Lata Mathew

Equivalency challenge: Evaluation of Lipodox® as the generic equivalent for Doxil® in a human ovarian cancer orthotropic mouse model

BACKGROUND The objective of this study was to evaluate the in vivo growth inhibition activity and tumor distribution of Doxil® compared to Lipodox® as its generic (GLD) in human ovarian cancer orthotopic mouse model. METHODS In the efficacy study 50 mice were randomized to: vehicle, Doxil® 5mg/kg or 10mg/kg, or GLD 5mg/kg or 10mg/kg for a total of three cycles with monitoring for response and toxicity with 10 mice in each arm. In the microdialysis(MD) study, 60 mice were randomized to: Doxil® 5mg/kg or 10mg/kg, or GLD 5mg/kg or 10mg/kg single dose (n=15 mice/arm). MD sample time points included total of 29 samples from baseline through 100h and were evaluated with a validated PaperSpray LC/MS assay. RESULTS There was 15.7% decrease (p<0.0001) in efficacy of GLD the 5mg/kg and 21.3% decrease (p<0.0001) in efficacy of the 10mg/kg dose of GLD when compared to equivalent doses of Doxil®. The intratumoral concentration for the GLD ranged from 1.0 to 25.5ng/mL (5mg/kg) and 2.9-35.6ng/mL (10mg/kg) compared to 2.7-42.2ng/mL (p<0.04, 5mg/kg) and 2.0-76ng/mL (p<0.02, 10mg/kg) for the Doxil®, respectively. CONCLUSION Significant differences in preclinical efficacy were observed between Doxil® and GLD. These may be due to significant pharmacodynamic effects of drug distribution and decrease uptake of GLD in tumor tissue. A prospective clinical comparison of these two products is warranted to determine equivalency.

Evaluation of Active Hexose Correlated Compound (Ahcc) on Phase IiDrug Metabolism Pathways and the Implications for Supplement-DrugInteractions

Background: The evaluation of active hexose correlated compound (AHCC) on hepatic metabolism mediateddrug interaction is critical in current clinical setting as there is little published information on the potential effect on drug efficacy and safety. The primary objective of this study was to evaluate the potential phase II hepatic metabolism pathways associated with the metabolism of AHCC and to determine potential drug/AHCC interactions. Methods: Four primary hepatic metabolism phase II pathways were evaluated: glutathione S-transferase (GST), quinone oxidoreductase (QOR), catechol-O-methyltransferases (COMT) and uridine diphosphate (UDP)- glucuronosyltransferase (UGT). Pooled human liver microsomes and human liver S9 fractions were utilized to evaluate QOR and UGT metabolism inhibition assays. The pool human liver S9 fractions were used to assess GST activity. Cryopreserved inducible human liver hepatocytes were used to evaluate potential induction of UGT and COMT metabolism. All experiments were carried out in triplicate. Results: Data demonstrated that AHCC is not an inhibitor of GST or UGT pathways, but may be a potential inhibitor of QOR pathway. Evaluation of induction of the phase II pathways demonstrated that AHCC showed potential induction of the UGT 1A3 and 1A6 pathways. There was no induction of the COMT pathway. Conclusion: Historically, drug interaction studies have only focused on Phase I metabolism pathways, so currently there is very limited information regarding the phase II metabolism of most commonly used medications. In conclusion, additional studies are warranted to determine potential of any phase II hepatic interactions with AHCC when administered with other medications or supplement that are substrates of these pathways.

Assessment of Dong Quai Hepatic Metabolism and Potential Interactions when Combined with Chemotherapy

Background: Dong Quai is a common herbal supplement classified as a “phytoestrogen” used for the improvement of female reproductive function. In the oncology setting, women often seek natural approaches for managing symptoms associated with decreased hormone levels either from surgery or chemotherapy-induced. Clinically, the concern is the safety of phytoestrogens in combination with chemotherapy. The objective of this study was to characterize the hepatic metabolism of Dong Quai to define the potential for drug interactions with selected chemotherapy agents and its impact on alterations in the cytotoxicity in panel of human cancer cell lines. Methods: In vitro high through-put cytochrome P450 (CYP450) inhibition assay was performed for CYP450 2C9, 2C8, 2D6 and 3A4 isoenzymesto evaluate phase I metabolism of Dong Quai alcohol-free extract. An ex vivo hepatic induction assay with human hepatocytes was used to determine whether Dong Quai is an inducer of CYP450 isoenzymes. The potential cytotoxic effects of Dong Quai alone and its effect when combined with selected chemotherapies were evaluated by a growth inhibition assay in a panel of eight human cancer cell lines. Results: No inhibition of CYP450 was observed in presence of Dong Quai. At an estimated clinical relevant concentration of 0.86 mg/mL, Dong Quai demonstrated Quai induced CYP3A4, 2C9, 2C8 and 2D6. Dong Quaidid not demonstrated cytotoxicity by itself in the panels of eight human cancer cell lines with 50% growth inhibition was not achieved. The 25% growth inhibition was achieved at concentrations ranging from 0.39 mg/mL to 4.48 mg/mL. Combination growth inhibition assays showed decreased cytotoxic activity of chemotherapy agents. Conclusion: This data suggests that Dong Quai is an inducer of the CYP450 pathways and also decreased cytotoxic activity of selected chemotherapy. Until confirmatory in vivo information available Dong Quai should be used with caution with chemotherapy.

Evaluation Fucoidan Extracts From Undaria pinnatifida and Fucus vesiculosus in Combination With Anticancer Drugs in Human Cancer Orthotopic Mouse Models

Objective: To determine the activity of fucoidan from Undaria pinnatifida (UPF) and Fucus vesiculosus (FVF) when given in combination of chemotherapy drugs using selected human breast or ovarian cancer orthotopic mouse models. Methods: Mice were inoculated with 1 × 106 cells of TOV-112d, MCF-7, or ZR-75 subcutaneously or SKOV3-GFP-Luc intraperitoneally on day 0. MCF-7 and ZR-75 mice were administered with estradiol valerate 2 mg/kg in 0.2 mL castor oil subcutaneously two days prior to cell inoculation. Mice were randomized to one of six arms (N = 10/arm) paclitaxel, UPF/paclitaxel, FVF/paclitaxel, tamoxifen, UPF/tamoxifen, or FVF/tamoxifen. Tumors were measured three times per week for 28 days. Results: Improved activity was observed with UPF or FVF in combination with tamoxifen in both the MCF-7 and ZR-75D breast cancer mouse models. Decreased activity of paclitaxel was observed when given in combination with UPF or FVF in both breast cancer mouse models. The combination of FVF/tamoxifen in the TOV-112d ovarian cancer mouse model had improved activity but no there was difference observed with the UPF/tamoxifen in either ovarian cancer mouse model. No difference was observed with combination of UPF or FVF with paclitaxel in human ovarian cancer SKOV3 or TOV-112d orthotopic mouse models. Conclusion: This study did confirm that UPF/FVF in combination with tamoxifen did not decrease tamoxifen activity in both breast and ovarian cancer, with some potential to improve activity compared to tamoxifen alone in breast cancers. Previous in vitro studies had suggested UPF and FVF had overall synergistic activity with paclitaxel; however, in the current in vivo human cancer mouse model studies there was no change in paclitaxel activity when given in combination with UPF or FVF in either of the two human ovarian cancer models. Furthermore, this study demonstrated that UPF or FVF given in combination with paclitaxel had a potential antagonistic effect in breast cancer models. Additional studies are warranted to delineate mechanisms contributing to variation in the in vivo activity when given in combination with paclitaxel. As a first step, a clinical pharmacokinetic study evaluating impact of FVF/UPF given in combination with chemotherapy in patients with solid tumors is underway.

Preclinical Evaluation of Safety of Fucoidan Extracts From Undaria pinnatifida and Fucus vesiculosus for Use in Cancer Treatment

Objectives: To evaluate potential hepatic metabolism-mediated drug interactions with fucoidan from Undaria pinnatifida (UPF) or Fucus vesiculosus (FVF) and potential growth inhibition activity with either fucoidan alone or with chemotherapy. In vivo studies were done to confirm safety and investigate fucoidan-mediated immune modulation. Methods: Cytochrome P450 (CYP450) 3A4, 2C8, 2C9, and 2D6 inhibition experiments were conducted in vitro followed by an ex vivo human hepatocytes model to evaluate the CYP450 induction potential of each fucoidan at highest theoretical concentrations. Four hepatic metabolism phase II pathways—glutathione S transferase (GST), quinone oxidoreductase (QOR), catechol-O-methyltransferases (COMT), and uridine di-phosphate (UDP)-glucuronosyltransferase (UGT)—were evaluated with validated immunoassays. Growth inhibition assays were performed with each fucoidan alone and in combination with chemotherapy agents in a panel of human cancer cell lines. In vivo studies evaluated safety and immune modualtion. Results: CYP450 inhibition was observed with FVF. The GST, QOR, and UGT pathways had no changes. UPF and FVF both interacted with COMT. No growth inhibitory activity in cancer cell lines was observed. UPF and FVF had synergistic activity with paclitaxel or tamoxifen and additive activity with topotecan. In vivo, FVF decreased HeLa human cervical tumor growth and both FVF and UPF decreased TOV-112D human ovarian tumor growth. Otherwise, no significant change in tumor growth was observed. FVF immune modulation of IgG and IL-6 was observed (p<0.03). Conclusion: At higher doses, UPF and FVF may have limited potential for drug-supplement interactions, with either CYP450 or COMT hepatic metabolism pathways. Additional studies are warranted to evaluate to confirm findings of fucoidans in combination with chemotherapy.

Evaluation of Active Hexose Correlated Compound (AHCC) in Combination With Anticancer Hormones in Orthotopic Breast Cancer Models

Objective. To determine the impact on antitumor activity when active hexose correlated compound (AHCC) in combination with anticancer hormonal agents in orthotopic mouse models of human estrogen receptor positive breast cancer and evaluate impact of AHCC on aromatase activity. Methods. The study consisted of 7 treatment arms (n=10) conducted in 2 breast cancer mouse models: MCF-7 and ZR-75. Treatment groups included untreated, vehicle, AHCC 50 mg/kg, AHCC 50 mg/kg + tamoxifen 10 mg/kg, tamoxifen 10 mg/kg, AHCC 50 mg/kg + letrozole 10 µg/mouse, or letrozole 10 µg/mouse. All treatments were administered daily by oral gavage for 12 weeks. Tumors were measured 3 times a week. In vitro estrone and 17β-estradiol enzyme immunoassay was used to evaluate aromatase activity. Results. There was no difference in the activity with the combination of AHCC + tamoxifen compared with tamoxifen (P = 0.29). In the ZR-75 model (catechol-O-methyltransferase [COMT] wild-type), there was no difference in activity with the letrozole + AHCC compared with letrozole. However, in the MCF-7 model (COMT variant), AHCC + letrozole resulted in a decrease in activity compared with letrozole (P < 0.01). Immunoassay data suggested that AHCC is a potential inducer of aromatase activity. In both tumor models, there was cytotoxicity observed with AHCC compared with untreated (P < 0.02). Conclusion. AHCC did not change the activity of tamoxifen. AHCC may have some interaction with letrozole in patients with COMT variant genotype. AHCC had cytotoxicity that warrents additional studies to evaluate its potential role for consolidation/prevention of breast cancer.

Abstract B78: Evaluation of letrozole for the prevention and inhibition of tumor growth in human cervical cancer xenograft mouse models

Purpose: To evaluate the efficacy of letrozole, a third generation aromatase inhibitor, for the prevention or delay of cervical cancer recurrence in either human papillomavirus (HPV) positive (HeLa) or HPV negative (C-33a) cervical cancer xenograft mouse models. Method: In this three arm study with two xenograft cervical cancer mouse models, HeLa (HPV 16 positive), and C-33a (HPV negative), the efficacy of letrozole was evaluated in prevention and tumor growth inhibition. Sixty mice were divided into six groups of ten mice each of treatment arm, vehicle control arm and no treatment arm for two cell lines. Mice in the treatment arm received letrozole 10 μg orally once daily every day beginning seven days before the inoculation with respective cancer cells and continued until day 90 of the study. Mice were injected subcutaneously on eighth day of the study with 0.5 million cells of respective cell line. Tumor growth was assessed three times a week. After 90 days of letrozole treatment, a 30 day period without drug was observed to evaluate tumor growth. At the end of the study, tumors were extracted and the expression of estrogen receptor alpha (ERα and estrogen receptor beta (ERβ was evaluated from the extracted protein by immunoblotting and RT-PCR was completed on DNA samples from extracted tumors to evaluate the HPV expression. Results: After 90 days of continuous treatment, letrozole inhibited microscopic cervical tumor growth in the HeLa mouse models demonstrated by 45% reduction in tumor volume in the HeLa letrozole treated mice compared to the no treatment control (p Conclusion: Although there was no change in ERα, ERβ, or HPV expression observed in this study, the response data in the HPV 16 positive HeLa tumor model compared to the HPV negative C-33a tumor model suggests HPV expression is associated with the potential activity of letrozole for the prevention of cervical cancer recurrence/progression. Additional studies are needed to elucidate the differences of letrozole activity in presence and absence of HPV expression. Overall, this preliminary data indicates a potential agent for the prevention and treatment of cervical cancer recurrence. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B78.

Abstract B79: Evaluation of active hexose correlated compound (AHCC) for the prevention or delay of tumor growth in human cervical cancer xenograft model

Purpose: Active hexose correlated compound (AHCC) is a mixture of polysaccharides, amino acids, lipids and minerals extracted from the culture of the basidiomycete mushroom Lentinula edodes (shiitake) that has been proposed to have many health benefits including both immunomodulatory and anti-tumor effects. In clinical studies AHCC has demonstrated numerous immunomodulating and potential restorative effects on natural killer (NK) cells, macrophages and cytokines. The objectives of this study were to evaluate if daily treatment with AHCC would eradicate human papillomavirus (HPV) 16/18 expression and prevent or delay cervical tumor growth using human xenograft mouse model. Methods: Selected cervical cancer cells, SiHa (HPV 16/18 positive), and C-33A (HPV negative) were treated in vitro with a single dose AHCC 0.42 mg/mL and incubated for 72 hours. In the second study AHCC dose was repeated once every 24 hours for total of seven days. This was followed by a three arm in vivo study in two xenograft cervical cancer mouse models, SiHa (HPV 16/18 positive), and C-33A (HPV negative), in which each cell line had ten mice for the treatment arm, vehicle control arm and no treatment arm. Mice in the treatment arm received 50 mg/kg AHCC in 0.25 mL of sterile water every day for seven days before the injection of the tumor cells and until the completion of the study. Tumors were measured three times per week. After 90 days of treatment, there was a 30 day observation period to evaluate the potential for recurrence of the HPV infection and the impact on tumor growth. At the end of the study, tumors were extracted and RT-PCR was completed on DNA samples from extracted protein to evaluate the HPV expression. Results: In vitro treatment with a single dose of AHCC for 72 hour incubation suppressed HPV expression in the first 24 hours but then HPV expression recovered by 48 hours. However, with continuous in vitro exposure, sustained HPV suppression was observed. In the in vivo animal studies, expression of HPV was eradicated with once daily AHCC dosing for 90 days and no detection of HPV expression was sustained after 30 days off treatment. In addition, AHCC daily treatment was associated with a 15.9% decrease in SiHa (HPV 16/18 positive) tumor growth compared to the untreated control (P Conclusion: In conclusion, these data suggest daily dosing of AHCC will eradicate HPV 16/18 infections and may have a role in the prevention of HPV-related cervical cancer. Furthermore, there is a potential for the addition of AHCC to primary treatment regimens for cervical cancer, which may potentially improve response rates and prevent recurrence. A confirmatory pilot study in HPV positive women is underway. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B79.

Abstract A29: Development of liposomal curcumin as a new potential anticancer agent

Previously, pre‐clinical dose finding tolerance study in a human xenograft pancreatic cancer model of intravenous liposomal curcumin determined 20 mg/kg TIW was to be the optimum dose and schedule for anticancer activity. Follow up pharmacokinetic and dose finding studies in a health rat model evaluated doses up to 40 mg/kg was not associated with weight loss, hematological, serologic, or dose‐limiting toxicity. In canine model, a single‐dose finding tolerance study ranging from 2 mg/kg up to 40 mg/kg revealed the maximum tolerated dose (MTD) of liposomal curcumin to be 20 mg/kg or 540 uMol/L of curcumin maximal blood concentration. Toxicity observed at this dose level was characterized by a brief single episode of reversible hematuria. The dose limiting toxicity was observed at a single dose of 40 mg/kg of liposomal curcumin. Following dose on day 1, life threatening toxicity followed within 48 hours with the dogs exhibiting irreversible acute hemolysis with hematuria, over 60% blood loss, and associated serologic abnormalities. A control cohort of dogs infused with the same quantity of liposome contained in the 40/mg/kg dose was without ensuing toxicity. These changes suggest the mechanism of hemolysis following 40mg/kg curcumin is due to an oxidant effect. Following acute hemolysis, the iron chelation activity of curcumin could contribute to unremitting anemia by blocking iron reutilization. In conclusion, at concentrations below the canine MTD of 20 mg/kg, curcumin acts as an anti‐oxidant, and acts as a pro‐oxidant at higher concentrations. The disparity between rodent and canine sensitivity to liposomal curcumin may be due to species differences in pharmacokinetics and/or curcumin metabolism. Ongoing study will define liposomal curcumin pharmacokinetic parameters at the MTD in canine mode as well define tolerability of multiple dosing in dogs. In addition hematological studies are being conducted to determine the mechanism of hemolysis that was observed at higher dose levels and identify potential biomarkers for predicting toxicity. Preliminary data suggests liposomal curcumin will have promising anticancer activity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A29.