Pharmacogenetics of Mitotane

Adrenocortical carcinoma (ACC) is a disease with an incidence of 0.5-2.0 per
million per year. Survival is low with a 5-year survival of 37-47%. Mitotane
(o,p`DDD) is the only registered drug in the treatment of ACC. It has
adrenalytic activity, mainly on the adrenal cortex. The exact mechanism of
action is unknown however. Mitotane is used in adjuvant setting as well as in
patients with metastatic disease.
Mitotane has a small therapeutic window of 14-20 mg/L. Survival benefit has
only been proven in patients with blood levels higher than 14mg/L whereas blood
levels higher than 20mg/L are associated with increased toxicity. Toxicity is
primarily gastro-intestinal and neurological and even leads to temporary or
final discontinuation of mitotane therapy in some cases.

Little is known about the pharmacokinetics and pharmacodynamics of mitotane.
Mitotane appears to have an absorption rate of approximately 35-40%. It is a
lipophilic drug, with a long half life of weeks to months. No clear
relationship is known between the oral dose of mitotane and the plasma
concentration. Current dosing schemes of mitotane are based on expert opinion.
Both high-dose and low-dose strategies are used for the build-up phase and
therapeutic levels can be reached with both. A recent pharmacokinetics (PK)
study showed that patients on high dose-strategy do not reach therapeutic
levels faster than patients on low-dose-strategy. Some patients reach
therapeutic levels in a few weeks while others need months or do not reach
therapeutic levels at all. All these unknown properties of mitotane make it
difficult to dose mitotane adequately for reaching therapeutic levels, without
increasing the risk of toxicity.

Drug therapy often excites large differences in response between individuals.
This leads to both therapeutic failures and adverse drug reactions, causing
increased suffering for patients and costs for society. This inter-individual
variability is caused by factors such as age, renal and liver function,
co-morbidity, drug-drug interactions, nutritional status, intoxications and
last but not least, pharmacogenetic differences.

Recent study by our group has shown considerably weak correlations between
weight, age, gender, height and the pharmacokinetics of mitotane. The
variability in mitotane requirement that can not be explained by these clinical
factors, in other words the residual variability, may very well be explained by
pharmacogenetic differences between patients. Recent research states that
patients with a certain genotype of the CYP2B6 gene show higher mitotane plasma
concentrations at three months of treatment, which supports our hypothesis.

Pharmacogenetics is the discipline searching for genetic polymorphisms in genes
involved in drug transport, metabolism and action. These polymorphisms often
elicit inter-individual differences in response to pharmacotherapy and in
adverse drug reactions by affecting pharmacokinetic parameters. The goal of
pharmacogenetics is to eventually be able to predict the behavior of a drug in
an individual, based on that person*s polymorphisms in drug metabolizing genes.
This would enable individualization of treatment, correcting for specific
polymorphisms in the choice and dose of drug. It has been suggested that basing
pharmacologic therapy on a person*s genotype would improve efficacy in 10-20%
of all drug therapy and decrease adverse drug reactions with 10-15%.

The clinical importance of genotype-based dosing depends on a number of
variables. First, genotype-based dosing can be especially important for drugs
with a small therapeutic range because in these drugs small dose adjustments
can have large effects on both response and adverse drug reactions. Mainly
drugs that are used chronically and drug therapies in which there is a large
time-delay between start of therapy and response can benefit from
genotype-testing, especially if this is associated with a high rate of
non-responders. Other important factors are the effect size of the clinical
outcome parameter and the allele frequency, since these determine the number of
subjects that could profit from dose adjustment.

All the properties named above are relevant for mitotane therapy: mitotane has
a small therapeutic range, it often takes more than three months to reach
therapeutic levels, long-term use is generally indicated and toxicity is
considerable. Moreover, there is no good alternative for mitotane, making
optimal treatment all the more important. These arguments make mitotane a good
candidate for genotype-based dosing, in the context of improving strategies for
reaching therapeutic levels as well as for decreasing the occurrence of serious
adverse drug reactions.

Not much is known about mitotane metabolism. The metabolites of mitotane are
excreted through both stool and urine. Mitotane is metabolized to it*s
metabolites 1,1-(o,p`-dichlorodiphenyl) acetic acid (o,p`DDA) and
1,1-(o,p`-dichlorodiphenyl)-2,2 dichloroethene (o,p`DDE) through respectively
β- and α-hydroxylation; the exact location for this process (i.e. adrenal
cortex or liver) is unknown. O,p`DDA appears to be the active metabolite.
Several studies have suggested that hepatic metabolization occurs through the
cytochrome P450 system, the exact enzyme remains unknown however. It has been
shown that mitotane induces CYP3A4 activity; induction of CYP2C9 has been
suggested in different studies as well. The role of p-glycoprotein ABCB1 in
mitotane metabolism has not yet been fully elucidated. The article of Haak et
al. suggests that the expression of P-glycoprotein in adrenocortical carcinoma
is not related to the response to mitotane therapy or the clinical
manifestations. A more recent study by D*Avalio shows a non significant
association between ABCB1 polymorphism and mitotane concentration. However, due
the low patient numbers in both studies, a possible interaction of
p-glycoprotein and mitotane, should not yet be completely dismissed.

The DMET (Drug Metabolizing Enzymes and Transporters) platform is a relatively
new DNA array, developed for the assessment of a patient*s genotype with regard
to drug metabolism. This is a standardized genetic set used to scan 1936
variants in 225 genes related to drug absorption, distribution, metabolism and
elimination (ADME). The assay can be seen as a pathway-based approach for
exploring pharmacogenetic variability. It is appropriate for this type of
research, because it contains the large majority of genes acting in drug
metabolism pathways. Other possible approaches are the candidate-gene approach
and the genome-wide analysis. However, the candidate-gene approach is not
possible because we do not know on which gene to focus and the genome-wide
analysis is too broad for the aim of this study.

Primary objective:
We hypothesize that genetic variability influences mitotane pharmacokinetics.
Therefore our aim is to explore the inter-individual differences in genes
coding for drug metabolizing enzymes in patients treated with mitotane and to
assess if these differences can explain (part of) residual variability in
mitotane dose necessary to maintain therapeutic levels in steady state.
First, genetic variants in ADME genes of mitotane will be detected. Secondly,
we will investigate if the detected SNP's in ADME genese have an association
with the mitotane dose in steady state.

Secondary objective:
We will assess if genetic variability influences the time needed to reach a
therapeutic mitotane concentration (>=14mg/).

Secondary objective:
We will asses if there is an association between polymorhisms in drug ADME
genes and the occurrence of serious adverse drug reactions due to mitotane
treatment.

Explorative pharmacogenetic study, pathway-based approach. Multi-centre.

Setting: Patients with histological confirmed adrenal cortical carcinoma that
are being treated with mitotane or have completed mitotane therapy in the past,
This may include mitotane as adjuvant treatment (after surgery to prevent
recurrence of the disease) or as a treatment for metastatic disease. Patients
must have been treated with mitotane for at least 24 weeks.
A power calculation has shown that is necessary to enroll DNA of 50 patients.
This DNA will be extracted from one EDTA blood sample per patient. The DNA of
patients is examined using a special technique, the so-called Drug Metabolizing
Enzymes and Transporters (DMET) array that allows the identification of > 2000
known variations in 225 genes involved in drug metabolism.
A relation between the individual variations in gene level and:
1. the required dosage and duration of the time to reach the first mitotane
measurement of >=14mg/l,
2. the dosage required tot maintain a stable mitotane plasma level,
will be analyzed

For study subjects from whom blood is already stored, there is no extra burden.
For study subjects for whom this is not the case, the expected burden from this
protocol is minimized to a venepuncture. The burden that has been described, is
that when the needle is inserted to draw blood, some people feel moderate pain,
while others feel only a prick or stinging sensation. Afterwards some patients
describe a slight aching. Rare risks for the minimal invasive venepuncture are:
excessive bleeding, fainting or feeling light-headed, hematoma or blood
accumulating under the skin, infection (a slight risk any time the skin is
broken) and multiple punctures to locate veins.