Evaluation of in Vitro Mitochondrial Toxicity Assays ... - ACS Publications

Dec 10, 2018 - measures mitochondrial respiration in the form of oxygen. Received: August 28 ..... inhibitor rotenone impaired respiration and cell vi...
0 downloads 0 Views 829KB Size
Subscriber access provided by YORK UNIV

Article

Evaluation of In Vitro Mitochondrial Toxicity Assays and Physicochemical Properties for Prediction of Organ Toxicity Using 228 Pharmaceutical Drugs Payal Rana, Michael D. Aleo, Mark Gosink, and Yvonne Will Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00246 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 12, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

Evaluation of In Vitro Mitochondrial Toxicity Assays and Physicochemical Properties for Prediction of Organ Toxicity Using 228 Pharmaceutical Drugs

Payal Rana*a, Michael D. Aleoa , Mark Gosinka, and Yvonne Willa

aDrug

Safety Research & Development, Pfizer, Eastern Point Road, Groton, CT 06340, USA

Key words: Mitochondrial toxicity, Organ toxicity, Physicochemical properties, In vitro assays, Drug-induced liver injury, Hepatotoxicity, Cardiotoxicity, Nephrotoxicity

1|Page ACS Paragon Plus Environment

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 51

Table of contents graphics:

2|Page ACS Paragon Plus Environment

Page 3 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

ABSTRACT Mitochondrial toxicity has been shown to contribute to a variety of organ toxicities such as liver, cardiac and kidney. In the past decades, two high throughput applicable screening assays (isolated rat liver mitochondria; glucose-galactose grown HepG2 cells) to assess mitochondrial toxicity have been deployed in many pharmaceutical companies and numerous publications have demonstrated its usefulness for mechanistic investigations. However, only two publications have demonstrated the utility of these screens as a predictor of human drug induced liver injury. In the present study, we screened 73 hepatotoxicants, 46 cardiotoxicants, 49 nephrotoxicants and 60 compounds not known to cause human organ toxicity for their effects on mitochondrial function(s) in the assays mentioned above. Predictive performance was evaluated using specificity and sensitivity of the assays for predicting organ toxicity. Our results show that the predictive performance of the mitochondrial assays are superior for

3|Page ACS Paragon Plus Environment

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 51

hepatotoxicity as compared to cardiotoxicity and nephrotoxicity (sensitivity 63% vs 33% and 28% with similar specificity of 93%) when the analysis was done at 100* Cmax (drug concentration in human plasma level). We further explored the association of mitochondrial toxicity with physicochemical properties such as cLogP (calculated Log Partition Coefficient), TPSA (Topological Polar Surface Area), ionization status and molecular weight of the drugs and found that cLogP was most significantly associated mitochondrial toxicity. Since these assays are amenable to higher throughput, we recommend that chemists use these assays to perform SAR (structure activity relationship) early in the drug discovery process when chemical matter is abundant. This assures that compounds that lack the propensity to cause mitochondrial dysfunction (and associated organ toxicity) will move forward into animals and humans.

INTRODUCTION Drug induced mitochondrial toxicity contributes to toxicities of many organs, such as the liver, heart, kidney, skeletal muscle and brain.1 In addition, it has been shown that

4|Page ACS Paragon Plus Environment

Page 5 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

mitochondrial toxicity at least in part contributed to the attrition of phenformin, troglitazone, nefazodone and cerivastatin.2,3 The knowledge that many drug classes (antidiabetic, anti-lipidemic, antivirals, antibiotics and anti-depressants) can exhibit mitochondrial liabilities has led to the development of high-throughput applicable mitochondrial assays4,5 that can be positioned early in the drug discovery screening process. These assays have thus far mostly been used for mechanistic evaluations of numerous drug classes.6,7,8,9 To date, few published studies have used mitochondrial toxicity assessment as a predictor of human liver injury. 10,11,12 Porceddu et al., tested 87 drugs known to cause hepatotoxicity and 37 drugs not reported to cause hepatotoxicity and reported a >90% positive predictive value for human drug induced liver injury using a multi-parameter assay conducted in mouse liver mitochondria.11 Aleo et al., 12 used 24 Most-DILI-, 28 Less-DILI-, and 20 No-DILI-concern drugs annotated in the United States National Center for Toxicological Research Liver Toxicity Knowledge Base (NCTRLTKB) and demonstrated mitochondrial toxicity was generally correlated across human DILI concern categories, such as death or black box warnings when combined with inhibition of the bile salt export protein.12 5|Page ACS Paragon Plus Environment

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 51

In this study, we utilized two routine assays for detection of mitochondrial toxicants deployed within Pfizer to advance our understanding of 1) predictivity towards major organ toxicity, other than hepatotoxicity, and 2) to investigate the physicochemical attributes that contribute to positive findings in these assays. The first assay utilizes cells grown in two types of media, a) high glucose and b) galactose containing media.13, 14, 15

Cells grown in high glucose-containing medium use glycolysis for ATP generation

and are resistant to mitochondrial insult. In contrast, cells grown in galactose-containing medium rely almost exclusively on mitochondria for their ATP production, and hence are very sensitive to mitochondrial insult.

The second assay, called RST (Respiratory

Screening Technology), measures mitochondrial respiration in the form of oxygen consumption in freshly isolated rat liver mitochondria using a time resolved fluorescent oxygen-sensitive probe.15,16,17,18 In the present study, we screened 73 hepatotoxicants, 46 cardiotoxicants, 49 nephrotoxicants and 60 compounds not known to cause human organ toxicity. It was our desire to advance the usage of mitochondrial toxicity assays as a tool for predicting organ toxicity and understand the physicochemical property space that drives these 6|Page ACS Paragon Plus Environment

Page 7 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemical Research in Toxicology

liabilities.

We addressed the following three questions:

(A) What percentage of

compounds in each organ toxicity class tested positive in the mitochondrial assays? (B) What is the predictive value towards particular organ toxicity (specificity/sensitivity)? (C) Do compounds that tested positive in the mitochondrial assays occupy a different physicochemical property space than those that tested negative?

7|Page ACS Paragon Plus Environment

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 51

EXPERIMENTAL PROCEDURES Mitochondrial Toxicity In vitro assays Methods for measurements of mitochondrial respiration in isolated rat liver mitochondria

4,11,16,17,18

and

assessment

of

mitochondrial

toxicity

using

glucose/galactose model 5,13,14,37,52 have been previously published. Statistical Analysis Concentration-response plots and IC50 values for each compound were generated either in Graph Pad Prism 5 (San Diego, CA) using a non-linear regression analysis or using SiGHTS (System Integrated Global High Throughput Screening) Pfizer’s internal proprietary data analysis software using a non-linear regression analysis. Statistical analysis was performed using the two way ANOVA between groups in Graph Pad Prism 5 (San Diego, CA). P value 3. 15 | P a g e ACS Paragon Plus Environment

Chemical Research in Toxicology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 51

There was a statistically significant (P 3, 3.87) compared to those compounds which tested negative in the mitochondrial assays (Mean cLogP