Assessing Mitochondrial Toxicity in Early Drug Development
Mitochondrial toxicity assessments are crucial to ensuring the safety and efficacy of candidate compounds during drug development.
However, the pharmaceutical industry needs more stringent approaches to screening than current assays provide.
This case study highlights a novel assay platform that examines mitochondrial health with more sensitivity, speed and efficacy.
Download this case study to discover:
- The importance of mitochondrial toxicity testing
- Real-time assays that offer unparalleled sensitivity and mechanistic insights
- Toxic compound screening methods that save time, money and reduce risk
The Growing Importance
of Mitochondrial Toxicity
Assessment in Drug Discovery
Agilent Case Study
"Primum non nocere." First, do no harm.
It's one of the guiding principles underlying the ethical practice of healthcare.
More than that, it's a cautionary tale – one that resonates throughout the
world of drug discovery and development. History has shown it's one thing
to develop a drug that makes a positive impact on the medical condition of
interest, it's quite another to ensure that a drug isn't also causing unintended
health issues elsewhere.
Mitochondrial toxicity is a case in point. Around the turn of the millennium,
several drugs were brought to the market only to be quickly pulled again, in
what turned out to be very high-profile failures for the companies involved.
The reason the drugs were withdrawn? Liver failure, which was eventually
determined to be associated with metabolic/mitochondrial toxicity. Among
these drugs was a promising antidiabetic medication called troglitazone,
which itself was eventually linked to more than 400 cases of acute hepatic
failure, resulting in hundreds of millions of dollars in legal costs for
The withdrawal of troglitazone and others brought heightened scrutiny to
the importance of mitochondrial toxicity as a core criterion of drug safety.
It was clear that traditional in vitro cytotoxicity tests were inadequate; these
assays relied on mammalian cells, which have the ability to circumvent
certain toxicities by accelerating glycolysis and inhibiting mitochondrial
function through a mechanism known as the Crabtree effect. The net result
was a disconnect between the in vitro and in vivo situations where potential
mitochondrial toxins could be missed under certain assay conditions.
Mitochondrial toxicity in the spotlight
At the time, the only available method for
directly measuring mitochondrial respiration
relied on a device known as the Clark
electrode. A breakthrough at the time of
its invention in the early 1960s, the Clark
electrode was nonetheless a single-chamber
technology that was unsuitable for the
throughput needs of the pharma industry.
The Glu/Gal assay, developed by Luxcel
Bioscience (now a part of Agilent) in
collaboration with Pfizer, was a milestone
in the assessment of mitochondrial
toxicity. This assay uses glucose- and
galactose-conditioned media in combination
with the redox-sensitive dye MTT. Although
it is reasonably effective and relatively
inexpensive to perform, the Glu/Gal assay
has shortcomings that limit its use as an
indicator of mitochondrial safety. One issue
is that it has relatively poor sensitivity; more
problematic is the inability of the assay
to differentiate between drugs that inhibit
mitochondrial function solely by uncoupling
the electron transport chain and drugs
that exert toxicity through multiple cellular
mechanisms. Thus, there is potentially
important mechanistic information that this
assay is simply not designed to deliver.
For Research Use Only. Not for use in diagnostic procedures.
This information is subject to change without notice.
© Agilent Technologies, Inc. 2022, 2023
Published in the USA, April 27, 2023
A number of companies, including Seahorse Bioscience
(now a part of Agilent), recognized the potential to assess
mitochondrial function using fluorescent probes that were
sensitive to oxygen and pH. This led to the introduction
of real-time assays of mitochondrial function that report
oxygen consumption and glycolytic flux with a high degree of
sensitivity, in a convenient 96-well microtiter plate format that
is scalable for practical use in drug development labs.
Tightening the net
The pharmaceutical industry has expressed a need for more
stringent approaches to mitochondrial toxicity screening
beyond what available assays have been able to provide,
in hopes of avoiding potentially huge development costs
(or worse). The introduction of the Agilent Seahorse XF
assay platform, accompanied by scientifically controlled and
validated real-time metabolic assays kit formulations, has
opened new avenues for examining mitochondrial health in a
timely and effective manner.
In their efforts to prevent potentially toxic compounds from
slipping through the net early in the drug discovery process,
a group of scientists at Genentech led by Dr. Tomomi Kiyota
and Dr. William Proctor have compared the performance
of Glu/Gal assays with results from Agilent Seahorse
XF‑based methods, particularly the XF Mito Stress Test
(MST) assay. This single assay analyzes multiple parameters
relevant to mitochondrial health, including basal respiration,
ATP-linked respiration, maximal and reserve capacities, and
The results from Kiyota and Proctor demonstrated that the XF
assay has the potential to identify mitochondrial toxins that
could not be identified using Glu/Gal methodology (Figure 1).
In fact, the Glu/Gal assay failed to identify troglitazone as an
inhibitor of mitochondrial function; in contrast, the MST assay
sensitively and clearly demonstrated dose-dependent toxicity
for this well-known mitochondrial poison.
Figure 1. Comparison of Agilent Seahorse OCR-based
XF assay and Glu/Gal total ATP assay. (A) The
XF assay showed clear mitochondrial toxicity for
troglitazone. (B) Mitochondrial toxicity was not
detected for troglitazone using the Glu/Gal method.
Troglitazone OCR data
Troglitazone Glu/Gal assay results
The MST assay provides essential insights into mitochondrial
dysfunction and allows users to investigate functional
differences among cell types, drug candidates, and genetic
or biochemical interventions. Building on the principle of the
MST assay, Agilent has developed a streamlined Seahorse
XF Mito Tox assay solution that can easily be integrated into
a drug discovery workflow with improved throughput and
data interpretability. In light of its combination of sensitivity,
specificity, and throughput, the XF Mito Tox assay is deemed
an ideal approach for confident identification of mitochondrial
liability, thus helping to de-risk the drug discovery pipeline.