P3-11-03: A Phase I Trial of D-Limonene in Newly Diagnosed Breast Cancer Patients: A Chemoprevention Study.
Authors: Lang JE , Miller JA , Nokes BT , LeBeau LG , Delbridge M , Chow H-HS 
Published: June 2012

Background: D-limonene (DL) is a highly lipophilic monoterpene found naturally in citrus that has been demonstrated in preclinical studies to have anticancer properties. Early phase clinical trials support the investigation of DL in the chemoprevention of breast cancer. We sought to evaluate whether DL and its presumed active metabolite perillic acid (PA) would distribute extensively to the breast tissue and reach an effective drug concentration. We hypothesized that the mechanism of DL is related to the induction of apoptosis in cancer cells.

Materials and Methods: We enrolled 40 patients with newly diagnosed Stage 0–2 breast cancer to take 2 grams daily of oral DL for 2–6 weeks prior to planned surgical intervention. Blood was drawn pre/post intervention to assess for toxicity and plasma concentration of DL and PA. Adverse effects related or possibly related to the study drug were noted. A small piece of breast tissue adjacent to the tumor mass was used to measure drug concentration for each patient. DL and PA levels in breast tissue were analyzed by gas chromatography and liquid chromatography, respectively, in tandem with mass spectrometry. Analysis of variance testing was used to determine if DL or PA preferentially concentrated in the breast tissue compared to plasma. We tested for a possible modulation of the biomarkers estrogen receptor (ER), progesterone receptor, HER2, Ki67 or grade pre/post treatment by comparing results from the core biopsy to the surgical pathology and applying pairwise Student's T tests. The caspase 3 and the annexin V assays were performed by plating 10,000 cells per well for the cell lines MCF7, MDA-231, BT474, and T47D and separately administering DL and PA acid in serial dilutions in their treatment concentration ranges in triplicate and read by microplate. Results: DL was found to preferentially concentrate in breast tissue versus plasma (tissue/plasma concentration ratio (TPCR) of 1297, p&lt;0.001) while PA did not concentrate (TPCR of 1.4, p=0.9). 20 patients (50%) reported Grade 1 eructation, which was the most common adverse effect. A slight decrease in white count from a mean of 7.1 to 6.6 (p=0.03) and a slight increase in ALT from a mean of 22.5 to 26.9 (p=0.03) were noted. No other statistically significant changes in laboratory values related to serum complete blood count, renal, hepatic or other studies were noted. No change in tissue biomarkers were noted post-treatment. DL produced a dose dependent increase in the apoptotic markers cleaved caspase 3 and annexin V for the ER positive cell lines MCF7, BT474, and T47D, however, no such response was noted for the triple negative cell line MDA-231. A PA dose response trend was noted with the annexin V but not the caspase 3 assay for each cell line.

Discussion: D-limonene preferentially concentrates in the breast tissue and is a candidate chemopreventive agent based on its favorable side effect profile. PA does not readily concentrate in the breast when administered as oral DL. Our correlative studies establish that DL induces apoptosis in ER positive breast cancer cell lines; no definite relationship between PA and apoptosis was found. Further clinical trials of DL are necessary to establish its potential role as a chemopreventive agent in breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-11-03.