P1252109245vasCD

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Aromatase Inhibitor-induced musculoskeletal
Symptoms (AIMSS) Identification of genetic
predictors and causative mechanisms Jason
Robarge M.S.1, Todd Skaar Ph.D.1,2, Djane Duarte
Ph.D.1, Michael Vasko Ph.D.1, David Flockhart
M.D. Ph.D.1,2 1Department of Pharmacology and
Toxicology, 2Department of Medicine, Division of
Clinical Pharmacology
RESULTS
ABSTRACT
METHODS
Background Aromatase inhibitor-induced
musculoskeletal symptoms (AIMSS) are a limiting
toxicity developed during breast cancer therapy
with aromatase inhibitors. Poorly understood,
these arthralgias are associated with improved
response, while negatively impacting patient
quality of life and drug compliance. We
hypothesize that aromatase inhibitor therapy
alters basal pain thresholds by inhibiting
peripheral aromatase and reducing 17ß-estradiol
signaling that modulates nociception. Methods
To generate a model to explore causative
mechanisms underlying AIMSS, we have utilized
behavioral pain models in rodents. Using
ovariectomized Sprague Dawley rats as a model for
the post-menopausal state, we have characterized
acute nociceptive responses following
administration of letrozole, an aromatase
inhibitor. Results Our preliminary studies
indicate letrozole reduces nociceptive thresholds
to mechanical stimuli. Experiments are underway
to determine the dose-dependence of this effect,
the effect of acute versus chronic dosing, and
the influence of genetic background on the
response.
Animals Behavioral experiments were performed on
female Sprague-Dawley rats (Harlan Laboratories,
USA). At approximately 150g, rats were
bilaterally ovariectomized by Harlan, shipped,
and used following 2 weeks of recovery. Animals
were housed in a controlled environment in the
Animal Laboratory Resource Center of the Indiana
University School of Medicine. Food and water
were available ad libitum. Experimental protocols
were approved by the Indiana University School of
Medicine, Institutional Animal Care and Use
Committee.   Administration of letrozole Letrozole
was dissolved in hydroxypropyl-ß-cyclodextrin
(HPßCD) (10 in sterile PBS). Letrozole was
administered to treatment group rats (N6) as a
single intraperitoneal injection (1.0 ml/kg) at a
dose of 1.0 mg/kg. Control rats (N5) received a
single intraperitoneal injection (1ml/kg) of
vehicle (10 HPßCD).   Behavioral
Experiments Baseline mechanical and thermal
nociceptive thresholds were measured 2 days prior
to drug administration. The measurement taken 24
hours prior to drug was used as the baseline
response. Thresholds were re-assessed 30min
following administration to detect acute changes
in response. Thresholds were measured again at
3, 5, 8, 12, and 21 days. von Frey
filaments Mechanical hypernociception was
evaluated using the von Frey filaments (Stoelting
Co.). Before each test, the animals were
acclimated in the test cage for approximately 30
min. The hind paw plantar surface was touched
with one of a series of filaments with
logarithmically incremental stiffness (0.626g).
A single trial consisted of three applications of
a particular filament, applied once every 34s. A
response was defined as withdrawal of the
stimulated paw. The updown method was used to
record the threshold.   Hargreaves test Paw
withdrawal latency to radiant heat was assessed
using a infra-red heat source (Ugo Basile,
Italy). Before each test, the animals were
acclimated in the test cage for approximately 30
min. The I.R. source was placed underneath the
mid-plantar surface of the hind paw. The
intensity of the heat source was chosen (30) to
yield baseline latencies ranging from 8s to 10s
in non-OVX animals of equivalent weight, and a
cut-off of 30s was used to avoid tissue damage at
the paw. A paw withdrawal in response to heat
was detected by a photocell, which switched off
the I.R. source and timer. The paw withdrawal
latency was taken to be the mean of three trials
from both hind paws, with at least 10s
in-between. Data analysis Group means and change
from baseline were analyzed using repeated
measures ANOVA. Data analysis was performed
using R, v2.7.1.
Figure2 A B. Time course of mechanical
hyperalgesia

A. Withdrawal latencies to a mechanical stimulus
(von Frey hair) to the plantar surface of the
paw. Responses were measured at baseline and
following I.P. administration of letrozole
(N5animals/10 paws) or vehicle (N5animals/10
paws). Each point represents mean latency
response SEM. Treatment group response was not
significantly different at baseline, while
overall mean change in response was different
between groups (p0.0427). At 8 days, the
response compared to baseline was significantly
lower in letrozole treated animals (Tukeys HSD
post hoc analysis, p0.00732). B. Responses
are represented as percent change from baseline.
Each point represents mean SEM.
INTRODUCTION
Third generation aromatase inhibitors (AIs) -
anastrozole (Arimidex), letrozole (Femara), and
exemestane (Aromasin) - are commonly used for the
treatment of hormone receptor-positive breast
cancer. Although they are highly effective,
their side-effects often limit their usefulness
one of the most significant is the
musculoskeletal arthralgia. The arthralgia
developed on AIs presents clinically as
non-inflammatory regional musculoskeletal
disorder and has been termed aromatase
inhibitor-induced musculoskeletal symptoms or
AIMSS. Patients often complain of stiff or
painful joints, morning stiffness, and
tendonitis.   AIMSS are frequent, and develop
soon after initiating therapy. An analysis
conducted by the COnsortium on BReast cancer
phArmacogenomics (COBRA) of breast cancer
patients receiving daily exemestane or letrozole
showed an incidence of 45.4 a rate now
corroborated by others.1,2 The median time to
onset was 1.6 months (range 0.4 10 months). 13
of the patients to discontinue therapy because of
the AIMSS.1   Unfortunately, AIMSS also appear to
be associated with improved survival
consequently, our inability to manage the AIMSS
means that breast cancer patients are often not
able to take a drug that would help them survive
longer.3 Therefore, understanding the mechanisms
contributing to or predictors of AIMSS may help
us to improve drug tolerability in those patients
who develop symptoms, potentially improving their
long term prognosis.   While AIMSS may limit
tolerance to AIs in some patients, there are no
clear predictors of who may get the symptoms, and
the causative mechanism remains unknown. The
mechanism seems to be a result of the estrogen
deprivation however, the specific target is
unknown. One hint may come from previous in-vitro
and rodent studies that have implicated estrogen
as a neuroendocrine modulator of pain processing
its action is thought to occur via a number of
mechanisms within the CNS and in peripheral
primary afferent neurons. The presence of
aromatase and estrogen receptor immunoreactivity
in neurons suggests that estrogens may be
produced locally and act to influence pain
pathways via autocrine or paracrine
signaling.4,5,6 Therefore, we hypothesized
that AI-induced depletion of neuronal estrogen
synthesis and activity cause altered pain
sensitivity. As an initial step to explore the
mechanism by which AIs may alter pain pathways,
we are developing a rodent model to test the
effect of aromatase inhibition on nociception.
To simulate the physiological conditions of AI
therapy in post-menopausal women, we treated
ovariectomized (OVX) rats with letrozole. The
primary outcome for these experiments was the AI
induced change in nociceptive threshold for
mechanical and thermal stimuli.
CONCLUSIONS AND FUTURE DIRECTIONS
Letrozole reduced the nociceptive threshold of
rats to a mechanical stimulus. However, it did
not alter the basal sensitivity to thermal
stimulus. These preliminary studies indicate
that letrozole sensitizes rats to mechanical
stimuli. To better understand these results, our
future studies will focus on the direct effects
of letrozole on neuron function. These
experimental models should provide insights into
the mechanisms of letrozole induced
musculoskeletal pain in breast cancer patients.
RESULTS
REFERENCES
Figure1 A B. Time course of thermal hyperalgesia

• 1. Henry, N.L. et al. Prospective
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• 2. Crew, K.D. et al. Prevalence of joint symptoms
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  Oncol. 25(25) 3877-83, 2007.
• 3. Cuzick, J., Sestak, I., Cella, D.
  Fallowfield, L. Treatment-emergent endocrine
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  recurrence a retrospective analysis of the ATAC
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• 4. Evrard HC, Harada N, Balthazart J.
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• 5. Evrard, H.C. and Erskine, M.S. Spinal estrogen
  synthesis alters nociception-related behaviors in
  male rat. Soc Neurosci Abstract. 746.16, 2005.
• 6. Evrard HC. Estrogen synthesis in the spinal
  dorsal horn a new central mechanism for the
  hormonal regulation of pain. Am J Physiol Regul
  Integr Comp Physiol. 291(2)R291-9, 2006.

A. Withdrawal latencies to a thermal nociceptive
stimulus applied to the plantar surface of the
paw. Response were measured at baseline and
following I.P. administration of letrozole (N6
animals/12 paws) or vehicle (N5 animals/10
paws). Each point represents mean latency
response SEM. There were no significant
differences between treatment means nor
significant changes from baseline in either
treatment group. B. Responses are represented as
percent change from baseline. Each point
represents mean SEM.