Development & validation of LC–MS/MS assay for the quantification of pegcantratinib Research Article
treatment of patients with germline mutations in the tumor suppressor gene CYLD, provides several chal- lenges in terms of development of an analytical assay for drug quantification. No assay has been published for this mini-PEGylated agent, with assay develop- ment for PEGylated drugs historically facing signifi- cant hurdles due to the heterogeneity and high MW of the PEG fraction [9]. In addition, as pegcantratinib is being developed in this clinical situation for topi- cal administration, an assay was required to allow the quantitation of drug levels extracted from tumor material collected by punch biopsies. We have developed a sensitive and specific HPLC–
MS/MS assay, coupled with in-source CID, for direct quantitative measurement of pegcantratinib in human skin tumors. The novel method involves HPLC separation of the PEGylated small molecule, followed by in-source fragmentation in the instru- ment ionization source, generating a small molecule pegcantratinib-specific fragment ion that can be used as surrogate for the quantitative analysis. Excellent sensitivity was achieved, with an LLOQ below values previously published for PEGylated molecules [10,11]. We have successfully validated the method according to the EMA guidance on bioanalytical method vali- dation [12] and demonstrated its applicability to deter- mine the drug’s tumor distribution in skin tumor punch biopsies obtained from patients with CYLD mutations participating in the current clinical trial.
Conclusion & future perspective The bioanalytical method described, based on simple tumor homogenization and acetonitrile extraction followed by HPLC–MS/MS coupled with in-source CID analysis, was validated for the quantitative mea- surement of the PEGylated molecule, pegcantratinib, in human tumor biopsies. The method requires small amounts of tumor matrix; it is selective, sensitive, precise and accurate. It was successfully employed to measure concentrations of pegcantratinib in human skin tumor biopsies of CYLD carrier patients enrolled in a Phase Ib open label clinical trial. The
Executive summary
Background • An LC method coupled with in-source collision-induced dissociation MS was developed and validated to quantify pegcantratinib in skin tumor biopsies of patients enrolled in an early phase clinical to determine tumor penetration.
Experimental • The sample preparation procedure required a 10 mg sample for tissue homogenization and involved protein precipitation with acetonitrile following addition of internal standard.
Results & discussion • The method is selective, precise, accurate and sensitive, with an LLOQ of 2.5 ng/ml in tumor. • The preliminary clinical data showed tumor drug absorption and penetration to inner tumor layers.
preliminary clinical data obtained showed tumor drug absorption and penetration to inner tumor layers. Data concerning the tumor exposition will provide useful information for investigating corre- lations between tumor exposure and response in an expanded Phase Ib/IIa clinical trial.
Disclaimer The views expressed in this publication are those of the au- thors and not necessarily those of the Wellcome Trust or the Department of Health.
Financial & competing interests disclosure This work was supported by the Wellcome Trust (WT- 097163MA); Wellcome Trust and Department of Health un- der the Health Innovation Challenge Fund (100935/Z/13/Z) and Cancer Research UK. This publication presents indepen- dent research commissioned by the Health Innovation Chal- lenge Fund (HICF-R7-395), a parallel funding partnership between the Wellcome Trust and the Department of Health. S Traversa is an employee of Creabilis Therapeutics. The au- thors have no other relevant affiliations or financial involve- ment with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of
this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate in- stitutional review board approval or have followed the prin- ciples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for inves- tigations involving human subjects, informed consent has been obtained from the participants involved.
Open access This article is distributed under the terms of the Creative Com- mons Attribution License 4.0 which permits any use, distribu- tion, and reproduction in any medium, provided the original author(s) and the source are credited. To view a copy of the license, visit
http://creativecommons.org/licenses/by/4.0/
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