PROCESS FOR THE PREPARATION OF ERYTHROCYTES LOADED WITH ONE OR MORE SUBSTANCES OF PHARMACEUTICAL INTEREST AND SO OBTAINED ERYTHROCYTES
The present invention relates to a process for preparing erythrocytes loaded with one or more substance of pharmaceutical interest. The present invention is also directed to loaded erythrocytes thus obtained and pharmaceutical compositions comprising said population of loaded erythrocytes as well as therapeutic application thereof, in particular in the treatment of Ataxia telangiectasia.
1 . A process for preparing a population of erythrocytes loaded with one or more substances of pharmaceutical interest comprising:
(a) swelling intact erythrocytes using a first hypotonic solution;
(b) further swelling the intact erythrocytes obtained in step (a), with a second hypotonic solution that is more hypotonic that the first hypotonic solution, wherein the further swelling is done without reaching lysis;
(c) concentrating the intact erythrocytes obtained in step (b),
(d) placing the concentrated erythrocytes in contact with a lysing solution comprising one or more substances of pharmaceutical interest;
(e) adding a sealing solution to the erythrocytes of step (d) to seal the one or more substances of pharmaceutical interest within the erythrocytes; and
thereby obtaining the population of erythrocytes loaded with the one or more substances of pharmaceutical interest.
2 . The process of claim 1 , which further comprises between steps (a) and (b), an additional step wherein at least part of the first hypotonic solution is removed before the addition of the second hypotonic solution.
3 . The process of claim 1 , wherein the concentration step (c) is carried out by a method comprising hemofiltration, haemodialysis or centrifugation of the intact erythrocytes obtained in step (b).
4 . The process of claim 1 , wherein the first hypotonic solution brings the intact erythrocytes to an osmolality between 230-150 mOsm/Kg.
5 . The process of claim 1 , wherein the first hypotonic solution brings the intact erythrocytes to an osmolality between 250-200 mOsm/Kg.
6 . The process of claim 1 , wherein the second hypotonic solution brings the intact erythrocytes to an osmolality between 200 and 170 mOsm/Kg.
7 . The process of claim 1 , wherein the lysing solution in step (d) brings the erythrocytes to an osmolality between 150 and 110 mOsm/Kg.
8 . The process of claim 1 , wherein the sealing solution in step (e) is a hypertonic solution from 300 to 5000 mOsm/kg.
9 . The process of claim 1 , the one or more substances of pharmaceutical interest are selected from the group consisting of: peptides, oligopeptides, polypeptides, proteins, oligonucleotides, nucleotide analogues, nucleosides, nucleoside analogues, hormones, immunosuppressant drugs, anti-tumor drugs, corticosteroids, glucocorticoids, anti-retroviral anti-inflammatory drugs, cytokines, toxins, substances with immunization activities, contrast media for diagnosis, particles, and nanoparticles.
10 . The process of claim 9 , wherein the nanoparticles are selected from the group consisting of: metal-containing nanoparticles, magnetic nanoparticles, and complex nanoparticle-active molecules.
11 . The process of claim 9 , wherein the particles comprise super-paramagnetic particles (SPIO).
12 . The process of claim 1 , wherein the one or more substances of pharmaceutical interest are selected from the group consisting of: 6-mercaptopurine, fludarabine phosphate, azidothymidine phosphate, dideoxycytidine, deoxyadenosine, glutathione, bisphosphonates, prednisolone, prednisolone phosphate, dexamethasone, dexamethasone phosphate, dexamethasone sodium phosphate, betamethasone, betamethasone phosphate, deflazacort, thymidine phosphorylase, phenylalanine ammonia lyase, and indocyanine green.
13 . The process of claim 1 , wherein the loaded population of erythrocytes encapsulate between 5 mg and 50 mg dexamethasone sodium phosphate.
14 . The process of claim 13 , wherein the loaded population of erythrocytes encapsulate between 9.9 mg and 29.4 mg dexamethasone sodium phosphate.
15 . The process of claim 3 , wherein the hemofiltration obtains a concentration of erythrocytes (hematocrit) above 30%.
16 . The process of claim 1 , wherein step (a) is carried out maintaining the erythrocytes at a concentration (hematocrit) of 3-7%.
17 . The process of claim 1 , wherein step (d) is carried out maintaining the erythrocytes at a concentration (hematocrit) of 30-65% in contact with the lysis solution containing the one or more substances of pharmaceutical interest.
18 . The process of claim 1 , wherein step (e) is carried out maintaining the erythrocytes at a concentration (hematocrit) of 15-40%.
19 . The process of claim 1 , wherein the erythrocytes having the one or more substances of pharmaceutical interest have an average percentage of phosphatidylserine exposure below 10% as measured with an annexin V assay.
20 . The process of claim 1 , wherein the erythrocytes having the one or more substances of pharmaceutical interest have an average amount of lactate produced for every 106 erythrocytes of greater than 0.100 nmol/h.