Liposomes and its use for enzyme delivery

1 . A liposome comprising

a) a phospholipid which is dipalmitoylphosphatidylcholine (DPPC);

b) cholesterol (chol);

c) a conjugate comprising a cholesterol moiety, a polyethylene glycol (PEG) moiety and a peptide moiety comprising a RGD sequence, wherein the PEG moiety is covalently attached to the cholesterol moiety by one end via a bond of the type alkyl ether and is covalently attached to the peptide moiety comprising the RGD sequence by the other end:

d) myristalkonium chloride (MKC) present in a percentage of between 0.4 and 4.3% mol with respect to the total mol of the components of the liposome a), b), c) and d); and

e) alpha-galactosidase enzyme (GLA) present in a ratio of micrograms of GLA in respect to the total milligrams of the components of the liposome a), b), c) and d) of between and including 2 μg/mg to 35 μg/mg.

2 . The liposome according to claim 1 wherein, in the alpha-galactosidase enzyme, each of its monomers is either of sequence SEQ ID NO: 1, or of sequence with at least 85% of sequence identity with SEQ ID NO: 1.

3 . The liposome according to claim 1 , wherein the alpha-galactosidase enzyme is obtained from a Chinese Hamster Ovary (CHO) cell culture.

4 . The liposome according to claim 1 , wherein the PEG moiety has a molecular weight from 50 to 600 Daltons.

5 . The liposome according to claim 1 , wherein the PEG, moiety has a molecular weight of 400 Daltons, the RGD sequence is SEQ ID NO: 2, the ratio of micrograms of GLA to milligrams of the components of liposome a), b), c), and d) is between 20-30 μg/mg, wherein, in the GLA, each of its monomers is either of sequence SEQ ID NO: 1, or of sequence with at least 85% of sequence identity with SEQ ID NO: 1, and the mol ratio DPPC:chol:chol-PEG-RGD is 10:6.5:0.5.

6 . The liposome according to claim 4 , wherein the PEG moiety has a molecular weight from 200 to 400 Daltons.

7 . A pharmaceutical composition comprising a therapeutically effective amount of liposomes as defined in claim 1 , together with pharmaceutically acceptable excipient(s), carriers, or vehicles.

8 . The pharmaceutical composition according to claim 7 further comprising glucose, sucrose or trehalose in an amount from 2 to 10% in weight with respect to the total volume of the composition.

9 . The pharmaceutical composition according to claim 7 , wherein GLA is present in an amount of at least 0.2 mg per mL of the pharmaceutical composition.

10 . A process for the production of a liposome according to claim 1 comprising the following steps:

a) preparing an aqueous solution which comprises the GLA enzyme;

b) preparing a solution comprising the conjugate, cholesterol and a phospholipid dissolved in an organic solvent;

c) adding MKC, either to the solution of the step a), or to the solution of the step b);

d) expanding the solution of step b) with a compressed fluid, wherein, when the MKC is added to the solution of step b), the expanding step occurs after the MKC has been added;

e) depressurizing the expanded solution resulting from the step d) over the resulting solution of the step a); and

f) diafiltrating and concentrating, in any order, the resulting solution obtained in step e).

11 . A method for the treatment of Fabry disease, which comprises administering the liposome according to claim 1 to a subject in need thereof.

12 . The method according to claim 11 , wherein the liposome is administered to a subject suffering Fabry disease as a single dose; or, alternatively, repeatedly at least once every two weeks.

13 . A method for the treatment of Fabry disease, which comprises administering the pharmaceutical composition according to claim 7 to a subject in need thereof.

14 . The method according to claim 13 , wherein the pharmaceutical composition is administered to a subject suffering Fabry disease as a single dose; or, alternatively, repeatedly at least once every two weeks.