Ex vivo methods for validating substance testing with human organs and/or tissues

• US 8,765,364 B2
• Filed: 05/18/2007
• Issued: 07/01/2014
• Est. Priority Date: 05/18/2007
• Status: Active Grant

• Current Assignee: Lifeline Scientific, Inc. (Shanghai Genext Medical Technology Co., Ltd.)
• Original Assignee: Lifeline Scientific, Inc. (Shanghai Genext Medical Technology Co., Ltd.)
• Inventors: Brassil, John, Kravitz, David, Curtis, Gerald
• Primary Examiner(s): SINGH, SATYENDRA K
• Application Number:
  US11/802,064
• Publication Number:
  US 20080286747A1
• Time in Patent Office: 2,601 Days
• Field of Search: None
• US Class Current: 435/1.2
• CPC Class Codes: G01N 33/5088 of vertebrates
• CPC Class Codes: G16H 10/40 for data related to laboratory analysis, e.g. patient specimen analysis
• CPC Class Codes: G16H 20/17 delivered via infusion or injection

Abstract

Methods of validating results of assessments of test substances using human-derived tissues and/or organs, particularly tissues and/or organs unsuitable for transplantation, include assessing the suitability of the organ and/or tissue for substance testing, as well as inter-organ variability and use of exogenous and/or endogenous controls.

Claims

1. An ex vivo method of validating results of substance testing using at least one human organ or tissue unsuitable for transplantation, wherein said human organ or tissue is a metabolically active human organ or tissue that has been permanently removed from its origin, the method comprising:

(a) assessing said human organ or tissue for suitability for testing prior to administration of a test substance by perfusing said organ or tissue under physiological and normothermic conditions with a perfusate that comprises about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine and adenosine monophosphate (AMP), but does not comprise the test substance, wherein assessing said human organ or tissue for suitability includes determining that said human organ or tissue is unsuitable for transplantation, and either

assaying the perfusate after it leaves said organ or tissue, or

monitoring a parameter of said organ or tissue;

(b) preparing a perfusate comprising said test substance, about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine and adenosine monophosphate (AMP), and perfusing said organ or tissue under said conditions with the perfusate containing the test substance;

(c) assessing a parameter relating to said test substance;

(d) assessing said organ or tissue for suitability for testing of said parameter relating to said test substance during and/or after administration of said test substance by

quantifying a functional state of said organ or tissue relative to said parameter being assessed, wherein said organ or tissue acts as its own control,

verifying that the organ or tissue is still having its effect, or being affected by, substances in connection with said parameter being assessed for the test substance, and

verifying that said parameter being assessed is not inappropriately affected by an extraneous change in the organ or tissue during the course of assessment of the parameter relating to said test substance; and

wherein quantifying the functional state of said organ or tissue relative to said parameter being assessed in step (d) comprises perfusing said organ or tissue under same conditions with the same perfusate comprising about 15 to about 20% (v/v) matched human erythrocytes, about 2 to about 6% human serum albumin, N-acetylcysteine and adenosine monophosphate (AMP), and additionally containing at least one control substance having a known qualitative or quantitative effect with respect to said parameter, and assessing the same parameter with respect to at least one said control substance.

2. The method of claim 1, wherein said control substance has a competitive relationship with said test substance relative to said parameter, and is perfused through the organ or tissue after the test substance has been perfused through the organ or tissue.

3. The method of claim 1, wherein said control substance has a competitive relationship with said test substance relative to said parameter, and is perfused through the organ or tissue simultaneously with the test substance.

4. The method of claim 1, wherein said control substance has a non-competitive relationship with said test substance relative to said parameter, and is perfused through the organ or tissue simultaneously with the test substance.

5. The method of claim 1, wherein step quantifying the functional state of said organ or tissue relative to said parameter being assessed in step (d) comprises perfusing said organ or tissue with the same perfusate additionally containing at least two control substances having a known qualitative or quantitative effect with respect to said parameter, and assessing the same parameter with respect to at least two said control substances.

6. The method of claim 5, wherein a value of said parameter for a first said control substance has previously been established to be significantly different from a value of said parameter for a second said control substance.

7. The method of claim 6, wherein the value of said parameter for the first said control substance has previously been established to be zero or near zero, and the value of said parameter for the second said control substance has previously been established to be substantially different from zero.

8. The method of claim 6, wherein said parameter is plasma clearance of the substance by liver, and the at least two control substances are propanolol and atenolol.

9. The method of claim 6, wherein said parameter is uptake of the substance by heart, and the at least one control substance is selected from the group consisting of dopa and dopamine.

10. The method of claim 1, further comprising determining a ratio between a first value of said parameter for said test substance and a second value of said parameter for said control substance.

11. The method of claim 10, comprising performing said method on a plurality of the same type of organ, establishing a normative range of said ratios, and discounting results of tests in which the ratio is outside of said normative range of ratios.

12. The method of claim 1, wherein said parameter is passive absorption of the substance by organs of the type tested.

13. The method of claim 12, wherein the organs of the type tested are intestines, and the at least one control substance is at least one member selected from the group consisting of antipyrine, terbutaline, mannitol and labeled dextrans.

14. The method of claim 1, wherein said parameter is active absorption of the substance by organs of the type tested, and said at least one control substance is perfused through the organ or tissue after the test substance has been perfused through the organ or tissue.

15. The method of claim 14, wherein the organs of the type tested are intestines, and the at least one control substance is at least one member selected from the group consisting of cephalexin and arginine.

16. The method of claim 14, wherein the organs of the type tested are lungs, and the at least one control substance is selected from the group consisting of salbutamol and ipratropium.

17. The method of claim 1, wherein said parameter is phase I metabolism of the substance by organs of the type tested.

18. The method of claim 17, wherein the organs of the type tested are intestines or kidneys, and the at least one control substance is at least one member selected from the group consisting of phenacetin, tolbutamide, s-methenyltoin, dextromethorphan, chloroxazone and methadone.

19. The method of claim 17, wherein the organs of the type tested are livers, and the at least one control substance is at least one member selected from the group consisting of phenacetin, tolbutamide, s-methenyltoin, dextromethorphan, chloroxazone, methadone, carbamazepine and 7-nitrazepam.

20. The method of claim 17, wherein the organs of the type tested are lungs, and the at least one control substance is at least one member selected from the group consisting of phenacetin, tolbutamide, s-methenyltoin, dextromethorphan, chloroxazone, methadone and carbamazepine.

21. The method of claim 17, comprising selecting said control substance as a function of a type of enzymatic metabolism suspected to occur with said test substance.

22. The method of claim 1, wherein said parameter is phase II metabolism of the substance by organs of the type tested.

23. The method of claim 22, wherein the organs of the type tested are selected from the group consisting of intestines, livers, lungs and kidneys, and the at least one control substance is at least one member selected from the group consisting of harmol and naphthol.

24. The method of claim 1, wherein said parameter is biliary excretion of the substance by liver, the at least one control substance is at least one member selected from the group consisting of tetrabomosulphophthalein, dibromosulphophthalein, indocyanine green and bile salts, and said at least one control substance is perfused through the organ or tissue after the test substance has been perfused through the organ or tissue.

25. The method of claim 1, wherein said parameter is receptor mediated endocytosis of the substance by liver, and the at least one control substance is at least one member selected from the group consisting of asialoglycoproteins.

26. The method of claim 1, wherein said parameter is active uptake of the substance by lung, and the at least one control substance is at least one member selected from the group consisting of putrescine, spermine and spermidine, and said at least one control substance is perfused through the organ or tissue after the test substance has been perfused through the organ or tissue.

27. The method of claim 1, wherein said parameter is glomerular filtration of the substance by kidney, and the at least one control substance is at least one member selected from the group consisting of inulin and creatinine.

28. The method of claim 27, wherein said control substance is radioactively labeled inulin.

29. The method of claim 1, wherein said parameter is active secretion of the substance by kidney, and the at least one control substance is p-aminohippuric acid, and said at least one control substance is perfused through the organ or tissue after the test substance has been perfused through the organ or tissue.

30. The method of claim 1, wherein said parameter is active reabsorption of the substance by kidney, and the at least one control substance is selected from the group consisting of tetra-ethylammonium acetate, sodium and glucose, and said at least one control substance is perfused through the organ or tissue after the test substance has been perfused through the organ or tissue.

31. The method of claim 1, wherein said parameter is Phase II metabolism of the substance by kidney, and the at least one control substance is at least one member selected from the group consisting of mercaptans and glutathione conjugates.

32. The method of claim 1, wherein said control substance is an endogenous substance.

33. The method of claim 1, wherein said control substance is an exogenous substance.

34. The method of claim 1, wherein said organ or tissue has been subjected to hypothermic storage before step (a).

35. The method of claim 1, wherein said organ or tissue has been subjected to disease or injury before step (a).

36. The method of claim 35, wherein said disease or injury occurred before harvest of said organ or tissue.

37. The method of claim 35, wherein said disease or injury occurred after harvest of said organ or tissue.

38. The method of claim 1, wherein said organ or tissue has been subjected to injury before step (a), and said injury resulted from prolonged warm ischemia.

39. The method of claim 1, wherein said organ or tissue has been subjected to injury before step (a), and said injury resulted from hypothermic storage.

40. The method of claim 1, wherein said at least one organ or tissue is a combination of organs.

41. The method of claim 1, wherein said at least one organ or tissue is an organ or tissue harvested from a deceased human.

42. The method of claim 1, wherein said parameter is assessed in at least one member selected from the group consisting of a perfusate sample, a biopsy from said at least one organ or tissue, and an effluent of said at least one organ or tissue.