Animals

1. 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 17β-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-β-cyclodextrin (HPβCD) (10% in sterile PBS). Letrozole was administered to treatment group rats (N=6) as a single intraperitoneal injection (1.0 ml/kg) at a dose of 1.0 mg/kg. Control rats (N=5) received a single intraperitoneal injection (1ml/kg) of vehicle (10% HPβCD). 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 (0.6–26g). A single trial consisted of three applications of a particular filament, applied once every 3–4s. A response was defined as withdrawal of the stimulated paw. The up–down 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 (N=5animals/10 paws) or vehicle (N=5animals/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 (p=0.0427). At 8 days, the response compared to baseline was significantly lower in letrozole treated animals (Tukey’s HSD post hoc analysis, p=0.00732**). 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 characterization of musculoskeletal symptoms in early stage breast cancer patients treated with aromatase inhibitors. Breast Cancer Res Treat. 111(2): 365-72, 2007. 2. Crew, K.D. et al. Prevalence of joint symptoms in postmenopausal women taking aromatase inhibitors for early-stage breast cancer. J Clin Oncol. 25(25): 3877-83, 2007. 3. Cuzick, J., Sestak, I., Cella, D. & Fallowfield, L. Treatment-emergent endocrine symptoms and the risk of breast cancer recurrence: a retrospective analysis of the ATAC trial. Lancet Oncol. 9(12):1143-8, 2008. 4. Evrard HC, Harada N, Balthazart J. Localization of estrogen-synthase (aromatase) in the rat spinal cord. Soc Neurosci Abstract. 27: 508.6, 2001. 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 (N=6 animals/12 paws) or vehicle (N=5 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.