ITRM20130280A1 - PROCEDURE FOR THE PREPARATION OF ERYTHROCYTES LOADED WITH ONE OR MORE SUBSTANCES OF PHARMACEUTICAL INTEREST. - Google Patents

Description

Procedure for the preparation of erythrocytes loaded with one or more substances of pharmaceutical interest.

DESCRIPTION

The present invention is directed to a process for preparing erythrocytes loaded with one or more active principles, to the population of loaded erythrocytes thus obtained and to pharmaceutical compositions comprising said population of loaded erythrocytes.

STATE OF THE PRIOR ART

Red blood cells also known as erythrocytes are described in the state of the art as pharmaceutical carriers capable of transporting and releasing into the circulation and / or efficiently directing active ingredients to the target sites of interest. The advantage in using erythrocytes as pharmaceutical carriers lies mainly in the fact that the active ingredient can be kept in circulation for periods of days or weeks and in any case for much longer periods than is normally the case using oral, intravenous formulations or delivery systems. prolonged mediated by liposomes or other pharmaceutical carriers. Furthermore, these vehicles, once they have carried out their function of transporting the active principle, are removed from the circulation according to the physiological route of elimination of native red blood cells in the liver and spleen.

Numerous procedures have been proposed to encapsulate active ingredients or contrast media in human or other mammalian red blood cells for biomedical and clinical purposes.

In particular, in the patent EP0882448 a process for encapsulating medicaments in erythrocytes in concentrations sufficient to allow the desired pharmacological effect to be obtained was described for the first time. The process described in the above mentioned prior patent comprises a series of operational steps which can be summarized as follows:

- a first step in which the erythrocytes are swollen;

- a second step in which the swollen erythrocytes are lysed to allow the opening on the membrane of said erythrocytes with sufficiently large pores to allow the entry into the intracellular space of the active principles of interest;

- a concentration step of the lysed erythrocytes

- an encapsulation step in which the erythrocytes are put in contact with the active ingredients, followed by a step of closing / resealing the erythrocytes, which has the purpose of trapping the active ingredients in the red blood cells themselves.

The procedure just described has made it possible to obtain erythrocytes loaded with active ingredients and suitable for use as carriers for medicaments. Currently, therefore, the most effective reference method for encapsulating drugs in red blood cells remains, for those skilled in the art, the one described above.

In using this procedure, however, it was observed that in the concentration transition of the lysed erythrocytes under the operating conditions defined in the above patent, the lysed erythrocytes are subjected to mechanical stress which can make the subsequent step of reconstitution of the loaded erythrocytes difficult.

Among the various known techniques for encapsulating active substances in erythrocytes, the one described by the patent EP1773452-B1 provides for a correction of the process parameters such as the change in the flow of the lysing solution and the adjustment of its osmolality, in order to obtain reproducibility in the encapsulation of the active substance as the patient's Osmotic Fragility (or osmotic globular resistance) varies.

The purpose of the present invention is to further improve the results, which are already satisfactory, achieved with the process described in EP 0882448 in order to obtain an improved process for encapsulating substances of pharmaceutical interest in the erythrocytes.

SUMMARY OF THE INVENTION

The present application refers to a process for the preparation of erythrocytes loaded with one or more substances of pharmaceutical interest which, compared to the analogous process described in the state of the known art, is improved in various aspects.

In particular, this procedure comprises a series of operative steps which are characterized by the fact that the erythrocyte concentration step is performed before the cell lysis step necessary to allow the encapsulation of pharmaceutically active molecules in the red blood cells. Precisely this lysis step is performed during the contact phase with a solution comprising the substance to be encapsulated.

The procedure of the invention provides that the starting erythrocytes are subjected to two successive passages of cellular swelling, without lysis, using suitable hypotonic solutions, passages which, in fact, replace the swelling and lysis passage according to the analogue process of the prior art.

Therefore, the object of the present invention is a process for preparing erythrocytes loaded with one or more substances of pharmaceutical interest, for example active principles, comprising steps in which:

a) the erythrocytes swell using a first hypotonic solution;

b) further swell, without reaching lysis, the erythrocytes obtained in step a) using a second hypotonic solution, said second solution being more hypotonic than said first solution;

c) the erythrocytes obtained in step b) are concentrated;

d) the erythrocytes thus concentrated are placed in contact with a lysing solution comprising one or more substances of pharmaceutical interest and subsequently e) a sealing solution is added in order to obtain a population of erythrocytes loaded with said and said substances of pharmaceutical interest . The procedure can advantageously include an intermediate step (a2) between steps (a) and (b) in which the first hypotonic solution is removed, at least in part, before adding the second hypotonic solution.

Further objects of the invention are the population of erythrocytes loaded with one or more active principles obtainable through the above process and the pharmaceutical compositions comprising a population of erythrocytes loaded as defined above.

The invention is based on the surprising discovery that it is possible to encapsulate active ingredients in erythrocytes subjected only to swelling and concentration of the same without previous induction of hemolysis. In fact, the opening of the pores (hemolysis) can be effectively obtained, after concentration, with the same solution containing the substances of interest. The new procedure is much more effective than the previous methods and generates a final product (erythrocytes containing at least one pharmaceutically active substance) very similar to native erythrocytes (not subjected to the procedure).

Advantages offered by the invention

The operational variations introduced in the new procedure allow both to better preserve the plasma membrane of red blood cells and to concentrate them more in order to make the encapsulation efficiency much higher.

In the process of the invention, the encapsulation of the substances of interest takes place with a better yield than the process described in the state of the known art. In particular, the inventors of the present process have shown that in order to reach the same levels of encapsulated active principle it is possible to use, in the process of the invention, a significantly lower quantity of starting active principle than that currently used with the process. standard. In particular, as also reported in the experimental section, to obtain an encapsulation of up to about 11 mg, for example of dexamethasone sodium phosphate, for the same amount of red blood cells subjected to the treatment, about 500 mg of starting drug are required with the procedure of the prior art and only 62.5 mg with the procedure described here.

Furthermore, this procedure has proved to be reproducible and reliable in encapsulating quantities of the substance of interest in the red blood cells in proportion to the initial quantity of the same substance: these characteristics allow the clinical use of different doses, allowing the administration of doses commensurate with the different clinical needs, varying only the amount of active ingredient added during the procedure.

The greater encapsulation efficiency has been demonstrated not only with active molecules having low molecular weights (for example dexamethasone sodium phosphate as shown in example 1) but also with molecules having high molecular weights. As reported in example 5, proteins having molecular weights of the order of 110 kDa (dimers of Hk hexokinase of yeast of 55 kDa) or of the order of 60 kDa (thymidine phosphorylase) have been effectively encapsulated.

Thanks to the modified sequence of the operating phases, important improvements have also been obtained in the physiological parameters relating to the population of erythrocytes loaded and obtained with the process of the invention. In particular, as shown in the examples, the erythrocytes loaded with the procedure described here show parameters such as mean cell volume and cell viability (metabolism) which are completely comparable to those of untreated erythrocytes. Overall, the experimental data show how the new procedure is able to better preserve the cell size and cellular content of the starting erythrocytes compared to the prior art procedure, allowing, in fact, to obtain a significantly more similar population of loaded erythrocytes from the physiological point of view to an untreated erythrocyte population.

Comparative experiments conducted among the population of erythrocytes loaded according to the invention or with the analogous procedure of the prior art have shown, for example, that the mean cell volume (MCV) of red blood cells is respectively about 86 femtoliters (present invention) and about 71 femtoliters (prior art) where the MCV value for untreated erythrocytes is between 80-97 femtoliters. In addition, the amount of mean corpuscular hemoglobin (MCH) measured in the red blood cells subjected to the procedure in question and that described in the state of the art was found to be respectively about 21.2 picograms (much closer to the norm) and about 14 picograms (prior art) with a value, for untreated erythrocytes, which normally varies between 27.6-33.3.

The improved overlap between erythrocytes loaded with this procedure and untreated erythrocytes is also confirmed by the increased cellular viability (metabolism) observed. In particular, as will be better described later, the viability of the erythrocytes treated according to the present invention is significantly better both in terms of increased metabolic capacity and in terms of reduced presence of senescence markers. As discussed in more detail below, the data relating to greater cell viability (greater metabolism and fewer senescence markers) allow us to state that the procedure described here is, in fact, capable of producing a population of charged erythrocytes having a ™ predictably longer half-life compared to erythrocytes obtained with the anterior art procedure. It follows that the erythrocyte population according to the invention allows to obtain the transport of the encapsulated substances and / or their release for a longer period of time than that allowed by the charged erythrocytes obtained according to the procedure described in the known art.

The present invention, thanks to the technical measures described, also allows to overcome the limits of the method described in EP1773452-B1 and to obtain an encapsulation of active substance that can be reproduced without correction of the process parameters for each individual patient, since the process is independent both by the different osmotic fragility of the patient's red blood cells (as shown in example 7) and by the initial hematocrit (as shown in example 8).

Description of the Figures

Figure 1: Figure 1 is a schematic representation of the process described here compared with the process of the prior art (EP0882448).

Figure 2. Figure 2 is a graph relating to the osmotic globular resistance (RGO) for two individuals, evaluated by measuring the total free hemoglobin as a function of osmolality. RGO is also expressed as osmolality at which 50% hemolysis or 50% free hemoglobin is observed.

Figure 3: representation of a kit suitable for carrying out the process of the invention when used in conjunction with a medical device as described in BO2010A000255.

DETAILED DESCRIPTION OF THE INVENTION

GLOSSARY

Some terms typical of the technical sector are clarified below.

For the purposes of the present invention, the expression `` erythrocyte swelling '' means an increase in the volume and sphericity of the erythrocytes due to an increase in internal pressure caused by the entry mainly of water, however without abnormal opening of the pores on the cell membrane or irreversible rupture of the same. Normally the swelling, in the understanding of the present patent, does not imply the leakage of cellular material.

For the purposes of the present invention, and in the specific technical field, with the terms â € œlysisâ € or â € œhaemolysisâ € or â € œpartial lysisâ € means the reversible opening of the pores on the cell membrane with consequent free passage in both directions of intra- and extracellular materials. Lysis, therefore, is a phenomenon of temporary and reversible permeabilization and not of total irreversible rupture of the cell membrane.

It follows that the expression â € œleysate erythrocyteâ € refers to an erythrocyte that has pores on the plasma membrane that can be closed in such a way that the integrity of the cell membrane is restored.

For the purposes of this description, the expression â € œsealing solutionâ € means a solution used capable of allowing the closure of the pores present on the plasma membrane of the erythrocytes mainly through the leakage of water. This solution allows the substance or substances of pharmaceutical interest to be encapsulated inside the erythrocytes thanks to the opening of said pores.

The expression â € œloaded erythrocytesâ € means in the present description erythrocytes (also referred to as red blood cells) which encapsulate within them variable quantities of one or more substances of interest.

For the purposes of the present invention, the expression â € œsealed erythrocytesâ € refers to red blood cells which, unlike the lysed erythrocyte, have a plasma membrane permeability comparable (superimposable) to that of untreated red blood cells.

To carry out the process of the invention, the starting erythrocytes can be obtained by taking and isolating red blood cells from a blood sample of an individual. Preferably the starting sample is treated with an anti-coagulant agent, for example heparin, in order to avoid coagulation.

Optionally, the erythrocytes, before being subjected to the treatment according to the invention, can be isolated and subjected to one or more washes with physiological solution in order to obtain a starting erythrocyte population in which they are not present, or are present in negligible concentrations, any contaminants, for example, plasma, platelets, lymphocytes, etc.

Step (a):

As indicated above, the procedure comprises a step a) in which the erythrocyte population is initially swollen by using a first hypotonic solution.

In one embodiment of the invention, the first hypotonic solution has an osmolality between 230-150 mOsm / Kg, for example a preferred osmolality of 180 mOsm / Kg. In any case, the osmolality and volume of the first solution are such that contact with this first solution brings the red blood cells to an osmolality included in the range from 250 to 200 mOsm / Kg. In particular, the first hypotonic solution can be obtained, for example, by mixing 5 volumes of physiological saline solution and 3 volumes of sterile distilled water. For purely illustrative and non-limiting purposes, step (a) can be performed by keeping the erythrocytes in about 300 mL of the first solution at a concentration (hematocrit) of about 3-7%, for about 5 minutes at room temperature. .

Step a) can optionally be followed by a step of removal, at least in part, of the first hypotonic solution from the swollen erythrocytes. Such removal can be achieved, for example, by gentle centrifugation of the treated erythrocytes and separation of the supernatant.

Step (b):

The swollen erythrocytes obtained as indicated above are subsequently subjected to further swelling through the use of a second hypotonic solution (step b). The second solution is characterized by being more hypotonic than the first solution. The tonicity of the second solution is chosen in such a way as to cause further swelling of the erythrocytes, without however provoking their lysis which would cause consequent leakage of intracellular material. The conditions of hypotonicity are controlled in such a way as to avoid the induction of an excessive cellular fragility in view of the subsequent passage of erythrocyte concentration. The osmolality values of the second hypotonic solution are experimentally determined in the laboratory and are constant in the procedure. The osmolality of the second solution is such as to bring the red blood cells into a state of swelling without however reaching the opening of pores on their surface, opening which would cause the initial leakage of cellular content and an excessive fragility of the erythrocytes.

The second hypotonic solution has an osmolality in the range from 80 to 170 mOsm / Kg. In a preferred embodiment, the osmolality of the second solution is about 120 mOsm / Kg. In any case, the osmolality and volume of the second solution are such that contact with this second solution brings the red blood cells to an osmolality included in the range from 200 to 170 mOsm / Kg.

The second hypotonic solution can be obtained, for example, by mixing 5 volumes of physiological saline and 7 volumes of sterile distilled water.

For purely illustrative purposes, step (b) is performed by maintaining the erythrocytes in about 64 mL of the second solution at a concentration (hematocrit) of about 8-15%, for about 5 minutes and at room temperature.

Step (c):

The swollen erythrocytes resulting from steps a) and b) above are then subjected to a concentration step c). Any known technology suitable for concentrating a sample of red blood cells, for example haemofiltration, centrifugation or dialysis, can be used to concentrate swollen red blood cells. In a preferred embodiment of the invention, the concentration is carried out by haemofiltration.

In particular, in haemofiltration, any haemofilter (or even a dialysis filter), known to experts in the art, can be used to separate the cellular part from the liquid in which it is suspended in order to reduce the volume of suspension and then concentrate the swollen erythrocytes. In general, the smaller the volume of the hemofilter (sizes for neonatal or pediatric use for example), the higher the level of blood concentration that can be reached.

Hemoconcentration is preferably carried out at room temperature for a time varying between 15 and 35 minutes. In general, the concentration of erythrocytes (hematocrit) obtained at the end of step c) is higher than 30%, for example 35%, 40%, 45%, 50%, 55%, 60%, 65%. In this phase of concentration the osmolality of the erythrocyte suspension is almost constant, varying only by a few units of mOsm / Kg with respect to the osmolality obtained after contact with the second hypotonic solution used in the previous step b).

Since the concentration transition is carried out on swollen red blood cells but essentially intact in their structure, i.e. not lysed, the procedure described here significantly reduces the risk of obtaining irreversibly damaged erythrocytes to the point that they can no longer be effectively used as a pharmacological carrier. In fact, the optimal purpose of the procedure is to obtain a population of loaded and â € œreconstitutedâ € erythrocytes with characteristics as close as possible to the physiological characteristics of the starting population and in any case able to perform the role of carrier in an efficient and for extended periods of time.

Step (d):

Subsequently, the erythrocytes thus concentrated are placed in contact with a lysing hypotonic solution comprising one or more substances of pharmaceutical interest (step d). This solution has the characteristic of lowering the osmolality of red blood cells to the point of causing their temporary lysis, ie the reversible opening of the pores on the cell membrane. The solution containing the active ingredients can be for example an aqueous solution with low osmolality.

In a preferred embodiment of the invention, the lysing solution has an osmolality ranging from 10 to 100 mOsm / Kg. In any case, the osmolality and the volume of the lysing solution are such that contact with the lysing solution brings the red blood cells to an osmolality included in the range from 150 to 110 mOsm / Kg.

This solution, in addition to being hypotonic, contains the substance or substances of interest to be encapsulated. The permeabilization of the plasma membrane of the erythrocytes will therefore favor their diffusion inside the cell.

By way of example, step (d) can be performed by keeping the erythrocytes, at a concentration (hematocrit) of 30-65%, in contact with the lysing solution containing the active substances, for about 10 minutes at room temperature.

Step (e):

In order to encapsulate the molecule or molecules of interest inside the erythrocytes, a sealing solution is used, aimed at restoring the parameters of the treated erythrocytes as close as possible to the physiological conditions. The sealing solution is a hypertonic solution with an osmolality in the range from 300 to 5000 mOsm / Kg.

In a specific embodiment of the invention the sealing solution employed is a Phosphate-Inosine-Glucose-Pyruvate-Adenine (PIGPA) solution. However, it is possible to obtain a similar closing effect through any hypertonic solution composed, for example, of distilled water and mineral salts or other nutrients used by red blood cells. However, the PIGPA hypertonic solution is preferred as it includes nutrients that help the cell to restore some of the lost content and cellular metabolic functions. In this sense, the closing of the pores is facilitated and the normal membrane structure re-established (reannealing).

By way of example, the sealing solution preferably has the following composition: 33 mM NaH2PO4; 1,606 M KCl; 0.194 M NaCl; 0.1 M inosine; 5 mM adenine; 20 mM ATP; 0.1 M glucose; 0.1 M pyruvate; and 4 mM MgCl2. By way of example, approximately 3 mL of the sealing solution can be used for a volume of approximately 35-55 mL of lysed erythrocytes at a concentration (hematocrit) of approximately 15-40%. In particular, the contact of the sealing solution with the erythrocytes can for example be carried out for about 30 minutes, preferably at a temperature of 37 ° C. In this phase, the suspension of red blood cells is brought to an osmolality at least equal to or greater than the physiological one. Although the temperature of 37 ° C is not essential, it contributes to the rapid and optimal restoration of metabolic processes within the cell.

The substances of pharmaceutical interest to be encapsulated, alone or in combination, in the red blood cells can be chosen from among those known according to the particular treatment requirements required.

In an embodiment of the invention, the substances of pharmaceutical interest are selected from the following groups: active principles selected from peptides, oligopeptides, polypeptides, proteins; active ingredients selected from oligonucleotides, nucletide analogues, nucleosides, nucleoside analogues; active ingredients selected from hormones, immunosuppressants, anti-tumor, corticosteroids, glucocorticoids, non-steroidal anti-inflammatory anti-retrovirals, cytokines, toxins, substances with vaccinating activity; contrast media for diagnostics; particles or nanoparticles selected from nanoparticles containing a metal, magnetic nanoparticles, superparamagnetic nanoparticles (SPIO), nanoparticle-active molecule complexes.

For example, the substances can be chosen from 6-mercaptopurine, fludarabine phosphate, azidothymidine phosphate, dideoxycytosine, dideoxyinosine, glutathione, bisphosphonates, prednisolone, prednisolone phosphate, dexamethasone, dexamethasone phosphate, betamethasone, phosphorone phosphate, phosphorine phosphate, phenyl phosphatone phosphorone, thymyl phosphatone indocyanine, super-paramagnetic particles.

In an embodiment of the present invention, the active ingredients may also comprise pro-drugs or precursors of biologically active ingredients. By way of non-limiting example, this pro-drug can be dexamethasone sodium phosphate (or dexamethasone-21 phosphate) which, once encapsulated in the erythrocyte cell and administered to the patient, is converted, by means of an endogenous activation mechanism ( dephosphorylation), in the form of the active anti-inflammatory drug dexamethasone. Alternatively, the conversion from pro-drug to drug can be obtained, if an endogenous activation mechanism is not present, by co-administration of the suitable activator, in the same erythrocyte.

A further object of the present invention is a population of erythrocytes loaded with one or more substances of pharmaceutical interest obtainable by means of the procedure described above.

As already indicated, the population of erythrocytes obtained with the process of the present invention shows a greater cellular viability (metabolism and survival) compared to the analogous population obtained with the procedure described in the literature. In particular, the erythrocytes treated according to the present invention have a cellular half-life, evaluated in terms of the percentage of phosphatidylserine measured with the Annexin V assay, very similar to that of native erythrocytes. Annesina Vâ € ™ s essay is carried out in laboratory practice by the technician of the sector, it has already been described in the literature (Canonico B. et al. 2010) and therefore does not require any particular in-depth analysis here. This assay measures the percentage of erythrocytes that express phosphaditylserine on the outer surface of the plasma membrane, the presence of which indicates damage and accelerated cellular senescence through the natural elimination mechanism. In general, red blood cells with exposed phosphatidylserine are prone to phagocytosis and are cleared from the bloodstream more rapidly than erythrocytes that do not have this protein on the outer membrane. It follows that, the lower the percentage of erythrocytes with phosphatidylserine exposed, the greater the half-life of the erythrocytes circulated in the human body will be expected. The increased half-life is reflected in longer drug release or circulation times. In the population of erythrocytes loaded with the process of the invention, average percentages of phosphatidylserine exposed below 10% are observed, for example 9%, 8%, 7%, 6%, 5%, 4%, 3% , 2%, while the corresponding value of phosphatidylserine exposed on erythrocytes treated with other procedures can exceed 20-40%. These results demonstrate that the process of the invention allows to obtain erythrocytes-carriers which, having a predictably almost natural half-life, transport and / or release the encapsulated substances into the circulation for a sufficiently long period of time to meet the most pharmacological needs. common.

The excellent vitality of the erythrocyte population described here is also confirmed by the evaluation of the metabolic capacity of the erythrocytes obtained. The evaluation of the metabolic capacity, as known to the skilled in the art, is an index of the cell's ability to preserve the biochemical functions necessary for its survival. Red blood cells are cells whose energy production is essentially based on the biochemical pathway of glycolysis of which lactate is the final product. For the erythrocyte population subject of this application, it has been shown that the average quantity of lactate produced for each 10 <6> erythrocytes is greater than 0.100 nmol / h, which is very similar to that of native erythrocytes.

The biochemical / molecular characteristics described above, relating to the erythrocyte population object of the present application, indicate that this population can be used as a carrier for active principles more efficiently than the erythrocyte population described in the prior art, since it is characterized by greater vitality and a predictably longer half-life.

A further object of the present invention is a pharmaceutical composition comprising charged erythrocytes obtained according to the invention and a pharmacologically acceptable excipient.

The compositions described herein are compositions suitable for the administration of erythrocytes and suitable for reaching the target site of pharmacological interest. Therefore they are compositions for parenteral administration preferably in physiological solution, but also, for example, aqueous suspensions (also glucose) or formulated according to what is described in the known art. By way of non-limiting example, water or buffers can be used as pharmacologically acceptable excipients, supplemented with preservatives, stabilizers, sugars and mineral salts, etc. Such compositions can also be in lyophilized form for storage and reconstituted in a suitable carrier before use.

The process object of the present application can be carried out with any known equipment suitable for haemfiltration with movement of different solutions and control of flows, osmolalities and volumes. Preferably, the equipment and the process are managed automatically on the basis of a suitable program, for example using an electro-medical device called Red Cell Loader.

An example of a kit for running the claimed process, to be used in conjunction with the equipment described in the previous Italian patent application BO2010A000255, is shown in Figure 3. The kit contains the following numbered structural elements:

(1) Spike connector for hypotonic solution 1

(2) Spike connector for hypotonic solution 2

(3) Spike connector for 2 Liter bag of saline solution for injection (Wash) (4) Connector for waste bag

(5) Luer connector for 50 mL patient blood inlet (50 mL syringe) (6) Final collection bag

(7) Waste bag

(8) Transfer bag

(9) Connector for pump to Red Cell Loader (machine right side, stand side) (10) Reservoir (for ultrafiltrate)

(11) Blood concentrator filter

(12) Bowl (separation and blood washing bowl)

(13) Pierceable point (for drug inlet and PIGPA resealing solution)

EXAMPLES

The invention is described below with all the experimental details in the following examples, which have a purely descriptive and not limitative purpose of the present invention.

Example 1: Procedure for loading erythrocytes

The erythrocyte loading procedure was carried out using the apparatus described in patent application (IT) BO2010A 000255 & US 61 / 373.018 as detailed below.

The erythrocytes separated starting from 50 mL of whole blood, by means of a centrifugal system of the â € œLatham Bowlâ € type rotating at 5600 rpm, are washed with 750 mL of saline solution at a washing speed of 225 mL / min and transferred to the transfer bag. To the transfer bag are added 300 mL of a first hypotonic solution having an osmolality of 200 mOsm / Kg which is then incubated on a stirring plate at room temperature for 5 minutes. Subsequently the first hypotonic solution is removed by centrifugation (Bowl) until it reaches about 80 mL of volume. The erythrocytes thus concentrated are transferred back to the transfer bag to which 64 mL of a second hypotonic solution with osmolality of 180 mOsm / Kg are then added.

The bag is then incubated at room temperature on a shaking plate for 5 minutes. At the end of the incubation the erythrocytes are concentrated through a hemofilter and about 80 mL of ultrafiltrate is collected in the reservoir to which a slight vacuum is applied by means of a vacuum pump. The concentrated erythrocytes are then recovered and transferred to a transfer bag. The drug of interest, in this example dexamethasone sodium phosphate (25 mg / mL), was pre-mixed with about 11 mL of water for injectable solutions (the preparation has an osmolality of about 20 mOsm / kg) and added to the erythrocytes. concentrates by injection into the transfer bag. This must be done in 5 minutes. The contents of the transfer bag are then incubated at room temperature on a shaking plate for 10 minutes. Subsequently 3 mL of a sealing solution are added (with osmolality 3800 mOsm / kg). This addition must be done in 5 minutes. The transfer bag is incubated for 30 minutes at 37 ° C ± 2 on the shaking plate. The erythrocytes are then transferred to the bowl and washed abundantly with 1100 mL of saline at a flow rate of 225 mL / min. Finally, the loaded erythrocytes obtained in this way are transferred to a final collection bag. The total time of the procedure is about 1h and 30 min.

Example 2: Encapsulation effectiveness

The process described in the present invention allows an encapsulation (introduction) efficiency of the active principle (dexamethasone sodium phosphate, in this example) almost 10 times higher than the known process (process 1), as shown in table 1.

In particular, 50 mL of whole blood was used as starting material. In the step of loading the active ingredient, (step d) of the method described above, 20 mL of DSP (dexamethasone sodium phosphate) 25 mg / mL are added for the known procedure (procedure 1), and only 2.5 mL of the same DSP solution added with 11 mL of water for injection in the process of the present invention (process 2). The analysis of the DSP content present in the erythrocytes loaded according to procedure 1 or 2 was carried out by HPLC instrumentation after extraction of the active principle from the inside of the red blood cells by boiling and diluting in water and methanol. The results are reported in Table 1.

Table 1

Known procedure Procedure object of the 500mg dose present application 62.5 mg initial DSP of initial dose of DSP

DSP

encapsulated a

mg / bag 8.9 11.2

end of procedure

(average)

Example 3: Lactate production in erythrocytes

A whole blood bag from a healthy donor was used. An initial aliquot of his red blood cells was used as an untreated sample.

50 mL of whole blood were processed using the process object of the present invention. At the end of the procedure, 30 mL of treated erythrocytes were collected and brought to 40% hematocrit by centrifugation. Subsequently, glucose was added to each sample and incubated at 37 <0> C for 3 hours, analyzing the accumulation of lactate in the supernatant every 30 minutes (transformation of glucose into lactate by the glycolytic pathway). The analysis was performed through the use of a blood gas analyzer.

The lactate production of untreated RBCs (erythrocytes) is comparable to the lactate production of RBCs produced by the procedure described here. This result indicates that the red blood cells obtained with the process object of the present invention are able to maintain the main metabolic functionality (glycolysis) by transforming glucose into lactate with an efficiency similar to untreated red blood cells (control), as shown in table 2 .

Table 2

Procedure described herein untreated RBC 250mg DSP initial quantity Lactate production nmol 106RBC / h 0.138 0.130

Example 4: Half-life of loaded erythrocytes

The presumed half-life of the erythrocytes loaded according to the procedure described here was evaluated by measuring the Annexin V on the cell surface, known to be a marker of cell death (senescence) as detailed below.

In particular, a bag of whole blood from a healthy donor was used. An initial aliquot of his red blood cells was used as an untreated sample. 50 mL of whole blood were processed using the process object of the present invention. 10 <6> erythrocytes were withdrawn from the final process product and the untreated sample; they were diluted in the reaction buffer for annexin V, added 3 µl of annexin V conjugated with the FITC fluorochrome and the analysis was carried out on the flow cytometer.

As shown in table 3 below, the increase in Annexin V from 0.75% of the untreated control to 6.26% of the red blood cells obtained with the procedure described here is an indication of a still high capacity of the latter to remain in circulation. for a long time. In fact, the literature reports for products based on red blood cells for transfusion use values of Annexin V similar to those obtained for loaded red blood cells obtained with the procedure described here (Relevy H. et al., 2008).

Table 3

Procedure described here

Untreated RBCs

250mg SDR initial amount Annexin V% 0.75 6.26

Example 5: Encapsulated dose variation

The process object of the present invention allows to encapsulate doses of active ingredient, such as DSP (dexamethasone sodium phosphate), in a very wide therapeutic range simply by varying the initial dose of drug used, as shown in Table 4 below.

In particular, 50 mL of whole blood was used for each experiment and for each initial quantity of DSP. For loading the DSP according to the known procedure, 20 mL of DSP 25 mg / mL were added.

In the case of the procedure described here, 10 mL, 5mL, 2.5 mL and 2 mL of DPS 25 mg / mL were added for the doses of 250, 125, 62.5 and 50, respectively, pre-mixed each with 11 mL of water for injection. The analysis of the DSP encapsulated in red blood cells first involves a 1:10 dilution in distilled water, a step of boiling the sample to denature the proteins, subsequent centrifugation and extraction in water and methanol. The analysis of the encapsulated DSP was performed by HPLC. The results are reported in Table 4.

Table 4

Known procedure Procedure described here Initial quantity SDR mg 500 250 125 62.5 50 SDR encapsulated dose mg 8.9 29.4 18.3 11.2 9.9

Example 6 Encapsulation of high molecular weight active ingredients

The process object of the present description also allows to encapsulate high molecular weight proteins such as Exokinase (HK) with an encapsulation efficiency higher than 15% of the initial product, as shown in Table 5 below.

In particular, 50 mL of whole blood from a healthy donor was used which underwent the encapsulation procedure described here. The encapsulated active ingredient was the exokinase protein. In the step of adding the active ingredient, 200 mg of HK dissolved in 14 mL of water for injection were added.

Table 5

Procedure described here

Initial amount of

IU / total 10000

HK protein

Exokinase (HK)

encapsulated at end of IU / total 1600

process

Example 7: Influence of globular osmotic resistance on erythrocyte loading.

Each individual has his own osmotic globular resistance (RGO) which could influence the outcome of the loading process. The data shown below indicate that donors with different osmotic resistances maintain very similar drug loading. Therefore, the process object of the present invention proves not to be significantly influenced by the patient's initial RGO as evident from the data in table 6, unlike what is indicated for similar known procedures.

The procedure described in the present invention was used to determine the variability of the product loading inside the red blood cells according to the variation of the RGO of various individuals, with an initial dose of DSP (dexamethasone sodium phosphate) equal to 50 mg. Five tests were performed starting from 50 mL of whole blood from 5 different individuals with different RGOs.

The osmotic globular resistance of each individual was measured by diluting a portion of their whole blood in solutions with decreasing concentration of NaCl (8 different osmolality values), measuring the free hemoglobin in each of the solutions and building the graph of the total free hemoglobin as a function of osmolality (see figure 1). The RGO value was subsequently obtained (corresponding to osmolality at 50% of hemolysis or 50% of free hemoglobin), by interpolation of these curves obtained. The hemoglobin released was quantified by Drabkin's reagent with a spectrophotometer reading (Drabkin DL. Med Sci 1949). The analysis of the DSP (dexamethasone sodium phosphate) loaded in the final erythrocytes was carried out after their lysis by boiling, extraction in water-methanol and HPLC method. The results are reported in table 6.

Table 6

RGO (Hemolysis RGO (Hemolysis

50%) 50%) DSP loading

Concentration

Sample mOsm / Kg of NaCl g / dL mg / bag

1 153 0.47 9.6

2 143 0.44 10.5

3 151 0.47 9.8

4 141 0.43 10.2

5 143 0.44 9.9

Average 146 ± 5 0.45 ± 0.02 10.0 ± 0.4

As shown in Table 6 above, in the procedure described here, the loading of dexamethasone sodium phosphate (DSP) into the red blood cells of individuals with different initial RGOs (from 141 to 153 mOsm / kg) does not show such changes that a pharmacologically different effect (average loading 10.0 ± 0.4 mg / bag). The variation of encapsulated DSP with the variation of the RGO of the individual described in these examples is negligible from the pharmacological point of view.

Example 8: Influence of initial hematocrit change on erythrocyte loading.

To determine the variability of the erythrocyte loading as the starting blood hematocrit varies, the procedure described in the present invention was used with an initial dose of DSP (dexamethasone sodium phosphate) equal to 62.5 mg. 10 tests were performed with 5 different individuals (1 test with hematocrit about 40% and 1 test with hematocrit about 50% for each donor). The standardization of the hematocrit for each donor was carried out by centrifugation or dilution of the initial blood. The analysis of the DSP loaded in the final erythrocytes was carried out after their lysis by boiling, extraction in water-methanol and HPLC method. THE

results are reported in Table 7.

Table 7

HCT adjusted initial blood 40% HCT adjusted initial blood 50% Sample

HCT HCT DSP loaded DSP loaded (mg / final bag)

(%) (%) (mg / final bag)

1 39.9 11.48 49.9 13.04

2 40.0 11.72 50.1 10.76

3 39.3 10.94 50.0 10.52

4 40.8 11.75 50.0 11.70

5 40.0 11.16 50.1 9.92

Average 40.0 ± 0.5 11.41 ± 0.65 50.0 ± 0.1 11.19 ± 1.22

As evident from the data shown in Table 7, the loading of dexamethasone sodium

phosphate in the red blood cells of individuals with different initial hematocrits (hematocrits from 40% to 50%) is extremely constant (from 11.41 to 11.19 mg / final bag) and not

shows variations with statistical significance (p> 0.05 with t-Student test for data

coupled) without requiring the variation of parameters of the procedure therein

described. The variation of encapsulated SDR at a variation of 10% of the initial hematocrit, which on the contrary is a highly significant variation (p <0.001 with t-Student test

for paired data) of the individuals described in these examples is pharmacologically negligible.

Claims (15)

CLAIMS 1. Procedure for preparing erythrocytes loaded with one or more active ingredients comprising the following steps: a) swell the erythrocytes using a first hypotonic solution; b) further swell, without reaching lysis, the erythrocytes obtained in step a) using a second hypotonic solution, more hypotonic than the first solution; c) concentrate the erythrocytes obtained in step b); d) placing the concentrated erythrocytes in contact with a lysing solution comprising one or more substances of pharmaceutical interest, and subsequently e) adding a sealing solution in order to obtain a population of erythrocytes loaded with said one or more active principles. 2. Process according to claim 1, wherein said first solution brings the erythrocytes to an osmolality comprised between 250-200 mOsm / Kg. 3. Process according to claim 1 or 2, in which said second hypotonic solution brings the erythrocytes to an osmolality of between 200 and 170 mOSm / Kg. Process according to any one of claims 1 to 3, which comprises between steps (a) and (b), an additional step in which the first hypotonic solution is at least partially removed before adding the second hypotonic solution. Process according to any one of claims 1 to 4, wherein said concentration step (c) is carried out by means of haemofiltration, hemodialysis or centrifugation. 6. Process according to any one of claims 1 to 5 wherein the lysing solution in step (d) brings the erythrocytes to an osmolality comprised in the range between 150 and 110 mOsm / Kg. Method according to any one of claims 1 to 6, wherein the sealing solution in step (e) is a hypertonic solution of 300 to 5000 mOsm / kg. Process according to any one of claims 1 to 7, in which the substances of pharmaceutical interest are selected from the following groups: active principles selected from: peptides, oligopeptides, polypeptides, proteins; active ingredients selected from: oligonucleotides, nucletide analogues, nucleosides, nucleoside analogues; active ingredients selected from: hormones, immunosuppressants, anti-tumor, corticosteroids, glucocorticoids, non-steroidal anti-inflammatory anti-retrovirals, cytokines, toxins, substances with vaccine activity; contrast media for diagnostics; particles and nanoparticles chosen from: nanoparticles containing a metal, magnetic nanoparticles, super-paramagnetic particles (SPIO), nanoparticle-active molecule complexes. 9. Process according to claim 8, wherein said substances of pharmaceutical interest are selected from 6-mercaptopurine, fludarabine phosphate, azidothymidine phosphate, dideoxycytosine, dideoxyinosine, glutathione, bisphosphonates, prednisolone, prednisolone phosphate, dexamethasone, phosphate phosphate, dexamethasone, phosphatone , thymidine phosphorylase, phenylalanine ammoniolyase, indocyanine green, super-paramagnetic particles. 10. Population of erythrocytes loaded with one or more substances of pharmaceutical interest obtainable through the process according to any one of claims 1 to 9. 11. Population according to claim 10, in which the percentage of phosphatidylserine produced by the erythrocytes, measured with the Annexin V assay is less than 10%. Population according to claims 10 or 11 wherein the amount of lactate produced for each 10 <6> erythrocytes is greater than 0.100 nmol / h. 13. Population according to any one of claims 10 to 12, wherein said one or more substances of pharmaceutical interest are selected from the following groups: active principles selected from: peptides, oligopeptides, polypeptides, proteins; active ingredients selected from: oligonucleotides, nucletide analogues, nucleosides, nucleoside analogues; active ingredients selected from: hormones, immunosuppressants, anti-tumor, corticosteroids, glucocorticoids, non-steroidal anti-inflammatory anti-retrovirals, cytokines, toxins, substances with vaccinating activity; contrast media for diagnostics; particles or nanoparticles chosen from: nanoparticles containing a metal, magnetic nanoparticles, superparamagnetic particles (SPIO), nanoparticle-active molecule complexes. 14. Population according to claim 13, in which said one or more substances of pharmaceutical interest are chosen among: 6-mercaptopurine, fludarabine phosphate, azidothymidine phosphate, dideoxycytosine, dideoxyinosine, glutathione, bisphosphonates, prednisolone, prednisolone phosphate, dexamethasone, dexamethasone phosphate, betamethasone, betamethasone phosphate ammalamine, superparocyanine phosphates, phenylamine phosphates, superparocyanine phosphates, phenylamine phosphates Pharmaceutical composition comprising a population of erythrocytes according to any one of claims 10 to 14 and one or more pharmacologically acceptable excipients.