ARGININE DEIMINASE ENCAPSULATED INSIDE ERYTHROCYTES AND THEIR USE IN TREATING CANCER AND ARGINASE-1 DEFICIENCY
20220193208 · 2022-06-23
Inventors
Cpc classification
A61K38/50
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K38/50
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
Abstract
The present invention is related to arginine deiminase encapsulated into erythrocytes for use in therapy. It is in particular related to the use thereof in treating arginase-1 deficiency. Also, it relates to novel pharmaceutical compositions comprising arginine deiminase from M. arginini encapsulated into erythrocytes and the use thereof in treating diseases that may benefit from arginine depletion, such as arginine dependent cancers, in particular arginine-auxotrophic cancers, and arginase-1 deficiency.
Claims
1.-14. (canceled)
15. A pharmaceutically acceptable suspension of erythrocytes encapsulating a therapeutically effective amount of a magnesium-independent arginine deiminase (ADI), wherein when administered to a patient in need thereof, the suspension of erythrocytes delivers to said patient a sustained reduction in blood arginine levels for at least 10 days post administration.
16. The suspension of claim 15, further comprising a preservative solution for erythrocytes.
17. The suspension of claim 15, wherein the ADI is a full length ADI, or a variant or fragment thereof retaining substantially the same enzymatic activity and magnesium independence as the ADI having the amino acid sequence as set forth in SEQ ID NO: 1.
18. The suspension of claim 15, wherein the ADI comprises an amino acid sequence that is at least 80% identical to the amino acid sequence as set forth in sequence SEQ ID NO: 1.
19. The suspension of claim 18, wherein the ADI comprises the amino acid sequence as set forth in SEQ ID NO: 1.
20. The suspension of claim 15, wherein the concentration of the ADI enzymatic activity comprises about 1 to about 400 U/ml, about 5 to about 400 U/ml, or about 5 to about 350 U/ml, and/or wherein the amount of ADI comprises about 0.5 to about 6.5 mg/ml, about 1 to 6 mg/ml, about 1 to about 5 mg/ml, about 1 to about 4 mg/ml, about 1 to about 3 mg/ml, about 1.2 to about 2.8 mg/ml, about 1.4 to about 2.6 mg/ml, or about 1.6 to 2.4 about mg/ml.
21. The suspension of claim 15, wherein extracellular hemoglobin levels do not exceed 0.2 g/dl for at least 72 hours when the suspension is maintained between 2 and 8° C.
22. The suspension of claim 15, wherein less than 1% hemolysis occurs in 72 hours when the suspension is maintained between 2 and 8° C.
23. The suspension of claim 15, having an osmolarity of between about 270 and about 350 mOsm/l.
24. The suspension of claim 15, wherein the erythrocytes are human erythrocytes or are generated from human stem cells capable of becoming erythrocytes.
25. The suspension of claim 24, wherein the stem cells have been transformed to express the magnesium-independent ADI.
26. The suspension of claim 15, wherein the reduction in blood arginine levels provides a clinical benefit in the treatment and/or prevention of any one or all of the following: arginase-1 deficiency (A1D), arginine-dependent cancers, septic shock, and angiogenesis-related diseases.
27. The suspension of claim 15, packaged as a single dose in a container suitable for blood transfusion, said dose having a volume of between about 150 ml to about 350 ml.
28. The suspension of claim 27, wherein the dose comprises between about 50 to about 3500 units (U) of ADI enzymatic activity per kilogram of patient body weight.
29. The suspension of claim 15, prepared according to a method comprising the following steps: 1) suspending erythrocytes in an isotonic solution; 2) exposing the suspension to a cooled hypotonic solution between +1 and +8° C., wherein the hypotonic solution and the suspension are separated by a dialysis membrane; 3) encapsulating the ADI at a temperature between +1 and +8° C.; and 4) resealing the erythrocytes in an isotonic or hypertonic solution at a temperature between about +30 and +42° C.
30. The suspension of claim 15, prepared according to a method comprising the following steps: 1) suspending a pellet of erythrocytes in an isotonic solution at a hematocrit level equal to or greater than 65%, between +1 and +8° C.; 2) lysing the erythrocytes at a temperature between +1 and +8° C. by passing the suspension into a dialysis device and exposing the erythrocytes to hypotonic conditions; 3) encapsulating the erythrocytes by adding the ADI to the suspension before and/or during the lysing step, at a temperature between +1 and +8° C.; and 4) resealing the erythrocytes under isotonic or hypertonic conditions at a temperature between +30 and +42° C.
31. The suspension of claim 15, prepared according to a method comprising the following steps: 1) encapsulating the ADI inside the erythrocytes by placing the erythrocytes into contact with a hypotonic medium, contacting the erythrocytes with the ADI, and resealing the erythrocytes; 2) preparing a suspension comprising the resealed erythrocytes of step 1 having an osmolality of between about 290 and about 330 mOsmol/kg; 3) incubating the suspension of step 2 at an osmolality of between about 290 and about 330 mOsmol/kg, for a period greater than or equal to 30 minutes; 4) removing the liquid medium of the incubated suspension of step 3; 5) suspending the erythrocytes obtained under step 4 into a solution suitable for administration of the suspension to a patient.
32. The suspension of claim 15, wherein the suspension is a stabilized erythrocyte suspension.
33. The suspension of claim 15, wherein the suspension is a ready-to-use stabilized erythrocyte suspension.
34. The suspension of claim 15, wherein the ADI is co-factor independent.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0206]
[0207]
[0208]
[0209]
[0210]
[0211]
[0212]
EXAMPLES
Example 1. Method for Obtaining and Characterizing Arginine Deiminase (ADI)
[0213] Production of the strain and isolation of a hyper-producing clone: The natural sequence of ADI from Mycoplasma arginini (GenBank: X54141) was optimized by modifying some codons because genetic codes between E. coli and M. arginini are different (a new plasmid was created coded C1124-ADM-02). The purification process has been described in Misawa and coll. (Misawa, S. et al, 1994, J. of Biotechno. 36, 1994, 145-155) with some modifications, i.e., the bacterial production HMS174 (DE3) T1R strain has been used instead of the JM101 strain. Other modifications are described below.
Fermentation:
[0214] The production was achieved in a fermenter with FED_Coli_9 batch medium, with stirring, controlled pressure and pH from the pre-culture 2 at an optical density of 0.05. The growth phase (at 37° C.) took place until an optical density (600 nm) of 100 was obtained and the expression induction was achieved at 32° C. by adding 1 mM IPTG into the culture medium. The cell sediment was harvested 26-27 h after induction in two phases: the cell broth was concentrated 5-10 times after passing over a 500 kDa hollow fiber and then the cell pellet was recovered by centrifugation at 15900×g and then stored at −20° C.
Purification:
[0215] ADI was produced as inclusion bodies (IB). The cell pellets were suspended in a lysis buffer to disrupt the cells. Then the disrupted cells were washed to collect the IBs and the IBs were stored at −20° C.
[0216] The purification of ADI started by thawing the IB pellet in a buffer composed of 50 mM TRIS base pH 8.5, 6M Guanidinium Hydrochloride, 10 mM Dithiothreitol. The solubilization was achieved with an incubation time of 1 h at 37±2° C. After clarification, the refolding step took place for 40-45 hours at room temperature in a buffer composed of 3 mM monopotassium phosphate (KH2PO4), 7 mM dipotassium phosphate (K2HPO4) pH 7,35. After a second clarification step, the medium was loaded onto a Q-sepharose column. Elution was performed with 250 and 500 mM NaCl and the elution fraction was submitted to a tangential flow filtration (TFF). Two polishing steps (using Sartobind Q column) and two TFF steps complete the purification of ADI. A final 0.2 μM filtration was performed before storage of ADI at −20° C.
[0217] Characterization: The specific activity of the enzyme was determined by measuring the produced Citrulline as described in example 2. The protein content was determined by reading absorbance at 280 nM. The purity was determined by SDS-PAGE. The osmolarity was measured with an osmometer (Micro-Osmometer Loser Type 15). The main characteristics of one produced batch of ADI are summarized herein below in table 2.
TABLE-US-00002 TABLE 2 Main characteristics of one produced batch of ADI ADI of M. arginini Formulation Liquid phase frozen at −80° C. Characteristics: 323 mOsm/Kg - 16,65 mg/mL 50 mM phosphate de sodium pH 6.5, Sucrose 40 mg/mL, Lysine 40 mM Specific activity ~47 U/mg Purity 97.5%
Example 2. ADI Specific Activity Assay Using Citrulline Measurement
[0218] This assay is based on a 2-step reaction (Boyde and Rahmatullah, 1980, Analytical Biochemistry, vol 107, p 424-431): [0219] First, L-Arginine is converted into citrulline and ammonia by ADI [0220] Second, in presence of diacetyl monoxime, iron (Ill) chloride, thiosemicarbazide, sulfuric and phosphoric acids, citrulline is converted into a colored chromophore readable at 530 nm.
[0221] A L-citrulline standard curve is prepared to determine the ADI enzymatic activity of all assay samples, by reading the absorbance at 530 nm. Specific activity (U/mg) is calculated using the enzymatic activity (U/mL) and the protein content (mg).
Example 3. Encapsulation of ADI in Murine Erythrocytes
[0222] Whole blood of C57BL/6 mice (Charles River) was centrifuged at 1000×g, for 10 min, at 4° C. to remove the plasma and buffy coat. The erythrocytes were washed three times with 0.9% NaCl (v:v). The frozen ADI was thawed and added to the erythrocyte suspension in order to obtain a final suspension with a hematocrit of 65%, containing an initial concentration of ADI of 2 to 7 mg/mL.
[0223] The suspension was then loaded on a hemodialyzer at a flow rate of 120 ml/h and dialyzed against a hypotonic solution (40-50 mOsmol/kg) at a flow rate of 15 ml/min as a counter-current. The suspension was then resealed with a hypertonic solution (1 600-2 100 mOsmol/kg) and then incubated for 30 min at 37° C. After three washes in 0.9% NaCl, 0.2% glucose, the suspension was taken up in a preservation solution AS3 supplemented with 20% decomplemented plasma.
[0224] The obtained products are characterized at the time point D0 (within the 2 h following their preparation) and at time point D1 (i.e. after ˜18 h-24 h of storage at 2-8° C.). The hematologic characteristics are obtained with a veterinary automaton (Sysmex, PocH-100iV).
Results:
[0225] ADI activity in the finished products was assayed with the method described in example 4 against an external calibration range of ADI in aqueous solution. These results show that ADI activity in the finished products increases with the amount of enzyme introduced into the RBC and that it is easily possible to encapsulate up to 2 mg of ADI per ml of finished product while maintaining good stability. The main characteristics of 6 different batches of ERY-ADI murine final products (ERY-ADI-1 to 6) are given herein below in Table 3.
TABLE-US-00003 TABLE 3 Main characteristics of 6 ERY-ADI murine final products as measured at the time point D0 (2 h following the preparation) ERY- ERY- ERY- ERY- ERY- ERY- Batches ADI-1 ADI-2 ADI-3 ADI-4 ADI-5 ADI-6 Hematological Hematocrit (%) 51.1 51.1 50.8 51.3 51.1 51.3 parameters Corpuscle volume (fl) 39.5 40.1 40.6 41.3 40.0 38.60 Corpuscle hemoglobin (g/dl) 26.6 26.3 25.3 26.7 28.2 25.7 Total hemoglobin (g/dl) 14.8 14.4 14.0 14.8 15.6 13.8 Extracellular Hb (g/dl) 0.3 0.4 0.3 0.8 0.3 0.3 ADI ADI concentration 3 4.5 4.5 4.5 3.5 5 parameters before process (mg/mL) Intra-erythrocyte concentration of 1.15 1.56 1.90 1.13 1.37 2.70 ADI (mg/ml of RBC-100% Ht) Extracellular activity (%) 4.8 5.7 4.8 6 4.0 4.1 Intracellular activity (%) 95.2 94.3 95.2 94 96.0 95.9 Encapsulation yield of ADI (%) 38 35 42 25 39 54
Example 4. Assay of Encapsulated ADI in the Erythrocytes
[0226] The assay of the ADI activity entrapped in red blood cells and in the supernatants, is based on a measurement of NH.sub.3 produced by ADI from L-Arginine. The NH.sub.3 ions were assayed indirectly by enzymatic action of glutamate dehydrogenase (GLDH) according to the kit marketed by Roche Diagnostics (11877984).
Example 5. Pharmacokinetics of Erythrocytes Encapsulating ADI in C57BL/6 Mice
[0227] The murine product ERY-ADI 6 was labeled with CFSE (fluorescent) and administered intravenously into C57BL/6 mice. At each time points (D0+15 min, D0+6 h, D1, D2, D5, D9, D13 and D16), 3 mice were sacrificed and the blood was collected on a lithium heparinate tube kept at +4° C. away from light for determining the pharmacokinetics. The proportion of red blood cells labeled with CFSE in the whole blood was determined by a flow cytometry method. Five microliters of whole blood were diluted in 1 ml of PBS 0.5% BSA and each sample was passed in triplicate (counting of 10,000 cells in FL-1; cytometer FC500, Beckman Coulter). The evaluation of the survival of red blood cells loaded with ADI was obtained by adding the proportion of erythrocytes loaded with ADI labeled with CFSE at different time points to the proportion of erythrocytes loaded with ADI labeled with CFSE at T0+15 min (100% control). The different obtained percentages for each time are indicated in the graph depicted in
[0228] Based on half-life calculation, CFSE-labeled erythrocytes encapsulating ADI have an estimated half-life comprised between 18 and 22 days.
Example 6. Pharmacodynamics of Erythrocytes Encapsulating ADI in C57BL/6 Mice
[0229] The product ERY-ADI 6 of erythrocytes encapsulating ADI enzyme was injected intravenously to C57BL/6 mice at a dose of 8 ml/kg. At each time points (D0+15 min, D0+6 h, D1, D2, D5, D9, D13 and D16), 3 mice are sacrificed and the blood is collected on lithium heparinate tubes stored at 4° C. for the determination of plasma L-Arginine levels.
[0230] As shown in
Example 7. Administration of Erythrocytes Encapsulating ADI to Arginase-Deficient Mice (1.SUP.st .Study)
[0231] An in vivo study was set up with an arginase-deficient mouse model (For a complete description see Sin et al, 2013, PLOS One, vol. 8 (11)). These mice are devoid of arginase 1 activity and exhibit severe pathobiochemical aspects of hyperargininemia commonly seen in humans. Hyperargininemia (or Arginase deficiency) is triggered by 5 injections of tamoxifen. Blood arginine concentrations start to rise few days after the last tamoxifen injection. To demonstrate an efficacy of murine product ERY-ADI 4 to decrease blood L-Arginine level in this mouse model, ERY-ADI 4 (4 and 8 mL/kg) and mock-loaded erythrocytes (8 mL/kg) were intravenously injected to 15 arginase-deficient mice 7 days after the last tamoxifen injection. Blood was collected the day (D1) and two days later (D3) after administration of the three products. Results of the in vivo study are presented in
TABLE-US-00004 TABLE 4 Summary of the results of the in vivo study of ERY-ADI-4 in an arginase-deficient mouse model. Indicated are the % depletion of blood Arginine levels. Negative numbers indicate an increase of Arginine levels. % depletion Mock loaded ERY-ADI 4 ERY-ADI 4 (Blood L-Arg) erythrocytes (8 mL/kg) (4 mL/kg) (8 mL/kg) D1 −10% 60% 92% D3 −30% 7% 73%
[0232] As shown in this table and in
[0233] Serum Ammonia was analyzed at the same time as the conversion of L-Arginine by ADI results in the production of Citrulline and Ammonia. As shown in
Example 8. Administration of Erythrocytes Encapsulating ADI to Arginase-Deficient Mice (Second Study)
[0234] To confirm the efficacy of murine product ERY-ADI to decrease blood L-Arginine level in this mouse model, a second study was set up on mice treated with tamoxifen in accordance with example 7. ERY-ADI product was intravenously injected 3 days after the last tamoxifen injection. One or two intravenous administration(s) of ERY-ADI (at 8 mL/kg) was scheduled (Groups 2 and 3 respectively). A control group was administered the free form of ADI enzyme (Group 4). A second control group composed of mice bearing arginase activity was part of the study (Group 1).
[0235] Blood was collected the day (D3) and one week later (Day 10). The day of sacrifice, mice blood was collected too (Day 13).
[0236] Results of the second in vivo study are presented in
[0237] First, blood arginine levels were lower in this second study because we changed the injections schedule related to the tamoxifen injections. In this study, the administration of ERY-ADI was planned 3 days instead of 7 days after last tamoxifen injection, resulting in a lower blood arginine level baseline.
[0238] As shown in Figure. 6, erythrocytes encapsulating ADI, when administered at a dose volume of 8 mL/kg, decreased blood L-Arginine by 81% and 77% compared to baseline levels for groups 2 and 3 respectively at Day 10 (i.e. 7 days after administration). The second administration of ERY-ADI was scheduled after the blood collection on day 10. Ten (10) days after administration, depletion of blood arginine is still very important with some percentages of depletion of 82% and 68% for group 2 and group 3 respectively compared to baseline levels. In contrast no blood arginine depletion was observed in mice injected with free form of ADI at Day 10 (Group 4). On the contrary the blood arginine concentration reached a concentration of 349 μM reflecting the blood arginine level increase in the model of arginase-deficient mice. No measurement of blood L-Arginine level could be performed at Day 13 for group 4 since all animals died further to the 2.sup.nd injection with free form of ADI.
[0239] This second study confirmed the results observed with the first study with some additional information about the pharmacodynamics of the ERY-ADI product when injected to arginase-deficient mice. One single administration allows a blood arginine depletion for at least 10 days and a second injection of ERY-ADI product did not result in any side effects on this mouse model.
Example 9. Administration of Erythrocytes Encapsulating ADI to Arginase-Deficient Mice (Third Study)
[0240] As no survival was observed when ERY-ADI administration was performed 3 days after the last tamoxifen injection, the third study was designed with an important change in test item administration schedule. ERY-ADI was intravenously injected the day before the first tamoxifen injection (Day 0). In this study, the last tamoxifen injection was carried out on Day 5. One or two intravenous administration(s) of ERY-ADI was scheduled (Groups 2 and 3, respectively). Group 1 was a control group composed of Arginase-deficient mice administered the mock RBC (i.e. processed RBC with no ADI). Blood was collected on Day 0, Day 11, Day 14 (before second administration of ERY-ADI) and when mice were sacrificed for excess body weight loss (identified as “end of study”).
[0241] As shown in
[0242] When ERY-ADI was reinjected at Day 14 (group 3), whole blood arginine level was measured few days later, at the time of sacrifice for ethical reasons (identified as “end of study”). The blood arginine was still depleted following the second administration of ERY-ADI, but arginase-deficient mice has to be sacrificed due to significant body weight loss.
[0243] This third study confirmed the results observed with the first two studies with some additional information about the pharmacodynamics of the ERY-ADI product when injected into arginase-deficient mice. A single administration yielded a blood arginine depletion for at least 11 days in this mouse model and a second injection of ERY-ADI yielded a sustained blood arginine depletion for at least 17 days (lifespan of the first mouse that had been sacrificed for ethical reasons).
[0244] However, due to the severity of this mouse model, no survival has been observed beyond 15 days after last tamoxifen injection, whatever the number of ERY-ADI administrations performed.
Example 10: Production of Human Red Blood Cells Encapsulating Arginine Deiminase
[0245] A pouch of leucocyte-depleted human RBCs (provided by the “Etablissement français du sang”) was subjected to three washes with 0.9% NaCl. The Arginine Deiminase (ADI) solution was gently thawed and added to the RBC suspension to obtain a final concentration with a hematocrit of 60% containing 3 or 5 mg/mL of ADI. The suspension was homogenized and loaded on a hemodialyzer at a flow rate of 90 mL/h and dialyzed against a hypotonic solution at 30 mOsmol/kg. The suspension was then resealed with a hypertonic solution and then incubated for 30 minutes a 30° C. After 3 washes in 0.9% NaCl, 0.2% Glucose, the suspension was taken up in a preservative solution AS3 (NaCl, NaH.sub.2PO.sub.4, Citric acid, Na-citrate, adenine and glucose. Osmolality is 288 mOs/kg and pH 5,88). The products obtained were characterized at Day 0, Day 1 and Day x. The hematologic characteristics were obtained with a veterinary automat (Sysmex, PocH-100iV).
[0246] It is important to note that no magnesium, iron or other enzyme co-factor was (or need be) added to the disclosed RBC-encapsulated arginine deiminase (ADI) compositions and suspensions. Unlike non-encapsulated ADI preparations, which may make use of magnesium or other co-factors present in a subject's bloodstream, the ADI of the present disclosure is generally limited to the contents of the RBCs. To solve this problem, Applicants specifically selected a co-factor-independent ADI (e.g. M. arginini) to ensure long-lasting in vivo ADI activity, without the need to supplement the products with magnesium or some other co-factor. Applicants envision that other co-factor-independent ADI may be used effectively in the practice of the disclosed invention.
[0247] As used herein, “Co-factor-independent ADI” means an ADI that does not depend upon enzyme co-factors such as vitamins, pro-vitamins, vitamin precursors, or metal ions (e.g. magnesium, iron, manganese, etc.). As used herein, “magnesium-independent ADI” means an ADI that does not depend upon magnesium to support its enzymatic activity.
[0248] Results. The hematologic and biochemical characteristics of 6 finished products at Day 0 (day of manufacturing) are compiled in Table 5 below: three were manufactured with an ADI concentration of 3 mg/mL and three with an ADI concentration of 5 g/mL, expressed with respect to the RBC suspension before dialysis. All the ERY-ADI products were prepared with the same batch of ADI. In vitro stability was assessed on Day 0, Day 1 (Table 6) and Day 7 (Table 7).
TABLE-US-00005 TABLE 5 Hematologic and biochemical characteristics of ERY-ADI I to VI (Day 0) ADI 3 mg/mL ADI 5 mg/mL ERY- ERY- ERY- ERY- ERY- ERY- Day 0 parameters ADI I ADI II ADI III ADI IV ADI V ADI VI Hemato- Hematocrit (%) 48.2 50.8 48.0 50.4 48.0 49.2 Logical Corpuscular volume (fL) 87.5 82.9 85.7 85.3 83.3 86.9 data Corpuscular 27.6 28.6 28.6 28.1 29.4 27.9 hemoglobin (g/dL) RBC count (10.sup.6/μL) 5.52 6.12 5.60 5.92 5.78 5.66 Total Hemoglobin (g/dL) 13.4 14.4 13.8 14.2 14.2 13.8 Extracellular Hb (g/dL) 0.1 0.1 0.1 0.1 0.1 0.1 ADI Intra-erythrocyte 0.88 0.94 1.01 1.55 1.36 1.46 concentration of ADI (mg/mL of RBC) Intra-erythrocyte 42.1 44.9 48.3 74.1 65.0 69.8 activity of ADI (U/mL) Extracellular activity (%) 1.2 0.0 3.0 0.6 0.8 4.0 Intracellular activity (%) 98.8 100.0 97.0 99.4 99.2 96.0 Entrapment yield of ADI (%) 29.4 31.5 33.7 31.0 27.3 29.3
[0249] Independent of the ADI concentration added before entrapment, the entrapment yield was very reproducible (from 27.3 to 33.7%).
TABLE-US-00006 TABLE 6 Hematologic and biochemical characteristics of ERY-ADI Ito VI (Day 1) ADI 3 mg/mL ADI 5 mg/mL ERY- ERY- ERY- ERY- ERY- ERY- Day 1 parameters ADI I ADI II ADI III ADI IV ADI V ADI VI Hemato- Hematocrit (%) 46.6 51.4 47.8 49.4 49.0 46.4 Logical Corpuscular volume (fL) 85.5 81.6 83.8 83.8 81.4 84.7 Data Corpuscular 28.8 30.3 29.1 28.3 29.6 29.1 hemoglobin (g/dL) RBC count (10.sup.6/μL) 5.46 6.28 5.70 5.90 6.02 5.48 Total Hemoglobin (g/dL) 13.4 15.6 14.0 14.0 14.4 13.4 Extracellular Hb (g/dL) 0.1 0.1 0.2 0.1 0.1 0.2 ADI Intra-erythrocyte 0.89 0.83 1.00 1.50 1.22 1.86 concentration of ADI (mg/mL of RBC) Intra-erythrocyte 42.5 39.7 47.8 71.7 58.3 88.9 activity of ADI (U/mL) Extracellular activity (%) 1.3 1.1 6.0 1.3 1.7 5.1 Intracellular activity (%) 98.7 98.9 94.0 98.7 98.3 94.9
TABLE-US-00007 TABLE 7 Hematologic and biochemical characteristics of ERY-ADI I to VI (Day 7) ADI 3 mg/mL ADI 5 mg/mL ERY- ERY- ERY- ERY- ERY- ERY- Day 7 parameters ADI I ADI II ADI III ADI IV ADI V ADI VI Hemato- Hematocrit (%) 47.6 51.4 50.4 50.4 48.0 47.6 Logical Corpuscular volume (fL) 86.4 82.8 86.6 85.3 82.6 87.6 data Corpuscular 27.4 28.8 27.3 28.2 29.6 27.6 hemoglobin (g/dL) RBC count (10.sup.6/μL) 5.52 6.22 5.82 5.90 5.82 5.44 Total Hemoglobin (g/dL) 13.0 14.8 13.8 14.2 14.2 13.2 Extracellular Hb (g/dL) 0.3 0.5 0.5 0.4 0.5 0.5 ADI Intra-erythrocyte 0.85 0.80 0.91 1.35 1.22 1.54 concentration of ADI (mg/mL of RBC) Intra-erythrocyte 40.6 38.2 43.5 64.5 58.3 73.6 activity of ADI (U/mL) Extracellular activity (%) 3.7 5.2 11.5 4.2 3.7 12.7 Intracellular activity (%) 96.3 94.9 88.5 95.8 96.3 87.3
[0250] Entrapment of Arginine Deiminase in human red blood cells proved to be a very reproducible process. The main parameters were stable between Day 0 and Day 1 (e.g. extracellular hemoglobin, intracellular and extracellular enzyme activity, hematocrit, corpuscular volume). At Day 7, the measured parameters indicated that the ERY-ADI product is very stable in vitro, independently of the ADI concentration added before the entrapment process.
[0251] Conclusion. Arginine Deiminase (ADI; EC number 3.5.3.6) entrapped in red blood cells (RBC), was obtained using the Erytech's proprietary ERYCAPS® technology platform. The entrapment of therapeutic enzymes into red blood cells can provide effective, long-acting therapeutic activity with reduced toxicity.
[0252] Entrapment of Arginine Deiminase inside Red Blood Cells (ERY-ADI product), greatly improves the pharmacological properties of the enzyme. In healthy mice, plasma L-arginine depletion was complete within 15 minutes after administration and was sustained for 13 days.
[0253] When injected to Arginase-deficient mice, ERY-ADI demonstrated a spectacular efficacy on the very high blood L-arginine concentrations displayed by these mice. Indeed, 24 h after administration, blood L-arginine concentration was reduced by 52 and 92% when 4 or 8 mL/kg were injected, respectively. Three days after administration, blood L-arginine level remained reduced by 19 and 73%. Moreover, despite the production of ammonia by Arginine Deiminase, the serum level stayed comparable to the mock-loaded RBC control.
[0254] These results were confirmed in a second study wherein a single administration yielded a blood arginine depletion for at least 10 days and a second injection of ERY-ADI was well tolerated by arginase-deficient mice.
[0255] Entrapment of Arginine Deiminase was successfully performed in human Red Blood Cells with a good reproducibility and in vitro stability.
[0256] Based on these results, ERY-ADI is envisioned to be capable of counteracting the primary biochemical defect of the rare genetic disorder of Arginase deficiency.
[0257] The invention will now be described by the following numbered paragraphs:
[0258] 1. A pharmaceutical composition comprising arginine deiminase (ADI) encapsulated into erythrocytes and optionally a pharmaceutically acceptable vehicle for its use in treating arginase-1 deficiency, preferably wherein the composition is capable of reducing pathological plasma or whole blood arginine levels in a patient or subject suffering from Arginase 1 (Arg1) deficiency to normal physiological or near-normal physiological plasma or whole blood arginine levels, more preferably wherein the composition is capable of reducing the pathological levels of arginine by at least about 20, 30, 40, 50, 60, 70 or about 80% for a period of at least about 6, 7, 8, 9, 10 or 11 days post administration of a single dose of the composition;
[0259] optionally wherein the ADI is any one, any combination, or all of the following: co-factor-independent, magnesium-independent, iron-independent, vitamin-independent, pro-vitamin-independent; and
[0260] optionally wherein the composition is characterized by any or all of the following ranges of values across the indicated parameters:
[0261] Hematocrit (%): about 48 to about 51;
[0262] Corpuscular volume (fL): about 82 to about 88;
[0263] Corpuscular hemoglobin (g/dL): about 27 to about 30;
[0264] RBC count (106/μL): about 5.5 to about 6.2;
[0265] Total Hemoglobin (g/dL): about 13.0 to about 14.5;
[0266] Extracellular Hb (g/dL): about 0.1 to about 0.2;
[0267] Intra-erythrocyte concentration of ADI (mg/mL of RBC): about 0.8 to about 1.6;
[0268] Intra-erythrocyte activity of ADI (U/mL): about 42 to about 49 or about 65 to about 74;
[0269] Extracellular activity (%): about 0.0 to about 4.0;
[0270] Intracellular activity (%): about 96 to about 100; and/or
[0271] Entrapment yield of ADI (%): about 27 to about 34.
[0272] 2. The pharmaceutical composition for the use according to paragraph 1, wherein said composition is a suspension having an osmolarity of between 270 and 350 mOsm/1.
[0273] 3. The pharmaceutical composition for the use according to paragraph 1 or 2, wherein the pharmaceutically acceptable vehicle is a preservative solution comprising NaCl and Adenine.
[0274] 4. The pharmaceutical composition for the use according to any one of paragraphs 1 to 3, wherein said ADI is from M. arginini.
[0275] 5. The pharmaceutical composition for the use according to any one of paragraphs 1 to 4, wherein the ADI comprises the amino acid sequence of SEQ ID NO: 1 or a variant or fragment thereof.
[0276] 6. The pharmaceutical composition for the use according to paragraphs 5, wherein said variant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence SEQ ID NO: 1.
[0277] 7. The pharmaceutical composition for the use according to paragraphs 5 or 6, wherein said variant or fragment retains the biological activity of the ADI having the amino acid sequence of SEQ ID NO: 1.
[0278] 8. The pharmaceutical composition for the use according to any one of paragraphs 1 to 7, wherein the concentration of encapsulated ADI is from 0.1 to 7 mg/ml.
[0279] 9. The pharmaceutical composition for the use according to any one of paragraphs 1 to 8, wherein the pharmaceutical composition is packaged in a dose having a volume from 10 to 250 ml.
[0280] 10. The pharmaceutical composition for the use according to paragraph 9, wherein the amount of ADI encapsulated in one dose for a patient is from 0.01 mg/kg to 500 mg/kg of encapsulated ADI per kg body weight of said patient.
[0281] 11. A suspension of erythrocytes encapsulating ADI from M. arginini.
[0282] 12. The suspension of paragraph 11, wherein said ADI comprises the amino acid sequence of SEQ ID NO: 1 or a variant or fragment thereof.
[0283] 13. The suspension of paragraph 12, wherein said variant comprises an amino acid sequence that is at least 80% identical to the amino acid sequence SEQ ID NO: 1.
[0284] 14. A pharmaceutical composition comprising a suspension according to any one of paragraphs 11 to 13, for its use in treating arginase-1 deficiency or arginine-dependent cancers, treating or preventing of septic shock, inhibiting angiogenesis and treating angiogenesis associated diseases.
[0285] 15. A method for treating arginase-1 deficiency or arginine-dependent cancers, treating or preventing of septic shock, inhibiting angiogenesis and treating angiogenesis associated diseases comprising administering to a patient or subject in need thereof the composition or suspension of any one of paragraphs 1 to 14.
[0286] 16. A method for reducing pathological plasma arginine levels to normal physiological plasma arginine levels in a patient or subject chronically suffering from said pathological plasma arginine levels, comprising the step of administering more than one dose of the composition or suspension of any one of paragraphs 1 to 14.
[0287] 17. The method of paragraph 16, wherein the pathological plasma arginine levels are in excess of about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590 or 600 μM.
[0288] 18. The method of paragraph 16 or 17, wherein the patient or subject has a known Arg1 mutation or exhibits less than about 5% arginase activity or substantially no arginase activity.
[0289] 19. The method of paragraph 16 or 17, wherein the pathological plasma arginine levels are at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more fold higher than healthy subjects not suffering from pathological plasma arginine levels.
[0290] 20. The method of any one of paragraphs 16 to 19, wherein one dose of the composition or suspension is sufficient to maintain non-pathological, normal physiological plasma arginine levels for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks.
[0291] 21. The method of any one of paragraphs 16 to 20, wherein the composition or suspension only needs to be administered once every 2 weeks or once every 4 weeks to maintain physiological plasma arginine levels.
[0292] The invention will now be described in the following non-limiting set of Claims.