Abstract A05: The anti-angiogenic activity and pharmacokinetic evaluation of a small molecule JFD-WS in preclinical testing
Authors: Kanagasabai Thanigaivelan , Dhandayuthapani Sivanesan , Subbarao Manasa , Alvarez Janelle , Bhalani Meera , Rathinavelu Appu 
Published: December 2015

VEGF signaling through activation of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), an endothelial cell-specific receptor tyrosine kinase is indispensable for developmental angiogenesis and cancer progression. Since VEGFR2 is the dominant angiogenic signaling receptor, it has become an important target in the development of anti-angiogenic therapies. As part of this strategy inducing apoptosis in cancer cells could make the therapeutic agents very effective. The in vitro anti-angiogenic activity of water soluble form of JFD named as JFD-WS was examined by ECMatrixTM gel assay with human umbilical vein endothelial cells (HUVEC) using 0.01-10µM concentrations at different time intervals (0, 4 and 8 hrs). A complete inhibition of in vitro angiogenesis was observed at 8 hrs after treating with 10µM concentration of JFD-WS. Furthermore, the VEGFR2 phosphorylation was inhibited by 38.65% in JFD-WS (1.0µM) treated HUVEC cells. Besides the in vitro anti-angiogenic activity, JFD-WS is also proven as a potent anti-cancer drug in this study by using GI-101A (human breast adenocarcinoma) xenograft implanted athymic nude mice. In our in vivo experiments, the intraperitoneal (i.p.) injection of JFD-WS (at a dose of 100 mg/kg body weight) was able to significantly inhibit the tumor growth compared to the control group. At the end of the treatment period nearly 35.19% inhibition of tumor growth was observed in JFD-WS treated animals along with prolongation of survival. The tumor inhibition was lot more effective when JFD-WS was combined with Taxol (10 mg/kg). Eventually, the serum levels of MUC1 proved a significant regression of the tumor burden in the experimental animals. In addition to the anti-angiogenic ability, the anti-tumor activity of JFD-WS seems to be further enhanced due to induction of apoptosis signals in the xenograft tumor implanted animals. The protein expression levels of key apoptotic signaling molecules such as Bax, Bcl2, cytochrome c, Apaf-1 and cleaved caspase-3 and p53 were analyzed in the tumor samples that were extracted from experimental animals. Interestingly, a significant increase in the expression of pro-apoptotic proteins p53 (3 fold), Bax (10.6 fold) and Apaf-1 (2.2 fold) were observed in JFD-WS treated animals. In support of the induction of apoptotic signals, the expression of anti-apoptotic protein Bcl2 was decreased by 1.8 fold. Consequently, the release of cytochrome c and the cleavage of caspase 3 found in the cytosolic fraction were significantly higher in JFD-WS treated group as compared with the untreated controls. Finally, the pharmacokinetics (PK) property of JFD-WS was analyzed by measuring the concentration in plasma and urine samples of Balb/c mice at different time intervals (Plasma: 2.5, 5, 10, 15, 30, 60, 120, 1440 min; and Urine: 15, 30, 60, 120, 1440 min), using a HPLC method. Our experiments showed that JFD-WS can reach peak plasma concentration after 15 mins and a maximum elimination in urine was observed after 30 mins of i.p. injection. The compound was undetectable in both plasma and urine after 24 hrs. Moreover, a two compartment model of absorption, distribution and elimination for JFD-WS, with the half-life (t1/2) of around 33 mins is suspected. Our results from both in vivo and in vitro experiments confirm the anti-angiogenic and pro-apoptotic effects of JFD-WS in xenograft tumor implanted athymic nude mice. Furthermore, our data clearly indicate that, JFD-WS is highly effective in causing tumor inhibition with minimal toxicity. (This project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida).

Citation Format: Thanigaivelan Kanagasabai, Sivanesan Dhandayuthapani, Manasa Subbarao, Janelle Alvarez, Meera Bhalani, Appu Rathinavelu. The anti-angiogenic activity and pharmacokinetic evaluation of a small molecule JFD-WS in preclinical testing. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A05.