Methods for assessing transendothelial barrier integrity

Patent metadata

Field	Value
Publication number	US-2021247385-A1
Application number	US-202117180406-A
Country	US
Kind code	A1
Filing date	Feb 19, 2021
Priority date	Aug 21, 2018
Publication date	Aug 12, 2021
Grant date	—

Abstract

This application relates to a method for identifying a drug candidate capable of increasing or decreasing barrier tissue integrity of endothelial cells. Moreover, this application relates to the use of a tight junction gene transcriptional reporter as a surrogate marker of transendothelial barrier integrity.

First claim

1 - 25 . (canceled)

26 . An in vitro method, comprising the steps of:
a) providing endothelial cells (ECs) comprising a reporter gene under the control of a tight junction gene promoter, wherein the ECs are enriched for cells expressing the reporter gene; b) contacting the ECs with a drug candidate; and c) measuring in vitro transendothelial barrier integrity (TBI) before and after contacting the ECs with the drug candidate, or measuring in vitro TBI of the ECs contacted with the drug candidate and in parallel measuring in vitro TBI of ECs not contacted with the drug candidate; wherein the method identifies a drug candidate capable of i) increasing in vivo transendothelial barrier integrity (TBI) or ii) decreasing in vivo TBI of endothelial cells (ECs); wherein: (i) a higher in vitro TBI of the ECs contacted with the drug candidate compared with the in vitro TBI of the ECs not contacted with the drug candidate is indicative of a drug capable of increasing in vivo TBI of ECs, and (ii) a lower in vitro TBI of the ECs contacted with the drug candidate compared with the in vitro TBI of the ECs not contacted with the drug candidate is indicative of a drug capable of decreasing in vivo TBI of ECs.

27 . The method of claim 26 , wherein step c) comprises measuring the transendothelial electrical resistance (TEER) wherein the measured TEER is indicative for in vitro TBI.

28 . The method of claim 26 , wherein step c) comprises measuring the expression of the reporter gene wherein the expression of the reporter gene is indicative for in vitro TBI.

29 . The method of claim 26 , wherein the tight junction gene is selected from the group consisting of CLDN5, ocludin (OCLN) and MARVELD3.

30 . The method of claim 26 , wherein the ECs are differentiated from pluripotent stem cells.

31 . The method of claim 26 , wherein a polynucleotide encoding the reporter gene is inserted at the 3′ end of the tight junction gene.

32 . The method of claim 31 , wherein (i) a tight junction gene reporter gene fusion protein is expressed, or (ii) the reporter gene is expressed from an internal ribosomal entry site (IRES), or (iii) a tight junction gene reporter gene fusion protein is expressed and subsequently processed to individual tight junction protein and reporter protein.

33 . The method of claim 32 , wherein (iii) a tight junction gene reporter gene fusion protein is expressed and subsequently processed to individual tight junction protein and reporter protein, wherein a polynucleotide encoding a self-cleaving peptide is introduced between the tight junction gene and the reporter gene.

34 . The method of claim 26 , wherein activation of the promoter of the tight junction gene leads to expression of the reporter gene.

35 . The method claim 26 , wherein the cells are enriched for cells expressing the reporter gene in step a) by fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS).

36 . The method of claim 26 , which is performed in a high-throughput format.

37 . The method of claim 26 , which is used to screen molecules in a drug development setting, in particular for high-throughput screening a drug candidate compound library.

38 . A cell culture produced according to step a) of claim 26 , wherein the fraction of cells expressing the tight junction gene is higher than 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

39 . A cell capable of expressing a reporter gene, wherein expression of the reporter gene is under the control of the promoter of a tight junction gene.

40 . The cell of claim 39 , wherein the tight junction gene is CLDN5.

41 . A method of treating a disease in an individual, comprising:
administering to said individual a therapeutically effective amount of a composition comprising 2-[3-(6-Methyl-2-pyridinyl)-1H-pyrazol-4-yl]-1,5-naphthyridine in a pharmaceutically acceptable form; or administering to said individual a therapeutically effective amount of a composition comprising 4-[4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide in a pharmaceutically acceptable form.

42 . The method of claim 41 , wherein said disease is associated with vascular complications.

43 . The method of claim 41 , wherein said disease is selected from the group consisting of diabetes Type-2 and Type-1, diabetic retinopathy, Wet AMD, Metabolic Syndrome, Severe Obesity, Hypercholesterolemia, Hypertension, coronary artery disease, nephropathy, retinopathy, kidney failure, tissue ischemia, chronic hypoxia, artherosclerosis, and tissue edema caused by drug-induced toxicity.

44 . The method of claim 41 , wherein said disease is diabetic retinopathy or Wet AMD.

Assignees

• Hoffmann La Roche

Inventors

• Cowan Chad A
• Meyer Claas Aiko
• Roudnicky Filip
• Zhang Jitao David

Classifications

• C12N2506/03

  from non-embryonic pluripotent stem cells · CPC title

• G01N33/5032

  on intercellular interactions · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• A61K31/4745

  condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines (yohimbine derivatives, vinblastine A61K31/475; ergoline derivatives A61K31/48) · CPC title

• C12N2503/02

  Drug screening · CPC title