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PDF file - 351K, Influence of reduced glutathione (GSH) and ABCC3 function on dasatinib transport.

PDF file - 144K, Influence of Abcc4 on aspirin transport.

PDF file - 399K, Influence of Abcc4 function on dasatinib disposition.

PDF file - 131K, Concentration-dependence of dasatinib transport by ABCC4.

Purpose: Several oral multikinase inhibitors are known to interact in vitro with the human ATP-binding cassette transporter ABCC4 (MRP4), but the in vivo relevance of this interaction remains poorly understood. We hypothesized that host ABCC4 activity may influence the pharmacokinetic profile of dasatinib and subsequently affect its antitumor properties.Experimental Design: Transport of dasatinib was studied in cells transfected with human ABCC4 or the ortholog mouse transporter, Abcc4. Pharmacokinetic studies were done in wild-type and Abcc4-null mice. The influence of Abcc4 deficiency on dasatinib efficacy was evaluated in a model of Ph+ acute lymphoblastic leukemia by injection of luciferase-positive, p185(BCR-ABL)-expressing Arf(−/−) pre-B cells.Results: Dasatinib accumulation was significantly changed in cells overexpressing ABCC4 or Abcc4 compared with control cells (P < 0.001). Deficiency of Abcc4 in vivo was associated with a 1.75-fold decrease in systemic exposure to oral dasatinib, but had no influence on the pharmacokinetics of intravenous dasatinib. Abcc4 was found to be highly expressed in the stomach, and dasatinib efflux from isolated mouse stomachs ex vivo was impaired by Abcc4 deficiency (P < 0.01), without any detectable changes in gastric pH. Abcc4-null mice receiving dasatinib had an increase in leukemic burden, based on bioluminescence imaging, and decreased overall survival compared with wild-type mice (P = 0.048).Conclusions: This study suggests that Abcc4 in the stomach facilitates the oral absorption of dasatinib, and it possibly plays a similar role for other orally administered substrates, such as acetylsalicylic acid. This phenomenon also provides a mechanistic explanation for the malabsorption of certain drugs following gastric resection. Clin Cancer Res; 19(16); 4359–70. ©2013 AACR.

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PDF file - 295K, In vitro transport and activity of dasatinib against p185(BCR-ABL)-expressing Arf(-/-) leukemia initiating pre-B cells (LIC).

PDF file - 88K, Expression of Abcb1 and Abcg2 in mouse intestine.

Purpose: Several oral multikinase inhibitors are known to interact in vitro with the human ATP-binding cassette transporter ABCC4 (MRP4), but the in vivo relevance of this interaction remains poorly understood. We hypothesized that host ABCC4 activity may influence the pharmacokinetic profile of dasatinib and subsequently affect its antitumor properties. Experimental Design: Transport of dasatinib was studied in cells transfected with human ABCC4 or the ortholog mouse transporter, Abcc4. Pharmacokinetic studies were done in wild-type and Abcc4-null mice. The influence of Abcc4 deficiency on dasatinib efficacy was evaluated in a model of Ph+ acute lymphoblastic leukemia by injection of luciferase-positive, p185(BCR-ABL)-expressing Arf(−/−) pre-B cells. Results: Dasatinib accumulation was significantly changed in cells overexpressing ABCC4 or Abcc4 compared with control cells (P < 0.001). Deficiency of Abcc4 in vivo was associated with a 1.75-fold decrease in systemic exposure to oral dasatinib, but had no influence on the pharmacokinetics of intravenous dasatinib. Abcc4 was found to be highly expressed in the stomach, and dasatinib efflux from isolated mouse stomachs ex vivo was impaired by Abcc4 deficiency (P < 0.01), without any detectable changes in gastric pH. Abcc4-null mice receiving dasatinib had an increase in leukemic burden, based on bioluminescence imaging, and decreased overall survival compared with wild-type mice (P = 0.048). Conclusions: This study suggests that Abcc4 in the stomach facilitates the oral absorption of dasatinib, and it possibly plays a similar role for other orally administered substrates, such as acetylsalicylic acid. This phenomenon also provides a mechanistic explanation for the malabsorption of certain drugs following gastric resection. Clin Cancer Res; 19(16); 4359–70. ©2013 AACR.

Purpose To assess the toxicity, pharmacokinetics, and pharmacodynamics of multikinase inhibitor sorafenib in combination with clofarabine and cytarabine in children with relapsed/refractory leukemia. Patients and Methods Twelve patients with acute leukemia (11 with acute myeloid leukemia [AML]) received sorafenib on days 1 to 7 and then concurrently with cytarabine (1 g/m2) and clofarabine (stratum one: 40 mg/m2, n = 10; stratum two [recent transplantation or fungal infection]: 20 mg/m2, n = 2) on days 8 to 12. Sorafenib was continued until day 28 if tolerated. Two sorafenib dose levels (200 mg/m2 and 150 mg/m2 twice daily) were planned. Sorafenib pharmacokinetic and pharmacodynamic studies were performed on days 7 and 8. Results At sorafenib 200 mg/m2, two of four patients in stratum one and one of two patients in stratum two had grade 3 hand-foot skin reaction and/or rash as dose-limiting toxicities (DLTs). No DLTs were observed in six patients in stratum one at sorafenib 150 mg/m2. Sorafenib inhibited the phosphorylation of AKT, S6 ribosomal protein, and 4E-BP1 in leukemia cells. The rate of sorafenib conversion to its metabolite sorafenib N-oxide was high (mean, 33%; range, 17% to 69%). In vitro, the N-oxide potently inhibited FLT3–internal tandem duplication (ITD; binding constant, 70 nmol/L) and the viability of five AML cell lines. On day 8, sorafenib decreased blast percentages in 10 of 12 patients (median, 66%; range, 9% to 95%). After combination chemotherapy, six patients (three FLT3-ITD and three FLT3 wild-type AML) achieved complete remission, two (both FLT3-ITD AML) had complete remission with incomplete blood count recovery, and one (FLT3 wild-type AML) had partial remission. Conclusion Sorafenib in combination with clofarabine and cytarabine is tolerable and shows activity in relapsed/refractory pediatric AML.

Fusarochromanone (FC-101a) has been identified as a potential, novel anti-cancer compound. Several studies revealed that FC-101a is an inhibitor of many human cancer cell lines, increases apoptosis of melanoma cell lines and significantly reduces tumor growth in mice. The long-term goal of this project is to elucidate how FC-101a inhibits cancer cell growth by identifying its molecular target(s). In this study, we used DNA microarray analysis and realtime PCR to identify genes that are differentially-expressed in response to FC-101a treatment in yeast and human bladder cancer cells. We hypothesize that changes in gene expression in response to FC-101a treatment will elucidate cellular mechanisms by which FC-101a inhibits cancer cell growth. Our study includes human cancer cell lines as a model system and budding yeast as a model organism to identify cellular pathways that are responsive to FC-101a treatment in multiple eukaryotic cell types. In this study, we have determined that among human skin, breast, prostate and bladder cancer cell lines, bladder cancer cells are the most sensitive to FC-101a treatment. Thus, we performed DNA microarray analysis using human bladder carcinoma cells treated with FC-101a for 24, 48 and 72 hours and yeast cells treated with FC-101a for 20 minutes. We identified hundreds of genes that are highly expressed and greater than two-fold up-regulated or down-regulated in FC-101a-treated cells compared with untreated cells. Of these FC-101a-responsive genes common to yeast and human bladder cancer cells, a majority of these genes regulate apoptosis, metabolic pathways, cell cycle progression, DNA replication, cell signaling and DNA recombination and repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A35.

Abstract 1073 Background: Aberrant receptor tyrosine kinase (RTK) signaling arising from genetic abnormalities, such as FLT3-internal tandem duplications (FLT3-ITD), is an important mechanism in the development and growth of acute myeloid leukemia (AML) and is often associated with a poor outcome. Hence, inhibition of RTK signaling is an attractive novel treatment option, particularly for disease that is resistant to conventional chemotherapy. We evaluated the clinical activity of the multikinase inhibitor sorafenib in children with de novo FLT3-ITD–positive AML or relapsed/refractory AML. Methods: Fourteen patients were treated. Six patients with newly diagnosed FLT3- ITD–positive AML (aged 9–16 years; median, 12 years) received 2 cycles of remission induction therapy and then started sorafenib (200 mg/m2 twice daily for 20 days) the day after completing induction II (low-dose cytarabine, daunorubicin, and etoposide). Nine patients (aged 6–17 years; median, 9 years) with relapsed AML (including one treated on the above regimen) received sorafenib alone (2 dose levels; 200 and 150 mg/m2) twice daily for the first week of therapy, concurrently with clofarabine and cytarabine on days 8–12, and then alone from days 13 to 28. Sorafenib pharmacokinetics were analyzed at steady-state on day 8 of sorafenib in patients with newly diagnosed AML and on day 7 in patients with relapsed AML. In patients with relapsed AML, the effect of sorafenib on signaling pathways in AML cells was assessed by flow cytometry. Results: All 6 newly diagnosed patients, including 2 whose AML was refractory to induction I, achieved a complete remission (CR) after induction II; 5 had negative minimal residual disease (MRD; 50% decrease in blast percentage and/or bone marrow cellularity after 1 week of sorafenib. After concurrent sorafenib and chemotherapy, 5 of the 9 patients with relapsed AML achieved CR (2 had negative MRD) and 2 achieved a partial remission (PR; 5%-25% AML cells in bone marrow); all 4 patients with FLT3-ITD had a CR or PR. After sorafenib treatment, 6 patients underwent HSCT while 2 with FLT3-ITD who could not receive HSCT were treated with single-agent sorafenib and have maintained CR for up to 8 months. Hand-foot skin reaction (HFSR) or rash occurred in all patients and improved with cessation of sorafenib. Dose-limiting toxicity (DLT, grade 3 HFSR and/or rash) was observed in 3 of the 6 patients with relapsed AML treated with 200 mg/m2 of sorafenib; no DLT was observed at 150 mg/m2. The effect of sorafenib on downstream RTK signaling was tested in the leukemic cells of 4 patients: in most samples, phosphorylation of S6 ribosomal protein and 4E-BP1 was inhibited. The mean (± SD) steady-state concentration (Css) of sorafenib was 3.3 ± 1.2 mg/L in the newly diagnosed group and 6.5 ± 3.6 mg/L (200 mg/m2) and 7.3 ± 3.6 mg/L (150 mg/m2) in those with relapsed AML. In both groups, the mean conversion of sorafenib to sorafenib N-oxide was 27%-35% (approximately 3 times greater than previously reported), and mean sorafenib N-oxide Css was 1.0–3.2 mg/L (2.1-6.7 μM). In a 442-kinase screen, the inhibitory profiles of sorafenib N-oxide and sorafenib were similar, and FLT3-ITD phosphorylation was potently inhibited by both forms (sorafenib N-oxide Kd = 0.070 μM; sorafenib Kd = 0.094 μM). Sorafenib N-oxide inhibited the growth of an AML cell line with FLT3-ITD (IC50 = 0.026 μM) and 4 AML cell lines with wild-type FLT3 (IC50 = 3.9–13.3 μM) at approximately half the potency of sorafenib. Conclusion: In children with de novo FLT3-ITD and relapsed/refractory AML, sorafenib given alone or with chemotherapy induced dramatic responses and inhibited aberrant RTK signaling in leukemic cells. Sorafenib and its active metabolite (sorafenib N-oxide) likely contribute to both efficacy and toxicity. These results warrant the incorporation of sorafenib into future pediatric AML trials. Disclosures: Inaba: Bayer/Onyx: Research Funding. Off Label Use: Sorafenib and clofarabine: both used for treatment of pediatric acute myeloid leukemia.

The PI3K/Akt/mTOR and RAS/RAF/MEK/ERK signaling pathways are activated in 50 to 75% of primary AML cases, which have been associated with poor long-term outcome. The class I PI3K isoforms α, β, δ are expressed in nearly all primary childhood AML blast samples (Ross, Blood 2004), providing a potential target to circumvent aberrant signaling in this pathway. However, because of cross-talk and redundancy of signal transduction pathways, rational combinations of inhibitors may represent an advantageous treatment strategy for AML. We have previously shown that sorafenib, a multikinase inhibitor in development for the treatment of AML, effectively inhibits phosho-ERK in AML cells, with limited inhibition of AKT signaling (Hu, Mol Cancer Ther, 2008). Here we evaluated the anti-leukemic activity of the dual PI3K/mTOR inhibitor BEZ235 as a single agent and in combination with sorafenib at in vivo relevant concentrations (≤ 1 µM BEZ235 and ≤ 10 µM sorafenib) in four AML cell lines (HL60, U937, THP-1, and OCI-AML3), and 9 primary childhood AML blast samples (7 with wild-type FLT3 and 2 with FLT3 internal tandem duplication mutations). We first evaluated the single-agent activity of BEZ235 in AML cells in suspension culture. After exposure to increasing concentrations of BEZ235 for 72h, IC50 values in a MTT assay ranged from 0.125 to 0.5 µM, but maximal growth inhibition did not exceed 40% of untreated controls. After exposure to BEZ235 1 µM for 72 h, an increase in apoptosis (Annexin V positive cells) to 30% was observed in U937 and OCI-AML3 cells, and inhibition of proliferation (EdU incorporation) was observed in U937 and THP-1 cells; no change in cell cycle distribution was observed. Phospho-Akt was inhibited by 50% to 80% of untreated controls in 3 cell lines (U937, THP-1, and HL-60) treated with BEZ235 1 µM for 1 h. We next examined the anti-leukemic activity of the combination of BEZ235 and sorafenib (simultaneous exposure for 72 h) in AML cell lines and primary blast samples that were co-cultured in direct contact with bone-marrow-derived mesenchymal stromal cells. In AML cell lines, sorafenib 10 µM induced apoptosis in twice as many cells (39% to 54%) than BEZ235 1.0 µM (17% to 23%); however, the combination increased apoptosis to 50 to 68% in all cell lines. Enhanced apoptosis was also observed in all primary childhood AML blasts samples treated with the drug combination compared to single agent treatment. In conclusion, BEZ235 has limited single-agent activity but enhances the activity of sorafenib in vitro in AML cell lines and primary blast samples. Studies are ongoing to evaluate the drug combination in a xenograft model of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4492.

Dasatinib, an orally available multikinase inhibitor, has been recently approved for imatinib- resistant chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. As with other tyrosine kinase inhibitors, dasatinib exhibits extensive interindividual pharmacokinetic variability, the causes of which are presently unknown. Previously, a notable degree of variability was observed with dasatinib oral absorption (time to peak concentration ranging from 0.5 to 6 h) and overall systemic exposure (CV, ∼50%) in patients (Demetri et al, Clin Cancer Res 2009). Identifying the factors underlying this variability may help enable clinicians to better manage the balance between dasatinib efficacy and toxicity. Here, we evaluated the role of ABCC4 (MRP4), a member of the ATP-binding cassette transporter family, as a factor regulating the oral absorption of dasatinib. Results from ABCC4-overexpressing inside-out vesicles indicated that dasatinib, at clinically relevant concentrations, was actively transported by ABCC4. Saturation of ABCC4-related transport was not seen even at a dasatinib concentration of 65 µM, suggesting high capacity transport. Dasatinib (1 µM) accumulation was increased ∼3-fold after a 5-min incubation in ABCC4-overexpressing vesicles compared to empty vector controls. The encouraging in vitro results prompted us to assess the in vivo role of ABCC4 in dasatinib absorption using Abcc4 knockout mice on a C57BL/6 background (Abcc4(-/-) mice) (Leggas et al, Mol Cell Biol 2004). Oral administration of dasatinib (10 mg/kg, in 50 mM sodium acetate buffer pH 4.6) to both wildtype mice and Abcc4(-/-) mice resulted in significant differences (P = 0.015) in dasatinib plasma concentrations at 4 out of 6 samples collected at serial time points. The largest difference was observed at 15 min, where dasatinib plasma concentrations in the Abcc4(-/-) mice were 5-fold decreased compared with wildtype mice. Although later time points also showed a decrease in dasatinib concentrations in Abcc4(-/-) mice (3.2-fold at 30 min; 2.2-fold at 1 h; 1.5-fold at 2 h), there was a convergence of the plasma curves by 4 h, suggesting that absorption rather than elimination pathways of dasatinib were altered in the Abcc4(-/-) mice. Indeed, pharmacokinetic analyses revealed a nearly 12-fold (P = 0.014) decrease in the dasatinib absorption rate constant in Abcc4(-/-) mice compared with wildtype mice, whereas the terminal half-life was unchanged (P = 0.41). The reduced absorption of dasatinib in Abcc4(-/-) mice was associated with a 4-fold (P = 0.019) decrease in peak concentration and a 2-fold decrease in AUC (P = 0.041). Collectively, these findings demonstrate that ABCC4 plays an important role in the intestinal absorption of dasatinib and reveal a new host factor that may contribute to enhanced interindividual pharmacokinetic variability in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1528.

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The primary goal of this research is to develop novel anti-cancer agents based on fusarochromanone (FC101a), a natural mycotoxin with potent anti-angiogenic and direct anti-tumor activity. This research may lead to new cancer chemotherapeutic agents that are more potent and less toxic than conventional chemotherapy. Like most other bioactive natural compounds, the potency of FC101a is compromised in-vivo, suggesting unfavorable bioavailability, distribution, and/or metabolism. Development of FC101a as a chemotherapeutic drug requires the completion of its total synthesis, as well as the synthesis of a series of structural analogs with greater in-vivo potency than the lead compound. Progress towards both the chemical and biological synthesis of FC101a will be presented. We have employed a new pathway for synthesizing the unique multifunctional four-carbon side chain in the parent compound. We have also been successful in synthesizing the 6-iodo-4-chromanone, a key intermediate that is needed for the coupling reaction with the side-chain. Additionally, we have synthesized two novel acetal analogs of FC101a and will present the rational for choosing these structural analogs for devising structure activity relationships for this series of compounds. For the biological synthesis, we have obtained a highly fluorescent metabolite from the rice culture of two Fusarium strains (#4482 and #8508) that resembles FC101a, based on TLC analyses. We have also developed an efficient HPLC purification protocol to obtain samples of this compound with high purity. We are currently working to scale up this separation and confirm the structure of this compound through 1H, 13C-NMR as well as high-resolution mass spectrometry analyses. We will report our preliminary results on the inhibition effects of FC101a on the growth of human aggressive skin cancer carcinoma (SCC) both in-vitro and in-vivo. We have also investigated the mechanism of the biological function of FC101a. We will report our results on the expression levels and phosphorylation states of key proteins involved in cell proliferation signaling pathways, cell sorting profiles, and FACS analyses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1363. doi:10.1158/1538-7445.AM2011-1363

Background Fusarochromanone (FC101) is a small molecule fungal metabolite with a host of interesting biological functions, including very potent anti-angiogenic and direct anti-cancer activity. Results Herein, we report that FC101 exhibits very potent in-vitro growth inhibitory effects (IC50 ranging from 10nM-2.5 μM) against HaCat (pre-malignant skin), P9-WT (malignant skin), MCF-7 (low malignant breast), MDA-231 (malignant breast), SV-HUC (premalignant bladder), UM-UC14 (malignant bladder), and PC3 (malignant prostate) in a time-course and dose-dependent manner, with the UM-UC14 cells being the most sensitive. FC101 induces apoptosis and an increase in proportion of cells in the sub-G1 phase in both HaCat and P9-WT cell lines as evidenced by cell cycle profile analysis. In a mouse xenograft SCC tumor model, FC101 was well tolerated, non-toxic, and achieved a 30% reduction in tumor size at a dose of 8 mg/kg/day. FC101 is also a potent anti-angiogenenic agent. At nanomolar doses, FC101 inhibits the vascular endothelial growth factor-A (VEGF-A)-mediated proliferation of endothelial cells. Conclusions Our data presented here indicates that FC101 is an excellent lead candidate for a small molecule anti-cancer agent that simultaneously affects angiogenesis signaling, cancer signal transduction, and apoptosis. Further understanding of the underlying FC101’s molecular mechanism may lead to the design of novel targeted and selective therapeutics, both of which are pursued targets in cancer drug discovery.