AHCC® Supplementation to Support Immune Function to Clear Persistent Human Papillomavirus Infections.

Objective: To determine the efficacy, safety, and durability of the use of AHCC supplementation for 6 months to support the host immune system to clear high-risk human papillomavirus (HPV) infections. The AHCC supplement is a proprietary, standardized extract of cultured lentinula edodes mycelia (AHCC®, Amino Up, Ltd., Sapporo, Japan) that has been shown to have unique immune modulatory benefits. Study Design: This was a randomized, double-blind, placebo-controlled study (CTN: NCT02405533) in 50 women over 30 years of age with confirmed persistent high-risk HPV infections for greater than 2 years. Patients were randomized to placebo once daily for 12 months (N = 25) or AHCC 3-g supplementation by mouth once daily on empty stomach for 6 months followed by 6 months of placebo (N = 25). Every 3 months, patients were evaluated with HPV DNA and HPV RNA testing as well as a blood sample collected to evaluate a panel of immune markers including interferon-alpha, interferon-beta (IFN-ß), interferon-gamma (IFN-γ), IgG1, T lymphocytes, and natural killer (NK) cell levels. At the completion of the 12-month study period, patients on the placebo arm were given the option to continue on the study to receive AHCC supplementation unblinded for 6 months with the same follow-up appointments and testing as the intervention arm. Results: Fifty women with high-risk HPV were enrolled, and 41 completed the study. Fourteen (63.6%) of the 22 patients in the AHCC supplementation arm were HPV RNA/HPV DNA negative after 6 months, with 64.3% (9/14) achieving a durable response defined as being HPV RNA/HPV DNA negative 6 months off supplementation. On the placebo arm, two (10.5%) of 19 patients were HPV negative at 12 months. In the twelve placebo arm patients who elected to continue on the unblinded study, 50% (n = 6) were HPV RNA/HPV DNA negative after 6 months of AHCC supplementation. At the time of completion of the study, there were a total of 34 patients (22 blinded and 12 unblinded) who had received AHCC supplementation with an overall response rate of 58.8% that cleared HPV persistent infections. At the time of enrollment, the mean IFN-ß level was 60.5 ± 37.6 pg/ml in women with confirmed persistent HPV infections. Suppression of IFN-ß to less than 20 pg/ml correlated with an increase in T lymphocytes and IFN-γ and durable clearance of HPV infections in women who received AHCC supplementation. Conclusion: Results from this phase II study demonstrated that AHCC 3 g once daily was effective to support the host immune system to eliminate persistent HPV infections and was well tolerated with no significant adverse side effects reported. The duration of AHCC supplementation required beyond the first negative result needs more evaluation to optimize success for durable outcomes. The suppression of the IFN-ß level to less than 20 pg/ml correlated with clearance of HPV infections and merits further evaluation as a clinical tool for monitoring patients with HPV infections. Clinical Trial Registration: clinicaltrials.gov/ct2/, identifier NCT02405533.

From Bench to Bedside: Evaluation of AHCC Supplementation to Modulate the Host Immunity to Clear High-Risk Human Papillomavirus Infections.

Objective: There is currently no effective medicine or supplement for clearance of high risk- human papillomavirus (HR-HPV) infections. We have taken a systematic approach evaluating the potential use of AHCC supplementation to support clearance of HR-HPV infections. The primary objective of this research was to evaluate AHCC supplementation to modulation of the host immune system to clear HR-HPV infections from bench to bedside. Methods: Cervical cancer cells, CaSki (HPV16+), HeLa(HPV18+), SiHa(HPV16/18+), and C-33A(HPV-), were treated in vitro with AHCC 0.42 mg/mL daily x7 days then observed x7 days with daily sample collection. A confirmatory study in cervical cancer mouse models, SiHa(HPV16/18+) and C-33A(HPV-), was conducted: mice were divided into three groups per cell line then dosed with AHCC 50 mg/kg/d (N = 10), or vehicle alone (N = 10), or no supplementation (N = 10) for a total of 90 days followed by 30 days of observation. Tumors were measured 3x/week and blood samples collected bi-weekly to evaluate interferon (IFN) alpha(α), beta(ß), and gamma(γ) and immunoglobulin G(IgG) by immunoassays. Tumors were evaluated for HR-HPV expression by PCR. Two pilot studies of 10 patients each were conducted in women with confirmed persistent HR-HPV+ infections. The 1st study evaluated AHCC 3g from 5 weeks up to 6 months and 2nd study evaluated AHCC 1g < 8 months. HR-HPV DNA status and the immune panel were monitored at each visit. Results: HR-HPV clearance was observed in vitro and confirmed in the animal studies as a durable response. Four of six (66.7%) patients had confirmed HR-HPV clearance after 3-6 months of AHCC 3g. Similarly, 4 of 9 (44%) patients had confirmed HR-HPV clearance after 7 months of AHCC 1g. Suppression of IFNß <25 pg/mL was observed in those clearing the HR-HPV infection. Conclusion: Pre-clinical in vitro and in vivo studies demonstrated durable clearance of HR-HPV infections. The preliminary data from the two pilot studies suggested that AHCC supplementation supports the host immune system for successful clearance of HR-HPV infections. A confirmatory phase II randomized, double-blinded, placebo-controlled study is ongoing.

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 17ß-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.

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.

Determination of minimum effective dose and optimal dosing schedule for liposomal curcumin in a xenograft human pancreatic cancer model.

BACKGROUND: Curcumin is a food chemical present in tumeric (Curcuma longa) that has pharmacological activity to suppress carcinogenesis and inhibits multiple signaling pathways such as nuclear factor kappaB (NF-kappaB), cyclooxygenase-2 (Cox-2) and interleukin-8 (IL-8). Oral curcumin has poor oral bioavailability limiting its clinical activity; however, a patent pending liposomal formulation of curcumin was developed to improve drug delivery and has demonstrated activity in multiple cancers. This study was designed to determine the minimum effective dose (MED) as well as the optimal dosing schedule of liposomal curcumin in a xenograft mouse model of human pancreatic cancer. MATERIALS AND METHODS: The MED determination and optimal schedule was evaluated in female athymic nude mice injected subcutaneously with MiaPaCa-2 cells. Dosing was initiated at an average tumor size of 5mm. For the MED, mice were treated with the following dose levels of liposomal curcumin: no treatment, liposome only, 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg and 40 mg/kg given by tail vein injection three times weekly for 28 days. For the optimum dosing schedule, three additional schedules were evaluated and compared to the control of three times weekly; daily (five days per week), every four days, and weekly for 28 days. All mice were weighed and tumor measurements taken three times weekly to evaluate toxicity and efficacy. RESULTS: The 20 mg/kg dose had the greatest decrease in tumor growth at 52% decrease in tumor growth when compared to no treatment control mice. MED was determined to be 20 mg/kg and was used for the optimal dosing schedule determination. Daily dosing and three times per week dosing had greater inhibition of tumor growth with no discernable difference than once weekly or every 4 day dosing. No toxicity was observed at any dose or schedule. CONCLUSION: The MED for liposomal curcumin is 20 mg/kg given once daily three times per week to achieve optimal tumor growth inhibition. This was dose recommended for additional preclinical studies to define safety and tolerability of liposomal curcumin in rat and dog models.