US2021405027A1PendingUtilityA1
In vitro nephrotoxicity screening assay
Est. expiryJun 17, 2036(~9.9 yrs left)· nominal 20-yr term from priority
G01N 33/5014C12Q 2600/158G01N 33/5008G01N 33/5044G01N 33/5005C12Q 1/6883G01N 33/6863G01N 2333/485C12Q 2600/142
62
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Claims
Abstract
The invention relates to methods for predicting the in vivo nephrotoxicity of a drug substance, in particular a nucleic acid molecule such as a siRNA or an antisense oligonucleotide using an in vitro cell based assay measuring the levels of extracellular EGF as toxicity biomarker, potentially in combination with other biomarkers like ATP and KIM-1.
Claims
exact text as granted — not AI-modified1 . An in vitro method for predicting in vivo nephrotoxicity of a drug substance in a mammal, said method comprising the steps of:
a. culturing cells expressing epidermal growth factor receptor (EGFR) in a suitable cell culture media containing at least 4 ng/ml of epidermal growth factor (EGF); b. administering the drug substance to said cell culture; c. incubating the cells for a period of time; and d. subsequently measuring the EGF level in the supernatant;
wherein an increase in EGF in the supernatant is indicative of a drug substance which is, or is predicted to be, associated with nephrotoxicity.
2 . The method according to claim 1 , wherein EGF level in the supernatant is compared to a reference value obtained from cells treated with vehicle control or a non-toxic reference drug substance, where the non-toxic drug substance has been validated as non-toxic in vivo.
3 . The method according to claim 2 , wherein the non-toxic reference drug substance is an oligonucleotide compound consisting of CGTcagtatgcgAATc (SEQ ID NO: 1), wherein lower case letters represent DNA units, bold upper case letters represent beta-D-oxy-LNA units, all LNA C are 5′methyl C and all internucleoside linkages are phosphorothioate linkage.
4 . The method according to claim 3 , wherein a level of EGF in the supernatant above 200% relative to the vehicle control or non-toxic reference value is predicative of nephrotoxicity of the drug substance.
5 . The method according to claims 2 to 4 , wherein EGF level in the supernatant is further compared to a second reference value obtained from cells treated with a nephrotoxic drug substance, where the nephrotoxic reference drug substance has been validated to cause nephrotoxicity in vivo.
6 . The method according to claim 5 , wherein the toxic reference drug substance is an oligonucleotide compound consisting of GCtgtgtgagcttGG (SEQ ID NO: 4), wherein lower case letters represent DNA units, bold upper case letters represent beta-D-oxy-LNA units, all LNA C are 5′methyl C and all internucleoside linkages are phosphorothioate linkage.
7 . The method according to claims 5 to 6 , wherein a toxicity grade is determined according to the following formula
(
(
[
EGF
]
drug
substance
-
[
EGF
]
non
-
toxic
reference
+
(
AW
/
1
00
)
)
[
E
G
F
]
toxic
reference
-
[
EGF
]
non
-
toxic
reference
+
(
AW
/
1
00
)
)
)
×
100
%
wherein AW is the assay window, which is determined as the difference between the non-toxic reference drug substance and the nephrotoxic reference drug substance.
8 . The method according claim 7 , wherein a toxicity grade above 6 is predictive of nephrotoxicity of the drug substance, such as above 20, such as above 50.
9 . The method according to any one of claims 1 to 8 wherein step d) further comprises the measurement of intracellular adenosine triphosphate (ATP) levels; wherein a decrease in intracellular ATP levels is indicative of a drug substance which is, or is predicted to be, associated with nephrotoxicity.
10 . The method according to claim 9 , wherein a level of intracellular ATP below 80% relative to the saline or non-toxic reference value is predicative of nephrotoxicity of the drug substance.
11 . The method according to any one of claims 1 to 10 , wherein step d) further comprises the measurement of extracellular kidney injury molecule-1 (KIM-1) protein or intracellular mRNA levels, wherein an increase in KIM-1 levels are indicative of a drug substance which is, or is predicted to be, associated with nephrotoxicity.
12 . The method according to claim 11 , wherein a level of above 200% relative to the saline or non-toxic reference value is predicative of nephrotoxicity of the drug substance.
13 . The method according to any one of claims 1 to 12 , wherein the cells expressing EGFR is selected from the group consisting of epithelial cell, endothelial cell, mesenchymal cells, neuroectodermal cells and hepatocytes.
14 . The method according to claim 13 , wherein the cell culture is a primary kidney epithelial cell culture selected from the group consisting of proximal tubule epithelial cells, distal tubule epithelial cells and collecting duct epithelial cells, in particular primary human PTEC or rat PTEC cells.
15 . The method according to claim 13 , wherein the cells expressing EGFR are cultured from an immortalized cell line, such as PTEC-TERT-1, ciPTEC, HK-2, NKi-2 or human A549 cell lines.
16 . The method according to any one of claims 1 to 16 , wherein the period of incubation with the drug substance is between 2 and 6 days, such as around 3 days.
17 . The method according to any one of claims 1 to 16 , wherein the drug substance is selected from the group consisting of nucleic acid based molecules, chemotherapeutic agents;
aminoglycosides; anti-bacterial agents, anti-viral agents; anti-fungal agents, anti-inflammatory agents and immunosuppressant agents.
18 . The method according to any one of claims 1 to 17 , wherein the drug substance is a nucleic acid molecule selected from a RNAi agents, an antisense oligonucleotide or an aptamer.
19 . The method according to any one of claims 18 , wherein the nucleic acid molecule comprises one or more 2′ sugar modified nucleosides, independently selected from the group consisting of 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabino nucleic acid (ANA), 2′-fluoro-ANA and LNA nucleosides.
20 . The method according to any one of claims 18 to 19 , wherein the nucleic acid molecule comprises at least one modified internucleoside linkage.
21 . The method according to claim 11 or 12 , wherein the increase in KIM-1 is predicative of nephrotoxicity for a nucleic acid molecule according to any one of claims 18 to 20 even if the EGF levels are not increased.
22 . A method for selecting one or more drug substances for in vivo administration to a mammal, from a library of drug substances, said method comprising the steps of
a. obtaining a library of drug substances; b. administering each member of the library of drug substances to a cell culture expressing epidermal growth factor receptor (EGFR) and where the medium contains at least 4 ng/ml of epidermal growth factor (EGF), such as between 8 and 15ng/ml, such as 10 ng/ml of epidermal growth factor (EGF); c. culturing the cells in vitro for a period of time, such as per any one of the claims 16 ; d. measuring the amount of intracellular EGF, and optionally other biomarkers, for each drug substance, such as per any one of the claims 1 to 12 ; and e. selecting one or more drug substances wherein the toxicity grade is below 6, when determined according to claim 7 .
23 . The method according to claim 22 , wherein the therapeutic index of the selected drug substance is decreased when compared to a toxic reference substance or parent drug substance.
24 . The method according to claim 22 or 23 , wherein the library of nucleic acid molecules is a library of nucleic acid molecule variants (child nucleic acid molecules) of a parent nucleic acid molecule, wherein the parent nucleic acid molecule is toxic, such as nephrotoxic, and wherein step d) identifies one or more nucleic acid molecule variants which are less toxic than the parent nucleic acid molecules; wherein the nucleic acid molecule variants retain the nucleobase sequence of the parent nucleic acid molecule.
25 . A drug substance obtained by the method according to any one of claims 22 to 24 .
26 . The nucleic acid molecule of claim 25 for use in a medicine.Join the waitlist — get patent alerts
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