IN VITRO DRUG METABOLISM REAGENT AND USES THEREOF
The present disclosure provides an in vitro reagent for evaluating xenobiotic metabolism in a cell culture based assay. The in vitro reagent is an admixture of metabolically competent cells and exogenous drug metabolizing enzyme co-factors follow by cryopreservation in the absence of cryopreservation agent so that the cells would be rendered permeable upon thawing due to plasma membrane disruption (while maintaining the integrity of organelles). The permeabilized plasma membranes allow ready diffusion of the exogenous cofactors into the cells to enhance the activities of cellular drug metabolizing enzymes. Addition of a xenobiotic test compound to the thawed in vitro reagent allows metabolism of the test compound by the metabolically competent cells, with metabolites readily diffusible outside the cells due to the permeabilized plasma membranes.
1 - 20 . (canceled)
21 . A method for manufacturing an in vitro reagent for evaluating xenobiotic metabolism, comprising:
a) permeabilizing metabolically competent cells;
b) adding exogenous drug metabolizing enzyme co-factors either before or after the metabolically competent cells are permeabilized; and
c) providing a buffered solution,
wherein the reagent does not comprise a cryopreservative agent.
22 . The method of claim 21 , wherein the reagent is further stored frozen at a temperature of −10° C. to about −80° C.
23 . The method of claim 21 , wherein the reagent is not stored frozen in liquid nitrogen.
24 . The method of claim 21 , wherein the metabolically competent cells are permeabilized by freezing and thawing in the absence of a cryopreservative agent.
25 . The method of claim 24 , wherein the permeabilized metabolically competent cells are not centrifuged prior to use.
26 . The method of claim 24 , wherein the permeabilized metabolically competent cells are not counted prior to use.
27 . The method of claim 21 , wherein the metabolically competent cells are hepatocytes.
28 . The method of claim 21 , wherein the metabolically competent cells are enterocytes.
29 . The method of claim 21 , wherein the metabolically competent cells are engineered to contain cytochrome P450 isoforms.
30 . The method of claim 21 , wherein the metabolically competent cells comprise drug metabolizing enzyme (DME) activities.
31 . The method of claim 21 , wherein the metabolically competent cells are human, rat, monkey, dog, mammals, avian or non-mammalian.
32 . The method of claim 21 , wherein the metabolically competent cells are primary cells.
33 . The method of claim 32 , wherein the metabolically competent cells are pooled from more than one donor
34 . The method of claim 21 , wherein the metabolically competent cells are a cell line.
35 . The method of claim 21 , wherein the drug metabolizing enzyme co-factors are selected from β-Nicotinamide adenine dinucleotide 2′-phosphate (NADPH), Uridine 5′-diphosphoglucuronic acid (UDPGA), 3′-Phosphoadenosine 5′-phosphosulfate (PAPS), N-acetyl coenzyme A, s-adenosyl methionine, amino acids, carnitine, and L-glutathione.
36 . A method for manufacturing an in vitro reagent for evaluating xenobiotic metabolism, comprising:
a) combining intact metabolically competent cells with exogenous drug metabolizing enzyme co-factors in a cell culture medium to form a cell mixture;
b) freezing the cell mixture at a temperature from about −10° C. to about −80° C., wherein the cell mixture does not comprise a cryopreservative agent; and,
c) thawing the cell mixture to form the in vitro reagent wherein cell membranes of the metabolically competent cells are permeabilized via thawing.
37 . The method of claim 36 , wherein the in vitro reagent is used to evaluate xenobiotic metabolism after thawing without the step of centrifuging or cell counting.
38 . The method of claim 36 , wherein the metabolically competent cells are hepatocytes.
39 . The method of claim 36 , wherein the metabolically competent cells are enterocytes.
40 . The method of claim 36 , wherein the metabolically competent cells are engineered to contain cytochrome P450 isoforms.
41 - 78 . (canceled)