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 erythrocytes loaded with one or more substances of pharmaceutical interest comprising the following steps:
(A) preparing intact erythrocytes for permeabilization and loading with one or more substances of pharmaceutical interest, said preparing comprising:
(a) swelling intact erythrocytes using a first hypotonic solution, wherein said first hypotonic solution brings the intact erythrocytes to an osmolality between 250-200 mOsm/Kg and prepares the intact erythrocytes for further swelling;
(b) further swelling the intact erythrocytes obtained in step (a), without reaching lysis, using a second hypotonic solution more hypotonic than the first hypotonic solution, wherein said second hypotonic solution brings the intact erythrocytes to an osmolality between 200 and 170 mOsm/Kg; and
(c) concentrating the intact erythrocytes obtained in step (b) to provide concentrated, intact erythrocytes prepared for permeabilization and loading with the one or more substances of pharmaceutical interest;
(B) concurrently (i) permeabilizing the concentrated, intact erythrocytes of step (c) and (ii) diffusing one or more substances of pharmaceutical interest into the erythrocytes thus permeabilized by placing the concentrated, intact erythrocytes obtained in step (c) in contact with a lysing solution comprising the one or more substances of pharmaceutical interest; and subsequently
(C) adding a sealing solution to seal the one or more substances of pharmaceutical interest within the erythrocytes.
2. The process according to claim 1 , which 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 according to claim 1 , wherein said concentration step (c) is carried out by hemofiltration, haemodialysis or centrifugation of the intact erythrocytes obtained in step (b).
4. The process according to claim 1 wherein the lysing solution in step (B) brings the erythrocytes to an osmolality between 150 and 110 mOsm/Kg.
5. The process according to claim 1 , wherein the sealing solution in step (C) is a hypertonic solution from 300 to 5000 mOsm/kg.
6. The process according to claim 1 , said 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.
7. The process according to claim 1 , wherein said 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, betamethasone, betamethasone phosphate, deflazacort, thymidine phosphorylase, phenylalanine ammonia lyase, and indocyanine green.
8. The process according to claim 6 , wherein said nanoparticles are selected from the group consisting of: metal-containing nanoparticles, magnetic nanoparticles, and complex nanoparticle-active molecules.
9. The process according to claim 6 , wherein said particles comprise super-paramagnetic particles (SPIO).
10. The process according to claim 3 , wherein said hemofiltration obtains a concentration of erythrocytes (hematocrit) above 30%.
11. The process according to claim 1 , wherein step (a) is carried out maintaining the erythrocytes at a concentration (hematocrit) of 3-7%.
12. The process according to claim 1 , wherein step (B) 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.
13. The process according to claim 1 , wherein step (C) is carried out maintaining the erythrocytes at a concentration (hematocrit) of 15-40%.
14. The process according to 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.
15. The process according to claim 1 , wherein the erythrocytes having the one or more substances of pharmaceutical interest have an average amount of lactate produced for every 10 6 erythrocytes of greater than 0.100 nmol/h.