Method of treating a mammal, including human, against cancer using methionine and asparagine depletion
a technology of asparagine and mammalian cells, applied in the direction of enzymes, biochemical equipment and processes, lysine, etc., can solve the problems of inability to obtain such an increase in cell mortality in vitro, inherently unpredictable effect of drug combination, etc., to achieve the effect of increasing cell mortality, assessing cytotoxic effects, and increasing cell mortality
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example 1
I. Abbreviations
[0183]CCK-8: Cell counting kit-8
DPBS: Dulbecco's Phosphate-Buffered Saline
IMDM: Iscove's Modified Dulbecco's
[0184]MGL: Methionine-γ-lyase
v / v: Volume to volume
II. Operating Conditions
II.1 Test Item
II.1.1. L-Asparaginase
Description: Medac® (Germany), E. Coli L-asparaginase 10 000 IU
[0185]One concentration of L-asparaginase (2.53 IU / mL) was prepared by serial dilutions in Dulbecco Phosphate Buffered Saline (DPBS) 1×. Concentration of L-asparaginase was diluted 11-fold to obtain final concentration of 0.23 IU / mL (IC50).
II.1.2. Methionine-γ-Lyase (MGL)
[0186]Description: P. Putida methionine-γ-lyase (MGL) produced in E Coli.
One concentration of MGL (2.09 IU / mL) was prepared by serial dilutions in Dulbecco Phosphate Buffered Saline (DPBS) 1×. Concentration of MGL was diluted 11-fold to obtain final concentration of 0.19 IU / mL (IC50).
II.2 Cell Lines
II.2.1. Description
[0187]Name: HL-60 cell line
Description: Human promyelocytic leukemia cell line (suspension)
Supplier and refe...
example 2
R ENCAPSULATION OF L-ASPARAGINASE IN MURINE ERYTHROCYTES
[0208]The L-asparaginase (Medac®E. Coli L-asparaginase) is encapsulated in murine erythrocytes (OF1 mice) by the method of hypotonic dialysis in a dialysis bag. The blood is centrifuged beforehand to remove the plasma, and then washed three times with 0.9% NaCl. The haematocrit is adjusted to 70% in the presence of the asparaginase, added to a final concentration of 400 IU / ml of erythrocytes or red blood cells (RBC) before starting the dialysis. The dialysis lasts 50 minutes at 4° C. against a lysis buffer of low osmolarity. The murine erythrocytes are then resealed through the addition of a high osmolarity solution and incubating 30 minutes at 37° C. After two washings with 0.9% NaCl and one washing with Sag-mannitol supplemented with bovine serum albumin BSA (6%), the erythrocytes are adjusted to haematocrit 50%. The erythrocytes encapsulating the L-asparaginase are called L-Aspa RBC. The encapsulation generates L-Aspa RBC at...
example 3
TION OF L-ASPARAGINASE IN HUMAN ERYTHROCYTES
[0216]The method described in WO-A-2006 / 016247 is used to produce a batch of erythrocytes encapsulating L-asparaginase. In accordance with the teaching of WO-A-2006 / 016247, the osmotic fragility is considered and the lysis parameters are adjusted accordingly (flow rate of the erythrocyte suspension in the dialysis cartridge is adjusted). The method is further performed in conformity with the physician prescription, which takes into account the weight of the patient and the dose of L-asparaginase to be administered. The specifications of the end product are as follows:[0217]mean corpuscular volume (MCV): 70-95 fL[0218]mean corpuscular haemoglobin concentration (MCHC): 23-35 g / dL[0219]extracellular haemoglobin≤0.2 g / dL of suspension[0220]osmotic fragility≤6 g / L of NaCl[0221]mean corpuscular L-asparaginase concentration: 78-146 IU / mL[0222]extracellular L-asparaginase≤2% of the total enzyme activity.
[0223]The suspension of erythrocytes so obta...
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