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    • 专利摘要:
    The invention relates to a pharmaceutical composition containing a PLP enzyme, and optionally its cofactor, pyridoxal phosphate (PLP), and / or a phosphated or non-phosphated precursor of PLP, its use as a medicament, its production method and a method of therapeutic treatment thereto. The pharmaceutical composition comprises erythrocytes and a pharmaceutically acceptable vehicle, the erythrocytes encapsulating the PLP enzyme. The PLP enzyme may be methioninase, tyrosine phenol-lyase, tyrosine aminotransferase or cystathionine beta-synthase.
    公开号:FR3017299A1
    申请号:FR1451100
    申请日:2014-02-12
    公开日:2015-08-14
    发明作者:Yann Godfrin；Vanessa Bourgeaux；Fabien Gay；Thomas Cortese
    申请人:Erytech Pharma SA；
    IPC主号:
    专利说明:

    [0001] The invention relates to a pharmaceutical composition containing a PLP enzyme, for example methioninase, and optionally its cofactor, pyridoxal phosphate (PLP), and / or a phosphated or non-phosphated precursor of PLP, its use as a medicament, and its method. of production and a method of therapeutic treatment thereto. Pyridoxal phosphate (PLP), a derivative of vitamin B6, is a cofactor used by a wide variety of enzymes. Called "PLP enzymes", they are a group of about 145 distinct enzymes involved mostly in the metabolic pathways of amino acid transformation. The reactions catalysed by these enzymes include decarboxylations, transaminations or elimination reactions (Percudani and Perrachi, EMBO reports vol.4 No. 9, 2003). Due to the large number of enzymes belonging to the group of PLP enzymes and reactions catalyzed by them, their potential for use in human therapy has been investigated. Among the various therapeutic intervention opportunities associated with PLP enzymes, their use in the treatment of cancer and cardiovascular pathologies has been the subject of numerous studies (EI-Sayed and Shindia Targets in Gene Therapy Prof. Yongping you ed ., 2011). More particularly, methioninase would have an interest in depleting plasma methionine and inducing apoptosis of auxotrophic tumor cells for this amino acid. It has been shown that many human tumor cells are unable to proliferate when methionine is replaced by its precursor homocysteine while normal cells have the ability to proliferate in such a medium. This dependence on methionine has been observed in particular for cell lines derived from cancers of the breast, lung, colon, kidney, bladder, melanoma and glioblastoma (Durando et al., Bull Cancer 2008; 95 (1): 69-76).
    [0002] Despite the therapeutic interest of PLP enzymes, the development of treatments based on a systemic administration of these enzymes faces significant limitations: PLP enzymes are mainly obtained from prokaryotic organisms and are therefore highly immunogenic when given to humans - their half-life in plasma is short, requiring frequent administrations or large doses to achieve sufficient activity - the low bioavailability of the PLP cofactor plasma causes a rapid drop in activity after administration. These limitations have been widely described in the case of methioninase. Sun et al. produced a recombinant methioninase in the Escherichia col bacteria from the gene encoding the enzyme extracted from the bacterium Pseudomonas putida. The enzyme thus obtained, called rMETase, was injected intravenously into immunodeficient mice.
    [0003] Twenty-four hours after injection, the plasma activity of the enzyme, determined in vitro without the addition of PLP, was undetectable, signaling its short duration of action (Sun et al., Cancer Research 63, 8377-8383, 2003). One year later, the same team published the results of rMETase administration in the macaque monkey (Yang et al., Clinical Cancer Research Vol 10, 2131-2138, 2004). In this study, doses of rMETase of 1000, 2000 and 4000 units / kg were administered intravenously to 6 monkeys. A second injection was performed 28 days after the first and led in 2 monkeys to anaphylactic shock resulting in the death of one of the two animals. The immunogenicity of rMETase also resulted in the development of IgM (majority) and IgM anti-rMETase antibodies for the majority of treated animals (4 out of 6). The neutralizing nature of these antibodies has been demonstrated in vitro. To overcome the limitations of short half-life and immunogenicity of their methioninase, the same authors then proposed to use the pegylation of their enzyme. Grafting of PEG groups is a known technique for increasing the half-life and decreasing the immunogenicity of therapeutic proteins. Activated PEG derivatives were brought into contact with rMETase to obtain PEG-rMETase. This modification of the enzyme increased its half-life in mice by 2 h for the free enzyme to 38 h for PEG-rMETase. This significant increase in half-life is accompanied by a decrease in immunogenicity (Sun et al., Cancer Research 63, 8377-8383, 2003). Although pegylation partially addressed the half-life and immunogenicity issues, a major problem with PLP enzymes remains: the low bioavailability of the cofactor in the plasma. PLP enzymes are catalytically active in the presence of their cofactor PLP, which is called a holoenzyme. After injection, the holoenzyme is rapidly converted to an inactive apoenzyme due to the loss of the PLP cofactor. Exogenously supplied PLP is rapidly unavailable for the enzyme, with the plasma half-life of free PLP being only about 15 minutes. This phenomenon has been demonstrated in the case of the PLP Tyrosine Phenol-Lyase (TPL) enzyme. Elmer et al. (Cancer Research 38, 3663-3667, 1978) purified TPL and injected it into normal mice. 5 hours after the injection, blood samples were taken to measure the activity of the BPD. This assay of activity was carried out according to 2 conditions: a part of the samples was assayed without addition of PLP, the other part was assayed with the addition of an optimal quantity of PLP in the reaction mixture for the assay ( these 2 conditions reflecting the activity effectively measured in the plasma and the potential activity of the enzyme if it had access to its cofactor PLP). The comparison of the results obtained shows that only 7% of the potential activity of the TPL is actually measured in the plasma. The same test was conducted with a group of mice in which, concomitantly with the injection of TPL, a large amount of PLP was administered, and then PLP reinjections were made every hour. In this case, the comparison of the assay results shows that 37% of the potential activity is actually measured in the plasma. The coadministration of PLP has thus made it possible to improve in a limited way the activity of TPL in plasma. However exogenously supplied PLP is rapidly unavailable for TPL, the plasma half-life of free PLP being about 15 minutes. Therefore, the elevation of PLP level in the plasma by repeated injections of PLP in solution is not feasible. Elmer et al. propose to provide PLP prolonged over time via an implant consisting of spermaceti and peanut oil injected intramuscularly at the hip. However, this solution is not convincing, it fails to restore the activity effectively measured in the plasma beyond 25% of the potential activity and it does not improve in a statistically significant way the anti-aging effect. TPL tumor in mice implanted with a B-16 melanoma tumor. Similar observations were made with methioninase. Sun et al. (Cancer Research 63, 8377-8383, 2003) find that, in vitro, the PLP-rMETase holoenzyme is relatively stable but that, in vivo, this complex dissociates rapidly leading to a loss of rMETase activity. The authors further show that the methionine depletion time obtained with rMETase as well as with PEG-rMETase can be improved by PLP supplementation via the implantation of a PLP pump (PLP-administering pump). continued). Nevertheless, this continuous administration device will invariably encounter the problem of the low bioavailability of PLP in plasma. Therefore, although the therapeutic potential of PLP enzymes has been the subject of much research that led in particular to the preparation of pilot clinical studies for methioninase, no demonstration of the clinical efficacy of these enzymes could be demonstrated. made. Thus in order to exploit the therapeutic potential of PLP enzymes, it would be advantageous to have a solution to maintain these enzymes in the presence of an optimal and available amount of PLP.
    [0004] Various methods have been described to allow the incorporation of active ingredients into erythrocytes. Among these methods, the so-called lysis-resealing technique is the most widespread. This technique includes three variants, hypotonic dialysis, hypotonic preswelling and hypotonic dilution, all based on the difference in osmotic pressure between the inside and the outside of the erythrocytes. These variants have in common the following five steps: a globular pellet is washed and centrifuged one or more times with a physiological buffer, the erythrocytes are brought into contact with a hypotonic liquid medium resulting in the opening of pores in the erythrocyte membrane, the active principle enters the erythrocytes, the pores are closed ("resealed") using a hypertonic buffer, enclosing the active ingredient inside the erythrocytes, then they are suspended in a preservative solution . The hypotonic dialysis technique is the most interesting and has been the subject of industrial developments. That described in EP 1 773 452 is the most efficient at present, it has the advantage of being reproducible and to improve the encapsulation efficiency of the active ingredient. The encapsulation of enzymes in erythrocytes, in order to limit the risks related to the immunogenicity of the enzyme and to prolong its half-life, has already been proposed in work that has been the subject of scientific publications. The encapsulation of an enzyme, L-asparaginase has been described in EP 1 773 452, as well as arginine deiminase in EP 1 874 341. Previous work does not concern a cofactor enzyme and does not address the complexity related to the kinetics of an enzyme to PLP and its cofactor PLP.
    [0005] An object of the invention is to provide a pharmaceutical composition containing a PLP enzyme, which makes it possible to limit the risks related to the immunogenicity of the enzyme, to prolong its half-life while putting the enzyme in the presence of an optimal and available amount of its PLP cofactor. The invention thus relates to a suspension of erythrocytes in a pharmaceutically acceptable vehicle or a pharmaceutical composition comprising erythrocytes and a pharmaceutically acceptable vehicle, the erythrocytes encapsulating a PLP enzyme. Hereinafter, a composition will be used to designate the suspension and the pharmaceutical composition without distinction. By "encapsulant" is meant that the active ingredient (enzyme and optionally cofactor and / or other molecule) is present essentially or totally inside. "Essentially" means that a minority proportion of the active ingredient can still be trapped in the membrane. The composition contains in particular from 0.01 to 30, preferably from 0.05 to 10 mg of PLP enzyme per ml of red blood cells. According to a first embodiment, the PLP enzyme is methioninase, also called, inter alia, L-methioninase, Methionine Gamma Lyase MGL, EC number 4.4.1.11, CAS number 42616-25-1. For methioninase sources that can be used according to the invention, reference may in particular be made to the publication El Sayed A, Applied Microbiol. Biotechnol. (2010) 86: 445-467. In a second embodiment, the PLP enzyme is Tyrosine Phenol-Lyase or TPL, EC 4.1.99.2, CAS 9059-31-8. One can refer to H. Kumagai et al., J. Biol. Chem. 245, 7: 1767-72 and 245, 7: 1773-7.
    [0006] In a third embodiment, the PLP enzyme is tyrosine aminotransferase (hTATase), EC 2.6.1.5, CAS 9014-55-5. Reference can be made to R. Rettenmeier et al., Nucleic Acids Res. 1990, 18, 13: 3583-61. According to a fourth embodiment, the PLP enzyme is cystathionine beta-synthase or synthase, EC 4.2.1.22, CAS 9023-99-8. Reference can be made to J. Kraus et al., J. Biol. Chem. 1978, 253, 18: 6523-8. The composition may further comprise an enzyme co-factor, PLP, and / or a precursor thereof, which may be a non-phosphate precursor, such as a non-phosphate form of vitamin B6, and / or a phosphate precursor, such as pyridoxine phosphate (PNP). Vitamin B6 exists in different forms, phosphates and non-phosphates. Pyridoxine phosphate (PNP), pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP) are the phosphatic forms. The corresponding nonphosphate forms are pyridoxine (PN), pyridoxal (PL), and pyridoxamine (PM). The non-phosphate forms of vitamin B6 can cross the erythrocyte membrane, which is difficult for phosphate forms. Following the predominant route, pyridoxine (PN) is transformed into PNP under the effect of PN kinase, PNP is then transformed into PLP under the effect of PNP-oxidase. PLP can then be transformed into pyridoxal (PL) under the effect of PLP-phosphatase. It is easily understood that the precursor provided is likely to undergo transformations in erythrocytes during the preparation process or during storage of the composition. By non-phosphated form of vitamin B6, we mean here one of the three "vitamers" of vitamin B6 or a mixture of 2 or 3 vitamins: PL, PN and PM. The PN form is preferred. They can be in the form of salt as well.
    [0007] In one embodiment, the composition therefore comprises pyridoxal phosphate (PLP) and / or a non-phosphated form of vitamin B6 and / or a phosphate precursor, pyridoxine phosphate (PNP) and / or pyridoxamine phosphate. The composition comprises in particular from 0.05 to 600, in particular from 0.5 to 100, preferably from 5 to 50 pmoles of PLP and / or PNP and / or PMP, encapsulated per liter (L) of red blood cells. The compositions according to the invention preferably have a hematocrit greater than or equal to 35%, 40% or 45%. According to one embodiment, the composition comprises, on the one hand, erythrocytes and a pharmaceutically acceptable vehicle, the erythrocytes encapsulating PLP enzyme, eg methioninase, and, on the other hand, vitamin B6 under a non-phosphated form, preferably PN, for simultaneous, separate or sequential administration. The composition may especially be in the form of a kit, comprising, separately, erythrocytes (suspension) and vitamin B6 in a non-phosphated form, preferably PN (solution). According to one embodiment, the pharmaceutically acceptable vehicle is a "preservation solution" for erythrocytes, namely a solution in which the erythrocytes encapsulating an active ingredient are suspended in their form adapted to be stored while waiting for their injection. A preservation solution preferably comprises at least one agent for the conservation of erythrocytes, in particular chosen from glucose, dextrose, adenine and mannitol. Advantageously, the preservation solution contains inorganic phosphate for the inhibition of the PLP-intra-erythrocyte phosphatase enzyme.
    [0008] The preservation solution may be an aqueous solution comprising NaCl, adenine and at least one of glucose, dextrose and mannitol. According to one characteristic, it further comprises inorganic phosphate. The preservation solution may comprise NaCl, adenine and dextrose, preferably AS3 medium. According to one characteristic, it further comprises inorganic phosphate. The preservation solution may comprise NaCl, adenine, glucose and mannitol, preferably SAG-Mannitol or ADsol medium. According to one characteristic, it further comprises inorganic phosphate. In particular, the composition or suspension, in preservation solution, is characterized by an extracellular hemoglobin level maintained at or below 0.5, in particular at 0.3, especially at 0.2, preferably at 0.15. more preferably at 0.1 g / dl at 72 h and stored at 2 to 8 ° C. In particular, the composition or suspension, in preservation solution, is characterized by an extracellular hemoglobin level maintained at or below 0.5, in particular at 0.3, especially at 0.2, preferably at 0.15. more preferably at 0.1 g / dl for a period of between 24 hours and 20 days, especially between 24 and 72 hours and storage at a temperature between 2 and 8 ° C. The extracellular hemoglobin level is advantageously measured by the manual reference method described in G. B. Blakney and A. J. Dinwoodie, Clin. Biochem. 8, 96- 102, 1975. Automata also exist which also make it possible to make this measurement, with a sensitivity of their own. In particular, the composition or suspension, in the form of a preservation solution, is characterized by a hemolysis rate maintained at or below 2, in particular at 1.5, preferably at 1% at 72 hours, and preservation at a temperature of between 2 and and 8 ° C.
    [0009] In particular, the composition or suspension, in preserving solution, is characterized by a hemolysis rate maintained at or below 2, especially at 1.5, preferably at 1% for a period of between 24 h and 20 days, especially between 24 and 72 h and at a temperature between 2 and 8 ° C. In particular, the hematocrit of the suspension is equal to or greater than 35%, 40%, 45%. According to a particular modality, the metabolism of vitamin B6 in erythrocytes is modified in order to increase the intra-erythrocyte concentration of PLP by the increase in intra-erythrocyte levels of PN-kinase and PNP-oxidase and / or the decrease in According to one characteristic, the composition comprises, in addition to the PLP enzyme, eg methioninase, and PLP or a precursor thereof, PN-kinase and / or lipopolysaccharide. PNP-oxidase and / or an agent inhibiting PLP-phosphatase. These enzymes or agents can be encapsulated in the erythrocytes or be in extra- and intra-erythrocyte. This or these enzymes or agents may also be administered separately, in particular being mixed with the non-phosphated vitamin B6 formulation when it is separated from the erythrocyte suspension.
    [0010] The invention thus relates to such compositions for use as a medicament. The subject of the invention is, in particular, a medicament making it possible to provide, to a patient in need thereof, a PLP enzyme and its cofactor, under conditions of good bioavailability, which means that the enzyme and its cofactor are available. for each other and in an amount effective for the enzyme to be active and effective in a therapeutic application. The drug is intended in particular to deplete or reduce the plasma or circulating concentration and / or at an organ, a substrate of the enzyme. According to a first sub-object, the drug comprises methioninase and makes it possible to deplete or reduce plasma or circulating methionine in a patient who needs it. The drug is an anticancer drug, it allows the treatment of cancer, including cancer including tumor cells auxotrophic for methionine, including breast cancer, lung, colon, kidney, bladder, melanoma and glioblastoma. According to a second sub-object, the drug comprises methioninase and can deplete or reduce plasma or circulating and / or hepatic homocysteine in a patient who needs it. The drug can treat homocysteinuria and / or hyperhomocysteinemia and / or associated conditions, such as cardiovascular disease, central nervous system, ocular system and / or skeletal system (EI-Sayed and Shindia Targets in gene therapy Prof. Yongping you ed., 2011). According to a third sub-object of the invention, the medicament contains TPL and makes it possible to deplete or reduce the plasma or circulating tyrosine in a patient who needs it. The drug is an anticancer drug, it allows the treatment of cancer, including cancer including tumor cells auxotrophic for tyrosine, including melanoma. According to a fourth sub-object of the invention, the medicament contains hTATase, and makes it possible to deplete or reduce the plasma or circulating and / or hepatic tyrosine in a patient who needs it. The drug can treat a rare disease related to a deficiency of this PLP enzyme, including Richner-Hanhart syndrome (tyrosinemia type II). According to a fifth sub-object of the invention, the medicament contains cystathionine beta-synthase and makes it possible to deplete or reduce plasma or circulating and / or hepatic homocysteine in a patient who needs it. The medicament makes it possible to treat homocysteinuria and / or hyperhomocysteinemia and / or associated pathologies, such as cardiovascular disease, of the central nervous system, of the ocular system and / or of the skeleton. The invention also relates to a process for the preparation of a pharmaceutical composition comprising erythrocytes encapsulating a PLP enzyme, eg methioninase, a pharmaceutically acceptable carrier, and pyridoxal phosphate (PLP) and / or a phosphated or non-phosphated precursor of PLP, the process comprising the following steps: optionally, and preferably, an erythrocyte pellet is washed and centrifuged one or more times with a physiological buffer; the erythrocyte suspension is brought into contact with a hypotonic liquid medium resulting in the opening of pores in the erythrocyte membrane; the erythrocyte suspension is contacted with the PLP enzyme, e.g. methioninase, before or after pore opening; the PLP enzyme, e.g. methioninase, enters the erythrocytes; the pores are closed again with the aid of an isotonic or hypertonic buffer, advantageously hypertonic, and a suspension of resealed erythrocytes containing the PLP enzyme, e.g. methioninase, is collected; optionally the erythrocyte suspension is incubated to remove the most fragile erythrocytes; the erythrocyte suspension is washed and conditioned with a preservation solution; process in which: - the PLP and / or, if appropriate, a phosphated precursor of PLP, is co-encapsulated with the PLP enzyme, eg methioninase, - if necessary, the non-phosphated precursor of PLP is added to the suspension of erythrocytes before or after the opening of the pores, and / or - if necessary, the non-phosphated precursor of PLP is added during the incubation or the preservation solution.
    [0011] The erythrocyte suspension is brought into contact with a hypotonic liquid medium resulting in the opening of pores in the erythrocyte membrane. We have seen that there are three variants in the lysis-resealing technique, which are hypotonic dialysis, hypotonic preswelling and hypotonic dilution, all based on the difference in osmotic pressure between the inside and the outside of the cells. erythrocytes. Hypotonic dialysis is preferred. The erythrocyte suspension encapsulating the PLP enzyme, eg methioninase, and optionally PLP and / or a PLP precursor is particularly likely to be obtained by the following method: 1 - suspending an erythrocyte pellet in an isotonic solution at a hematocrit level equal to or greater than 65%, refrigeration between + 1 and + 8 ° C, 2 - lysis procedure, at a temperature maintained between + 1 and + 8 ° 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 ° C, in a dialysis device, such as a blood sausage or a cartridge of dialysis (the cartridge is preferred); 3 - encapsulation procedure by addition, preferably progressive, of the active ingredient (s) to be encapsulated (especially in pre-reconstituted solution) in the suspension before or during lysis, at a temperature maintained between + 1 and + 8 ° C; and 4 - resealing procedure conducted in the presence of an isotonic or hypertonic solution, advantageously hypertonic, at a higher temperature, especially between + 30 and + 42 ° C. In a preferred variant, one can draw inspiration from the method described in WO-A20 2006/016247 (EP 1 773 452): 1 - suspending an erythrocyte pellet in an isotonic solution at an equal hematocrit level or greater than 65%, refrigeration between + 1 and + 8 ° C, 2 - measurement of osmotic fragility from a sample of erythrocytes of this same pellet, 3 - lysis procedure, at a temperature maintained between + 1 and + 8 ° C, comprising passing the suspension of erythrocytes at a hematocrit level equal to or greater than 65% and a refrigerated hypotonic lysis solution between + 1 and + 8 ° C, in a device of dialysis, such as a coil or a dialysis cartridge (the cartridge is preferred); the lysis parameters being adjusted according to the previously measured osmotic fragility; in particular, depending on the measured osmotic fragility, the flow rate of the suspension of erythrocytes passing through the dialysis device is adjusted or the osmolarity of the lysis solution is adjusted; and 4 - encapsulation procedure by addition, preferably progressive, of the active ingredient (s) to be encapsulated (especially in pre-reconstituted solution) in the suspension before or during lysis, at a temperature maintained between + 1 and + 8 ° C; and 5 - resealing procedure conducted in the presence of an isotonic or hypertonic solution, advantageously hypertonic, at a higher temperature, especially between + 30 and + 42 ° C. In particular, for dialysis, the erythrocyte pellet 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 ° C, preferably between + 2 and + 6 ° C, typically around + 4 ° C. According to a particular modality, the hematocrit content is between 65 and 80%, preferably between 70 and 80%.
    [0012] When measured, the osmotic fragility is advantageously measured on the erythrocytes just before the lysis step, in the presence or absence, preferably in the presence, of the active ingredient (s) to be encapsulated. 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 carried out is quickly exploited, that is to say that the lysis procedure is carried out shortly 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. Regarding the manner of operating the lysis-resealing procedure, with measurement and taking into account of the osmotic fragility, the skilled person can refer for more details to WO-A-2006/016247. This document is incorporated herein by reference. An improvement of encapsulation techniques has been described in FR 1354204 filed May 7, 2013, to which the skilled person can refer and which is incorporated herein by reference. Thus, according to one embodiment, the erythrocytes encapsulating the active principle, namely PLP enzyme, eg methioninase, and optionally one or more other active ingredients such as PLP and / or a PLP precursor, are obtained by a process comprising encapsulation of the active ingredient inside erythrocytes by lysis-resealing, obtaining a suspension or a pellet comprising erythrocytes incorporating the active principle and a solution with an osmolality greater than or equal to 280 mOsmol / kg, in particular between about 280 and about 380 mOsmol / kg, preferably between about 290 and about 330 mOsmol / kg, incubating the pellet or suspension as is or after adding an incubation solution, at an osmolality greater than or equal to 280 mOsmol / kg, in particular between about 280 and about 380 mOsmol / kg, preferably between about 290 and about 330 mOsmol / kg. Incubation is carried out especially for a duration greater than or equal to 30 minutes, in particular greater than or equal to 1 hour. The liquid medium is then removed from the incubated suspension and the suspended erythrocytes are suspended in a solution that makes it possible to inject the suspension into a patient, preferably a preservation solution that allows the suspension of the suspension to be injected. a patient. The osmolality indicated is that of the solution in which the erythrocytes are in suspension or in pellet at the moment considered. According to a particular modality, during the production or storage process or in the final formulation, a non-phosphated precursor of PLP is provided, in particular a non-phosphated form of vitamin B6. This compound may for example be incorporated in the incubation solution or in the preservation solution, or in the formulation before injection when a pre-injection dilution is carried out. According to one characteristic, during the production or storage process or in the final formulation, it is especially possible to provide from 0.1 to 250, preferably from 1 to 50 mM, of PN and / or PL and / or PM. As described above a fraction of these non-phosphate derivatives of vitamin B6 will be converted to PLP in red blood cells. By "stabilized erythrocyte suspension" is meant in particular a suspension having an extracellular hemoglobin content which remains less than or equal to 0.2 g / dL until it is used in humans, which can be used in particular to 1 to 72 hours after the production of the batch of erythrocytes incorporating the active ingredient. By "stabilized erythrocyte suspension ready-to-use" is meant the stabilized suspension in a solution for injection to a patient, especially in preservative solution. Its hematocrit is generally equal to or greater than 35%, 40% 20 or 45%. By "erythrocyte pellet" is meant a concentrate or concentrate of erythrocytes collected after separation of erythrocytes from the liquid medium in which they were previously suspended. Separation can be by filtration or centrifugation. Centrifugation is the means generally used for such separation. A pellet 25 comprises a certain proportion of the liquid medium. Generally, the pellet has a hematocrit of between 70 and 85%. By "incubation solution" is meant the solution in which the erythrocytes encapsulating an active ingredient are present during the incubation step. The incubation can be done over a wide range of hematocrit, especially between 10 and 85% hematocrit. "Fragile erythrocytes" means erythrocytes derived from the incorporation procedure which are capable, once in suspension in a preservation solution, of being lysed when the suspension is stored at 2 to 8 ° C., especially at room temperature. from 1 to 72 hours. By "initial hematocrit" is meant the hematocrit before cell loss due to lysis of fragile erythrocytes during incubation. The method may especially comprise the following steps: (a) encapsulation of the active ingredient (s) to be encapsulated (PLP enzyme, eg methioninase, and optionally PLP and / or a PLP precursor) within erythrocytes, comprising the contacting the erythrocytes with a hypotonic medium (allowing the opening of pores in the erythrocyte membrane), bringing it into contact with the active principle (to allow its entry into the erythrocytes), resealing the erythrocytes, in particular at the using an isotonic or hypertonic medium, advantageously hypertonic, (b) obtaining or preparing a suspension or a pellet comprising erythrocytes incorporating the active ingredient and a solution with an osmolality greater than or equal to 280 mOsmol / kg, in between about 280 and about 380 mOsmol / kg, preferably between about 290 and about 330 mOsmol / kg, (c) incubating the pellet or suspension of step (b) in the state or at least by addition of an incubation solution, to an osmolality greater than or equal to 280 mOsmol / kg, in particular between about 280 and about 380 mOsmol / kg, preferably between about 290 and about 330 mOsmol / kg, for a greater duration or equal to 30 minutes, especially greater than or equal to 1 hour, (d) removal of the liquid medium from the incubated suspension of step (c), (e) suspension of the erythrocytes obtained under (d) in a solution allowing injecting the suspension into a patient, preferably a preservation solution for injecting the suspension into a patient.
    [0013] Vitamin B6 in a non-phosphated form may be added during the encapsulation step in step (a) or during the incubation in step (c) or in the preservation solution. According to a first modality, the step following the encapsulation by lysis-resealing, in particular step (b), comprises at least 1 washing cycle, preferably 2 or 3 washing cycles, by dilution of the suspension or the pellet. obtained in the lysis-resealing step or step (a) in a solution, with an osmolality greater than or equal to 280 mOsmol / kg, in particular between about 280 and about 380 mOsmol / kg, preferably between about 290 and about 330 mOsmol / kg, then obtaining an erythrocyte pellet or suspension. This pellet or this suspension comprises erythrocytes incorporating the active ingredient and a solution with an osmolality greater than or equal to 280 mOsmol / kg, in particular between about 280 and about 380 mOsmol / kg, preferably between about 290 and about 330 mOsmol / kg. . The following steps are then applied, e.g. (c), (d) and (e). According to a second modality, in the lysis-resealing step or step (a), the resealing of the erythrocytes using an isotonic or hypertonic medium produces the suspension of erythrocytes that can then be incubated, eg the suspension of step (b) in a solution with an osmolality greater than or equal to 280 mOsmol / kg, in particular between about 280 and about 380 mOsmol / kg, preferably between about 290 and about 330 mOsmol / kg. In other words, the lysis-resealing step or step (a) comprises a step of resealing erythrocytes in which the suspended erythrocytes encapsulating an active ingredient, are mixed with an isotonic or hypertonic resealing solution, advantageously hypertonic, producing an erythrocyte suspension at an osmolality greater than or equal to 280 mOsmol / kg, in particular between about 280 and about 380 mOsmol / kg, preferably between about 290 and about 330 mOsmol / kg. In this modality, the incubation step or step (c) comprises incubating the suspension resulting from resealing. The incubation is carried out for a duration greater than or equal to 30 minutes, in particular greater than or equal to 1 hour. The following steps are then applied, e.g. (d) and (e). The steps following lysis-resealing, eg (b) to (e), are conducted under conditions resulting in the lysis of fragile erythrocytes, or a majority of them, especially more than 50, 60, 70, 80 or 90%, or more. To do this, we can play on the incubation time, the incubation temperature and the osmolality of the solution in which the erythrocytes are in suspension. The higher the osmolality, the longer the incubation period can be. The lower the osmolality, the shorter the incubation can be to achieve the same effect. Similarly, the higher the temperature, the shorter the incubation period, and vice versa. One or more wash cycles will then remove cell debris and extracellular hemoglobin, as well as the extracellular active ingredient.
    [0014] According to the invention, a washing cycle comprises diluting the suspension or the erythrocyte pellet and then separating the erythrocytes from the washing solution. Preferably, a washing step preferably comprises 2 or 3 dilution-separation cycles. The separation can be carried out by any suitable means, such as filtration and centrifugation. Centrifugation is preferred.
    [0015] Incubation is not limited by the hematocrit of the suspension. Thus it is possible to incubate a suspension having an initial hematocrit generally between 10 and 85%, especially between 40 and 80%. We are talking about 70% pellet and suspension below this value. The elimination step or (d) is intended to "eliminate the liquid part of the incubated suspension or pellet, in order to eliminate in particular cell debris and extracellular hemoglobin, as well as, consequently, the active principle extracellular. According to a first modality of the elimination step or (d), a separation is carried out, in particular centrifugation, this being notably applicable to a suspension. This separation may be followed by one or more, for example 2 or 3, washing cycles, by dilution in isotonic solution, then separation, in particular by centrifugation. According to a second embodiment of the elimination step or (d), dilution is carried out before separation, in particular centrifugation, this being applicable to a suspension or a pellet. The dilution can be carried out in particular with an isotonic washing solution or with a preservation solution. The final step or (e) is to prepare the final suspension such that it can be administered to the patient without further treatment.
    [0016] According to a first modality of this step, a dilution of the erythrocyte pellet resulting from the elimination step or (d) with the injection solution, in particular preservation. According to a second modality of this step, one or more washing cycles of the erythrocyte pellet resulting from the elimination step or (d) with the injection solution, especially preservation, by dilution followed by separation. After washing, the erythrocytes are resuspended in the injection solution, especially for preservation. The method of the invention may furthermore comprise one, several or all of the following characteristics: the incubation step or (c) is carried out at a temperature of between about 2 and about 39 ° C., for a sufficient duration to ensure the lysis of fragile erythrocytes; the incubation step or (c) is carried out at a low temperature, in particular between about 2 and about 10 ° C., in particular between about 2 and about 8 ° C., and lasts from about 1 hour to about 72 hours. in particular from about 6 hours to about 48 hours, preferably from about 19 hours to about 30 hours; the incubation step or (c) is carried out at a higher temperature of between about 20 and about 39 ° C., especially at room temperature (25 ° C. ± 5 ° C.) and lasts about 30 minutes at about 10 hours, especially about 1 hour to about 6 hours, preferably about 2 hours to about 4 hours; it is possible to work at a temperature even higher than the ambient temperature, but this may have a negative impact on the cellular yield, the P50 and / or the 2,3-DPG content; in the incubation stage or (c), the suspension is at an initial hematocrit of between 10 and 85%, in particular between 40 and 80%; a pellet from separation having, for example, a hematocrit of between 70 and about 85 (±) / 0, or a diluted pellet having a hematocrit of between about 40 and 70% can be incubated; the incubation step comprises stirring the suspension; the incubation step does not include stirring; as a solution for the washing and / or the incubation, an aqueous solution of NaCl is used to obtain the desired osmolality; for example, a solution may thus comprise 0.9% NaCl; this solution may also comprise, in particular in addition to NaCl, glucose, in particular glucose monohydrate, monosodium phosphate dihydrate, disodium phosphate dodecahydrate; for example, a composition comprises: 0.9% NaCl, 0.2% glucose monohydrate, 0.034% monosodium phosphate dihydrate, 0.2% disodium hydrogen phosphate dodecahydrate; washing in the final stage or (e) is carried out with the preservation solution; the osmolality of the solution (liquid part) in the ready-to-use suspension or that can be injected to the patient is between about 280 and about 380 mOsmol / kg, preferably between about 290 and about 330 mOsmol / kg ; - the hematocrit of the suspension ready-to-use or injectable to the patient is equal to or greater than 35%, 40% or 45%; all the washing steps are carried out, incubation with the preservation solution; the washing solution of step (b) and / or the washing solution of step (e) and the preservation solution are of the same composition and comprise one or more compounds that promote the conservation of erythrocytes; the storage solution (and the washing solution or solutions, if any) is an aqueous solution comprising NaCl, adenine and at least one of glucose, dextrose and mannitol; the storage solution (and the washing solution or solutions, if appropriate) comprises NaCl, adenine and dextrose, preferably AS3 medium; the preservation solution (and the washing solution or solutions, if any) comprises NaCl, adenine, glucose and mannitol, preferably SAG-Mannitol or ADsol medium.
    [0017] The methods according to the invention comprise in particular the following step: (a) encapsulation of an active ingredient inside erythrocytes, comprising contacting with a hypotonic medium allowing the opening of pores in the erythrocyte membrane contacting with the active principle to allow it to enter the erythrocytes, resealing erythrocytes using an isotonic or hypertonic medium. Note that the active ingredient may be present in the suspension of erythrocytes before lysis thereof, or be added during lysis or after lysis, but always before resealing. In one embodiment of this step (a), the method comprises the following substeps: (a1) providing a suspension of erythrocytes at a hematocrit of 60 or 65% or higher, (a2) the osmotic fragility of the erythrocytes in this suspension, (a3) lysis and internalization procedure of the active principle (s), comprising the passage of the suspension of erythrocytes in a dialysis device, in particular a cartridge of dialysis, countercurrent to a lysis solution, adjusting the flow rate of the erythrocyte suspension or adjusting the flow rate of the lysis solution or adjusting the osmolarity of the lysis solution, depending on the osmotic fragility measured under (a2), (a4) procedure for resealing erythrocytes.
    [0018] Another subject of the invention is a method of therapeutic treatment intended to provide, to a patient who needs it, a PLP enzyme and its cofactor, under conditions of good bioavailability, which means that the enzyme and its cofactor are available for each other and in an amount effective for the enzyme to be active and effective in a therapeutic application. This method aims in particular to deplete or reduce the plasma or circulating concentration and / or at an organ, a substrate of the enzyme. This method comprises the administration of an effective amount of a composition according to the invention or the use of a kit according to the invention. According to a first sub-object, the invention is a method of therapeutic treatment for depleting or reducing plasma or circulating methionine in a patient who needs it. This method comprises the administration of an effective amount of a composition according to the invention or the use of a kit according to the invention, comprising methioninase and its cofactor. The method is a method of treating cancer, including a cancer comprising tumor cells auxotrophic for methionine, including breast cancer, lung, colon, kidney, bladder, melanoma and glioblastoma. According to a second sub-object, the invention is a therapeutic treatment method for depleting or reducing plasma or circulating and / or hepatic homocysteine in a patient who needs it. This method comprises the administration of an effective amount of a composition according to the invention or the use of a kit according to the invention, comprising methioninase and its cofactor. The method is a method of treating homocysteinuria and / or hyperhomocysteinemia and / or conditions associated with hyperhomocysteinemia, such as cardiovascular disease, central nervous system, ocular system and / or skeleton.
    [0019] According to a third sub-object of the invention, the invention is a method of therapeutic treatment for depleting or reducing plasma or circulating tyrosine in a patient who needs it. This method comprises the administration of an effective amount of a composition according to the invention or the use of a kit according to the invention, comprising TPL and its cofactor. The method is a method of treating a cancer, including a cancer comprising tumor cells auxotrophic for tyrosine, including melanoma. According to a fourth sub-object of the invention, the invention is a method of therapeutic treatment for depleting or reducing plasma or circulating and / or hepatic tyrosine in a patient who needs it. This method comprises the administration of an effective amount of a composition according to the invention or the use of a kit according to the invention, comprising hTATase and its cofactor. The method is a method of treating a rare disease related to a deficiency of this PLP enzyme, including Richner-Hanhart syndrome (tyrosinemia type II). According to a fifth sub-object of the invention, the invention is a method of therapeutic treatment for depleting or reducing plasma or circulating and / or hepatic homocysteine in a patient who needs it. This method comprises the administration of an effective amount of a composition according to the invention or the use of a kit according to the invention, comprising cystathionine beta-synthase and its cofactor. The method is a method of treating homocysteinuria and / or hyperhomocysteinemia and / or conditions associated with hyperhomocysteinemia, such as cardiovascular disease, central nervous system, ocular system and / or skeleton. The composition used in these therapeutic applications may further comprise the co-factor of this PLP enzyme, namely PLP, and / or a precursor thereof, which may be a non-phosphated precursor, such as a non-phosphate form. phosphated vitamin B6, and / or a phosphate precursor, such as pyridoxine phosphate (PNP). The composition may also comprise PN-kinase, PNP-oxidase, a PLP phosphatase inhibiting agent. More generally, the method of treatment may comprise the administration of a composition or a kit as described above. The patient, in one or more doses, in particular one or two, per month of treatment, is administered with 50 to 300 ml of suspension or composition of hematocrit greater than or equal to 35%, 40% or 45% in one or more injections. In particular, it is administered by intravenous or intraarterial injection, in particular by infusion. Alternatively, an effective amount of a composition comprising erythrocytes encapsulating PLP enzyme, eg methioninase, and an effective amount of a solution containing a non-phosphate form of vitamin B6, are separately administered to the same patient. , preferably PN. This non-phosphate form of vitamin B6 can be administered by injection, simultaneously or separately with the suspension of erythrocytes, or by any other route, especially orally. In a first embodiment of this method, the patient is injected with a suspension of erythrocytes encapsulating the active principle (s) prepared (s) between 1 and 72 hours, especially between 10 and 72 hours before injection. This suspension has a hematocrit equal to or greater than 35%, 40% or 45%. It is in a preservation solution. The level of extracellular hemoglobin is equal to or less than 0.5, in particular to 0.3, especially to 0.2, preferably to 0.15, more preferably to 0.1 g / dl, and / or the hemolysis equal to or less than 2, especially 1.5, preferably 1%. The suspension is not washed or the like before injection.
    [0020] The invention will now be described in more detail with the aid of embodiments given as non-limiting examples and referring to the drawing in which: FIGS. 1 and 2. Comparison of the intra-erythrocyte concentrations (FIG. extracellular (FIG. 2) of PLP after incubation of a suspension of GR-MGL-PLP with pyridoxine (PN) at different concentrations. The suspension of GR-MGL-PLP incubated for 3 h and 24 h at ambient temperature in the absence of pyridoxine (0 mM) has a basal PLP level of approximately 3.9 μM. Incubation of the suspensions with pyridoxine at 2 mM and 4 mM makes it possible to increase the intra-erythrocyte concentration of PLP to 8 μM after 3 hours of incubation (light gray bars) and makes it possible to reach considerably higher levels ( 11 μM and 14 μM respectively) after 24h incubation (dark gray bars). Figure 3. Pharmacokinetics of GR loaded into MGL-PLP complex. The GR-MGL-PLP2 product is obtained by lysis-resealing of a suspension containing 3 mg / ml of MGL and -30 μM of PLP. The GR-MGL-PLP3 product is obtained by lysis-resealing of a suspension containing 3 mg / ml of MGL and -125 μM of PLP. The fluorescent labeling of products (CFSE) allows a traceability of GR in vivo. The products injected intravenously to the CD1 mice (8 ml / kg) show excellent stability with a survival of the RBCs injected greater than 80% at 120h, ie 5 days after their administration. Figure 4. Pharmacodynamics of GR-MGL-PLP. The GR-MGL-PLP2 product is obtained by lysis-resealing of a suspension containing 3 mg / ml of MGL and -30 μM of PLP. The GR-MGL-PLP3 product is obtained by lysis-resealing of a suspension containing 3 mg / ml of MGL and -125 μM of PLP. Both products are administered intravenously (IV) to CD1 mice (8 ml / kg) with pyridoxine supplementation IV after 6h for mice receiving GR-MGL-PLP2. Plasma L-methionine level is measured by HPLC-MS-MS. The level of L-Met in untreated CD1 was evaluated at 82 μM. Both GR-MGL-PLP2 and GR-MGL-PLP3 lead to rapid depletion 15 min after administration reducing L-Met to 22 pM and about 30 pM respectively, then maintain more moderate depletion at 35 pM but stable between 48h and 120h. EXAMPLE 1. Method of Obtaining and Characterizing Methionine Gamma Lyase (MGL) Production of the strain and isolation of a hyper-producer clone: the natural MGL sequence of Pseudomonas putida (GenBank: D88554.1) was optimized by modification of the rare codons (in order to adapt the sequence derived from P. putida to the Escherichia coli production strain) and suppression of three putative bacterial promoters in the coding sequence (box -35, box -10 and a binding site of a transcription factor at position 56). The E. coli strain HMS174 (DE3) was transformed with the expression vector pGTPc502_MGL (T7 promoter) containing the optimized sequence and a producer clone was selected. The producing clone is precultured in GY + 0.5% glucose + kanamycin medium for 6-8 h (preculture 1) and 16 h (preculture 2) at 37 ° C. Fermentation: the production is then carried out in a fermenter in GY medium, with stirring, pressure and pH controlled from preculture 2 at an optical density of 0.02.
    [0021] The growth phase proceeds until an optical density of 10 is obtained and the induction of expression is carried out by adding 1 mM IPTG in the culture medium. The bacterial growth is monitored over 20 h and a pellet is collected after passage over hollow fiber 500 kDa and centrifugation at 15900 x g. Purification: The cell pellet is then lysed in two stages by high pressure homogenization (1000, then 600 bar) in lysis buffer at 10 ° C. The cell lysate is then clarified at 10 ° C. by addition of 0.2% PEI and centrifugation at 15900 × g and ammonium sulphate precipitation at 6 ° C. over 20 h. Two crystallization steps are carried out on the resolubilized pellet by precipitation with 10% PEG-6000 + 10% ammonium sulfate and then with 12% PEG-6000 + 0.2M NaCl at 30 ° C. After centrifugation at 15900 × g, the pellet containing the MGL protein is resuspended in solubilization buffer and passed through a 0.45 μm filter before undergoing two ion exchange chromatograms (DEAE sepharose FF). The purified protein is then passed on Mustang Q (polishing) chromatography capsule to remove the various contaminants (endotoxin, HCP host cell protein). Finally, the purified MGL is concentrated and diafiltered in a 10 kDa stream tangential filtration cassette and freeze-dried under pressure and controlled temperature at a level of -50 mg of protein per tube. Characterization: The specific activity of the enzyme is determined by measuring the NH 3 product as described in Example 4. The purity is determined by SDS-PAGE. The level of PLP after recovery in water was evaluated according to the method described in Example 5.
    [0022] Osmolarity is measured with an osmometer. The following table summarizes the main characteristics of the produced MGL: P. putida MGL Freeze-dried formulation (quantity per tube: 49.2 mg). Characteristics after recovery in 625 biL water: 78.7 mg / mL, -622 pM PLP, 50 mM Na phosphate pH 7.2, Osmolarity 300 mOsmol / kg. Specific Activity 13.2 IU / mg Purity> 98% Example 2. Co-encapsulation of MGL and PLP in murine erythrocytes. Whole blood of CD1 mice (Charles River) is centrifuged at 1000 x g, 10 min, 4 ° C to remove plasma and buffy coat. The GRs are washed three times with 0.9% NaCl (v: v). The lyophilized MGL is resuspended in water at a concentration of 78.7 mg / ml and added to the erythrocyte suspension to obtain a final 70% hematocrit suspension containing different concentrations of MGL and PLP. The suspension is then loaded onto a hemodialyzer at a flow rate of 120 ml / h and dialyzed against a hypotonic solution at a flow rate of 15 ml / min in countercurrent. The suspension is then resealed with a hypertonic solution and incubated for 30 min at 37 ° C. After 3 washes in NaCl 0.9% glucose 0.2%, the suspension is taken up in a SAG-Mannitol preservation solution supplemented with 6% BSA. The products obtained are characterized by OJ (within 2 hours of their preparation) and by D1 (ie after -18h-24h storage at 2-8 ° C. Hematological characteristics are obtained with a veterinary automaton (Sysmex, PocH-100iV) .
    [0023] The activity of MGL found in finished products increases with the amount of MGL introduced into the process. It can therefore easily encapsulate up to 32 IU of MGL per ml of finished product while maintaining good stability. Three murine finished products GR-MGL-PLP1, GR-MGL-PLP2 and GR-MGL-PLP3 were prepared according to the following modalities: GR-MGL-PLP1: co-encapsulation of MGL and PLP from a suspension containing 3 mg / ml MGL and -30 μM PLP. The final product was taken up in SAG-Mannitol BSA 6% supplemented with 10 μM final PLP. GR-MGL-PLP2: co-encapsulation of MGL and PLP from a suspension containing 3 mg / ml of MGL and -30 μM of PLP. The finished product was taken up in SAG-Mannitol BSA 6%.
    [0024] GR-MGL-PLP3: co-encapsulation of MGL and PLP from a suspension containing 3 mg / ml of MGL and -124 μM of PLP. The final product was taken up in SAG-Mannitol BSA 6%. The hematological and biochemical characteristics of the three finished products at OJ (after their preparation) are detailed in the table below. The encapsulation yields are satisfactory and vary from 18.6% to 30.5%. GR-MGL-PLP1 GR-MGL-PLP2 GRMG LPLP3 Data Hematocrit (%) 50.0 49.6 50.0 Blood volume (fL) 46.3 46.5 46.8 Corpuscle hemoglobin (g / dL) 24.7 24.0 24.2 hematologic RBC concentration (106 / pL) 6.5 6.9 6.6 Total hemoglobin (g / dL) 14.8 15.4 15.0 extracellular Hb (g / dL) 0.1 0.1 0.1 MGL Intraerythrocyte concentration of MGL (mg / mL RBC) 0.97 0.94 0.67 Intraerythrocyte activity of MGL 12.8 12.4 8.8 (IU / mL RBC) ) * Extracellular activity (%) 0.92% 0.97% 1.32% Intracellular activity (%) 99.08% 99.03% 98.68% Yield of encapsulation of MGL (%) 18.6% 30 , 5% 19.9% PLP Intra-erythrocyte concentration of PLP (pmol / L RBC) Not determined 13.4 71.4 Intracellular PLP fraction (%) Not determined 99.5 98.7 Extracellular PLP fraction (%) Not determined 0.5 1.3 PLP encapsulation yield (%) Not determined 44.8 57.4 * Calculated with the specific activity of 13.2 IU / mg Example 3. Production of encapsulating human RBCs Methionine Gamma Lyase and PLP according to the industrial method A pocket of human RBC depleted in leucocytes (provided by the French Blood Establishment) is subjected to a cycle of 3 washes in 0.9% NaCl (Cobe 2991 scrubber). The lyophilized MGL is resuspended with 0.7% NaCl and added to the erythrocyte suspension to obtain a final 70% hematocrit suspension containing 3 mg / ml MGL and -30 μM PLP (from the MGL formulation. ). The suspension is homogenized and the encapsulation is carried out according to the process described in EP 1 773 452. The suspension resulting from resealing is then incubated for 3 hours at room temperature in order to eliminate the most fragile RBCs. The suspension is washed 3 times with a solution of 0.9% NaCl 0.9% glucose (Cobe 2991 scrubber) and then resuspended with 80 ml of preservation solution (AS-3). The level of encapsulated MGL is assayed as in Example 4. OJ J1 J7 Hematocrit (%) 52.0 51.6 52.7 Cellular volume (fL) 91.0 92.0 88.0 Hemoglobin corpuscular (g / dL ) 30.3 29.8 31.6 RBC concentration (106 / μL) 6.00 5.92 5.98 Total hemoglobin (g / dL) 16.4 16.2 16.6 extracellular Hb (g / dL) 0.119 0.197 0.280 Osmotic fragility (g / L) 1.17 Hemolysis (%) 0.7% 1.2% 1.7% Total concentration MGL (mg / mL) 0.36 0.35 MG supernatant concentration (mg / mL) mL) 0.01 0.01 Intra-erythrocyte concentration MGL (mg / mL 100% Ht) 0.68 0.67 Extracellular Activity (%) 1.3% 1.4% Intracellular Activity (%) 98.7% 98, 6% Encapsulation efficiency (%) 19.7% Example 4. Assay of GR-encapsulated MGL The assay of MGL activity in cell suspensions (total RBCs) and in supernatants is based on a measurement of NH3 produced by the MGL. The NH3 ions are indirectly measured by enzymatic action of glutamate dehydrogenase (GLDH) according to the kit marketed by Roche Diagnostics (11877984).
    [0025] Preparation of standards: MGL standards at different concentrations are prepared in matrices (total GR or supernatants) or in aqueous solution. For standards in aqueous solution, the MGL is prepared at concentrations ranging from 0 to 12 μg / ml in the presence of 20 μM PLP in 100 mM phosphate buffer, pH 7.2. For total GR template standards, 10 μl of GR-LR are lysed with 90 μl of a solution containing 260 μM PLP and MGL at concentrations ranging from 0 to 100 μg / ml. The "total GR" STDs are then diluted 20-fold with 100 mM phosphate buffer, pH 7.2. For supernatant matrix standards, 10 μl of GR-LR supernatants are lysed with 50 μl of a solution containing 6.4 μM PLP and MGL at concentrations ranging from 0 to 20 μg / ml.
    [0026] Pre-treatment of the samples: the samples to be assayed (10 μl) are pre-treated in the same way as the standards (addition of PLP and identical dilutions but without addition of MGL). Assay of MGL: 7.5 μl of standards (STD) or samples are introduced into the wells of a UV plate. 94 μl of reagent R1 (Roche kit) and 56 μl of reagent R2 (Roche kit) containing a-ketoglutarate in buffered solution, NADPH and GLDH are added to remove endogenous NH3 ions from the samples. After 10 min of incubation, 75 μl of 78.3 mM L-methionine are introduced and the reaction mixtures are incubated for 30 min. The degradation of NADPH to NADP + is monitored continuously by measuring the optical density at 340 nm. For standards and samples, the ADO / min value is calculated on the linear domain of the OD curves obtained at 340 nm. A calibration curve ADO / min = f (concentration or activity of MGL in the standards) is then plotted. The regression parameters are used to determine the concentration of MGL in the samples. This result can be expressed in mg / ml or in IU / ml (the specific activity of the MGL is evaluated for each batch). The level of intra-erythrocyte MGL is obtained by calculation with the following formula: [MGL] intra-erythrocyte = ([MGL] total ([MGL] supernatants x (1 hematocrit / 100)) / (hematocrit / 100). 5. Assaying the PLP in the HPLC Sanitary Samples The PLP assay in cell suspensions (total RBCs) and in the supernatants is an adaptation of the method described by Van de Kamp et al., Nutrition Research 15, 415-422, 1995. The assay is carried out by RP-HPLC (UFLC Shimadzu) with fluorimetric detection (RF-10AXL instrument, excitation: 300 nm, emission: 400 nm) .The PLP contained in the samples is extracted with trichloroacetic acid. After centrifugation (15,000 xg, 10 min), the supernatants are collected and then diluted in mobile phase A. A volume of 50 μl of sample is injected onto a 5 μ column C-18 Gemini, 250 μl. x 4.6 mm (Phenomenex) Mobile phase A is composed of 33 mM potassium phosphate monobasic, d 8 mM sodium 1-octanesulfonate supplemented with sodium bisulfite (0.5 g / L) to intensify the signal of PLP and mobile phase B of 33 mM potassium phosphate monobasic and 17% (v: v) of 2 propanol. The gradient used is mobile phase A (100%) with increasing proportions of mobile phase B: increase from 0% to 8% of B over a period of 8 min. The flow rate through the column is maintained at 1m1 / min. The concentration of PLP in the samples is determined with an external standard range of PLP subjected to the same TCA treatment as the samples. The retention time of the PLP is -3.4 min. The level of intra-erythrocyte PLP is obtained by calculation with the following formula: [Lintra-erythrocyte PLP = ([PLP] total ([PLP] supernatants X (1 hematocrit / 100)) / (hematocrit / 100).
    [0027] Example 6 Increase of the PLP Level in the GRs by Co-Encapsulation of PLP with MGL Suspensions of murine GR are subjected to the encapsulation process of MGL and PLP as described in Example 2. The assay of Intracellular PLP is carried out according to the method described in Example 5 A suspension of human GR is subjected to the MGL and PLP encapsulation process as described in Example 3. Before the incubation step at room temperature, a portion of the human GR-MGL-PLP is removed to perform an intracellular PLP assay according to the method described in Example 5.
    [0028] The following table compares the physiological levels of PLP in human or murine erythrocytes with the rate achieved by co-encapsulation of the latter with MGL. Murine GR human GRs Physiological rate of PLP 0.11 μM 2.4 μM * (Fonda) (Natta & Reynolds) PLP level Conditions before - 3.90 μM - 13.4 μM in GR-MGL-PLP1 or GR- MGL-PLP2 dialysis: - 3 mg / mL MGL - - 30 μM PLP PLP level Conditions before - - 71.4 μM in GR-MGL-PLP3 dialysis: - 3 mg / mL MGL - - 125 μM PLP * detail Calculation: 7.5 nmol / g Hb - 2.4 μM (taking an MCH of 32 g / dL). Example 7. Demonstration of the increase in the concentration of PLP in GR-MGL by in vitro incubation with pyridoxine. A suspension of human GR is subjected to the method of encapsulation of the MGL as described in Example 3. Before the incubation step of 3h, a portion of the GR is removed and separated in three for a volume volume incubation with pyridoxine at different concentrations (0 mM, 2 mM and 4 mM). After homogenization, these suspensions are incubated at room temperature (RT). After 3h and 24h incubation, samples of cell suspensions and supernatants (obtained after centrifugation suspensions at 1000 xg, at 4 ° C, for 10 min) are prepared and frozen for a measurement of the concentration of PLP by HPLC as described in Example 5. The results obtained are shown in FIGS. 1 and 2. In the absence of pyridoxine, the level of intra-erythrocyte PLP is 3.9 μM (PLP resulting from the co-encapsulation of MGL and PLP). This concentration of PLP remains constant after 3h and 24h incubation. A slight decrease in the PLP concentration is observed at 24 h and is concomitant with an appearance of extracellular PLP which can be explained by haemolysis at the end of incubation. In the presence of pyridoxine (2 mM or 4 mM), GR-MGL are enriched in PLP with intra-erythrocyte concentrations increased by a factor of 2 after 3h of incubation (-8 pM of PLP) and close to a factor of 3 after 24 hours of incubation with appearance of a dose effect (11 μM and 14 μM for pyridoxine concentrations of 2 mM and 4 mM, respectively). This result shows that an incubation of a GR suspension encapsulating a PLP-dependent enzyme with pyridoxine is able to increase the level of intracellular PLP in a sustainable manner. Example 8. Pharmacokinetics of GR encapsulating MGL-PLP in mice. The murine products GR-MGL-PLP2 and GR-MGL-PLP3 prepared and characterized as in Example 2 and labeled with CFSE (fluorescent) are injected intravenously into CD1 mice at a dose of 8 ml / kg. After different times (T + 24h, T + 48h and T + 120h), the mice are sacrificed and the blood is collected on a lithium heparin tube stored at + 4 ° C in the dark for the determination of pharmacokinetics . The proportion of CFSE-labeled red blood cells in whole blood is determined by a flow cytometry method. Five microliters of whole blood are diluted in 1mL PBS 0.5% BSA and each sample is triplicated (counting 10,000 cells in FL-1, FC500 cytometer, Beckman Coulter). The evaluation of the survival of red blood cells loaded with MGL is obtained by comparing the proportion of GRs marked with CFSE at the different times (T + 24h, T + 48h and T + 120h) to the proportion of GRs marked with CFSE at TO + 15 min (100% control). The different percentages obtained for each time are plotted on a graph (FIG. 3) representing the proportion of RBCs loaded with MGL in circulation as a function of time.
    [0029] Determination of the proportion of CFSE-labeled RBCs in circulating blood at different times shows excellent stability of the GR-MGL-PLP2 and GR-MGL-PLP3 products in vivo in mice up to 120 h post injection (83, 5 ± 0.6% and 94.7 ± 0.6% survival, respectively). EXAMPLE 9 Depletion of L-Methionine at 24 Hours The murine products GR-MGL-PLP1, GR-MGL-PLP2 and GR-MGL-PLP3 prepared and characterized as in Example 2 are injected intravenously into CD1 mice at a dose of 8 ml / kg. After 6 hours, 0.09 mg of pyridoxine (ie 150 μl of a 2.9 mM solution of pyridoxine hydrochloride) were injected into the mice receiving GR-MGLPLP2. The plasma level of L-Met was evaluated at 24 hours by HPLC-MS-MS (Piraud M. et al., Rapid Commun., Mass Spectrum 19, 3287-97, 2005). The following table shows the depletion obtained in the different groups of injected mice. Administered Product Plasma Coenzyme Supply Modalities Plasma L-Met Plasma (pM)% Depletion - Food 82.7 ± 22.5 - GR-MGL- Food 46.3 ± 3.5 44% PLP1 - PLP in the finished product (- 5 pmoles / L GR) - PLP in the finished product preservation solution (10 μM) GR-MGL- - Feed 22.3 ± 4.9 73% PLP2 - PLP encapsulated in the product Finished (- + pyridoxine 13.4 pmoles / L GR) - Injection IV of -0.09 mg of pyridoxine GR-MGL- - Feed 29.7 ± 4.6 64% PLP3 - PLP encapsulated in the finished product (- 71 , 4 pmol / L GR) The plasma L-Met level was evaluated at 82.7 ± 22.5 μM in the control mice. The GR-MGL-PLP1 product containing MGL encapsulated with a low concentration of PLP leads to a 44% depletion of L-Met, 24 hours after administration of the product. We hypothesized that the PLP added in the product preservation solution is not available for the MGL enzyme because 1) it is mostly bound to the BSA present in the preservative solution and 2) it can not pass the GR membrane. The results show that a greater supply of PLP in the red blood cell either by IV injection of pyridoxine (GR-MGL-PLP2) or by encapsulation of PLP at a higher concentration makes it possible to obtain depletions of L-Met -1, 5 times larger (73% and 64% respective depletion). Example 10. Pharmacodynamics of GR-MGL The murine products GR-MGL-PLP2 and GR-MGL-PLP3 prepared and characterized as in Example 2 are injected intravenously into CD1 mice at a dose of 8 ml / kg. After 6h, -0.09 mg of pyridoxine (ie 150 μl of a 2.9 mM solution of pyridoxine hydrochloride) were injected into the mice receiving GR-MGL-PLP2. The plasma level of L-Met was evaluated at 15 min, 24h, 48 h, and 120 h by HPLC-MS-MS (Piraud M. et al.,). Figure 4 shows the depletion obtained in the different groups of injected mice. The results show in both experimental groups a depletion of L-methionine stabilized at 35 μM and maintained over time (from 48 h to 120 h post injection). These results indicate that supplementation with PLP or its precursor (pyridoxine) makes it possible to maintain GR-encapsulated MGL activity for at least 120 h after injection in mice.
    权利要求:
    Claims (14)
    [0001]
    REVENDICATIONS1. A pharmaceutical composition comprising erythrocytes and a pharmaceutically acceptable vehicle, the erythrocytes encapsulating an enzyme with PLP (pyridoxal phosphate).
    [0002]
    2. A composition according to claim 1 comprising methioninase, tyrosine phenol lyase, tyrosine aminotransferase or cystathionine beta-synthase.
    [0003]
    3. Composition according to claim 1 or 2, comprising from 0.01 to 30, preferably from 0.05 to 10 mg of PLP enzyme per ml of red blood cells.
    [0004]
    4. Composition according to any one of claims 1 to 3, further comprising PLP and / or a phosphated or non-phosphated precursor of PLP.
    [0005]
    5. Composition according to claim 4, comprising from 0.05 to 600, in particular from 0.5 to 100, preferably from 5 to 50 pmoles, of PLP and / or PNP (pyridoxine phosphate) and / or PMP (phosphate). pyridoxamine), encapsulated per liter (L) of red blood cells.
    [0006]
    6. Composition according to claim 5, comprising, as non-phosphated form of PLP precursor, pyridoxal, pyridoxine and / or pyridoxamine, encapsulated and / or unencapsulated.
    [0007]
    A composition according to any one of the preceding claims, further comprising pyridoxine kinase (PN kinase), pyridoxine phosphate oxidase (PNP-oxidase), and / or a PLP-phosphatase inhibiting agent.
    [0008]
    8. A composition according to claim 1 or 2 comprising, on the one hand, erythrocytes and a pharmaceutically acceptable vehicle, the erythrocytes encapsulating a PLP enzyme and, on the other hand, a non-phosphate form of vitamin B6.
    [0009]
    9. A composition according to any one of the preceding claims for use as a medicament.
    [0010]
    10. Composition according to claim 9, comprising methioninase, as an anticancer drug, in particular against a cancer comprising tumor cells auxotrophic for methionine, or as a medicament for treating a homocysteinuria or a hyperhomocysteinemia or an associated cardiovascular disease.
    [0011]
    11. A composition according to claim 9, comprising tyrosine phenol-lyase, as anticancer drug, especially against a cancer comprising auxotrophic tumor cells for tyrosine.
    [0012]
    12. A composition according to claim 9, comprising tyrosine aminotransferase, as a drug against Richner-Hanhart syndrome (tyrosinemia of type li).
    [0013]
    13. A composition according to claim 9, comprising cystathionine betasynthase, as a medicament for treating homocysteinuria or hyperhomocysteinemia or an associated cardiovascular disease.
    [0014]
    A process for the preparation of a pharmaceutical composition comprising erythrocytes encapsulating a PLP enzyme, a pharmaceutically acceptable carrier, and pyridoxal phosphate (PLP) and / or a phosphated or non-phosphated precursor of PLP, the process comprising the following steps: optionally an erythrocyte pellet is washed and centrifuged one or more times with a physiological buffer; the erythrocyte suspension is brought into contact with a hypotonic liquid medium resulting in the opening of pores in the erythrocyte membrane; the erythrocyte suspension is contacted with the PLP enzyme before or after the opening of the pores; the enzyme enters the erythrocytes; the pores are closed with a hypertonic buffer and a suspension of resealed erythrocytes containing the enzyme is collected; optionally the erythrocyte suspension is incubated to remove the most fragile erythrocytes; the erythrocyte suspension is washed and conditioned with a preservation solution; process in which: - the PLP and / or, if appropriate, a phosphated precursor of PLP, is co-encapsulated with the PLP enzyme, - if necessary, the non-phosphated precursor of PLP is added to the suspension of erythrocytes before or after the opening of the pores, and / or - if necessary, the non-phosphated precursor of PLP is added during the incubation or the preservation solution.
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    同族专利:
    公开号 | 公开日
    IL246941A|2019-09-26|
    ES2808849T3|2021-03-02|
    KR102354103B1|2022-01-24|
    IL246941D0|2016-09-29|
    US20210008114A1|2021-01-14|
    RU2019123935A3|2020-03-26|
    AU2015217045A1|2016-08-18|
    AU2018201237A1|2018-03-15|
    RU2016133315A|2018-02-16|
    EP3104875A2|2016-12-21|
    JO3522B1|2020-07-05|
    CL2016002020A1|2017-06-23|
    WO2015121348A3|2015-10-08|
    RU2016133315A3|2018-09-26|
    SG11201606264SA|2016-08-30|
    RU2744659C2|2021-03-12|
    RU2019123935A|2019-10-03|
    EP3104875B1|2020-05-06|
    AU2015217045B2|2018-03-08|
    JP6563958B2|2019-08-21|
    SG10201907367QA|2019-09-27|
    US20160361361A1|2016-12-15|
    KR20160121532A|2016-10-19|
    FR3017299B1|2018-05-18|
    JP2017506260A|2017-03-02|
    US10046009B2|2018-08-14|
    AU2018201237B2|2019-09-26|
    RU2697086C2|2019-08-12|
    MX2016010235A|2016-10-28|
    CA2938469A1|2015-08-20|
    US20180344771A1|2018-12-06|
    CN111358941A|2020-07-03|
    CN106255506A|2016-12-21|
    EP3718562A1|2020-10-07|
    CN106255506B|2020-04-14|
    WO2015121348A2|2015-08-20|
    US10780126B2|2020-09-22|
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    法律状态:
    2016-02-18| PLFP| Fee payment|Year of fee payment: 3 |
    2017-02-08| PLFP| Fee payment|Year of fee payment: 4 |
    2018-01-26| PLFP| Fee payment|Year of fee payment: 5 |
    2020-01-13| PLFP| Fee payment|Year of fee payment: 7 |
    2021-01-22| PLFP| Fee payment|Year of fee payment: 8 |
    2022-01-12| PLFP| Fee payment|Year of fee payment: 9 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1451100A|FR3017299B1|2014-02-12|2014-02-12|PHARMACEUTICAL COMPOSITION COMPRISING ERYTHROCYTES ENCAPSULATING A PLP ENZYME AND ITS COFACTOR|
    FR1451100|2014-02-12|FR1451100A| FR3017299B1|2014-02-12|2014-02-12|PHARMACEUTICAL COMPOSITION COMPRISING ERYTHROCYTES ENCAPSULATING A PLP ENZYME AND ITS COFACTOR|
    MX2016010235A| MX2016010235A|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes encapsulating a plp-dependent enzyme and its cofactor.|
    KR1020167022067A| KR102354103B1|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes encapsulating a plp-dependent enzyme and its cofactor|
    SG10201907367QA| SG10201907367QA|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes encapsulating a plp-dependent enzyme and its cofactor|
    SG11201606264SA| SG11201606264SA|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes encapsulating a plp-dependent enzyme and its cofactor|
    JOP/2015/0030A| JO3522B1|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes encapsulating a PLP-dependent enzyme and its cofactor|
    EP15705563.3A| EP3104875B1|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes encapsulating a plp-dependent enzyme and its cofactor|
    RU2016133315A| RU2697086C2|2014-02-12|2015-02-12|Pharmaceutical composition containing erythrocytes in which an enzyme dependent on pyridoxalphosphate is enclosed, and cofactor thereof|
    AU2015217045A| AU2015217045B2|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes encapsulating a PLP-dependent enzyme and its cofactor|
    PCT/EP2015/052962| WO2015121348A2|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes encapsulating a plp-dependent enzyme and its cofactor|
    CA2938469A| CA2938469A1|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes encapsulating a plp-dependent enzyme and its cofactor|
    JP2016568136A| JP6563958B2|2014-02-12|2015-02-12|Pharmaceutical composition comprising red blood cells encapsulating PLP-dependent enzyme and its cofactor|
    EP20173043.9A| EP3718562A1|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes encapsulating a plp-dependent enzyme and its cofactor|
    RU2019123935A| RU2744659C2|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes containing enzyme dependent on pyridoxalphosphate and cofactor thereof|
    CN202010182496.4A| CN111358941A|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes encapsulating a PLP-dependent enzyme and its co-factor|
    US15/117,588| US10046009B2|2014-02-12|2015-02-12|Method of treatment using a pharmaceutical composition comprising erythrocytes encapsulating a PLP-dependent enzyme and its cofactor|
    ES15705563T| ES2808849T3|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes that encapsulate a PLP-dependent enzyme and its cofactor|
    CN201580007812.1A| CN106255506B|2014-02-12|2015-02-12|Pharmaceutical composition comprising erythrocytes encapsulating a PLP-dependent enzyme and its co-factor|
    IL246941A| IL246941A|2014-02-12|2016-07-25|Pharmaceutical composition comprising erythrocytes encapsulating a plp-dependent enzyme and its cofactor|
    CL2016002020A| CL2016002020A1|2014-02-12|2016-08-10|Pharmaceutical composition consisting of erythrocytes that encapsulate a dependent plp envelope and its cofactor|
    AU2018201237A| AU2018201237B2|2014-02-12|2018-02-21|Pharmaceutical composition comprising erythrocytes encapsulating a PLP-dependent enzyme and its cofactor|
    US16/102,171| US10780126B2|2014-02-12|2018-08-13|Pharmaceutical kit comprising erythrocytes encapsulating a PLP-dependent enzyme and, a non-phosphate PLP precursor|
    US17/028,383| US20210008114A1|2014-02-12|2020-09-22|Pharmaceutical composition comprising erythrocytes encapsulating a plp-dependent enzyme and, a non-phosphate plp precursor|
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