Erythrocytes containing arginine deiminase
09968663 ยท 2018-05-15
Assignee
Inventors
Cpc classification
A61K38/50
HUMAN NECESSITIES
A61P7/00
HUMAN NECESSITIES
International classification
Abstract
Use of erythrocytes containing arginine deirainase for the preparation of a medicinal product for lowering the plasma concentration of arginine in vivo. The use relates in particular to the treatment of arginine-dependent tumors, such as hepatocarcinoma and malignant melanoma, or inhibition of the synthesis of nitric oxide, and the prevention and/or treatment of septic shock.
Claims
1. A pharmaceutical composition comprising erythrocytes encapsulating arginine deiminase (ADI) in suspension in a pharmaceutically acceptable saline solution, wherein the encapsulation confers prolonged in vivo half-life beyond 24 hours to the arginine deiminase encapsulated in the erythrocytes, wherein the hematocrit in the suspension is from 40% to 70%, wherein the volume of the suspension is from 10 to 250 ml, and wherein the erythrocytes contained in the composition have an ADI enzymatic activity of from 16.7 to 180 IU per milliliters of pure erythrocytes.
2. The pharmaceutical composition of claim 1, wherein the saline solution comprises NaCl and at least one ingredient selected from the group consisting of glucose, dextrose, adenine, mannitol, and a mixture thereof.
3. The pharmaceutical composition of claim 1, wherein the saline solution is SAG-mannitol.
4. The pharmaceutical composition of claim 1, wherein the saline solution is ADsol.
5. The pharmaceutical composition of claim 1, further comprising a preservative for erythrocytes.
6. The pharmaceutical composition of claim 5, wherein the preservative is L-carnitine.
7. The pharmaceutical composition of claim 1, wherein the hematocrit of the suspension of erythrocytes is between 45 and 55%.
Description
(1) The invention will now be described in more detail on the basis of embodiments that are used as non-limiting examples, referring to the drawings in which:
(2)
(3)
(4)
(5)
(6) RBC or BC is used herein to designate red blood cells.
EXAMPLE 1
Installation
(7) Reference will first be made to
(8) The removable assembly includes a bag 2 that is flexible in volume, containing a suspension of erythrocytes to be lysed. Said bag 2 is equipped with a flexible tube 3, in a loop, operating in conjunction with pump P1, to provide circulation from and to the bag to maintain the erythrocytes in suspension. Said bag is in addition connected at its base to a flexible tube connected to the inlet of the blood compartment of a dialysis cartridge 5. Said tube 4 operates in conjunction with pump P2, which provides circulation of the suspension from the bag to the cartridge. A driven syringe pump PS1 is connected to tube 4 upstream of cartridge 5, and said syringe pump permits the enzyme to be introduced into the circulation of erythrocytes. The outlet of the blood compartment of cartridge is connected to an outlet flexible tube 6, which opens onto the exterior of module 1. A second driven syringe pump PS2 is connected to tube 6, and this syringe pump permits the enzyme to be introduced into the circulation of lysed erythrocytes. A bottle 7 containing a lysis solution is arranged in module 1, and is connected to the dialyzate inlet of cartridge 5 by a flexible tube 8, which operates in conjunction with pump P3 to provide circulation of the lysis solution through cartridge 5. Finally, the lysis solution leaving the cartridge is removed from module 1 by a flexible discharge tube 9, which ends in a bottle 10 located outside of module 1.
(9) Outlet tube 6 goes into a second module 11 with the overall shape of a parallelepiped, with a glass-covered front (not shown), arranged so that it can be opened and closed. At the back of this module there are means, not shown, for receiving elements forming part of the removable assembly that has just been described partially. Said elements comprise a flexible bag 12, connected to tube 6, and in which the lysed suspension will be stored. A driven syringe pump PS3 is connected to tube 6, for injecting the resealing product.
(10) The removable assembly is made entirely of flexible, transparent plastic, so that the process is completely visible.
(11) The device is further provided with various means that are not shown: means for cooling the interior of module 1 and regulating its temperature between +2 and +4 C., comprising, among other things, a temperature sensor located on tube 3 for measuring the temperature of the suspension circulating therein, and a temperature sensor for measuring the temperature T1 inside module 1, module 11 is further provided with means of heating the interior of module 11 and regulating the temperature T2 therein between +37 and +38 C.; a temperature sensor is fitted inside the module. means (for example ultrasonic or colorimetric) for detecting the presence of erythrocytes in the tubes, at D1 and D2, means PR1 for measuring the pressure at the inlet of the dialysis cartridge. electronic device receiving on the one hand data arriving from the temperature and pressure sensors and the detecting means, and on the other hand data relating to the settings of the lysis parameters; on the basis of said data, the device controls pumps P1, P2 and P3. A process flow chart is shown in
(12) The electronic device comprises a computer, designed for executing the above flow chart.
(13) Implementation of said device leads to the recovery, at 12, of a bag containing a suspension of erythrocytes containing the enzyme.
EXAMPLE 2
Production of Erythrocytes Encapsulating Arginine Deiminase
(14) 400 ml of blood is taken from the patient. The blood, maintained at 4 C., is deleukocytized and washed with a saline solution to remove the plasma, and placed in a flexible bag with a volume of 250 ml, at a hematocrit adjusted to 80%.
(15) An aqueous solution of arginine deiminase is added to the suspension of erythrocytes so as to obtain a concentration of 400 IU of enzyme per ml and a hematocrit of 70%.
(16) Take 1 ml of the suspension at 4 C. and place it in the instrument for measuring osmotic fragility described in J. Didelon et al. 2000 cited previously, the operating principle of which was described above. Measurements are taken for 10 minutes. The instrument makes it possible to determine the salinity that gives 50% hemolysis. This salinity is generally between 3 and 5.5 g NaCl per liter.
(17) The 250-ml capacity flexible bag 2, containing the suspension of erythrocytes and the enzyme, is placed in the installation of Example 1, and the suspension and the hypotonic lysis solution are admitted gradually into the respective compartments of the dialysis cartridge. The flow rate of the suspension of erythrocytes in the dialyzer is controlled between 15 and 30 ml/min, as a function of the salinity parameter determined in the preceding stage (osmotic fragility or resistance).
(18) The resealing solution is added in line at 10% v/v to the suspension of lysed erythrocytes just upstream of bag 12. The suspension is incubated for 30 min at 37 C. in the bag. It is then washed with a saline solution, a preserving solution is added to it (SAG-mannitol), then the bag is stored at +4 C. until it is used.
(19) The method makes it possible to obtain erythrocytes having an enzymatic activity between 80 and 180 IU per ml of pure erythrocytes.
(20) The total volume of the suspension is administered to the patient by intravenous perfusion in accordance with the usual practice of blood transfusion.
EXAMPLE 3
In Vitro Assay
(21) Arginise deimainase is an arginase catabolizing enzyme transforming arginine into citrulline and ammoniac. The aim of the study was to observe and confirm the depletion activity of arginine deiminase obtained from Pseudomonas aeruginosa once encapsulated into red blood cells. In this purpose, arginine deiminase-loaded red blood cells were incubated with arginine containing buffer. Citrulline and arginine levels were subsequently assessed by HPLC MS MS method.
(22) Preparation of Arginine Deiminase Loaded Red Blood Cells (RBC)
(23) Solution of recombinant SeMet-containing L-Arginine deiminase (ADI) (120 Ul/ml) originated from Pseudomona aeruginosa.
(24) Fresh heparinized OF1 mouse blood was obtained from Charles River laboratories and centrifuged (800 g. 10 min at 4 C.) to remove plasma and buffy coat. Packed erythrocytes were washed 3 times (1:1 v/v) with NaCl 0.9% (800 g, 10 min at 4 C.). After the final washing erythrocytes were mixed with (CGR-ADI) or without (CGR-LR) 20 Ul/ml arginine deiminase and the haematocrit of the RBC suspension was adjusted to 70% (using ADI solution or saline).
(25) Lysis of erythrocytes was obtained by a continuous flow dialysis process into a dialysis bag (cutoff 10 Kd). The hypotonic step was performed at 4 C. during 60 minutes against an adequate volume of lysis buffer at 40 mOsm/1 (NaH.sub.2PO.sub.4, 2H.sub.2O 0.73 g/l; Na.sub.2HPO.sub.4, 12H.sub.2O 5.035 g/l; glucose 0.36 g/l). 100 ml of hypotonic solution were added for 1 ml of erythrocytes introduced. After collection, the suspension of lysed erythrocytes was incubated at 37 C. for 10 min. The cells were then resealed and annealed by incubation at 37 C. during 30 minutes in a 1/10 (v/v) of resealing solution (adenine 0.39 g/l; inosine 15.6 g/l; sodium pyruvate 6.4 g/l; NaH.sub.2PO.sub.4, H2O 4.9 g/l; NaHPO.sub.4, 12H.sub.2O 10.9 g/l, glucose 11.5 g/l; NaCl 50 g/l). After resealing the erythrocytes were washed 3 times (800 g, 10 min at 4 C.) in Tris 310 mOsm/1 pH 7.4, BSA 4%.
(26) Whole blood, RBC suspension before and after dialysis step were monitored for haematocrit (Ht), mean cell volume (MCV), mean cell haemoglobin (MCHC) and mean corpuscular haemoglobin concentration (MCHC) using a Cobas Micros 601 CS 14/12 cell counter. CGR-ADI and CGR-LR suspension were at 25% haematocrit after dialysis.
(27) Aliquots of RBC suspension (with or without ADI) were collected before and after dialysis step for subsequent ADI activity measurement.
(28) Assay of ADI Activity
(29) Assay of ADI activity was performed on aliquots of RBC suspension (CGR-ADI and CGR-LR) collected before and after dialysis.
(30) Haematocrit of CGR-ADI and CGR-LR aliquots before dialysis were adjusted from 70% to 40% by an adequate dilution in NaCl 0.9%. Haematocrit of CGR-ADI and CGR-LR aliquot suspension after dialysis was of 25% and wasn't modified. The rate of ADI encapsulated was determined by measurement of ADI activity in whole blood or in supernatant. To determine ADI activity in whole blood, one third of RBC suspension aliquot was frozen in liquid nitrogen during 5 minutes and warmed at 37 C. and 10 l of a 10 fold dilution in 50 mM MES of frozen RBC was then used for enzymatic assay activity. To determine the enzymatic activity outside red cells, the other two-third of RBC suspension were centrifuged at 4 C. during 10 minutes and 10 l of a 2 fold dilution in 50 mM MES of supernatant was used for enzymatic assay. The amount of citrulline formed in 10 min was quantified by the colorimetric assay of Prescott and Jones. The standard assay mixture contained 900 l MES buffer 0.1M pH 6.0, MgCl.sub.2 20 mM and 10 l of supernatant or frozen RBC sample. The reaction was started by addition of 1 ml 10 mM L-arginine and was allowed to continue for 5, 10, 15 or 20 min at 37 C. The reaction was stopped at theses different times by the addition of 1 ml of an antipyrine-diacetylmonoxime solution. The mixture was boiled during 20 minutes and the absorbance at 466 nm was measured. Standard curves were constructed by appropriately diluting a stock solution of citrulline. Activity of ADI between 15 and 20 min was defined as micromoles of citrulline formed per min of enzyme. All measures were done in duplicates.
(31) In Vitro Functionality Assay
(32) In vitro assay was completed by incubating arginine deiminase-loaded red blood cells (CGR-ADI) in a buffer containing arginine and observing the levels of both arginine and citrulline aminoacid. Control of the reaction was realized with red blood cells loaded without arginine deiminase (CGR-LR) incubated in the same conditions.
(33) 1 ml of prewarmed arginine deiminase-loaded red cells (CGR-ADI) were mixed with 1 ml of buffer containing 300 M arginine, 20 mM MgCl.sub.2 in Tris pH 7.4. Tris buffer was prepared at 320 mOsm/kg, pH 7.4. The control assay was performed by mixing in the same conditions an equivalent amount of CGR-LR with arginine containing buffer. Mixing was realized by upside down movements. 400 l of sample, representating time 0, were immediately collected in an ependorf tube and placed at 4 C. The rest of the mixture was incubated during 30 minutes at 37 C. At the end of the reaction, 400 l representing time 30 (for 30 minutes) were collected in an ependorf and placed at 4 C. For all the samples of 400 l collected, three quarter (300 l) were centrifuged at 4 C. during 10 min. After centrifugation, an aliquot of 100 l of supernatant was collected and frozen at 20 C. The other quarter was directly frozen in liquid nitrogen during five minutes. After warming at 37 C., an aliquot of 50 l of sample was collected and frozen at 20 C.
(34) Arginine and citrulline levels in each sample were then assessed by HPLC/MS/MS method.
(35) Results: see
(36) Within 30 minutes at 37 C., the RBC entrapping ADI are able to deplete the arginine contained in the medium (supernatant) from about 135 mol/L to about 42 mol/L. In the same time, citrulline concentration in the medium (extra-erythrocytes) is produced from about 17 to about 110 mol/L. The activity of ADI in the supernatant was under the limite of detection (<0.1 Ul/Ml). The activity measured in the RBC pellet was 2.56 Ul/mL. It proves that the arginine deimination is provided by the intra erythrocyte ADI, and that arginine enter into the erythrocyte to be digested into them. In addition, it strongly suggests the citrulline produced into the erythrocyte is going to the extra RBC medium though the red cell membrane.
(37) Concerning the Control RBCs, which are processed erythrocytes where ADI is replaced by saline, arginine from the extra RBC medium decreases only from about 165 mol/L to about 157 mol/L within 30 minutes at 37 C., and citrulline reachs only from about 1 to about 2 mol/L. This low depletion can be explained by the endogenous activity of arginase contained into the RBCs. We underlined again arginase do not produce citrulline while arginine digestion, which is specific to ADI.
EXAMPLE 4
Kinetic Study of Arginine Concentration in Mice Plasma in Response to Injections of Two Formulations of Arginine Deiminase
(38) The aim of the study was to follow the plasma pharmacokinetic of arginine and citruline in OF1 mice in response to injection of arginine deiminase-loaded erythrocytes
(39) Preparation of Test and Control Substances
(40) Recombinant SeMet-containing L-Arginine deiminase (ADI) (120 UI/ml) originated from Pseudomona aeruginosa.
(41) Free arginine deiminase (ADI+RBC) was diluted in washed mouse red blood cells (RBC) and Sag-mannitol 1/3 (v/v) (Haemonetics) in order to obtain a final concentration at 10 UI/ml of packed RBC at hematocrit of 50%. Sag-mannitol was supplemented with 10 mM MgCl.sub.2 prior to addition.
(42) Test substance (ADILE) consisted in arginine deiminase loaded red blood cells (RBC). The procedure of preparation of arginine deiminase loaded red blood cells was determined as described for in vitro functionality test of ADI. Before dialysis ADI was mixed with washed packed erythrocytes in order to have a final concentration 50 UI/ml. After dialysis, encapsulated RBC were mixed with Sag-Mannitol 1/3 (v/v) supplemented with 10 mM MgCl.sub.2. Final hematocrit was adjusted to 50%. The ADI activity obtained after encapsulation was of 8.35 UI/ml of encapsulated RBC at hematocrit of 50%.
(43) A third sample with no enzyme (CGR) was prepared with washed RBC resuspended with Sag Mannitol (supplemented with 10 mM MgCl.sub.2) at final hematocrit of 50%. Saline solution was added in replacement of ADI.
(44) During whole experiment, whole blood, RBC suspension before and after dialysis step were monitored for haematocrit (Ht), mean cell volume (MCV), mean cell haemoglobin (MCHC) and mean corpuscular haemoglobin concentration (MCHC) using a Cobas Micros 601 CS 14/12 cell counter. Aliquots of RBC suspension were collected before and after dialysis step for subsequent ADI activity measurement. ADI activity measurement was determined as described previously.
(45) Animals
(46) 64 OF1 female mice, 5-6 week-old and weighing 18-22 g were obtained from Charles River Laboratories (L'Arbresle, France). Animals were observed for 7 days in a specific-pathogen-free (SPF) animal care before treatment. The experimental protocols were approved by the French Ministries of Agriculture and Research. The 64 healthy OF1 mice were randomized in 1 group of 4 mice and 3 groups of 20 mice. Treated mice received a single injection by intravenous route in an injection volume of 250 l.
(47) Treatment Schedule
(48) The treatment schedule was chosen as followed: mice from group 1 were not treated; mice from group 2 were treated by mouse red blood cells washed at hematocrit of 50% (RBC); mice from group 3 received a single injection of arginine deiminase loaded-erythrocytes (ADILE); mice of group 4 received a single injection of free arginine deiminase (ADI+RBC) in suspension in mouse RBC washed at hematocrit of 50%. The different product samples were administered in double blind.
(49) After treatment, mice were sacrificed by cardiac puncture. Isoflurane Forene (Centravet, Bondoufle, France) was used to anaesthetize the mice before sacrifice. The sacrifice of mice was performed as described below in the table. Approximately 800 l of whole blood were collected in heparin lithium glass tubes and kept immediately in ice-water bath after collection. Blood samples were immediately centrifuged at 2,500 g for 10 min at +4 C. to obtain plasma. About 200 l of plasma were transferred into propylene tubes, immediately frozen at 20 C. The remaining blood cell pellet was transferred into propylene tubes, immediately frozen at 20 C. until analysis. The levels of arginine and citrulline were measured in one vial of plasma and blood cell pellet.
(50) Amino Acids Analyses
(51) Concentration of arginine and citrulline in plasma and blood cell pellets were measured after extraction of arginine and citrulline by HPLC/MS/MS method.
(52) TABLE-US-00001 Dose ADI Administration Sampling times Treatments (UI/kg) route (hours) No mice None 0 NA NA 4 RBC 0 IV 3 4 (10 ml/kg) 6 4 12 4 24 4 48 4 ADILE 100 IV 3 4 (10 ml/kg) 6 4 12 4 24 3 48 3 ADI + RBC 100 IV 3 4 (10 ml/kg) 6 4 12 4 24 4 48 4 NA: Not Applicable
Results: see
(53) This prove the entrapment of ADI into RBC is possible by lysis/resealing steps, and that the ADI loaded into RBC is much longer efficient than ADI in free solution.