Granted Patent B2

US 12,650,422 · App. 15/320,710 · Granted Jun 9, 2026

Inventors: Kenneth R. Brouwer (Chapel Hill, NC); Christopher Black (Cary, NC); Jonathan Jackson (Raleigh, NC)

Assignee: Qualyst Transporter Solutions, LLC

G01N33/5008C12Q1/26G01N33/5067G01N2500/10

Abstract

Claims (24)

1 . A method of evaluating disposition of a candidate compound in an in vitro culture and/or suspension to predict in vivo disposition of the candidate compound, the method comprising:

(a) providing a cell culture and/or suspension, wherein the cell culture and/or suspension comprises a first sandwich culture of hepatocyte cells and providing a second sandwich culture and/or suspension of hepatocyte cells, each first and second sandwich culture comprising an artificial membrane system and at least one bile canaliculus, wherein the artificial membrane system comprises a cell transfected or knocked-out for transporters so as to be adapted to mimic cells, the first sandwich culture having an intact bile canaliculus and the second sandwich culture having a disrupted bile canaliculus;

(b) exposing a candidate compound to the first sandwich culture and to the second sandwich culture for a time sufficient to allow uptake of the candidate compound;

(c) simultaneously exposing the first and second sandwich cultures and/or suspension and the candidate compound to a medium containing proteins that are present extracellularly in liver at physiological concentrations to approximate the in vivo environment of hepatocytes in the liver, wherein the concentration of the proteins contained in the media is a physiologic concentration or a concentration having a characteristic similar to a physiological concentration;

(d) washing and lysing the first and second cell cultures and/or suspensions; and

(e) determining an amount of the candidate compound taken up in the first and second sandwich cultures and/or suspension, comprising determining an intracellular concentration of the candidate compound, to thereby evaluate disposition of the compound to predict in vivo disposition of the candidate compound without adjusting using protein binding data from a separate experiment.

2 . The method of claim 1 , wherein the artificial membrane system mimics a cell in co-culture with a supporting cell, wherein the supporting cell comprises a fibroblast cell and/or a Kupffer cell.

3 . The method of claim 1 , wherein the cell mimicked by the artificial membrane system is selected from the group consisting of a vesicle, a hepatocyte cell, a liver-derived cell, a renal cell, a gastrointestinal cell, a pancreatic cell, a cardiac cell, a neuronal cell, a myocyte cell, a lipocyte cell, and a pulmonary cell.

4 . The method of claim 1 , wherein the cell mimicked by the artificial membrane system comprises a cell line, wherein the cell line is selected from the group consisting of hepatocytes, Caco-2, and MDC.

5 . The method of claim 1 , wherein the cells are isolated from a source selected from the group consisting of mouse, rat, rabbit, human, monkey, ape, cat, dog, pig, hog, cattle, oxen, sheep, horses, turkeys, chickens, fish, ducks and geese.

6 . The method of claim 1 , wherein the culture and/or suspension further comprises a long-term culture and/or suspension.

7 . The method of claim 1 , wherein the first sandwich culture and the second sandwich culture and/or suspension further comprise a long-term sandwich culture and/or suspension.

8 . The method of claim 1 , wherein the protein component is selected from the group comprising an albumin, β-lipoprotein, alpha-1-acid glycoprotein, plasma or serum derived from mouse, rat, rabbit, human, monkey, ape, cat, dog, pig, hog, cattle, oxen, sheep, horses, turkeys, chickens, fish, ducks or geese, bilirubin, and combinations thereof.

9 . The method of claim 1 , wherein the method is carried out in at least one well of a multi-well plate.

10 . The method of claim 1 , further comprising screening a plurality of candidate compounds simultaneously.

11 . The method of claim 1 , wherein the medium containing proteins that are present extracellularly comprises another compound that modulates properties of the candidate compound.

12 . The method of claim 1 , wherein any combination of any of the exposing steps can occur in any order or simultaneously.

13 . The method of claim 12 , wherein the protein component is an albumin or serum derived from mouse, rat, rabbit, human, monkey, ape, cat, dog, pig, hog, cattle, oxen, sheep, horses, turkeys, chickens, fish, ducks or geese.

14 . The method of claim 1 , wherein determining an amount of the candidate compound taken up in the culture and/or suspension to thereby evaluate disposition comprises:

(a) determining hepatic accumulation;

(b) determining biliary excretion; and/or

(c) determining biliary clearance.

15 . The method of claim 1 , wherein the protein component comprises a mixture of protein components.

16 . The method of claim 1 , wherein the medium comprises a bile acid or mixture of bile acids.

Assignments (4)

Continuity (2)

References Cited (190)