominal pain and/or frequency of abdominal pain measured after 6 months of treatment compared to pre-treatment values.

According to an aspect of some embodiments of the present invention, a therapeutically effective amount of recombinant human α-galactosidase maintains stability or attenuates deterioration in the Mainz Severity Score Index (MSSI) in a subject.

According to an aspect of some embodiments of the present invention, the reduction in MSSI is measured after 6 months of treatment compared to pre-treatment values.

According to an aspect of some embodiments of the present invention, recombinant human α-galactosidase has a circulating half-life (T _1/2 ) of at least 5 hours after administration.

According to an aspect of some embodiments of the present invention, recombinant human α-galactosidase has a circulating half-life (T _1/2 ) of at least 20 hours after administration.

According to an aspect of some embodiments of the present invention, recombinant human α-galactosidase has a circulating half-life (T _1/2 ) of at least 50 hours after administration.

According to an aspect of some embodiments of the present invention, recombinant human α-galactosidase has a C _max of at least 5000 ng/mL following administration of 1 mg/kg of recombinant human α-galactosidase.

According to an aspect of some embodiments of the present invention, recombinant human α-galactosidase has a C _max of at least 8000 ng/mL after administration of 2 mg/kg.

According to an aspect of some embodiments of the present invention, recombinant human α-galactosidase has a bioavailability (AUC _0-∞ ) of at least 100,000 ng·h/mL following administration of 1.0 mg/kg of recombinant human α-galactosidase.

According to an aspect of some embodiments of the present invention, recombinant human α-galactosidase has a bioavailability (AUC _0-∞ ) of at least 400,000 ng h/mL following administration of 2.0 mg/kg of recombinant human α-galactosidase.

According to an aspect of some embodiments of the present invention, there is provided a unit dosage form containing 2.0-500 mg of recombinant human α-galactosidase formulated for administration to a human subject.

According to an aspect of some embodiments of the present invention, the unit dosage form contains 10 mg of recombinant human α-galactosidase.

According to an aspect of some embodiments of the present invention, the unit dosage form contains 50 mg of plant recombinant human α-galactosidase.

According to an aspect of some embodiments of the present invention, the unit dosage form contains 100-180 mg of recombinant human α-galactosidase.

According to an aspect of some embodiments of the present invention, the unit dosage form contains 150 mg of recombinant human α-galactosidase.

According to an aspect of some embodiments of the present invention, the unit dosage form is formulated as a liquid.

According to an aspect of some embodiments of the present invention, the unit dosage form is formulated for intravenous administration.

According to an aspect of some embodiments of the present invention, recombinant human α-galactosidase monomers are covalently linked to each other via a poly(alkylene)glycol linking moiety.

According to an aspect of some embodiments of the present invention, the poly(alkylene)glycol linking moiety contains at least 20 alkylene groups.

According to an aspect of some embodiments of the present invention, the recombinant human α-galactosidase is a plant recombinant human α-galactosidase.

According to an aspect of some embodiments of the present invention, the plant recombinant human α-galactosidase is a plant recombinant human α-galactosidase cross-linked with bis-NHS-PEG ₄₅ .

According to an aspect of some embodiments of the present invention, the recombinant human α-galactosidase comprises a human α-galactosidase protein with an amino acid sequence as set forth in any one of SEQ ID NOs: 1-3.

According to an aspect of some embodiments of the present invention, the human α-galactosidase protein is as shown in SEQ ID NO: 2 or 3.

Unless otherwise indicated, all technical and/or scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present invention pertains. While methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of the present invention, exemplary methods and/or materials are described below. In the event of a conflict, this patent specification, including the definitions, shall control. Moreover, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

Brief description of the drawings

Some embodiments of the present invention are described herein by way of example only, with reference to the accompanying drawings. With specific reference to the drawings in detail below, it is emphasized that the details shown are by way of example and for the purpose of illustrative discussion of embodiments of the present invention. In this regard, the description, considered with the drawings, makes it obvious to experts in the present field of technology how the embodiments of the present invention can be implemented.

On graphics:

Figure 1 is a graph showing the pharmacokinetic profile of plant recombinant human α-GAL cross-linked with bis-NHS-PEG ₄₅ (pegunigalsidase alfa) measured in patients' plasma up to 14 days after administration, at the start of treatment ( day 1), which is expressed as ng pegunihalsidase alfa per ml of plasma. The enzyme concentration was measured in blood samples taken at 0 hours (before administration/infusion), 1 hour (1 hour after the start of administration), at the end of administration (infusion) (EOI) and after 1, 4, 8, 24, 48 , 72, 96 hours after EOI and 2 weeks (14 days) after administration ("From _{2 weeks} ") before subsequent administration of pegunigalsidase alfa. The graph represents the mean plasma values (ng/mL) for all cohorts at different sampling periods over a 14-day period on a logarithmic scale. Triangles (light gray line) represent 1.0 mg/kg dose and squares (black line) represent 2.0 mg/kg pegunigalsidase alfa;

Figure 2 is a graph from publicly available pharmacokinetic (PK) data showing the pharmacokinetic profile of commercially available agalsidase beta (r-αhGalA, recombinant human α-GAL A, Fabrazyme™, Genzyme Corp, Cambridge MA) over approximately 10 hours (600 min ) after the start of administration (infusion), on a logarithmic scale. Data taken from American Journal of Human Genetics, 68, 711-722, 2001. Note the short-term (approximately 10 hours) bioavailability of all concentrations of agalsidase beta compared to the bioavailability of pegunigalsidase alfa for a similar dose (measured in days) (see Fig. 1);

в часах) (фигура 3В) и рассчитанного общего доступного количества фермента (площадь под кривой, AUC_0-∞, в мкг⋅мин/мл) (фигура 3С), по сравнению с имеющимися опубликованными данными для агалсидазы бета (r-αhGalA, Fabrazyme™) и агалсидазы альфа (Replagal™, Shire Human Genetic Therapies (HGT), Inc., Cambridge, MA). Дозировки представленной пегунигалсидазы альфа составляют 1,0 и 2,0 мг/кг, дозировка агалсидазы бета составляет 1,0 мг/кг, и агалсидазы альфа составляет 0,2 мг/кг. Следует обратить внимание на более высокую фармакокинетику пегунигалсидазы альфа по всем параметрам (C_max;

и AUC_0-∞) по сравнению с агалсидазой альфа или бета;Figures 3A-3C are histograms illustrating the pharmacokinetic parameters of pegunihalsidase alfa expressed as maximum plasma concentration (C _max , in ng/mL) (Figure 3A), half-life (

in hours) (Figure 3B) and calculated total enzyme available (area under the curve, AUC _0-∞ , in µg⋅min/mL) (Figure 3C), compared with available published data for agalsidase beta (r-αhGalA, Fabrazyme ™) and agalsidase alfa (Replagal™, Shire Human Genetic Therapies (HGT), Inc., Cambridge, MA). The dosages of pegunigalsidase alfa presented are 1.0 and 2.0 mg/kg, the dosage of agalsidase beta is 1.0 mg/kg, and agalsidase alfa is 0.2 mg/kg. Attention should be paid to the higher pharmacokinetics of pegunihalsidase alpha in all respects (C _max ;

and AUC _0-∞ ) compared with agalsidase alpha or beta;

Figures 4A-4B are graphical representations of a simulation of enzyme availability over a 4-week period obtained from a single infusion of pegunigalsidase alfa compared to an extended schedule (2 mg/kg, every 4 weeks) availability simulation of pegunigalsidase alfa with a standard schedule simulation. agalsidase beta (fabrazyme) (1 mg/kg every 2 weeks). Fig. 4A is a schematic illustration showing extrapolated enzyme availability over a four week period after a single infusion;

Fig. 4B is a simulation comparing the pharmacokinetic parameters of 2 mg/kg pegunigalsidase alfa administered once over a 4-week interval and 1 mg/kg agalsidase beta (fabrazyme) administered twice over the same 4-week interval;

Figure 4B compares 4 weeks of enzyme availability (partial AUC calculated for each subsequent week) for 2 mg/kg pegunigalsidase alfa administered once every 4 weeks with a partial AUC of 1 mg/kg agalsidase beta (fabrazyme) administered once every 2 weeks. Attention should be paid to the actual absence of a partial AUC of the enzyme agalsidase beta during the week after administration (see weeks 2 and 4 for agalsidase beta) compared to the predicted significant availability of the enzyme after a single administration of pegunigalsidase alfa for all 4 weeks after administration.

Detailed description of the invention

The present invention, in some embodiments, relates to a stabilized covalently linked human alpha-galactosidase homodimeric enzyme, and more specifically, but not exclusively, to a covalently stabilized covalently linked plant recombinant human alpha-galactosidase enzyme, effective treatment regimens for its use in the treatment of Fabry disease in human subjects using enzyme replacement therapy.

Before a detailed explanation of at least one embodiment of the invention, it should be understood that the present invention is not necessarily limited in its application to the details set forth in the following description or exemplified in the examples. The present invention is capable of other embodiments or may be practiced or carried out in various ways.

Deficiencies in lysosomal protein (eg, defects in lysosomal protein or lack of lysosomal protein) can cause significant harm to the health of the subject (lysosomal storage disease). Enzyme replacement therapy (ERT), in which a deficient protein is administered to a patient, has been used in attempts to treat lysosomal storage diseases. However, administration of a deficient protein does not necessarily result in significant and/or persistent activity of the administered protein in vivo.

Fabry disease is an example of an X-linked recessive (hereditary) lysosomal storage disease that can cause a wide range of symptoms. Deficiency of the lysosomal enzyme α-galactosidase A due to mutations causes the accumulation of a glycolipid known as globotriaosylceramide (also known as Gb3 or ceramidetrihexoside) in blood vessels, other tissues and organs. This accumulation leads to disruption of their proper functioning. Two enzyme replacement therapies (ERTs) are available to functionally compensate for α-galactosidase deficiency. Both agalsidase alfa (Replagal®, Shire) and agalsidase beta (Fabrazyme®, Genzyme-Sanofi) are recombinant forms of the human α-galactosidase A enzyme. Both agalsidase alfa and agalsidase beta have the disadvantage of having a short half-life, resulting in limited bioavailability, leading to unsatisfactory clinical results.

Due to the need to address the problem of impaired activity of α-galactosidases, stabilized forms of α-galactosidase (α-GAL) have been developed that exhibit enhanced activity and/or longer activity in both lysosome and serum conditions, indicating a potential long-term enhanced protein activity in vivo under clinically relevant conditions. In addition, the covalently linked human α-galactosidase of the present invention exhibited enhanced activity and/or longer activity in serum from patients with Fabry disease.

Using the stabilized covalently linked human α-galactosidase according to the present invention, the present inventors have developed an effective novel therapeutic dose and regimen for enzyme replacement therapy of Fabry disease in humans. Clinical experience with the covalently-stabilized human α-galactosidase of the present invention has shown that ERT with the covalently-stabilized human α-galactosidase of the present invention, in novel treatment regimens, is safe and effective for the treatment of Fabry disease in humans. The covalently stabilized human α-galactosidase of the present invention showed significantly improved pharmacokinetics, maintaining plasma concentrations for more than 10 days after administration, C _max greater than 5000 ng/ml at a dose of 1.0 mg/kg (7900-23000 ng/ml ) and greater than 8000 ng/mL at 2.0 mg/kg (13900-46500 ng/mL) and improved bioavailability (AUC) compared to currently available ERTs.

Treatment with the stabilized covalently bound human α-galactosidase of the present invention reduced the number of ceramidetrihexoside inclusions in the peritubular capillaries of the kidneys, significantly attenuated the characteristic deterioration of kidney function, improved kidney function and gastrointestinal symptoms, reduced patient pain index scores, improved or stabilized the patient's heart function and improved physical activity and overall quality of life. Clinical improvement, such as that seen in clinical studies, with an effective 2 mg/kg dosing regimen using wider infusion intervals due to the improved pharmacokinetics and bioavailability of the covalently stabilized human α-galactosidase of the present invention may provide ERT coverage for a specific disease patient population Fabry, such as, but not limited to, mild to moderate patients, young, early diagnosed and/or stable patients, which provides greater convenience of therapy and patient compliance while controlling the patient's symptoms, resulting in a significant improvement in the patient's quality of life and delayed the risk of disease complications.

Therefore, according to an aspect of some embodiments of the present invention, there is provided a method of treating Fabry disease in a human subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a covalently linked plant recombinant human α-galactosidase, wherein said therapeutically effective amount of said covalently linked plant recombinant α -galactosidase human is 0.2-2.0 mg/kg, which allows you to treat Fabry's disease in the subject, and the specified introduction is carried out at intervals of more than two weeks - every 4 weeks, and moreover, the monomers of the specified covalently linked plant recombinant α- human galactosidases are covalently linked to each other via a poly(alkylene)glycol linking moiety.

In some embodiments, the covalently linked plant recombinant human α-galactosidase is a covalently linked homodimeric plant recombinant human α-galactosidase.

In some embodiments, the covalently linked homodimeric plant recombinant human alpha-galactosidase protein has higher stability than native α-galactosidase and/or higher initial activity than native α-galactosidase, as described in detail below. In some embodiments, the covalently stabilized human α-galactosidase protein is a plant recombinant human α-GAL cross-linked with bis-NHS-PEG ₄₅ .

It should be noted that, in some embodiments, the method may be carried out using a covalently stabilized human α-galactosidase protein comprising a recombinant human α-galactosidase protein derived from non-plant (e.g., mammalian) cells, such as the commercially available agalsidase alfa (Replagal ®, Shire) and agalsidase beta (Fabrazyme®, Genzyme), a recombinant alpha-galactosidase produced in non-mammalian cells (plant, bacteria, insects, fungi, etc.) or a suitable alpha-galactosidase of any other origin.

As used herein, the term "Fabry disease" refers to any alpha-galactosidase A deficiency. "Alpha-galactose A deficiency" refers to any deficiency in a patient's natural alpha-galactosidase A activity that results in abnormal accumulations of glycolipids (e.g., globotriaosylceramide), mainly in capillary endothelial cells, kidney cells and/or cardiac myocytes. Deposits of this material can lead to severe neuropathic pain (eg, acroparesthesia and pain with vomiting), severe kidney and cardiovascular disease, and/or stroke. The accumulation of glycolipids can cause severe symptoms, which are usually observed in individuals suffering from Fabry disease. As a rule, more severe symptoms are observed in male patients, but can also be observed in heterozygous female carriers of the defective gene. This disease is known to be poorly diagnosed, especially in female patients. Affected people are characterized by a significantly reduced life expectancy; death usually occurs from renal, cardiac and/or cerebrovascular complications around the fourth and fifth decades of life.

As used herein, the term "monomer" in relation to α-galactosidase refers to a single polypeptide subunit of α-galactosidase. A polypeptide may include non-peptide substituents (eg, one or more saccharide moieties).

As used herein, the term "native" with respect to α-galactosidase encompasses proteins containing an amino acid sequence that is substantially identical (i.e., at least 95% homology, optionally at least 95% identity, optionally at least 99 % homology, optionally at least 99% identity, and optionally 100% identity) to the amino acid sequence of a naturally occurring α-galactosidase protein. Native α-galactosidase may be a protein isolated from a natural source or a recombinantly produced protein (eg, derived from human cells, mammalian cells, plant cells, yeast cells, bacterial cells, insect cells, etc.).

The term "native" when used in relation to the Quaternary structure of α-galactosidase (eg, dimer α-galactosidase), in addition, includes Quaternary structure, essentially identical to the structure of the naturally occurring protein.

As used herein, the phrase "naturally occurring protein" refers to a protein in a form that occurs naturally (eg, in the body), with respect to the amino acid sequence of the protein, as well as the protein's quaternary structure if the protein is in a stabilized, homodimeric form.

Post-translational modifications (eg, glycosylation) of naturally occurring α-galactosidase proteins (eg, in an organism that expresses a naturally occurring α-galactosidase protein) may be present, absent, or modified in the native form of α-galactosidase referred to herein. The native form of α-galactosidase (e.g., recombinantly produced α-galactosidase) may optionally contain post-translational modifications other than those of naturally occurring α-galactosidase, provided that the native form of α-galactosidase retains an amino acid sequence and structure substantially similar to the naturally occurring in nature α-galactosidase, as described herein above.

As used herein, a native form of a protein may refer to a monomeric structure (eg, an α-galactosidase monomer) and/or a multimeric structure (eg, an α-galactosidase dimer). For example, a dimeric protein can be described as the native form of α-galactosidase, and a monomeric polypeptide in a dimeric protein can be described as the native form of the α-galactosidase monomer.

Optionally, the multimeric protein structure described herein is a dimeric structure, as is the native form of α-galactosidase.

Alternatively, the covalently stabilized human α-galactosidase of the present invention contains more than two α-galactosidase monomers. For example, a multimeric protein structure may be a tetramer, hexamer, or octamer composed of α-galactosidase monomers.

The stabilized covalently linked human α-galactosidase of the present invention described herein contains covalent bonds that bind the α-galactosidase monomers therein and which are not present in the native form of α-galactosidase.

Thus, for example, the linking moiety is optionally a moiety that is covalently attached to the side chain, N-terminus, or C-terminus, or moiety associated with post-translational modifications (e.g., saccharide moiety) of the α-galactosidase monomer, as well as the side chain , N-terminus, or C-terminus, or a moiety associated with post-translational modifications (eg, a saccharide moiety) of another α-galactosidase monomer. Such illustrative linking fragments are described in detail herein below.

Optionally, the linking moiety is devoid of a disulfide bond. However, a linking moiety that includes a disulfide bond at a position that does not form a bond between monomers (eg, cleavage of a disulfide bond does not cleave a bond between monomers) is within the scope of this embodiment of the present invention. A potential advantage of a linker moiety lacking a disulfide bond is that it is not susceptible to cleavage by mildly reducing conditions like disulfide bonds.

A linking fragment is also referred to herein as a cross-linking fragment. The linking of α-galactosidase monomers by a linking moiety is referred to herein as "cross-linking".

In some embodiments, relatively short linkers may be less effective than longer linkers in cross-linking between different α-galactosidase monomers.

Therefore, in some embodiments, the linking moiety is not a covalent bond, chemical atom, or group, but rather is a bridging moiety.

Therefore, in some embodiments, the linking moiety is at least 10 atoms long, optionally at least 20 atoms long, optionally at least 30 atoms long, optionally at least 50 atoms long, optionally at least 100 atoms in length, optionally at least 200 atoms in length, optionally at least 300 atoms in length, optionally at least 400 atoms in length, optionally at least 500 atoms in length, optionally at least 600 atoms in length, optionally at least 700 atoms in length, optionally at least 800 atoms in length, and optionally at least 1000 atoms in length. In some embodiments, the length of the linking fragment (expressed in number of atoms) is in the range of 10-1000 atoms, 15-800 atoms, 20-600 atoms, 50-500 atoms, 65-400 atoms, 75-350 atoms, and 80- 200 atoms. In specific embodiments, the length of the linking fragment (expressed in number of atoms) is 20-600 atoms. According to another specific embodiment, the length of the linking fragment (expressed in the number of atoms) is 140 atoms.

In this document, the length of the linking fragment (when expressed as the number of atoms) refers to the length of the main chain of the linking fragment, i.e. the number of atoms forming a linear chain between the residues of each of the two monomers linked through a linking fragment.

In some aspects of the present invention, the covalently stabilized human α-galactosidase monomers of the present invention are covalently linked to each other via a poly(alkylene)glycol linking moiety, e.g., in some embodiments, the linking moiety comprises a poly(alkylene glycol) chain.

As used herein, the phrase "poly(alkylene glycol)" includes the family of polyether polymers which are characterized by the following general formula: -O-[(CH ₂ ) _m -O-] _n - where m is the number of methylene groups present in each alkylene glycol unit, and n represents the number of repeating units, and therefore represents the size or length of the polymer. For example, when m=2, the polymer is called polyethylene glycol, and when m=3, the polymer is called polypropylene glycol.

In some embodiments, m is an integer greater than 1 (eg, m=2, 3, 4, etc.).

Optionally, m varies among the units of the poly(alkylene glycol) chain. For example, a poly(alkylene glycol) chain may contain both ethylene glycol (m=2) and propylene glycol (m=3) units linked together.

The poly(alkylene glycol) optionally contains at least two functional groups (eg, as described herein), with each functional group forming a covalent bond with one of the α-galactosidase monomers. The functional groups are optionally the terminal groups of the poly(alkylene glycol) so that the entire length of the poly(alkylene glycol) is between the two functional groups.

The phrase "poly(alkylene glycol)" also encompasses analogs thereof in which the oxygen atom is replaced by another heteroatom such as, for example, S, -NH- and the like. This term further encompasses derivatives of the above in which one or more of the methylene groups constituting the polymer are substituted. Illustrative substituents on methylene groups include, without limitation, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, hydroxy, oxo, thiol, and thioalkoxy, and the like.

As used herein, the phrase "alkylene glycol unit" embraces a -(CH ₂ ) _m -O- group, or an analogue thereof, as described herein above, which forms the backbone of a poly(alkylene glycol) wherein (CH ₂ ) _m (or an analogue thereof ) is bonded to a heteroatom belonging to another alkylene glycol unit or to a fragment of an α-galactosidase monomer (in the case of a terminal unit), and O (or its heteroatom analog) is bonded to (CH ₂ ) _m (or its analog) of another alkylene glycol unit or functional a group that forms a bond with the α-galactosidase monomer.

The alkylene glycol unit may be branched such that it is linked to 3 or more adjacent alkylene glycol units, where each of the 3 or more adjacent alkylene glycol units is part of a poly(alkylene glycol) chain. Such a branched alkylene glycol unit is bonded through its heteroatom to one adjacent alkylene glycol unit, and each of the heteroatoms of the remaining adjacent alkylene glycol units is bonded to a carbon atom of the branched alkylene glycol unit. In addition, a heteroatom (eg, nitrogen) can bind more than one carbon atom of the alkylene glycol unit of which it is a part, thus forming a branched alkylene glycol unit (eg, [(-CH ₂ ) _m ] ₂ N- and the like).

In exemplary embodiments, at least 50% of the alkylene glycol units are identical, eg they contain the same heteroatoms and the same m values as the other. Optionally at least 70%, optionally at least 90% and optionally 100% of the alkylene glycol units are identical. In exemplary embodiments, the heteroatoms associated with identical alkylene glycol units are oxygen atoms. According to additional exemplary embodiments, m is 2 for identical units.

In one embodiment, the linker is a single straight chain linker, such as polyethylene glycol (PEG).

As used herein, the term "poly(ethylene glycol)" describes a poly(alkylene glycol) as defined herein above, wherein at least 50%, at least 70%, at least 90% and preferably 100% of the alkylene glycol units are -CH ₂ CH ₂ -O-. Similarly, the phrase "ethylene glycol units" is defined herein as -CH ₂ CH ₂ O- units.

In optional embodiments, the binder moiety contains a poly(ethylene glycol) or analogue thereof, characterized by the general formula:

-X ₁ -(CR ₁ R ₂ -CR ₃ R ₄ -Y) _n -X ₂ -

where each X ₁ and X ₂ represents a functional group (eg, as described herein) that forms a covalent bond with at least one α-galactosidase monomer;

Y is O, S or NR ₅ (optionally O);

n is an integer, optionally from 1 to 200 (optionally from 5 to 150 and optionally from 40 to 70), although higher values of n are also contemplated; And

each of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ is independently selected from the group consisting of the following: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, hydroxy, oxo, thiol and thioalkoxy.

In some embodiments, n is at least 5, optionally at least 8, optionally at least 15, optionally at least 25, and optionally at least 40.

In some embodiments, n is no greater than 200, optionally no greater than 150, and optionally no greater than 70.

The poly(ethylene glycol) or equivalent may optionally contain a copolymer, for example, in which not all CR ₁ R ₂ -CR ₃ R ₄ -Y units in the above formula are identical to each other.

In some embodiments, at least 50% of the CR ₁ R ₂ -CR ₃ R ₄ -Y units are identical. Optionally at least 70%, optionally at least 90% and optionally 100% of the CR ₁ R ₂ -CR ₃ R ₄ -Y units are identical.

Optionally, the linking moiety is branched, for example such that for one or more CR ₁ R ₂ -CR ₃ R ₄ -Y units in the above formula, at least one of R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ is -(CR ₁ R ₂ -CR ₃ R ₄ -Y) _p -X ₃ -, where R ₁ -R ₄ and Y are as defined herein above, p is an integer as defined in herein for n (eg, from 1 to 200), and X ₃ is as defined herein for X ₁ and X ₂ .

The functional groups may optionally form a bond, such as, without limitation, an amide bond, an amine bond, an ester bond, and/or an ether bond.

For example, the functional group may optionally contain a carbonyl group that forms an amide bond to a nitrogen atom in a polypeptide (e.g., at a lysine or N-terminal residue), or an ester bond to an oxygen atom in a polypeptide (e.g., at a serine, threonine, or tyrosine residue) .

Alternatively or additionally, the functional group may optionally contain a heteroatom (eg, N, S, O) that forms an amide bond, ester bond, or thioether bond with a carbonyl group in the polypeptide (eg, at a glutamate or aspartate residue or at the C-terminus).

Alternatively or additionally, the functional group may contain an alkyl or aryl group attached to the polypeptide (eg, to a heteroatom in the polypeptide).

Alternatively or additionally, the functional group may optionally contain a nitrogen atom that forms an amine bond with an alkyl group in the α-galactosidase monomer, or the α-galactosidase monomer may optionally contain a nitrogen atom that forms an amine bond with an alkyl group in the functional group. Such an amine bond may be formed by reductive amination (eg, as described herein below).

In some embodiments, at least one of the functional groups forms an amide bond with the polypeptide (eg, with a lysine residue therein).

The functional groups may be identical to each other or different.

In some embodiments, at least one of the functional groups is attached to one functional group of the polypeptide (for example, the amino group of the lysine residue or N-terminus), and at least one of the functional groups is attached to another functional group of the polypeptide (for example, the thiol group of the cysteine residue ).

In some embodiments, the stabilized covalently linked human α-galactosidase may contain additional PEG fragments associated with only one subunit by binding to a lysine residue in that subunit.

In some embodiments, the stabilized covalently linked human α-galactosidase of the present invention is cross-linked with bis-NHS-PEG ₄₅ . The preparation and characterization of such plant recombinant human α-galactosidase cross-linked with bis-NHS-PEG ₄₅ suitable for use in the present invention is described in detail in the international patent application according to the PCT WO 2011/107990 of the present patent owner. The stabilized covalently linked plant recombinant human α-galactosidase cross-linked with bis-NHS-PEG ₄₅ described herein is also referred to herein as "pegunigal sidase alpha".

The stabilized covalently linked plant recombinant human α-galactosidase cross-linked with bis-NHS-PEG ₄₅ described herein is biologically active, characterized by α-galactosidase activity. The α-galactosidase activity described herein is a biological activity that is characteristic of α-galactosidase (eg, catalytic activity characteristic of α-galactosidase, such as hydrolysis of the terminal α-galactosyl moiety of a substrate).

In some embodiments, the catalytic activity of α-galactosidase is characterized by the rate of catalysis at saturation (ie, the value of V _max ).

Alternatively, the α-galactosidase activity is a therapeutic activity (eg, an enzymatic activity having a therapeutic effect), such as a therapeutic activity in the context of Fabry disease. Optionally, therapeutic activity is determined in experimental animals (eg mice with Fabry disease) and optionally in human patients with Fabry disease.

Techniques for determining α-galactosidase activity will be known to those skilled in the art. Typically, α-galactosidase (ie, the native form or multimeric protein structure described herein) is contacted with a compound recognized in the art as a substrate for α-galactosidase, and the degree of activity is then quantified. Compounds that provide particularly convenient detection of α-galactosidase activity are known in the art and are commercially available.

In some embodiments, α-galactosidase activity is determined by a 4-methylumbelliferyl-α-D-galactopyranoside hydrolysis assay. In other embodiments, α-galactosidase activity is determined by a p-nitrophenyl-α-D-galactopyranoside hydrolysis assay.

When comparing the activity of the stabilized covalently linked human α-galactosidase protein described herein with the activity of native α-galactosidase, in some specific embodiments, the native α-galactosidase preferably contains substantially identical α-galactosidase monomers (e.g., in terms of amino acid sequence and glycosylation pattern) to the α-galactosidase monomers of the stabilized covalently linked recombinant human α-galactosidase protein.

In some embodiments, the stabilized covalently linked human α-galactosidase of the present invention has a circulating half-life in the physiological system of a human subject that is higher (e.g., at least 20% higher, at least 50% higher, at least 100% higher, at least 400% higher, at least 900% higher, at least 1500% higher, at least 2000% higher, at least 2500% higher, at least 3000% higher higher, at least 3500% higher, at least 4000% higher, at least 5000% higher, at least 7500% higher, at least 8000% higher, up to 10000% higher, up to 20,000% higher, up to 50,000% higher, 100,000% higher, 200,000% higher, or more) than the circulating half-life of non-cross-linked α-galactosidase [eg, agalsidase alfa (Replagal®, Shire) and agalsidase beta (Fabrazyme ®, Genzyme)].

As described herein, when tested in a clinical context, the circulating half-life of the stabilized covalently linked human α-galactosidase of the present invention after intravenous (IV) infusion was significantly increased. Thus, according to some aspects of the present invention, the covalently stabilized human α-galactosidase of the present invention has a circulating half-life (T 1/2) of at least 5 hours, at least 10 hours, at least 20 hours, at least 50, at least 60, at least 70, at least 80, or at least 90 hours after the IV infusion.

In some embodiments, the circulating half-life of the stabilized covalently bound human α-galactosidase of the present invention is such that the plasma concentration of the stabilized covalently bound human α-galactosidase of the present invention 14 days after intravenous administration of 2 mg/kg body weight has the same order of magnitude as the maximum plasma concentration of commercially available recombinant human α-galactosidase (agalsidase beta, Fabrazyme®) 2 hours after infusion at a dose of 1 mg/kg body weight).

In some embodiments, the circulating half-life (T _1/2 ) of the covalently stabilized human α-galactosidase of the present invention, when administered intravenously to a subject, is at least 5, 10, 20, 30, 40, at least 50, by at least 60, at least 70, at least 80 or at least 90 hours after the IV infusion. In some embodiments, the circulating half-life (T _1/2 ) following iv infusion of the stabilized covalently bound human α-galactosidase of the present invention is in the range of 3-100 hours, in the range of 5-70 hours, in the range , which is 5-50, 10-45, 15-40, 20-35 40-69 60-80, 60-90 and 25-50 hours. In specific embodiments, the circulating half-life (T _1/2 ) of the stabilized covalently linked human α-galactosidase of the present invention is 5-10 hours, 10-20 hours, 20-50 hours, or 50-80 or 50-90 hours.

In some embodiments, the bioavailability of the stabilized covalently linked human α-galactosidase of the present invention may reflect the total amount of enzyme available after administration, expressed as "area under the curve" (AUC _0-∞ ), in units of µg⋅min/mL or ng⋅h/ ml. Thus, in some embodiments, the bioavailability (area under the curve) of the stabilized covalently bound human α-galactosidase of the present invention following intravenous administration of 1.0 mg/kg of the stabilized covalently bound human α-galactosidase of the present invention to a subject is in the range of 10,000- 500,000 ng⋅h/mL, 50,000-250,000 ng⋅h/mL, at least 10,000 ng⋅h/mL, at least 25,000 ng⋅h/mL, at least 50,000 ng⋅h/mL, at least 75,000 ng⋅h/ml, at least 100,000 ng⋅h/ml, or at least 200,000 ng⋅h/ml. In specific embodiments, the bioavailability (area under the curve) of the stabilized covalently linked human α-galactosidase of the present invention is 100,000 ng h/mL following administration of 1.0 mg/kg of said recombinant human α-galactosidase.

In other embodiments, the bioavailability (area under the curve) of the stabilized covalently linked human α-galactosidase of the present invention following intravenous administration of 2.0 mg/kg of the stabilized covalently linked human α-galactosidase of the present invention to a subject is in the range of 50,000-800,000 ng⋅ h/mL, 100,000-600,000 ng⋅h/mL, 150,000-500,000 ng⋅h/mL, at least 50,000 ng⋅h/mL, at least 75,000 ng⋅h/mL, at least 100,000 ng⋅h/mL ml, at least 200,000 ng⋅h/ml, at least 300,000 ng⋅h/ml, at least 400,000 ng⋅h/ml, or at least 600,000 ng⋅h/ml. In specific embodiments, the bioavailability (area under the curve) of the stabilized covalently linked human α-galactosidase of the present invention is 400,000 ng h/mL following administration of 2.0 mg/kg of said recombinant human α-galactosidase.

An increased circulating half-life may optionally be associated with higher in vivo stability (eg, metabolic resistance), higher uptake and/or target organ activity.

Circulating half-lives can be determined by taking samples (eg, blood samples) from physiological systems (eg, humans, laboratory animals) at various intervals and determining the content of α-galactosidase in the sample using techniques known in the art.

Tissue half-lives can be determined by taking samples (eg, tissue samples) from physiological systems (eg, humans, laboratory animals) at various intervals and determining the content of α-galactosidase in the sample using techniques known in the art.

Optionally, the half-life is calculated as the final half-life (for example, as described in the Examples section), where the half-life is the time required for the concentration (for example, blood concentration) to decrease by 50% after reaching pseudo-equilibrium distribution. The finite half-life can be calculated from the finite linear part of the logarithm of concentration versus time by linear regression of the logarithm of concentration versus time (see, for example, Toutain & Bousquet-Melou [J Vet Pharmacol Ther 2004, 27:427-39]). Thus, the terminal half-life is a measure of the decrease in plasma concentration of a drug due to the rate of drug elimination, rather than its decrease for other reasons, and is not necessarily the time required to halve the amount of drug administered.

Determination of the content of α-galactosidase (for example, stabilized covalently linked human α-galactosidase according to the present invention or non-cross-linked α-galactosidase) may include detecting the physical presence of α-galactosidase (for example, via an antibody to α-galactosidase) and/or detecting the level of α-galactosidase activity (eg, as described herein).

As used herein, "human α-galactosidase" refers to a plant recombinant α-galactosidase containing an amino acid sequence substantially identical (eg, as described herein above) to the amino acid sequence of an α-galactosidase protein that occurs naturally in humans.

Examples of α-GALs suitable for use in the present invention include, without limitation, α-GALs with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. Optionally, according to specific embodiments α-GAL is characterized by an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3. In some embodiments, α-GAL may be any of SEQ ID NO: 2 or SEQ ID NO: 3. According to In other embodiments, the stabilized covalently linked human α-galactosidase of the present invention comprises a plant recombinant α-galactosidase population comprising a combination of plant recombinant α-galactosidase proteins comprising both α-GAL according to SEQ ID NO: 2 and α-GAL according to SEQ ID NO: 3.

As used herein, the term "α-galactosidase" refers to any protein that exhibits enzymatic activity (eg, hydrolysis) on galactose moieties in Gb ₃ (eg, α-galactosidase A). Optionally, "α-galactosidase" refers to E.C. 3.2.1.22. As used herein, the term "acid α-galactosidase" refers to an α-galactosidase characterized by the ability to hydrolyze terminally bound α-galactose moieties from galactose-containing oligosaccharides under acidic pH conditions (e.g., approximately pH 4.2-5) such as those found in lysosome.

Optionally, the α-galactosidase protein further comprises at least one mannose-6-phosphate (M6P) moiety. The M6P fragment (or fragments) may be linked to one or more α-galactosidase monomers of the α-galactosidase protein (eg, via a linker).

Techniques and reagents for introducing M6P-containing fragments into a biomolecule (eg, polypeptide) are described in International Patent Publication WO 2009/024977.

As detailed herein, intravenous administration of the stabilized, covalently linked human α-galactosidase of the present invention resulted in improvement/stabilization of clinical parameters in human patients, including assessments of renal function, cardiac function, and experienced suffering (eg, pain).

Accumulation of the Gb3 glycolipid is characteristic of the natural course of Fabry disease, and decreased accumulation is a clinical parameter in ERT for patients with Fabry disease. In one embodiment, the method of the present invention can be used to reduce glycolipid accumulation. Thus, in some embodiments, IV (intravenous) administration of a therapeutically effective amount of the stabilized covalently linked human α-galactosidase of the present invention reduces plasma Gb3 and/or lyso-Gb3 concentrations. In some embodiments, intravenous administration of a therapeutically effective amount of a stabilized covalently linked human α-galactosidase of the present invention reduces Gb3 (glycolipid) in the peritubular capillaries of the kidneys of subjects. In specific embodiments, the reduction is a reduction of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more as measured by the BLISS score. In some embodiments, the reduction is measured as a BLISS score. The reduction in Gb3 and/or lyso-Gb3 response to administration of the stabilized covalently linked human α-galactosidase of the present invention is measured after 1 month, 2 months, 3 months, 5 months, 6 months, one year of treatment or more. In some embodiments, the reduction in Gb3 is at least 50% as measured 6 months after the start of treatment compared to pre-treatment Gb3.

Patients with Fabry disease also suffer from pain, and in particular neuropathic pain, and the method and stabilized covalently linked human α-galactosidase of the present invention can be used to alleviate pain associated with Fabry disease in patients. In some embodiments, administering a therapeutically effective amount of a stabilized, covalently linked human α-galactosidase of the present invention reduces pain in subjects. A variety of pain assessment questionnaires are available for assessing pain reduction, a non-limiting list includes the McGill Pain Assessment Inventory, the Fabry Disease Pain Assessment Inventory, and the Brief Pain Assessment Inventory. In some embodiments, pain reduction is measured according to a short pain assessment questionnaire. In some embodiments, the pain reduction according to the questionnaire is at least 30%, at least 40%, at least 50%, or more. In specific embodiments, the reduction in pain is a reduction of at least 50%. Pain reduction in response to administration of the stabilized covalently linked human α-galactosidase of the present invention is measured after 1 month, 2 months, 3 months, 5 months, 6 months, one year of treatment or more. In other embodiments, the reduction in pain parameters is measured 6 months after the start of treatment and/or compared to pre-treatment scores.

In some embodiments, pain reduction is measured at 1 week, 2 weeks, and 3 weeks in each treatment cycle. In some embodiments, pain relief is detected in the second week after administration. In some embodiments, a reduction in pain is detected in the third week after administration.

In other embodiments, administration of a therapeutically effective amount of a stabilized, covalently linked human α-galactosidase of the present invention provides an improvement in perceived overall quality of life in subjects. Many quality of life assessment questionnaires are available to assess treatment outcomes, a non-limiting list includes the McGill Quality of Life Assessment Inventory, the Quality of Life Assessment Scale (QOLS), the WHO Quality of Life Assessment Inventory (WHOQOL), and the EQ-5D-5L questionnaire. In some embodiments, quality of life is measured in accordance with the EQ-5D-5L questionnaire, which contains 5 aspects of health: mobility, self-care ability, ability to perform ordinary activities, pain and discomfort, and anxiety and depression. There are 5 variant levels under each domain. In some embodiments, the improvement according to the questionnaire is at least 30%, at least 40%, at least 50%, or more. In specific embodiments, the improvement in quality of life is an improvement of at least 50%. Improvement in quality of life in response to administration of the stabilized covalently linked human α-galactosidase of the present invention is measured after 1 month, 2 months, 3 months, 5 months, 6 months, one year of treatment or more. In other embodiments, the improvement in quality of life is measured 6 months after the start of treatment and/or compared to pre-treatment scores.

Renal failure is another common clinical complication of Fabry disease, mainly caused by glycolipid accumulation in the renal tubules and characterized by progressive worsening over the course of the disease leading to end-stage renal disease. The method and the covalently stabilized human α-galactosidase of the present invention can be used to slow the progression of nephropathy and achieve stabilization of kidney function or to maintain a stable level of kidney function in a patient. Thus, in some embodiments, administering a therapeutically effective amount of the stabilized covalently linked human α-galactosidase of the present invention using the methods of the present invention attenuates Fabry nephropathy in subjects.

Thus, in accordance with other embodiments, administration of a therapeutically effective amount of a stabilized covalently linked human α-galactosidase of the present invention using the methods of the present invention maintains a stable level of nephropathic kidney function in subjects.

Methods for assessing kidney function in patients with Fabry disease include, but are not limited to, laboratory markers (proteinuria, microalbuminuria, assessment of chronic kidney disease (CKD), etc.), gross hematuria, biopsy, electron microscopy of biopsy, impaired renal function, and ultrasonography kidneys and anomalies detected in MRI. In some embodiments, kidney function (and impaired kidney function or nephropathy) is measured according to glomerular filtration rate and/or proteinuria. The glomerular filtration rate (GFR) is the volume of fluid filtered from the renal glomerular capillaries into the Bowman's capsule per unit of time. There are several different techniques used to calculate or estimate the glomerular filtration rate (GFR or eGFR (estimated glomerular filtration rate)). In clinical practice, GFR is estimated using creatinine clearance or estimates of creatinine clearance based on serum creatinine. The most commonly used methods for estimated GFR (eGFR) using the Modification of Diet in Renal Disease (MDRD) formula (using four variables: serum creatinine, age, ethnicity, and sex) and the CKD-EPI formula (Collaborative in the Epidemiology of Chronic kidney disease) designed to provide a formula that is more accurate than the MDRD formula.

In some embodiments, an amelioration of deterioration in kidney function is observed. Attenuation of deterioration in renal function in response to intravenous administration of the stabilized covalently linked human α-galactosidase of the present invention in accordance with some aspects of the present invention is measured after 3 months, 6 months, 12 months, 24 months of treatment or more. In other embodiments, the improvement in Fabry disease-associated kidney deterioration is measured 6 months after initiation of treatment and/or compared to pre-treatment scores. In specific embodiments, renal function is maintained at a constant level, the same or similar to when treatment begins, as measured using estimated glomerular filtration rate and/or proteinuria score after 6 months, 12 months, 24 months of treatment, or more.

In some embodiments, administration of a therapeutically effective amount of a stabilized, covalently linked human α-galactosidase of the present invention improves at least one gastrointestinal parameter in subjects. In some embodiments, the gastrointestinal tract parameter includes, without limitation, abdominal pain and/or frequency of abdominal pain. In some embodiments, there is an improvement in the frequency of signs and symptoms associated with the gastrointestinal tract. In other embodiments, the reduction in gastrointestinal parameters is measured 6 months after the start of treatment and/or compared to the state before treatment.

In specific embodiments, gastrointestinal parameters associated with Fabry disease remain at the same constant level as measured after 2, 4, 6, 12 months of treatment or more.

In some embodiments, administration of a therapeutically effective amount of a stabilized covalently linked human α-galactosidase of the present invention improves/stabilizes at least one cardiac parameter in subjects. Cardiac parameters include, without limitation, ECG components, cardiac function (eg, left ventricular ejection fraction), arrhythmias, valvular dysfunction, and cardiac hypertrophy. In other embodiments, the cardiac parameters are LVM and LVMI measured by MRI. Improvement/stabilization of cardiac parameters in response to intravenous administration of the stabilized covalently linked human α-galactosidase of the present invention in accordance with some aspects of the invention is measured after 6 months, 12 months, 24 months of treatment or more. In other embodiments, the reduction in LVM or LVMI measured by MRI is measured 6 months after the start of treatment and/or compared to pre-treatment values.

In specific embodiments, cardiac parameters remain at a constant level as measured at the start of treatment, after 6 months, 12 months, 24 months of treatment, or more.

In other embodiments, a patient's Fabry disease status can be assessed using the Mainz Severity Scoring Index (MSSI), which is a tool for quantifying the burden of Fabry disease, cumulative signs and symptoms of the disease. Thus, the MSSI can be used to assess disease status in patients treated with the alpha-galactosidase protein and the methods of the present invention. Thus, in some embodiments, administration of a therapeutically effective amount of a stabilized, covalently linked α-galactosidase of the present invention maintains a stable or improved MSSI score in subjects. MSSI in response to administration of the stabilized covalently linked human α-galactosidase of the present invention can be measured after 6 months, 12 months, 24 months of treatment, or more, in accordance with some aspects of the invention. In other embodiments, the reduction in MSSI is measured 6 months after the start of treatment and/or compared to pre-treatment scores.

In other specific embodiments, the patient's Fabry disease status as measured by MSSI remains at a constant level as measured at the start of treatment, after 6, 12, 24 months of treatment, or more.

According to another aspect of the embodiments of the present invention, a pharmaceutical composition is provided which comprises a covalently stabilized human α-galactosidase of the present invention as described herein and a pharmaceutically acceptable carrier. In some embodiments, the stabilized covalently linked human α-galactosidase of the present invention comprises a stabilized covalently linked plant recombinant human alpha-galactosidase protein.

As used herein, "pharmaceutical composition" refers to a formulation of one or more of the stabilized covalently linked human α-galactosidase protein of the present invention described herein with other chemical components such as pharmaceutically acceptable and suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of a compound to the body.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to the body and does not interfere with the biological activity and properties of the administered compound. Non-limiting examples of carriers are: propylene glycol, saline, emulsions and mixtures of organic solvents with water, and solid (eg powder) and gaseous carriers.

As used herein, the term "adjuvant" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of a compound. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

The pharmaceutical composition optionally contains an additional ingredient that further stabilizes the α-galactosidase of the stabilized covalently linked human α-galactosidase of the present invention. Optionally, the additional ingredient is galactose.

Alternatively, a galactose derivative (eg, a galactose-containing glycoside) may be used instead of galactose. Optionally, a non-reducing galactose derivative is used.

Formulation and administration techniques can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Pharmaceutical compositions of the present invention may be prepared by methods well known in the art, for example, by conventional mixing, dissolving, granulating, drageeing, triturating, emulsifying, encapsulating, trapping, or lyophilizing methods.

Thus, pharmaceutical compositions for use according to the present invention can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers containing excipients and excipients that facilitate the processing of the covalently stabilized human α-galactosidase of the present invention into formulations that can be used pharmaceutically. . The correct composition depends on the chosen route of administration.

For injection or infusion, the covalently stabilized human α-galactosidase of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution or physiological saline buffer with or without organic solvents such as propylene glycol, polyethylene glycol.

The covalently stabilized human α-galactosidase of the present invention can be formulated as part of an aqueous liquid suspension or solution for intravenous administration.

The stabilized covalently linked human α-galactosidase of the present invention described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Formulations for injection or infusion may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, optionally with an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles and may contain adjuvants such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the stabilized covalently linked human α-galactosidase of the present invention in a water-soluble form. In addition, suspensions of the stabilized covalently linked human α-galactosidase of the present invention may be prepared as suitable oily injection suspensions and emulsions (eg, water-in-oil, oil-in-water, or water-in-oil-in-oil emulsions). Suitable lipophilic solvents or carriers include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the stabilized covalently linked human α-galactosidase of the present invention, allowing highly concentrated solutions to be obtained.

The compositions of the present invention may be used with the methods described herein or with other treatments for Fabry disease. The stabilized covalently linked human α-galactosidase formulations of the present invention may be further diluted prior to administration to a subject. In some embodiments, formulations are diluted with saline and stored in IV bags or syringes prior to administration to a subject.

In exemplary embodiments, the covalently stabilized human α-galactosidase of the present invention is formulated according to conventional procedures into a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where appropriate, the pharmaceutical agent may also include a solubilizing agent and a local anesthetic such as lignocaine to relieve pain at the injection site. Typically, the ingredients are supplied either singly or mixed in a single dosage form, for example, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container such as an ampoule or sachet, indicating the amount of active agent. If the pharmaceutical agent is to be administered by infusion, it may be dispensed, for example, in an infusion bottle containing sterile pharmaceutical grade water or saline. If the pharmaceutical agent is administered by injection, for example, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration.

Alternatively, the covalently stabilized human α-galactosidase of the present invention may be in powder form for reconstitution with a suitable vehicle, eg sterile, pyrogen-free water, prior to use.

The pharmaceutical compositions described herein may also contain suitable carriers or excipients in solid phase or gel phase. Examples of such carriers or adjuvants include, without limitation, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Pharmaceutical compositions suitable for use in the context of the present invention include compositions in which the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of a stabilized covalently linked human α-galactosidase of the present invention effective to prevent, alleviate or ameliorate the symptoms of a disease, improve the quality of life, or prolong the survival of the subject being treated.

For any stabilized covalently linked human α-galactosidase used in the methods of the present invention, a therapeutically effective amount or dose can be initially estimated from activity assays in animals. For example, the dose can be formulated in animal models to achieve a range of circulating concentrations that includes the IC ₅₀ as determined by activity assays (e.g., the concentration of the stabilized covalently bound human α-galactosidase protein of interest that achieves a half-maximal increase in biological activity stabilized covalently linked human α-galactosidase protein). Such information can be used to more accurately determine applicable doses for humans.

As shown in the following Examples section, a therapeutically effective amount of a stabilized covalently linked human α-galactosidase according to embodiments of the present invention can range from 0.1 mg/kg body weight to about 5.0 mg/kg body weight. In some embodiments, a therapeutically effective amount for a stabilized covalently linked human α-galactosidase of the present invention may be any of 0.1, 0.2, 0.5, 0.75, 1.0, 1.25, 1.50, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0 3.25, 3.50, 3.75, 4.0, 4.25, 4.50, 4.75 or 5.00 mg/kg body weight. In specific embodiments, a therapeutically effective amount of the stabilized covalently linked human α-galactosidase of the present invention is 1.0 mg/kg body weight. In other specific embodiments, a therapeutically effective amount for a covalently stabilized human α-galactosidase of the present invention is 2.0 mg/kg body weight. In other specific embodiments, a therapeutically effective amount for a covalently stabilized human α-galactosidase of the present invention is 5.0 mg/kg body weight. In general, the toxicity and therapeutic efficacy of the stabilized covalently linked human α-galactosidase of the present invention described herein can be determined using standard pharmaceutical procedures in experimental animals, for example, by determining the EC ₅₀ , IC ₅₀ and LD ₅₀ (lethal dose, causing death in 50% of the test animals) for the protein structure under study. The data obtained from these activity assays and animal studies can be used in formulating a dosage range for human use.

The dosage may vary depending on the dosage form used and the route of administration used. The exact composition, route of administration and dosage can be chosen by the attending physician, taking into account the condition of the patient. (See, for example, Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", ch. 1, p. 1).

The amount of dosage and the interval between doses can be adjusted individually to provide plasma levels of the active ingredient that are sufficient to maintain the desired effects. In some embodiments, the stabilized covalently linked human α-galactosidase of the present invention is administered by intravenous infusion at a rate of 20-200 ml/hour. In specific embodiments, for example, the covalently stabilized human α-galactosidase of the present invention is delivered in a total volume of 150 ml, and for dosages less than 2.0 mg/ml, the infusion rate may be 37.5 or 75 ml. /hour for patients weighing less than 75 kg and 25.2 ml/hour for patients weighing more than 75 kg. In other embodiments, the covalently stabilized human α-galactosidase of the present invention is delivered in a total volume of 350 ml, and for dosages of 2.0 mg/kg body weight, the infusion rate may be 58.2 ml/hour for patients with weighing less than 90 kg, with individually determined infusion rates for patients weighing more than 90 kg. In other embodiments, the stabilized covalently bound human α-galactosidase of the present invention is delivered in a volume of 150 ml, for example, to patients weighing up to 70 kg, in a total volume of 250 ml, for example, to patients with a body weight of 70 to 100 kg and a total volume of 500 ml per infusion, for example, for patients weighing more than 100 kg, at a rate of 0.83 ml/min (50 ml/hour) for a 150 ml infusion, 1.38 ml/min (82.2 ml/hour) hour) for a 250 ml infusion and 2.78 ml/min (167 ml/hour) for a 500 ml infusion.

The timing of the infusion can be changed after monitoring patient tolerance to treatment. In some embodiments, the infusion time may be gradually reduced to 1.5 hours based on the judgment of the administering physician.

In some embodiments, the infusion time may be up to 8 hours from start to completion of the desired dosage of the stabilized covalently linked human α-galactosidase of the present invention. In some embodiments, the infusion time is 1, 2, 3, 4.5, 5, 6, 7, or 8 hours. In some cases, the infusion time may exceed 8 hours in accordance with individual circumstances and the needs of individual subjects.

In some embodiments (e.g., when the dosage of covalently bound human α-galactosidase of the present invention is 2.0 mg/kg), intravenous infusions may be administered along with a premedication protocol including, but not limited to, an H1 blocker (diphenhydramine, hydroxyzine, cetrizine, loratadine, desloratidine) and H2 blocker (ranitidine, cimetidine, famotidine) at standard doses 12 hours and/or 2 hours prior to infusion.

Surprisingly, when administered to patients with Fabry disease, the stabilized covalently linked human α-galactosidase of the present invention exhibited superior enhanced bioavailability (see Examples III-V herein below) compared to conventionally available alpha-galactosidase preparations used in Fabry disease ERT. . Thus, the covalently stabilized human α-galactosidase of the present invention will have prolonged bioavailability not only during the demonstrated 2 week post-administration period, but up to 3 and 4 weeks after intravenous administration.

Thus, in specific embodiments, the covalently stabilized human α-galactosidase of the present invention is administered intravenously as an infusion at dosing intervals of greater than two weeks (14 days ± 3 days). In some embodiments, the covalently stabilized human α-galactosidase of the present invention is administered intravenously as an infusion at dosing intervals of greater than two weeks (14 days ± 3 days) up to four weeks (28 days ± 3 days). In some embodiments, the interval may be any of the following: 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days (3 weeks), 22 days, 23 days, 24 days, 25 days, 26 days, 27 days or 28 days (4 weeks), 29 days, 30 days, 31 days (month), 32 days, 33 days, 34 days, 35 days (5 weeks), 36 days, 37 days, 38 days, 39 days, 41 days, 42 days (6 weeks), 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days (7 weeks), 50 days, 51 days, 52 days, 53 days, 54 days, 55 days or 56 days (8 weeks). In some embodiments, the covalently stabilized human α-galactosidase of the present invention is administered intravenously as an infusion at dosing intervals of greater than two weeks (14 days ± 3 days) up to eight weeks (56 days ± 3 days). In some embodiments, the stabilized covalently bound human α-galactosidase of the present invention is administered intravenously as an infusion at dosing intervals of 17 days-8 weeks, 17 days-6 weeks, 17 days-5 weeks, 3 weeks-6 weeks, 3 weeks - 5 weeks, 3 weeks - 4 weeks, 4 weeks - 6 weeks or 4 weeks - 5 weeks. In specific embodiments, the stabilized covalently linked human α-galactosidase of the present invention is administered intravenously at intervals ranging from once every 6 weeks ±3 days. In other embodiments, the interval is once every 6 weeks. In specific embodiments, the covalently stabilized human α-galactosidase of the present invention is administered intravenously at intervals ranging from once every 5 weeks±3 days. In other embodiments, the interval is once every 5 weeks. In specific embodiments, the stabilized covalently linked human α-galactosidase of the present invention is administered intravenously at intervals ranging from once every 4 weeks±3 days (eg, every month). In other embodiments, the interval is once every 4 weeks. In one embodiment, the stabilized covalently linked human α-galactosidase of the present invention is administered at intervals of three weeks±3 days between administrations.

According to another embodiment, the stabilized covalently linked human α-galactosidase of the present invention is administered at intervals of three weeks between administrations. In other embodiments, the covalently stabilized human α-galactosidase of the present invention is administered at a dosage of 1.0 mg/kg body weight, with intervals of three weeks between administrations. In other embodiments, the covalently stabilized human α-galactosidase of the present invention is administered at a dosage of 2.0 mg/kg body weight, with intervals of three weeks between administrations. In other embodiments, the stabilized covalently linked human α-galactosidase of the present invention is administered at a dosage of 1.0 mg/kg body weight, with intervals of 4 weeks between administrations. In other embodiments, the stabilized covalently linked human α-galactosidase of the present invention is administered at a dosage of 2.0 mg/kg body weight, with intervals of 4 weeks between administrations.

In order to facilitate the administration of the covalently bound human α-galactosidase of the present invention according to the method (e.g., schedule) of the present invention, the covalently stabilized human α-galactosidase of the present invention may be provided as a unit dosage form formulated for intravenous administration or in the form of a pharmaceutical composition formulated for intravenous administration (infusion).

Thus, according to an aspect of the present invention, there is provided a unit dosage form containing 2.0-500 mg of the stabilized covalently linked human α-galactosidase of the present invention formulated for administration to a human subject. It should be understood that this range is for a minimum dose administered at intervals greater than 2 weeks up to 4 weeks up to a maximum dose administered at intervals greater than 2 weeks up to 4 weeks in patients of body weight from 10 to 250 kg.

In an embodiment, the unit dosage form contains 2.0-500 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 5.0-470 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 10.0-450 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 17.0-425 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 21.0-400 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 35.0-370 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 55.0-340 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 75.0-300 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 90.0-270 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 100.0-225 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 100.0-200 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 110.0-190 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 120.0-175 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 130.0-150 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 10.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 30.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 50.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 75.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 100.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 125.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 150.0 mg of a stabilized cross-linked plant recombinant human alpha-galactosidase protein formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 175.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 200.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 250.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 300.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 350.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 400.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 430.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 480.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

In an embodiment, the unit dosage form contains 500.0 mg of a stabilized covalently linked human α-galactosidase of the present invention, formulated for administration to a human subject.

The method of treating Fabry disease in a human subject according to the present invention may be provided as a stand-alone treatment for Fabry disease, or may be combined with additional treatment options, conventional or non-traditional therapies. Patients with Fabry disease suitable for treatment with the method or the covalently stabilized human α-galactosidase of the present invention may also receive before, during or after treatment with the method or protein of the present invention, for example, treatment with a non-specific ERT of a therapeutic such as, but not limited to: mygalstat hydrochloride (Chaperone Amicus Therapeutics), ibiglustat (INN) (glucosylceramide synthase (GCS) inhibitor) (Genzyme Corp), lucerastat (INN) - piperadine derivative (Actelion Ltd), NP-003 (inhibitor glycoprotein-1 (MDR-1 or ABCB1)) (Neuraltus Pharmaceuticals, Inc), SBLSD-4- (gene therapy (in vivo gene editing) - Sangamo BioSciences, Inc.), Genz-78132 (glucosylceramide synthase (GCS) inhibitor) ( Genzyme Corporation), miglustat (a glucosylceramide synthase (GCS) inhibitor from GlaxoSmithKline Plc and Actelion Ltd).

Fabry disease patients suitable for treatment with the method or covalently bound human α-galactosidase of the present invention may also be treated before or after treatment with the method or covalently bound human α-galactosidase of the present invention treated with ERT-related therapeutic agents and regimens such as, but not limited to, Fabrazyme® and Replagal® or pegunigalsidase alfa in a conventional regimen.

Suitable patient populations for treatment with the method or protein of the present invention include Fabry disease patients of all ages, both male and female, and Fabry disease patients with or without severe symptoms, slowly progressing patients, early diagnosed as well as patients with established Fabry disease, younger patients and patients with resistant Fabry disease.

As used herein, the term "resistant" Fabry disease patients refers to patients with Fabry disease who maintain levels of at least one of the Fabry disease parameters for a predetermined period of time and/or maintain a similar rate of deterioration of that parameter (e.g., cardiac, renal parameter [such as decrease in eGFR], pain parameter, gastrointestinal parameter and biomarkers [such as Gb3 and lyso-Gb3 in plasma or urine] and the like). In some embodiments, resistant Fabry patients may include patients from populations containing newly diagnosed Fabry disease patients with mild to moderate symptoms, previously untreated patients, and patients previously treated for Fabry disease. According to some embodiments, resistant patients with Fabry disease include patients who were resistant prior to treatment with the methods and/or compositions of the present invention.

The amount of the composition to be administered will, of course, depend on the subject being treated, the severity of the lesion, the route of administration, the judgment of the prescribing physician, and the like.

The compositions of the present invention may, if desired, be presented in a package or dispenser such as an FDA/EMA (US Food and Drug Administration/European Medicines Agency) approved kit, which may contain one or several single dosage forms containing the active ingredient. The package may, for example, comprise a metal or plastic foil such as, but not limited to, a blister pack or a sealed container. The package or dispenser may be accompanied by instructions for use. The package or dispenser may also be accompanied by a notice associated with the container in the form prescribed by the government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the form of the compositions for human or veterinary use. Such notice, for example, could be a label approved by the US Food and Drug Administration for prescription drugs, or an approved product insert, or an EMA, or any other regulatory body. Compositions containing a protein structure according to embodiments of the present invention, formulated in a compatible pharmaceutical carrier, can also be prepared, placed in an appropriate container and labeled for the treatment of a specified condition or diagnosis, as described in detail herein.

Thus, according to an embodiment of the present invention, depending on the covalently bound human α-galactosidase stabilized according to the present invention selected, the pharmaceutical composition described herein is packaged in a packaging material and identified in printed form, in or on the packaging material, for use in the treatment of a condition in which the activity of a cross-linked protein structure is useful, as described herein above.

In some embodiments, the packaging material, package, or dispenser, kit, or container may contain or be accompanied by instructions for administering the stabilized covalently linked human α-galactosidase, or compositions containing it, to a subject in need thereof. In some embodiments, instructions for administration may be included on a label affixed to containers and/or vials containing stabilized covalently linked human α-galactosidase or compositions containing it according to the present invention.

Instructions for use and administration may include, inter alia, specific indications (e.g., Fabry disease), instructions for preparing covalently bound human α-galactosidase or compositions containing it for administration, details for administering covalently stabilized α- human galactosidase or compositions of the present invention and protocol after administration, as well as details regarding dosage and regimen of treatment with covalently bound human α-galactosidase stabilized or compositions containing it of the present invention. In specific embodiments, the instructions may include instructions for intravenously administering the stabilized covalently linked human α-galactosidase of the present invention as an infusion at dosing intervals of greater than two weeks (14 days ± 3 days), once every three weeks. , or once every 4 weeks ±3 days (e.g. monthly). In other embodiments, the instructions include instructions for administration with a dosing interval of once every 4 weeks. In a further embodiment, the instructions include instructions for administering the stabilized covalently linked human α-galactosidase of the present invention at intervals of three weeks±3 days between administrations.

As used herein, the term "about" refers to ±10%.

The terms "comprises," "comprising," "includes," "including," "characterized," and their related words in root and meaning, mean "including without limitation."

The term "consisting of" means "including and limited thereto".

The word "illustrative" is used herein to mean "serving as an example or illustration". Any embodiment described as "illustrative" is not necessarily to be construed as being preferred or advantageous over other embodiments and/or precluding the inclusion of features from other embodiments.

The word "optional" is used herein to mean "provided in some embodiments and not provided in other embodiments." Any particular embodiment of the present invention may include a plurality of "optional" features, as long as such features do not conflict.

As used herein, the singular forms include reference to plural forms unless the context clearly dictates otherwise. For example, the term "compound" or "at least one compound" can include a variety of compounds, including mixtures thereof.

In the present application, various embodiments of the present invention may be presented in range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the present invention. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered as specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6 and so on, as well as individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

Whenever a numeric range is specified herein, it is intended to include any cited number (fractional or integer) within the specified range. The phrases "range/ranges between" the first specified number and the second specified number and "range/ranges from" the first specified number "to" the second specified number are used interchangeably herein and are intended to include the first and second specified numbers and all fractional and integers in between.

As used herein, the term "method" refers to methods, means, techniques, and procedures for accomplishing a given task, including, without limitation, those methods, means, techniques, and procedures that are known or that can be readily developed from known methods, means. , technician and procedure practitioners in the chemical, pharmacological, biological, biochemical and medical engineering fields.

As used herein, the term "treatment" includes reversing, substantially inhibiting, slowing or reversing the progression of a condition, substantially alleviating the clinical or aesthetic symptoms of a condition, or substantially preventing the onset of clinical or aesthetic symptoms of a condition.

It should be understood that certain features of the present invention, which are described in the context of separate embodiments for clarity, may also be provided in combination in one embodiment. Conversely, various features of the present invention, which for brevity are described in the context of one embodiment, may also be provided separately or in any suitable subcombination or, as the case may be, in any other described embodiment of the present invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment operates without those elements.

The various embodiments and aspects of the present invention described herein above and claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which, together with the above descriptions, illustrate certain embodiments of the present invention in a non-limiting manner.

Example I

Clinical Trial Protocol: Phase 1/2, Open-label Study to Determine Optimal Dosage to Evaluate Safety, Tolerability, Pharmacokinetics, and Exploratory Efficacy Parameters of Pegunihalsidase Alfa Administered by IV Infusion Every 2 Weeks in Adult Patients with Fabry Disease

This study evaluates the safety, tolerability, pharmacokinetics, and exploratory efficacy of plant recombinant human α-galactosidase cross-linked with bis-NHS-PEG ₄₅ (prh-alfa-GAL-I-CL45) (pegunigalsidase alfa) in a target population. Subjects must have had an accurate diagnosis of Fabry disease based on alpha-GAL-A activity (males) or genetic testing (females) with evidence of the disease, and must not have been treated with ERT in the last 6 months. The parameters selected as endpoints were those most relevant to the disease and allow for a meaningful and up-to-date evaluation of safety, pharmacokinetics, and efficacy endpoints. The study of the three dose levels provided important information about the three dose levels in terms of safety, tolerability and clinical outcome.

Selection of the study population

Inclusion Criteria:

1. Symptomatic adult patients with Fabry disease (≥18 years, male and female)

2. Men: plasma and/or leukocyte alpha-galactosidase activity (using activity assay) below the lower limit of normal (plasma LLN = 3.2 nmol/h/mL, leukocyte LLN = 32 nmol/h/mg/protein )

3. Women: results of retrospective genetic tests consistent with Fabry mutations

4. The concentration of globotriaosylceramide (Gb3) in the urine is more than 1.5 times the normal range

5. Patients who have never received enzyme replacement therapy (ERT) in the past, or patients who have not received ERT in the past 6 months and have a negative pegunigalsidase alfa antibody test

6. eGFR≥60 ml/min/1.73 ^m2

7. Signing informed consent

8. Female patients and male patients whose partners of childbearing age have agreed to use a medically acceptable method of contraception, not including the calendar method

Exclusion Criteria

The presence of any of the following excluded subject enrollment:

1. Participation in any investigational medicinal product trial within 30 days prior to the Screening phase of the study

2. Treatment with any of Fabrazyme® (agalsidase-beta), Replagal® (agalsidase-alpha), or any other Fabry disease investigational drug

3. Chronic renal failure stages 3-5 (CKD 3-5)

4. History of dialysis or kidney transplant

5. Angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) therapy started or dose changed 4 weeks prior to screening

6. Severe myocardial fibrosis based on MRI (≥2 positive left ventricular segments with late enhancement [LE]) (Weidemann et al. 2009)

7. History of clinical stroke

8. Pregnancy or breastfeeding

9. Presence of HIV and/or HBsAg and/or hepatitis C infections

10. Known Allergies to ERT

11. Known allergy to gadolinium-based contrast agents

12. The presence of any medical, emotional, behavioral, or psychological condition that, in the opinion of the Investigator and/or the Medical Director, could affect the patient's compliance with study requirements.

The introduction of drugs

All infusions were administered during hospitalization, with a post-infusion follow-up period with the possibility of outpatient monitoring where appropriate.

Three treatment groups comprised the study population. Treatment group I: 0.2 mg/kg every 2 weeks. Treatment group II: 1 mg/kg every 2 weeks. Treatment group III: 2 mg/kg every 2 weeks.

Subjects (4-8 patients per group) received intravenous infusions every 2 weeks (14 days).

Pharmacokinetic (PK) parameters

To determine the profile of the investigational drug based on the profile of plasma concentration versus time, the following PK parameters are obtained: C _max , t _1/2 , T _max , AUC _0-t and AUC _0-∞ . Samples were collected on day 1 of treatment (first infusion), at the infusion at the 3-month visit, at the infusion at the 6-month visit, and at the infusion at the 12-month visit. Blood for PK was taken at the following time points: before infusion (baseline); 1 hour after the start of the infusion; at the end of the infusion; 1, 4, 8, 24, 48±3, 72±3, 96±3 hours and 2 weeks ±3 days after infusion (the last blood sample was taken immediately before the patient's next infusion)

Efficiency variable(s)

Performance parameters were assessed for endpoint analysis as follows:

Концентрации Gb3 в плазме и осадке мочи на исходном уровне и при каждой инфузии во время исследования

Plasma and urinary sediment Gb3 concentrations at baseline and at each infusion during the study

Концентрация глоботриаозилсфингозина (лизо-Gb3) в плазме крови на исходном уровне и при каждой инфузии во время исследования

Plasma concentrations of globotriaosylsphingosine (lyso-Gb3) at baseline and with each infusion during the study

Оценка желудочно-кишечных симптомов на исходном уровне и при последней инфузии

Assessment of gastrointestinal symptoms at baseline and last infusion

Функции почек (рСКФ и протеинурия) на исходном уровне и при последней инфузии

Kidney function (eGFR and proteinuria) at baseline and last infusion

Краткая форма опросника оценки боли (BPI) на исходном уровне и при последней инфузии

Brief Form of the Pain Assessment Inventory (BPI) at Baseline and Last Infusion

The following additional procedures were additionally performed at baseline:

Биопсия почки для концентрации Gb3

Kidney biopsy for Gb3 concentration

Пункционная биопсия кожи для определения концентрации Gb3

Needle biopsy of the skin to determine the concentration of Gb3

МРТ сердца и мозга

MRI of the heart and brain

Индекс оценки степени тяжести Майнца (MSSI)

Mainz Severity Assessment Index (MSSI)

Оценка сердечной функции (эхокардиография и ЭКГ под нагрузкой)

Assessment of cardiac function (echocardiography and exercise ECG)

Security Variables

Safety was assessed by the frequency, severity, and duration of treatment-related AEs (adverse events), including clinically significant laboratory abnormalities, ECG changes from baseline, physical examination findings, and injection site evaluation following study drug administration.

Anti-pegunigalsidase alfa antibodies were assessed pre-dose: at baseline, every month, at the last infusion, and 1 and 3 months after the last infusion.

Clinical laboratory evaluation

The following clinical parameters were evaluated during administration of plant recombinant human α-galactosidase cross-linked with bis-NHS-PEG ₄₅ (prh-α-GAL-I-CL45, pegunigalsidase alfa):

Hematology: complete blood count; total leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, and basophils), total erythrocyte count (hemoglobin, hematocrit, mean erythrocyte volume, mean erythrocyte hemoglobin, and mean erythrocyte hemoglobin concentration) and platelets.

Coagulation profile: prothrombin time (PT) and partial thromboplastin time (PTT).

Biochemistry: sodium, potassium, glucose, blood urea nitrogen, creatinine, calcium, phosphate (inorganic), uric acid, total protein, albumin, bilirubin (total), alkaline phosphatase, aspartate transaminase, alanine transaminase, gamma-glutamyl transferase, lactate dehydrogenase and creatine phosphokinase.

Complete urinalysis: indicator strip for determining the content of blood, glucose, ketones and protein.

Antibodies to pegunihalsidase alfa

Anti-pegunigal sidase alpha antibodies were evaluated, including neutralizing antibodies in subjects with a positive IgG antibody response.

Adverse Events (AE) and Serious Adverse Events (SAE)

An adverse event (AE) is defined as any adverse medical event in a subject participating in a clinical trial, including any adverse and unintended event, symptom, or disease temporally associated with the use of pegunigalsidase alfa, whether or not considered to be related to the investigational drug. means. AEs were collected from the start of treatment until 90 days post-dose at the last visit. AE also includes accidental injury, reasons for any change in drug therapy (drug and/or dose) other than a scheduled dose adjustment, reason for hospitalization, or reason for surgical procedures (excluding minor elective surgery for a pre-existing condition).

Example II

Pegunigalsidase alfa is safe and effective in reducing the symptoms of Fabry disease

Patients were monitored for safety (adverse effects) and improvement in clinical parameters following intravenous administration of 0.2 mg/kg, 1.0 mg/kg and 2.0 mg/kg pegunigalsidase alfa. Safety data indicated that pegunigalsidase alfa was well tolerated, with a low incidence of adverse effects (AE) among 18 patients. Pegunigalsidase alfa was also effective in reducing plasma globotriaosylceramide and renal peritubular capillary globotriaosylceramide (Gb3).

A significant decrease in plasma Gb3 and lyso-Gb3 was also observed in male patients when monitored after 6 months of treatment with pegunigalsidase alfa. Kidney function (eGFR and urinary protein/creatinine) remained stable in all patients during 12 months of treatment with pegunigalsidase alfa. Thus, biweekly treatment with pegunigalsidase alfa is clearly safe and effective in patients with Fabry disease.

Example III

Pharmacokinetic profile of pegunihalsidase alfa:

Cross-linking enhances the bioavailability of pegunihalsidase alfa

Pharmacokinetic parameters were monitored in patients treated with 0.2 mg/kg, 1.0 mg/kg, and 2.0 mg/kg pegunigalsidase alfa on day 1 of treatment (first infusion), by intravenous infusion at visit through 3 months, by IV infusion at the 6-month visit and by IV infusion at the 12-month visit, based on plasma levels of pegunigalsidase alfa. Blood for PK was taken immediately before administration and for 14 days between doses.

Significant plasma enzyme concentration (measured by immunoassay using antibodies to pegunigalsidase alpha) was clearly detectable for all 14 days after administration for all dosages (see examples in Fig. 1) in a dose-dependent manner. At all doses, plasma concentrations peaked shortly after administration and showed a slow, steady decline over the next 14 days. In FIG. 2 shows the plasma concentration pharmacokinetic profile of clinically approved commercial agalsidase beta (r-αhGalA, recombinant human mammalian cell α-GAL A, Fabrazyme™, Genzyme Corp, Cambridge MA) after administration. In contrast to the pharmacokinetic profile of pegunigalsidase alfa, agalsidase beta, as can be seen from the graph, is barely detectable in patients' plasma 10 hours after administration. Thus, pegunigalsidase alfa appears to have a significantly improved bioavailability lasting beyond the 14 days shown herein and clearly superior to that of the clinically approved Fabrazyme™.

пегунигалсидазы альфа при каждой дозировке был почти на два порядка величин выше, чем у клинически одобренной, коммерчески доступной агалсидазы бета (Fabrazyme™, Genzyme Corp, Cambridge MA) и агалсидазы альфа (Replagal™, Shire Human Genetic Therapy (HGT), Inc., Cambridge, MA, USA) (фиг. 3B).When evaluating specific pharmacokinetic variables, the benefits of pegunihalsidase alfa become even more apparent. Both the maximum plasma concentration of the enzyme after administration (C _max , Fig. 3A) and the total bioavailability (area under the plasma concentration-time curve, extrapolated to infinity, AUC _0-∞ , expressed as mass (for example, μg or ng) of enzyme per time (e.g. min or h) per ml plasma, Figure 3C) is dose dependent and consistent with the overall pharmacokinetic profile shown in Figure 1. Compared to agalsidase beta, both C _max and AUC _0-∞ pegunihalsidases alpha are clearly superior to the commercially available enzyme. Half-life

pegunigalsidase alfa at each dosage was nearly two orders of magnitude higher than clinically approved, commercially available agalsidase beta (Fabrazyme™, Genzyme Corp, Cambridge MA) and agalsidase alfa (Replagal™, Shire Human Genetic Therapy (HGT), Inc., Cambridge, MA, USA) (Fig. 3B).

Table I below provides detailed pharmacokinetic parameters for pegunihalsidase alfa.

Extrapolation of plasma concentration 14 days after the 14 day period, taking into account the half-life of pegunihalsidase alfa (see Figure 3B), indicates that even at or after days 21 and 28, significant potential therapeutic amounts are expected to pegunigalsidases alfa remain in the plasma of patients.

Example IV

Immunogenicity and bioavailability of cross-linked prh-α-GAL-I-CL45:

Serum samples were collected once a month for the first 4 months and then once every 2 months. 144 samples were analyzed, of which 123 were negative for anti-drug antibodies (ADA). Confirmed positive samples were further characterized for neutralizing activity, anti-glycan and anti-PEG antibodies.

A low incidence of antibodies to cross-linked prh-α-GAL-I-CL45 has been reported. Three patients (19%) were identified as positive for treatment-induced ADA on at least one visit, with two of the three containing neutralizing antibodies. The presence of ADA was transient and none of these patients were found to have ADA after 6-15 months of treatment. Notably, after 12 months of treatment, none of the patients in the prh-α-GAL-I-CL45 cross-linked 2.0 mg/kg cohort developed treatment-induced antibodies to prh-α-GAL-I cross-linked -CL45.

Without being limited to one hypothesis, these results suggest that re-treatment with cross-linked prh-α-GAL-I-CL45, which due to its improved stability provides a permanent effect, may lead to the induction of immune tolerance.

Example V

Improved pharmacokinetics of cross-linked pegunigalsidase alfa and an extended dosing regimen: a multicenter, open-label, switchover study

Conventional enzyme replacement therapy for patients with Fabry disease requires administration of the drug at maximum intervals of two weeks (14 days) between administrations. Extending the interval between doses without significantly reducing the clinical efficacy of treatment or increasing treatment-related adverse events can be of great clinical importance, reducing the number of interventions and improving patient compliance. Given the enhanced pharmacokinetics observed in patients with Fabry disease receiving biweekly infusions of pegunigalsidase alfa, the effect of an extended dosing regimen can be assessed.

Patients may be selected for study according to a variety of inclusion and exclusion criteria, including but not limited to: treatment of patients with Fabry disease without severe symptoms, a relatively slowly progressing patient, patients with early diagnosis, patients with persistent symptoms of the disease or with a similar level deterioration over a selected period of time.

Illustrative study inclusion criteria include, but are not limited to, patients with confirmed Fabry disease, as determined, for example, by alpha-galactosidase activity and genetic tests, with mild to moderate symptoms, as determined, for example, by the slope of rCK (estimated reserve coronary flow) and eGFR of the patient, patients aged 16-65 years, at least 3 years on ERT.

Clinical laboratory evaluation, monitoring of safety and efficacy variables and adverse events, and recording of pharmacokinetic parameters are performed as detailed in Example I, with modifications necessary for extended dosing intervals (e.g., samples are taken during the first and last infusions at the following times time: before infusion (baseline), 1 hour after the start of the infusion, at the end of the infusion, after 1, 4, 8, 24, 48±3, 72±3, 96±3 hours, 2 weeks ±3 days and 3 weeks ±3 days post-infusion (last blood sample will be taken just before the patient's second infusion) Optionally, samples are also taken between the 2nd and 3rd weeks post-infusion and optionally at times greater than 3 weeks post-infusion infusion, for example, 4 (four) weeks after the infusion ± 3 days and / or in the interval between the 3rd and 4th weeks after the infusion.

Sustained therapeutic plasma levels of pegunigalsidase alfa in patients receiving a three-weekly or four-weekly dose, or any dosage interval greater than two weeks post-infusion, without significant adverse effects and with satisfactory or similar levels of clinical parameters, such as and in previous ERT treatment, clinical parameters throughout the treatment period may form the basis of a new improved extended dosing protocol for enzyme replacement therapy for Fabry disease with pegunihalsidase alfa. Optimal intervals between infusions can be determined (or approximated) from the analysis of clinical data and pharmacokinetic parameters.

Example VI

Predictive analysis for 2mg/kg pegunihalsidase alfa versus 1mg/kg Fabrazyme®:

A predictive analysis was performed to determine the possible pharmacokinetic characteristics of pegunigalsidase alfa when the dosing interval was extended from 2 weeks to 3 or 4 weeks, and to compare the predicted exposure of pegunigalsidase alfa to that of Fabrazyme.

Kinetic characteristics of cross-linked prh-α-GAL-I-CL45 (pegunigalsidase alfa)

Data from patients treated with 2 mg/kg pegunigalsidase alfa (n=4) shown in Example 1 were used for the bulk of the predictions for pegunigalsidase alfa. Predictions tend to be linearly proportional to dose. The starting point for these forecasts was 3-month data. Half-life values of 3 months ranged from 70.1 to 105 hours for four subjects.

Because there were measurable pre-dose concentrations at 3 months due to a previous dose of pegunihalsidase alfa, pre-dose concentrations were extrapolated to each sample time using the final elimination rate (λz) value for each subject. These extrapolated concentrations were subtracted from the measured concentrations to obtain concentrations that were due to the dose administered at 3 months. Adjusted 3-month data for each subject were matched to a 2-compartment model using Phoenix WinNonlin software. Mean scores for model variables were used to simulate three consecutive doses of 2 mg/kg at intervals of 2, 3, or 4 weeks. The hypothetical infusion time was 6 hours. The values of C _max , AUC _tau (area for time between doses) and C _min (concentration before the next dose) are shown in table II.

Fabrazyme pharmacokinetics

Fabrazyme pharmacokinetic parameters were obtained from the package insert and Eng et al., 2001 (Am J Hum Genet, 68:711-722), FIG. 2.

Partial AUC and _Cave

Table III shows a comparison of predicted pharmacokinetic parameters following various dosing regimens of pegunigalsidase alfa and fabrazyme at a dose of 1 mg/kg every 2 weeks.

Data for pegunihalsidase alfa were obtained from an ongoing phase I/II study. Information on the pharmacokinetic characteristics of Fabrazyme is available in the package insert and in Eng et al., 2001 (Am J Hum Genet, 68:711-722). Perspective modeling was performed using Phoenix WinNonlin software (version 6.3).

Weekly partial AUC and C _ave calculations allowed comparison/analysis of drug availability at weeks 1, 2, 3 and 4 of pegunigalsidase alfa administration and comparison with Fabrazyme every two weeks.

Data are calculated per week and presented sequentially. The thick lines represent the time of reintroduction. Thus, for the 2nd week interval, the 3rd, 5th, and 7th lines represent dosing, for the 3rd week interval, the 4th, 7th, and 10th lines represent dosing, and so on. ). Gray and white rectangles visually define different infusion intervals. Mean weekly enzyme concentrations, at 168 hours (1 week) and 336 hours (2 weeks), were interpolated or extrapolated to estimate weekly drug coverage. As shown in the table: for pegunigalsidase alfa, 2 mg/kg every 2, 3 or 4 weeks, the weekly C _ave values (709, 87 and 11 ng/mL, respectively) are significantly higher than the negligible C _ave values at week 2 for factory.

Fabrazyme C _ave levels 10 hours post-infusion are similar to predicted pegunigalsidase alfa C _ave levels at week 4 after 2 mg/kg every 4 weeks (11 ng/mL).

Figure 4A graphically illustrates the extrapolated enzyme availability (plasma concentration) over a four week period following a single infusion of 2 mg/kg pegunigalsidase alfa. When the predicted AUC following treatment with pegunigalsidase alfa over a four-week period is compared with this value of Fabrazyme administered biweekly over the same period, maintenance of enzyme availability for pegunigalsidase alfa is clearly superior to that of Fabrazyme (see Figure 4B, especially the insert).

The results show that, in contrast to Fabrazyme, significant levels of the enzyme are expected to remain in the circulation throughout the treatment period, available for reaching target organs, following administration of 2 mg/kg pegunigalsidase alfa at 3 and 4 week intervals between infusions. .

и существенно более высокая общая биодоступность (AUC_0-∞), наблюдаемые для всех дозировок пегунигалсидазы альфа, отражают присутствие доступного активного фермента в течение 2 недель между инфузиями (введениями). Улучшенный фармакокинетический профиль указывает на то, что она подходит для эффективной долгосрочной ферментозаместительной терапии при болезни Фабри и ее можно вводить с интервалами, составляющими по меньшей мере 2 недели - каждые 4 недели.Taken together, the results presented herein indicate that the pharmacokinetic properties of the cross-linked alpha-galactosidase pegunigalsidase alfa are different and superior to those of both mammalian cell-derived, currently commercially available ERTs for Fabry disease agalsidase beta. (Fabrazyme™, Genzyme Corp, Cambridge MA) and agalsidase alfa (Replagal™, Shire Human Genetic Therapy (HGT), Inc., Cambridge, MA, USA). Longer half-life

and significantly higher overall bioavailability (AUC _0-∞ ) observed for all dosages of pegunigalsidase alfa reflect the presence of available active enzyme within 2 weeks between infusions (introductions). The improved pharmacokinetic profile indicates that it is suitable for effective long-term enzyme replacement therapy in Fabry disease and can be administered at intervals of at least 2 weeks to every 4 weeks.

Example VII

Clinical Trial Protocol: Phase 3, Open-label Switch Study to Evaluate the Safety, Efficacy, and Pharmacokinetics of Pegunihalsidase Alfa by IV Infusion Every 4 Weeks in Adult Fabry Disease Patients Currently Receiving ERT: Fabrazyme® (agalsidase beta) or Replagal ® (agalsidase alfa)

This study evaluates the safety, efficacy, and pharmacokinetics of plant recombinant human α-galactosidase crosslinked with bis-NHS-PEG ₄₅ (prh-α-GAL-I-CL45) (pegunigalsidase alfa) in patients with Fabry disease who are are currently being treated with a commercially available ERT (agalsidase alfa or agalsidase beta). Subjects must have an accurate diagnosis of Fabry disease based on alpha-GAL-A activity (males) or genetic testing (females) with evidence of the disease, and be treated with ERT for at least 3 years. Plant recombinant human α-galactosidase cross-linked with bis-NHS-PEG ₄₅ (prh-α-GAL-I-CL45) (pegunigalsidase alfa) will be administered at a dose of 2 mg/kg every four (4) weeks, within 52 weeks. The parameters selected as endpoints are the parameters most relevant to the disease and allow meaningful and up-to-date evaluation of safety, pharmacokinetics, and efficacy endpoints.

Dosage and administration of the drug:

Once a patient has been determined to be eligible to participate in the study, patients should be switched to pegunigalsidase alfa 2 mg/kg administered intravenously every 4 weeks. The timing of pegunigalsidase alfa infusion varies according to the patient's body weight, taking into account: patient tolerance, investigator judgment and approval of the sponsor's/MD's medical supervisor, and after attempting a gradual dose reduction.

Pharmacokinetic (PK) parameters

The following PK parameters are derived from plasma concentration-time profiles to determine the PK profile of the study drug: C _max , t _1/2 , T _max , AUC _0-t and AUC _0-∞ . Blood for PK analysis will be taken on day 1 and at the end of treatment (week 52). On blood draw days, blood samples are taken at the following time points: before infusion (baseline); 1 hour after the start of the infusion; at the end of the infusion; 1±0.25, 2±0.25, 4±0.25, 8±0.25, 24±0.5, 48±3 and 96±3 hours after infusion and after 14±3, 21±3 and 28±3 days post-infusion (last blood sample will be taken just before the patient's next infusion) (total 13 time points over 28 days).

Efficiency variable(s)

The performance parameters assessed for the endpoint analysis are as follows:

1. Estimated glomerular filtration rate (eGFRCKD-EPI)

2. Mass index of the left ventricle (g/m ² ) by echocardiogram

3. Plasma concentration of globotriaosylsphingosine (lyso-Gb3)

4. Plasma Gb3 concentrations

5. Lyso-Gb3 in urine

6. The ratio of protein / creatinine in a single portion of urine

7. Frequency of use of pain medications

8. Exercise tolerance (ECG under stress)

9. Brief Form of the Pain Assessment Inventory (BPI)

10. Mainz Severity Index (MSSI)

11. Quality of Life Questionnaire EQ-5D-5L

Security Variables

Safety is assessed by changes from baseline: clinical laboratory tests, physical examination, assessment of the injection site after drug administration, electrocardiogram (ECG), frequency, severity and duration of AEs (adverse events) occurring during treatment, ability to reduce premedication before infusion , the need for the use of premedication in general to monitor infusion reactions and treatment-induced anti-pegunigalsidase alfa antibodies.

Previous results indicated a longer half-life (t _1/2 ) and a significantly greater overall bioavailability (AUC _0-∞ ) for pegunigalsidase alfa at all doses in a biweekly infusion schedule (administration). Extended dosing intervals can have a significant impact on convenience, cost, compliance, and adverse effects of ERT for Fabry disease. To assess the suitability of plant recombinant human α-galactosidase cross-linked with bis-NHS-PEG ₄₅ (prh-α-GAL-I-CL45) (pegunigalsidase alfa) for effective long-term enzyme replacement therapy in Fabry disease, pegunigalsidase alfa is administered at intervals every 4 weeks.

In an open-label, switchover study evaluating the safety, efficacy, and pharmacokinetics of treatment with pegunigalsidase alfa 2 mg/kg every 4 weeks in patients previously treated with ERT, agalsidase alfa, or agalsidase beta for at least 3 years and at a stable dose (>80% of labeled dose/kg) for at least the past 6 months, will be enrolled in the trial and switched from their current ERT to intravenous (IV) infusions of pegunigalsidase 2 mg/kg every 4 weeks for 52 weeks (total 14 infusions).

Substantial evidence for the retention of improved pharmacokinetic properties of cross-linked alpha-galactosidase pegunigalsidase alfa over the 4-week infusion interval would help establish that cross-linked alpha-galactosidase pegunigalsidase alfa is different and superior to other currently approved, commercially available ERTs for disease Fabry agalsidase beta (Fabrazyme™, Genzyme Corp, Cambridge MA) and agalsidase alfa (Replagal™, Shire Human Genetic Therapies (HGT), Inc., Cambridge, MA, USA).

Although the invention has been described in connection with its specific embodiments, it is obvious that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims are intended to be covered.

All publications, patents, and patent applications referred to in this specification are incorporated herein in their entirety by reference in this specification to the same extent as if each individual publication, patent, or patent application were specifically and individually incorporated in this document by reference. In addition, the citation or indication of any reference in this application should not be construed as an admission that such a reference is available as prior art for the present invention. To the extent that section headings are used, they should not be considered necessarily limiting.

Claims (17)

1. Use of a unit dosage form in the treatment of Fabry disease, wherein the unit dosage form contains a plant recombinant human α-galactosidase cross-linked with bis-NHS-PEG45, wherein the monomers of said recombinant human α-galactosidase are covalently linked to each other via a poly(alkylene ) a glycol binder moiety containing at least 20 alkylene groups, said unit dosage form being formulated as an intravenous liquid and the administration being provided at a dosage of 2 mg/kg at 4 week intervals. 2. Use according to claim 1, wherein said plant recombinant human α-galactosidase comprises a human α-galactosidase protein having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-3. 3. Use according to claim 1 or 2, containing 20 or 30 or 40 or 50 or 75 or 100 or 125 or 150 or 175 or 200 or 250 mg of said plant recombinant human α-galactosidase . 4. Application according to any one of paragraphs. 1-3 containing 20 mg of said plant recombinant human α-galactosidase. 5. Application according to any one of paragraphs. 1-4 containing 30 mg of said plant recombinant human α-galactosidase. 6. Application according to any one of paragraphs. 1-4 containing 40 mg of said plant recombinant human α-galactosidase. 7. Application according to any one of paragraphs. 1-4 containing 50 mg of said plant recombinant human α-galactosidase. 8. Application according to any one of paragraphs. 1-4 containing 75 mg of said plant recombinant human α-galactosidase. 9. Application according to any one of paragraphs. 1-4 containing 100 mg of said plant recombinant human α-galactosidase. 10. Application according to any one of paragraphs. 1-4 containing 125 mg of said plant recombinant human α-galactosidase. 11. Application according to any one of paragraphs. 1-4 containing 150 mg of said plant recombinant human α-galactosidase. 12. Application according to any one of paragraphs. 1-4 containing 175 mg of said plant recombinant human α-galactosidase. 13. Application according to any one of paragraphs. 1-4 containing 200 mg of said plant recombinant human α-galactosidase. 14. Application according to any one of paragraphs. 1-4 containing 250 mg of said plant recombinant human α-galactosidase. 15. Application according to any one of paragraphs. 1-14, said recombinant human α-galactosidase having a circulating half-life (T1/2) of at least 50 hours after administration. 16. Application according to any one of paragraphs. 1-15, wherein said recombinant human α-galactosidase has a Cmax of at least 8000 ng/mL after administration of 2 mg/kg. 17. Application according to any one of paragraphs. 1-16, wherein said recombinant human α-galactosidase has a bioavailability (AUC0-∞) of at least 400,000 ng h/mL following administration of 2.0 mg/kg of said recombinant human α-galactosidase.

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