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Enterolactone glucuronide and β-glucuronidase in antibody directed enzyme prodrug therapy for targeted prostate cancer cell treatment

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Abstract

Evidence from preclinical and animal studies demonstrated an anticancer effect of flaxseed lignans, particularly enterolactone (ENL), against prostate cancer. However, extensive first-pass metabolism following oral lignan consumption results in their systemic availability primarily as glucuronic acid conjugates (ENL-Gluc) and their modest in vivo effects. To overcome the unfavorable pharmacokinetics and improve their effectiveness in prostate cancer, antibody-directed enzyme prodrug therapy (ADEPT) might offer a novel strategy to allow for restricted activation of ENL from circulating ENL-Gluc within the tumor environment. The anti-prostate-specific membrane antigen (PSMA) antibody D7 was fused with human β-glucuronidase (hβG) via a flexible linker. The binding property of the fusion construct, D7-hβG, against purified or cell surface PSMA was determined by flow cytometry and Octet Red 384 system, respectively, with a binding rate constant, K d, of 2.5 nM. The enzymatic activity of D7-hβG was first tested using the probe, 4-methylumbelliferone glucuronide. A 3.8-fold greater fluorescence intensity was observed at pH 4.5 at 2 h compared with pH 7.4. The ability of D7-hβG to activate ENL from ENL-Gluc was tested and detected using LC-MS/MS. Enhanced generation of ENL was observed with increasing ENL-Gluc concentrations and reached 3613.2 ng/mL following incubation with 100 μM ENL-Gluc at pH 4.5 for 0.5 h. D7-hβG also decreased docetaxel IC50 value from 23 nM to 14.9 nM in C4-2 cells. These results confirmed the binding and activity of D7-hβG and additional in vitro investigation is needed to support the future possibility of introducing this ADEPT system to animal models.

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Acknowledgements

This work was funded by the Saskatchewan Health Research Foundation (SHRF). We sincerely thank Dr. Ron Geyer for making available of the Octet Red 384 system and his student Yongpeng Fu for helping with the kinetic experiments. We acknowledge Deborah Michel for the technical support for flow cytometry and LC-MS/MS.

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Authors and Affiliations

  1. Drug Discovery and Development Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada

    Yunyun Di, Ed S. Krol & Jane Alcorn

  2. Laboratory of Molecular Cell Biology, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

    Shaoping Ji

  3. Department of Urology, Medical Center, University of Freiburg, Freiburg, Germany

    Philipp Wolf

  4. College of Pharmacy and Nutrition, University of Saskatchewan, 104 Clinic Place, Saskatoon, Saskatchewan, S7N 2Z4, Canada

    Jane Alcorn

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  1. Yunyun Di
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  2. Shaoping Ji
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  5. Jane Alcorn
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Correspondence to Jane Alcorn.

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Di, Y., Ji, S., Wolf, P. et al. Enterolactone glucuronide and β-glucuronidase in antibody directed enzyme prodrug therapy for targeted prostate cancer cell treatment. AAPS PharmSciTech 18, 2336–2345 (2017). https://doi.org/10.1208/s12249-017-0721-5

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  • DOI: https://doi.org/10.1208/s12249-017-0721-5

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