WO2007042647A1

WO2007042647A1 - Erythrocytes containing 5-fluorouracil (5-fu) and/or oxaliplatinum

Info

Publication number: WO2007042647A1
Application number: PCT/FR2006/002249
Authority: WO
Inventors: Yann Godfrin; Michel Rivoire
Original assignee: Erytech Pharma
Priority date: 2005-10-06
Filing date: 2006-10-06
Publication date: 2007-04-19
Also published as: FR2891843A1

Abstract

The invention concerns erythrocytes containing 5-fluorouracil, optionally associated with other active principles such as oxaliplatinum or another platinum derivative for anticancer therapy. The erythrocytes may have been subjected to a chemically or thermally denaturing treatment to promote their recognition by erythrophagocytic cells. The invention also concerns a drug produced from same, useful for treating primitive or secondary 5-FU-sensitive cancers, in particular secondary cancers present in the liver, the spleen, the lungs, the lymph nodes.

Description

ERYTHR YTES CONTAINING 5-FLUOROURACIL (5-FU) AND / OR LOXALIPLATIN

The present invention relates to the treatment of primary and secondary cancers responsive to 5-FU and a novel pharmaceutical form of 5-FU adapted for this purpose.

5-fluorouracil (5-FU, CAS: 51-21-8) is used in digestive adenocarcinomas, especially advanced or metastatic forms, or colorectal cancers in adjuvant situation, after resection (Dukes stage C); in mammary adenocarcinomas, particularly after locoregional treatment (adjuvant treatment), or during relapses; in squamous cell carcinoma of the upper aerodigestive tract and the esophagus.

5-FU is particularly used in chemotherapy of liver metastases originating from colorectal cancer.

5-FU has general toxicity and cardiac toxicity. ECG disorders, heart failure and acute myocardial infarction can be observed.

This molecule has a very short half-life, of the order of 15 to 20 minutes. It is no longer detectable in the blood 2-4 hours after IV administration. In order to increase the intratumoral concentration and decrease the systemic levels, the administration is by perfusion via a catheter placed surgically in the gastroduodenal artery, generally after ligation of the pyloric artery and cholecystectomy, and after control by angioscintigraphy . Response rates range from 30 to 80% and are higher than after systemic infusion. Multicenter randomized trials have demonstrated a survival benefit and improved quality of survival.

However the complications related to these methods are important. This is hepatitis in 50 to 70% of cases of sclerosing cholangitis in 15% of cases of gastro-duodenitis and arterial thrombosis occurring in 50% of cases before the ^6th month. Moreover, comparative studies conducted on the different modes of treatment with 5-FU point to a benefit in terms of hepatic toxicity for continuous administration compared to the administration of a bolus (Sotaro Sadahiro et al., Jpn J. Clin, Oncol, 2003, 33, 8: 377-381). Bolus administration of large doses of 5-FU may cause severe liver problems, as opposed to continuous administration. The infusion pattern of 5-FU in the gastroduodenal artery remains controversial at this time.

5-FU is currently used in adjuvant colorectal cancer treatment protocols in patients with primary tumor resection and / or secondary adenocarcinoma to control hepatic metastases. These protocols (eg FOLFOX 4 or LV5FU2) include repeated cycles with bolus administration (400 mg of 5-FU per m ² ) followed by continuous infusion over 22 hours (600 mg of 5-FU per m ² ), performed in a hospital. The cumulative effects of the aging of the population and the increasing control of resection surgery are increasing the number of immobilized beds for patients in treatment or palliative care in already problematic proportions.

An objective of the invention is to propose, in clinical practice, a new mode of administration of 5-FU which makes it possible at the same time to optimize the anti-tumor activity of this molecule while reducing its toxic effects, and allowing to target more easily and efficiently certain tumors such as liver metastases. The objective would be to have a method of administration to avoid the use of surgical procedures related to the establishment of conventional arterial pathways.

Another objective of the invention is to propose a mode of administration simplifying the treatment protocols integrating 5-FU and in particular reducing the residence time in a hospital environment. A 5-FU treatment in ambulatory mode is thus an objective of the invention. Erythrocytes have been considered as a vehicle for anti-cancer agents and in particular to target the reticuloendothelial system which is the major site of destruction of aged or abnormal erythrocytes. It has thus been envisaged to use erythrocytes to carry such anti-cancer agents directly in the organs of the RES (liver, spleen, lung, lymph nodes). It has even been envisaged to subject the erythrocytes chemical treatments (eg glutaraldehyde or neuraminidase) or thermal, promoting their recognition and destruction by erythrophagocytic cells (macrophages). For a complete recent review, reference will be made to the publication of CG. Millan et al., Journal of Controlled Release 95, 2004: 27-49. This review reports the incorporation of actinomycin D, methotrexate, bleomycin, adriamycin or doxorubicin, daunomycin, etoposide and carboplatin, with mixed results, none of which has led to use of erythrocytes as a vector of antitumor agents in the human clinic.

Despite the theoretical potential of erythrocytes as a vector that a relatively large number of studies point out, major drawbacks have not led to clinical applications. Among these disadvantages, mention will be made with CG. Millan et al, storage difficulties, risks of contamination, and especially the difficulty of encapsulating substances in erythrocytes and the lack of a recognized industrial process for their preparation. Indeed, the human clinic requires the availability of batches of reproducible quality drugs, and it appears that the techniques used so far are laboratory experimental methods that do not meet the specifications required for the routine production of drugs. .

In addition, as P. Labrude et al. (1985 Lyon Pharmaceutical 1985, 36, 4: 181-187), CG. Millan et al. conclude that the use of erythrocytes as vehicles of therapeutic agents requires specific therapeutic studies for each therapeutic agent to confirm the real capacity of erythrocytes to serve as a vehicle.

Thus, despite all the interest that the scientific community has focused on the targeting of liver tumors by erythrocytes incorporating an anti-cancer molecule, this mode of treatment has not found a market opportunity, certainly fault and in particular, an active molecule adapted to this mode of delivery and the absence of a method for reproducibly encapsulating it.

The work of K. K. Sawant et al. on the incorporation of 5-Fu in erythrocytes remained without result. The incorporation of 5-Fu was carried out by the Presswell dilution method, the erythrocytes were then washed and then treated with glutaraldehyde. The resulting erythrocytes targeted the lung and spleen.

The Applicant has developed a very efficient encapsulation method for efficiently encapsulating molecules in erythrocytes and in particular with encapsulation efficiencies sufficiently constant to consider use in the human clinic.

The invention firstly relates to erythrocytes containing 5-FU. By "container" is meant the presence of the active ingredient inside the red blood cell and / or trapped in the red blood cell membrane. Preferably, these erythrocytes have the property of targeting the liver preferentially. This means that they tend to concentrate in the liver where they are phagocytosed by macrophages from which a targeted release of 5-FU results. The erythrocytes of the invention are capable of being produced by the lysis / resealing method specifically described herein, followed by hepatic targeting treatment, particularly with Giutaraldehyde, as described below.

The so-called lysis-resealing encapsulation technique is described in patents EP-A-101 341 and EP-A-679 101. According to this technique, the primary compartment of a dialysis element is continuously fed with a suspension of erythrocytes, while the secondary compartment contains a hypotonic aqueous solution relative to the suspension of erythrocytes to lyse the erythrocytes; then in a resealing unit, resealing erythrocytes is induced in the presence of the molecule by increasing the osmotic and / or oncotic pressure, and then a suspension of erythrocytes containing it is collected.

The present invention advantageously adds to this basic technique, taking into account the osmotic fragility of erythrocytes used for encapsulation, for adapt the dialysis parameters, which is done in a short time after this measurement. This allows, from one batch of erythrocytes to another, to ensure reproducibility of the encapsulation efficiency compatible with a use in human medicine. The erythrocytes according to the invention are therefore particularly likely to be obtained by:

1 - suspending a red blood cell in an isotonic solution at a hematocrit level equal to or greater than 65%, refrigeration between + 1 and + 8 ⁰ C,

2 - measurement of the osmotic fragility on a sample of the suspension obtained in step 1 or on a suspension sample prepared as in step 1, 3 - lysis and internalization procedure of 5-FU, at the inside the same chamber, at a temperature constantly maintained between + 1 and + 8 ⁰ C, including the passage of the suspension of erythrocytes at a hematocrit level equal to or greater than 65% and a hypotonic lysis solution refrigerated between + 1 and 8 ⁰ C, in a dialysis cartridge; the lysis parameters being adjusted according to the osmotic fragility measured previously; and

4 - resealing procedure conducted in a second chamber within which the temperature is between + 30 and + 40 ⁰ C, and in the presence of a hypertonic solution.

By "internalization" is meant penetration of 5-FU into erythrocytes and / or entrapment in the erythrocyte membrane.

The subject of the invention is also a suspension of these erythrocytes in a pharmaceutically acceptable saline solution (for example standard medium for red blood cells, in particular a solution containing NaCl and one or more ingredients chosen from glucose, dextrose, adenine and mannitol, eg SAG-mannitol or ADsol). This solution is able to ensure the conservation of erythrocytes, and may include a preservative additive, such as L-cernitine. This suspension can be packaged ready for use or diluted before use. The final hematocrit content of the suspension ready for use (after optional extemporaneous dilution) is between 40 and 70%, preferably between 45 and 55%, better 50% or of the order of 50%. It can be administered intravenously, preferably by infusion. As opposed to direct infusion into the gastroduodenal artery, the The suspension according to the invention can advantageously be administered (perfused) systemically.

Such a suspension or any administrable formulation containing these erythrocytes constitutes in itself a drug or a pharmaceutical composition object of the invention. This drug may be packaged for example in the form of a soft bag for infusion, or in another form for administration by injection.

According to one characteristic of the invention, the medicament comprises a suspension of erythrocytes at a hematocrit level as indicated above. It is preferably packaged in a volume of 10 to 250 ml, preferably 50 to 200 ml. The preferred form of presentation is the infusion bag. The amount of encapsulated 5-FU corresponding to the medical prescription is preferably completely contained in the blood bag or other form of presentation, of the volume just indicated.

The erythrocytes of the invention may contain at least one other active ingredient, in particular chosen from the other anti-cancer agents, and / or at least one compound having an adjuvant effect. Alternatively or in addition, the drug contains at least a second population of erythrocytes, containing this other active ingredient or adjuvant.

In a particular embodiment, the erythrocytes or the drug contains a platinum derivative for anti-cancer use, particularly oxaliplatin, e.g. Eloxatin®. The invention thus describes erythrocytes containing oxaliplatin, as well as the corresponding suspensions and anti-cancer drugs.

The drug or pharmaceutical composition is useful in the treatment of all forms of cancer susceptible to 5-FU. 5-FU is thus used in digestive adenocarcinomas, including liver metastases (eg from colorectal cancer, breast adenocarcinoma, squamous cell carcinoma of the upper aerodigestive tract and esophagus). The main use in the context of the present invention is the treatment of tumors and in particular secondary cancers (metastases) present in the organs of the RES, namely the liver, spleen, lung, lymph nodes, and more particularly, the liver ( including liver metastases caused by colorectal cancer, colon cancer, usually after resection of primary tumor and / or secondary tumor).

The kinetics of the drugs according to the invention can be modified by an appropriate treatment favoring their recognition and their destruction by the erythrophagocytic cells (macrophages), leading to the rapid and massive release of 5-FU into the organs of the RES and improving the bioavailability of this molecule. This treatment can be chosen to obtain a rapid release effect of the totality or quasi-totality of the 5-FU in a predetermined time interval, in particular less than 24 hours, for example less than 16 hours, in the liver and / or the others. bodies of the RES.

Depending on whether the erythrocytes are treated with such a denaturing treatment, and depending on the conditions of the denaturing treatment used, different release profiles may be available, which makes it possible to adapt to the optimal treatment scheme for a pathological condition. given.

Thus, according to a first aspect, the release profile is of the release type of all or substantially all of the active ingredient over a limited period of time (rapid release). In this case, denatured erythrocytes are chosen under conditions adapted to this duration, for example for release in less than 24 or 16 hours, as mentioned above.

According to a second aspect, the profile is spread over time (slow release), in particular over several days, or several weeks. A peak of release by the most fragile erythrocytes can be observed soon after administration. Undenatured erythrocytes or moderately weakened by a denaturing treatment are then more appropriate. In a third aspect, the protocol combines the use of fast release profile erythrocytes and slow release profile erythrocytes.

According to a first modality, the denaturing treatment is chemical, eg by glutaraldehyde or by neuraminidase. This treatment is performed on erythrocytes containing 5-FU. This treatment can be performed as follows. The erythrocytes are incubated for 1 to 20 minutes, eg about 10 minutes, in 2 to 10 volumes, eg about 5 volumes, of a solution containing from 0.05 to 1%, eg 0.1% to 0.5%. about glutaraldehyde (or neuraminidase) and about 0.9% NaCl. The incubation is preferably carried out at room temperature (20-25 ^° C.). Then, the erythrocytes are washed in about 0.9% NaCl solution, eg in about 50 volumes of this saline solution, and preferably at least two times. Such treatment, especially with glutaraldehyde, promotes liver targeting.

According to a second modality, the treatment is thermal, eg heating between 40 and 60 ⁰ C, preferably between 45 and 50 ⁰ C. The heating time can be between 20 min and 1 hour, preferably between 30 and 45 min.

The invention also relates to a method for treating primary cancers and / or secondary tumors just mentioned. This method provides for the administration to a patient in need of an effective amount of such a drug, especially intravenously, by injection or infusion, preferably by infusion.

In the case of secondary cancers of one or more organs of the RES, in particular liver metastases, the method uses denatured or non-denatured erythrocytes, according to the desired slow or fast release profile. According to a particular modality, the erythrocytes are denatured so as to promote rapid and massive release of the active ingredient in the affected organ, in particular the liver, as described above.

According to an interesting modality, the patient is treated after surgical excision of the primary tumor and / or the secondary tumor. When its condition permits, the patient can be treated with his own erythrocytes, after these have been treated to encapsulate 5-FU. Alternatively, the erythrocytes come from one or more donors.

The method may thus include taking one or more blood samples, for example a blood bag (s), a patient or one or more donors, preparing an erythrocyte pellet, incorporating 5-FU according to the invention and the production of a batch of erythrocytes incorporating the active ingredient, a possible controlled denaturation, then the administration of the suspension or drug to the patient, intravenously.

Typically, a volume of treated erythrocyte suspension providing the required dose of 5-FU _{1 is} generally administered to the body weight of the patient. According to one characteristic of the invention, 10 to 250 ml, preferably 50 to 200 ml of a suspension of erythrocytes at a hematocrit level of between 40 and 70%, preferably between 45 and 55%, are administered. better by 50%.

The method according to the invention is adaptive in that it makes it possible to provide the amount of 5-FU required by an existing protocol and in that it can do so according to the desired kinetics. By way of example, the 1000 mg / m ² (bolus + continuous infusion over 22 h) of an infusion cycle according to the FOLFOX 4 or LV5FU2 protocols can be provided by the medicament according to the invention. Similarly, to take into account more widely the existing protocols, it may be specified that the method provides the patient with a prescribed dose of between 200 and 2400 mg / m ² .

According to a particular modality, such a suspension is administered at a frequency of between 15 days and three months, preferably monthly, for a sufficient duration.

During the lysis / resealing process, 5-FU may be present in the starting slurry. In this case, it is advantageous to measure the osmotic fragility in step 2 on the suspension sample containing 5-FU. According to a characteristic of the invention, the packed red cell is suspended in an isotonic solution at a high hematocrit level, equal to or greater than 65%, and preferably equal to or greater than 70%, and this suspension is refrigerated between + 1 and + 8 ⁰ C, preferably between + 2 and + 6 ⁰ C, typically around + ^{40 °} C. According to a particular modality, the hematocrit content is between 65 and 80%, preferably between 70 and 80%. and 80%.

In accordance with an important feature of the invention, osmotic fragility is measured on erythrocytes just prior to the lysis step. The erythrocytes or the suspension containing them are advantageously at a temperature close to or identical to the temperature selected for lysis. According to another advantageous characteristic of the invention, the measurement of the osmotic fragility is quickly exploited, that is to say that the lysis procedure is carried out at short notice after taking the sample. Preferably, this time interval between sampling and beginning of lysis is less than or equal to 30 minutes, more preferably less than or equal to 25 and even 20 minutes.

The two parameters for controlling dialysis are the time of presence of the cells in the dialyzer (depending on the characteristics of the latter) and the osmolarity of the dialysate. Both parameters should be adjusted according to the characteristics of resistance, or conversely of fragility, osmotic red blood cells conditioned to undergo the lysis / resealing steps. This osmotic resistance can be characterized by at least one of the following parameters: a. the osmolarity of the medium for which hemolysis occurs, that is, the beginning of the formation of pores. b. The velocity V of hemolysis, determined by the slope of the linear part of the curve% hemolysis = f (osmolarity of the medium). vs. The percentage of hemolysis for a given osmolarity. d. Osmolarity which allows to obtain 50% of hemolysis (H ₅₀ ). e. The time to obtain a certain percentage of hemolysis (for example

50%).

In preferred embodiments, the osmotic resistance is characterized by the parameters b, d or b and d. The osmotic fragility must therefore be measured in a short time, compatible with the short time between sampling and early lysis. According to one characteristic of the invention, one or more of these hemolysis parameters are measured against a hypotonic solution of known isotonicity, eg water (distilled water or the like), through a semi-permeable membrane. A manual method can be considered. However, according to a preferred embodiment of the invention, the osmotic fragility is measured with the aid of an automatic measuring apparatus designed to measure the osmotic fragility of a sample of erythrocytes in less than 15 minutes, more particularly in less than 12 minutes and preferably in less than 10 minutes, and the result obtained is used in a short time to adjust the lysis parameters, and start the latter.

Measurement of osmotic fragility can be achieved using an apparatus that at least partially automates the manual technique described by JV Dacie in Practical Haematology, 2 ^nd edn, Churchill, London 1956. An example of such a device is described in the article by J. Didelon et al., Clinical Hemorrheology and Microcirculation 23 (2000) 31-42. The principle is based on the use of a device bringing together, on either side of a semi-permeable membrane, the sample of the suspension of erythrocytes to be evaluated, and a hypotonic solution, of isotonicity known, eg distilled water, in suitable volumes, so as to generate slow hemolysis of erythrocytes as the NaCl ions diffuse to the solution, eg distilled water. The evolution of hemolysis over time is monitored by measurement of transmittance (see also J. Didelon et al., Biorheology 37, 2000: 409-416) using laser radiation having a wavelength of 808 nm. A photocell measures changes in the light transmitted through the suspension. For example, the measurements are performed over 10 minutes. The apparatus makes it possible to obtain one or more of the parameters a - e mentioned above.

According to a first modality, measurement of the osmotic fragility is carried out on a sample whose initial temperature is between + 1 and + 8 ^° C., preferably with distilled water also at this temperature, under conditions in which the temperature evolution is not detrimental to the. measured. According to a second modality, the measurement of the osmotic fragility is carried out on a sample maintained at the temperature between + 1 and + 8 ^° C. Thus, the measuring apparatus described in J. Didelon et al. supra can be modified to allow regulation of the temperature. Preferably this temperature is close to or identical to the lysis temperature.

Once one or more of these parameters have been determined, a relationship can be applied taking into account this or these parameters in order to determine either the flow rate of the cells in the dialyzer or the osmolarity of the dialysate, which is adequate to obtain red blood cells. encapsulating 5-FU and / or the desired amount thereof: Red blood flow = [A x (H ₅₀ )] + [B x (V)] + K

- A and B = adaptable variables according to the dialyzer and osmolarity of the lysis solution

- K = adjustment constant. Osmolarity dialysat = [C x (H ₅₀ )] + [D x (V)] + K

- C and D = adaptable variables according to the dialyzer and the flow of red blood cells in the dialyzer

- K = adjustment constant.

According to a preferred embodiment, the concentration of NaCl in g / L which causes 50% hemolysis is measured (parameter d) and the flow rate of the erythrocyte suspension in the dialysis cartridge are adjusted according to the concentration values. of NaCl measured.

According to one aspect of the invention, one starts the lysis procedure when the temperature of the suspension of erythrocytes is between +1 and + 8 ⁰ C, and the osmotic fragility has been measured and the lysis parameters recorded.

According to an advantageous characteristic, the initial suspension to be treated is placed in the lysis-internalization chamber mentioned above. According to one embodiment of the invention, the method uses a refrigerated module provided with a temperature regulation, it is placed in this module a bag of the suspended erythrocyte refrigerated between + 1 and + 8 ⁰ C, connected, or that is connected to a sterile removable disposable assembly comprising a dialysis cartridge, tubing connecting the cartridge on the one hand to the bag and on the other hand to the lysis solution, the module further comprising means capable of ensuring the circulation of the erythrocyte suspension and the lysis solution, module inside which the temperature is stabilized between + 1 and + 8 ^° C. The refrigerated module is sized to house the pocket and the disposable removable assembly. Providing the bag, the dialysis cartridge, the lysis solution, connected by the various tubings, in such a single refrigerated module is an advantageous characteristic of the method according to the invention.

The term "pocket" refers to the flexible pouches commonly used in the field of blood transfusion and blood derivatives.

In an important aspect of the invention, care is taken to maintain the erythrocytes in homogeneous suspension in the pouch, so as to maintain the hematocrit level of the suspension passing through the dialyser stable. According to a feature of the invention, the bag is thus provided with a looped external circulation capable of ensuring circulation of the suspension from and to the bag.

Dialysis cartridge means an element comprising two compartments separated by a dialysis wall through which an ion exchange can be carried out which makes it possible to modify in a controlled manner the osmotic pressure of an aqueous solution located in one of the compartments. introducing into the other compartment an aqueous solution comprising a salt. This type of cartridge is widely used in the medical field. According to a preferred embodiment, a hollow-fiber dialysis cartridge is used, for example such a cartridge having the following specifications: inner diameter of the fibers between 100 and 400 μm, total external surface of the fibers between 0.3 and 2 m ² , fiber length between 10 and 40 cm, ultrafiltration coefficient between 1, 5 and 8 mL / h.mmHg.

As mentioned above, the lysis procedure can be started when the temperature of the suspension in the bag is between + 1 and + ^80C . According to an interesting modality, the temperature of the suspension is controlled by means of a sensor placed on the outside loop circulation.

According to the osmotic fragility noted, it is possible to play on two main parameters, the flow rate of the erythrocyte suspension in the dialysis cartridge and the osmolarity of the lysis solution, it being understood that it is preferable to fix in both case, a constant flow rate for the lysis solution. The value of the flow is not critical.

Typically, for a hollow fiber dialysis cartridge, as described above, the flow rate of the lysis solution is set between 50 and 300 mL / min, preferably between 150 and 250 mL / min.

The lysis solution is a hypotonic saline solution compared to the suspension of red blood cells. When set at a constant value, its osmolarity can typically be between 30 and 110 mOsm, preferably between 40 and 100 mOsm, for example of the order of 90 mOsm.

By way of example, the lysis solution may comprise Na ₂ HPO ₄ θt / or NaHaPO ₄ and a sugar such as glucose.

According to a first modality, the flow rate of the erythrocyte suspension is adapted through the dialysis cartridge, while flow and osmolarity of the lysis buffer are set. The higher the osmotic fragility, the higher the flow rate of the suspension. Typically, for a cartridge whose specifications have been indicated supra, the flow rate will be varied within the range of 5 to 200 ml / min, preferably 10 to 40 ml / min.

In a second embodiment, the osmolarity of the lysis solution is adapted, while the suspension rates and the lysis solution are set. The higher the osmotic fragility, the greater the osmolarity of the lysis solution. Typically, the osmolarity will be varied within the range of 10 to 200 mOsm / l, preferably 20 to 150 mOsm / l. According to a third modality, both the flow rate of the erythrocyte suspension through the dialysis cartridge and the osmolarity of the lysis solution are adapted.

The 5-FU to be encapsulated may be present in the suspension bag and / or be introduced, preferably progressively, into the suspension circulation upstream or downstream of the dialysis cartridge. The volumes introduced being low, refrigeration of the active ingredient is optional.

Preferably, the suspension of red blood cells is produced from a packed blood group bloodcolland compatible with the recipient, leukocyte depleted, without identified pathogen, in particular presented in pocket, for example 500 ml The red blood cells may have been irradiated when they are intended for highly immunocompromised patients capable of expressing a "graft / host" immunological reaction (RJ, Davey Immunol Invest, 1995, 24 (1-2): 143-149).

According to one particularity of the invention, the initial red blood cell used to prepare the suspension has undergone prior treatment to remove elements of the blood other than erythrocytes. This type of treatment, for example washing with saline to remove the plasma or a preservative solution, is known to those skilled in the art.

According to one particular modality, the washing is carried out in the presence of 5-FU to be encapsulated.

The washing can be carried out by any usual technique, such as the quadruple pocket technique or 4 bags for the washing of red blood cells (MacoPharma method and transfer bag). It is also possible to use an automatic red blood cell washer of the COBE 2991 CeII Processor type.

Preferably, the temperature during the lysis step is maintained between + 2 and + 60 ^° C., and even more preferably around + ^{40 °} C. The resealing procedure is preferably carried out by reheating the lysed suspension and adding a hypertonic resealing solution. The resealing temperature may be between + 30 and + 40 ° C. It is preferably between + 35 and + 38 ^° C., for example approximately 37 ^° C. Incubation may typically be 15 to 45 minutes.

Preferably, the suspension exiting the dialysis cartridge and a hypertonic resealing solution are introduced, preferably continuously, into an intermediate bag. The suspension is warmed, and incubated at the desired temperature for a sufficient time to ensure resealing. According to a particular aspect, the intermediate bag is placed in a heated module or enclosure, the internal temperature of which is regulated at the chosen temperature.

Alternatively, the suspension is fed into an intermediate bag, as well as the resealing solution. When all the suspension has been collected in this pocket, it is sealed and transferred to a module for heating and incubation at the desired temperature.

The resealed red cell suspension may then undergo one or more wash steps with saline to remove unwanted or poorly resealed cells, residues, and extracellular hemoglobin.

The eventual treatment of controlled denaturation of erythrocytes can be carried out at this stage.

According to another characteristic, the erythrocytes are conditioned in a solution for the preservation of red blood cells, for example containing L-carnitine.

The erythrocytes produced are preferably stored at a temperature of between + 1 and + 80 ^° C., preferably between + 2 and + 60 ^° C., typically at approximately + ^{40 °} C.

The final hematocrit content of the product is preferably between 40 and 80%. The internalization of a platinum derivative, especially oxaliplatin, can be carried out as just described with regard to 5-FU.

The invention will now be described in more detail with the aid of embodiments taken as non-limiting examples and referring to the drawing in which:

- Figure 1 is a schematic representation of a lysis-resealing device according to the invention;

FIG. 2 is a basic block diagram of the method;

FIG. 3 presents tomography images showing the distribution of Tc 99 labeled erythrocytes, according to whether or not they are treated with glutaraldehyde.

Example 1: Installation

FIG. 1 is firstly referred to. A first dashed frame shows a first module 1, having a generally parallelepipedic shape, comprising a glazed front face that is not shown and that is shaped so that it can be opened and closed. On the bottom of this module are located peristaltic pumps P1, P2 and P3, and receiving means, not shown, a removable assembly which will now be described. The pumps P1 and P3 are at a predetermined constant rate. The pump P2 is controlled to vary the flow rate.

The removable assembly comprises a flexible pouch 2 of volume, containing a suspension of erythrocytes to be lysed. This pocket 2 is equipped with a flexible tubular 3 loop, cooperating with the pump P1, to ensure circulation to and from the pocket to keep the erythrocytes in suspension. This bag is further connected at its base to a flexible tubing 4 connected to the entrance of the "blood" compartment of a dialysis cartridge 5. This tubing 4 cooperates with the pump P2, which ensures the circulation of the suspension from the pocket to the cartridge. A pilot pump PS1 pilot is connected to the tubing 4 upstream of the cartridge 5, the syringe driver for the introduction of 5-FU into the erythrocyte circulation. The output of the "blood" compartment of the cartridge 5 is connected to a flexible outlet tube 6 opening out of the module 1. A second controlled PS2 syringe pump is connected to the tubing 6, which pushes syringe allowing the introduction of 5-FU into the circulation of lysed erythrocytes. A vial 7 containing a lysis solution is placed in the module 1, and connected to the "dialysate" inlet of the cartridge 5 by a flexible tubing 8, cooperating with the pump P3 ensuring the circulation of the lysis solution through the cartridge 5. Finally, the lysis solution leaving the cartridge is removed from the module 1 by a flexible evacuation pipe 9, opening into a bottle 10 located outside the module 1.

The outlet pipe 6 enters a second module 11 having a generally parallelepiped shape, having a glazed front face not shown and shaped so as to be open and closed. On the bottom of this module are arranged reception means, not shown, of elements forming part of the removable assembly which has just been partially described. It is a flexible pouch 12, connected to the tubing 6, and in which is stored the lysed suspension. A pilot pump PS3 pilot is connected to the tubing 6, and can inject the resealing product.

The removable assembly is made entirely of flexible and transparent plastic, allowing a complete visibility of the process.

The device is further provided with various means not shown:

means for cooling the inside of the module 1 and regulating the temperature between + 2 and + ^{40 °} C., comprising, inter alia, a temperature probe placed on the pipe 3 to measure the temperature of the suspension circulating therein, a temperature probe for temperature measurement T1 inside module 1,

- The module 11 is further provided with means for heating the interior of the module 11 and to regulate the temperature T2 between + 37 and + 38 ⁰ C; a temperature sensor is placed inside the module. detection means (for example ultrasound or colorimetric) of the presence of erythrocytes in the tubings, in D1 and D2,

PR1 means of pressure measurement at the entrance of the dialysis cartridge.

an electronic device receiving, on the one hand, the information coming from the temperature, pressure and detection means probes, and on the other hand the information relating to the settings of the lysis parameters; from these data, the device controls the pumps P1, P2 and P3. A process block diagram is shown in FIG.

The electronic device consists of a computer designed to operate the mimic above.

The implementation of this device leads to recover in 12 a bag containing a suspension of erythrocytes containing 5-FU.

Example 2: Production of erythrocyte encapsulating 5-FU

400 mL of blood is drawn from the patient. The blood, kept at 4 ° C, is leukocyte-washed and washed with saline to remove plasma, and placed in a flexible bag of 250 mL volume at 80% adjusted hematocrit.

An aqueous solution of 5-FU is added to the erythrocyte suspension so as to obtain the desired concentration of 5-FU in the suspension at 70% hematocrit.

1 ml of the suspension is taken at 4 ° C. and placed in the apparatus for measuring the osmotic fragility described in J. Didelon et al. 2000 above, whose operating principle has been described supra. The measurements are made over 10 minutes. The apparatus makes it possible to determine the salinity allowing to have 50% of hemolysis. This salinity is generally between 3 and 5.5 g of NaCl per liter.

The flexible pouch 2 volume 250 mL, containing the suspension of erythrocytes and 5-FU, is placed in the plant of Example 1, and the suspension and the hypotonic lysis solution are gradually admitted into the compartments. of the dialysis cartridge. Depending on the salinity parameter determined in the previous step (brittleness or osmotic resistance), the flow rate of the erythrocyte suspension in the dialyser is adjusted to between 15 and 30 ml / min. The resealing solution is added in line at 10% vol / vol to the suspension of lysed erythrocytes just upstream of the bag 12. The suspension is incubated for 30 min at 37 ° C in the bag. It is then washed with a saline solution, a preservation solution is added (SAG-mannitol), then the bag is kept at + 4 ° C until it is used.

The administration of the total volume of the suspension to the patient is performed by intravenous infusion according to the usual practices of blood transfusion.

Example 3: Targeting

A comparative study was conducted in rabbits to determine the impact of the lysis / resealing process of the invention and treatment with glutaraldehyde.

I. Making tampons

A. Buffer HBSS pH 8, 300 mOsmol / kg

Volume manufactured about 921ml Potassium phosphate monobasic anhydrous KH ₂ PO ₄ 60 mg

D-Anhydrous Glucose 1 g

CaCl ₂ 96% 131, 4 mg

MgSO ₄ 97.6 mg

KCI 400 mg NaCl 8 g

Sodium phosphate dibasic dodecahydrate Na ₂ HPO ₄ , 12H ₂ O 119.8 mg

The powders are weighed and taken up in 600 ml of sterile water. The pH is brought to 8 with 1N sodium hydroxide. The osmolarity is then measured. The volume of water is added to reach an osmolarity of 300 mosmol / kg. Storage at 4 ° C.

B. HBSS Wash Solution 0.2% Glucose, pH 8, 300 mOsmol / kg For 1000 ml of buffer: 1 g of glucose; QSP 1000ml with HBSS buffer, pH 8, 300 mOsmol / kg. Leave on ice.

C. Wash Solution HBSS Glucose 0.2%, pH 8, 300 mOsmol / kg, glycine 10mM

For 50ml of buffer: 40 mg of glycine; QSP 50 ml with 0.2% HBSS Glucose buffer, pH 8, 300 mOsmol / kg. Mix. Leave on ice.

D. 3.6% Glutaraldehyde, 0.9% NaCl (Stock Solution)

In a 50 ml Falcon, covered with aluminum foil, mix 3.6 ml of 50% glutaraldehyde with 46.4 ml of 0.9% NaCl.

Treatment

A. Denaturation of red blood cells (RGC) by glutaraldehyde 0.3% final

The RGCs (Hematocrit 70-80%) are taken up in 5 times their volume of glutaraldehyde 0.36%. This volume is prepared in a 50 ml falcon by diluting to 1/10 the 3.6% glutaraldehyde in NaCl solution. The mixture is mixed and then incubated for 15 minutes at Bain Marie 22 ° C.

B. Washes

1. Make a first volume-to-volume wash against HBSS wash buffer, 0.2% glucose, cold Glycine 1 OmM. Glycine can neutralize the action of glutaraldehyde.

2. Centrifuge 150 g, 4 ⁰ C for 5 minutes.

3. Remove the supernatant

4. Add volume to volume of HBSS buffer, glucose 0.2%

5. Resuspend the pellet 6. Repeat twice from Step 2.

III. Marking

A batch of red blood cells treated with glutaraldehyde, and an untreated batch, are then labeled with Technecium 99, washed and then each injected into a rabbit.

IV. Results

Rabbits are observed 4 hours after the injection using a gamma camera (single Photon Emission Computer Tomograph). The images are shown in Figure 3.

To the left of the figure, we see the image of the rabbit treated with Tc 99 erythrocytes without glutaraldehyde. The heart-lung block is very apparent, demonstrating that Tc 99 erythrocytes are in the bloodstream. Note their presence in the kidneys, which is explained by the strong vascularization of the kidneys and the urinary elimination of free Tc 99.

On the right of the figure, we see the image of the rabbit treated with Tc 99 erythrocytes with glutaraldehyde. Radioactivity is almost absent in the bloodstream and in the lungs and spleen. Some radioactivity appears in the kidney, translating the urinary elimination of free 99 Tc. The radioactivity, and thus the treated erythrocytes, are remarkably concentrated in the liver.

Claims

1. Erythrocyte containing 5-fluorouracil (5-FU), obtainable by:

2. Erythrocyte according to claim 1, obtainable by the implementation of steps 1 to 4, the 5-FU being added to the suspension in step 1.

3. Erythrocyte according to claim 1 or 2, containing another active ingredient.

4. Erythrocyte according to claim 3, containing oxaliplatin or another platinum derivative for anti-cancer use.

5. Erythrocyte according to any one of claims 1 to 4, having undergone chemical or thermal denaturing treatment intended to promote its recognition by erythrophagocytic cells.

6. Erythrocyte according to one of claims 1 to 5, treated with glutaraldehyde or neuraminidase.

7. Suspension comprising erythrocytes according to one of claims 1 to 6, in a pharmaceutically acceptable salt solution.

8. Suspension according to claim 7, having a hematocrit of between 40 and 70%.

9. Suspension according to claim 7 or 8, comprising erythrocytes containing 5-FU and erythrocytes containing another active ingredient.

The suspension of claim 9 wherein the other active ingredient is oxaliplatin or another platinum derivative for anti-cancer use.

11. Medicament containing erythrocytes according to one of claims 1 to 6 or a suspension according to one of claims 7 to 10.

12. The medicament as claimed in claim 11, for the treatment of primary or secondary cancers sensitive to 5-FU.

13. The medicament as claimed in claim 12, for the treatment of secondary cancers present in the liver, the spleen, the lungs and the lymph nodes.

14. The medicament as claimed in claim 12, for the treatment of a tumor of the liver, in particular liver metastasis.

15. Soft bag for infusion, containing a medicament according to any one of claims 11 to 14.

16. Use of erythrocytes according to one of claims 1 to 6 or a suspension according to one of claims 7 to 10 for the manufacture of an anti-cancer drug.

17. Use according to claim 16 for targeting the liver.

18. Erythrocyte population containing 5-fluorouracil and oxaliplatin or erythrocytes containing 5-fluorouracil and erythrocytes containing oxaliplatin.

19. Erythrocytes containing oxaliplatin.