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Simultaneous Determination of 1-Methyltryptophan and Indoleamine 2,3-Dioxygenase Biomakers of Tryptophan and Kynurenine in Mice Tumors by HPLC–MS/MS

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Abstract

Indoleamine 2,3-dioxygenase (IDO), an immune checkpoint protein, can cause the depletion of tryptophan (Trp) and accumulation of its metabolite of kynurenine (Kyn) in cancer cells, and generates the immunosuppressive microenvironment that supports tumor cell growth. A novel immunoregulatory prodrug micelle based on polyethylene glycol-derivatized an IDO-selective inhibitor of 1-methyltryptophan (1-MT), PEG-Fmoc-1-MT, was developed for inhibiting the IDO activity of the conversion of Trp to Kyn in tumor microenvironments. To investigate the 1-MT distribution and Trp/Kyn ratios in mice tumors with PEG-Fmoc-1-MT prodrug micelles treatment, a HPLC–MS/MS method for simultaneous determination of 1-MT and IDO biomakers of Trp and Kyn in mouse tumors was developed and validated. Triple-quadrupole mass spectrometry with positive electrospray ionization as source ionization in multiple reaction monitoring at m/z 219.0 → 160.1, 205.0 → 118.2, 209.0 → 146.1 and 249.3 → 148.3 was used for determination of 1-MT, Trp, Kyn and matrine (internal standard). The method demonstrated good linearity at the concentrations ranging from 10 to 10,000 ng/mL and lower limits of quantitation of 1 ng/mL for 1-MT, Trp and Kyn, respectively. The validated method was successfully applied to 1-MT tumor biodistribution and Trp/Kyn ratio studies in 4T1 tumor bearing mice i.v. with PEG-Fmoc-1-MT prodrug micelles. The mice tumors with PEG-Fmoc-1-MT prodrug micelles treatment exhibited higher 1-MT accumulation and lower Trp/Kyn ratio, in comparison with those of mice with 1-MT solution treatment. The developed PEG-Fmoc-1-MT prodrug micelles could be a promising IDO immunoregulatory prodrug micelles for cancer immunotherapy.

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Acknowledgements

This work was supported by the Nature Science Foundation of Ningxia (NO. 2020AAC03137).

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Author notes

  1. Liyue Zhou, Rongyue Zhu and Yang Lan contributed equally.

Authors and Affiliations

  1. Department of Pharmaceutics, School of Pharmacy, Ningxia Medical University, No. 1160, Shengli Street, Yinchuan, 750004, China

    Liyue Zhou, Rongyue Zhu, Yang Lan, Jiayu Yang, Yue Sun, Yanhui Hou, Xueqin Ma & Yanhua Liu

  2. Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education, Ningxia Medical University, Yinchuan, 750004, China

    Rongyue Zhu, Jiayu Yang, Yanhui Hou, Xueqin Ma & Yanhua Liu

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  1. Liyue Zhou
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  2. Rongyue Zhu
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  3. Yang Lan
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  4. Jiayu Yang
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  5. Yue Sun
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Contributions

LZ, RZ and YL, contributed equally. All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by LZ, RZ and YL. The first draft of the manuscript was written by YL and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Xueqin Ma or Yanhua Liu.

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The authors declare the absence of conflicts of interest.

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The study protocol was approved by the Ningxia Medical University. All animal experiments were conducted under protocols approved by the Ministry of Health of the People's Republic of China and following Guidelines for the Care and Use of Laboratory Animals of Ningxia Medical University, and informed consent was obtained from all study participants.

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Zhou, L., Zhu, R., Lan, Y. et al. Simultaneous Determination of 1-Methyltryptophan and Indoleamine 2,3-Dioxygenase Biomakers of Tryptophan and Kynurenine in Mice Tumors by HPLC–MS/MS. Chromatographia 84, 623–634 (2021). https://doi.org/10.1007/s10337-021-04043-w

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