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. 2021 Mar 13;14(3):259.
doi: 10.3390/ph14030259.

Validation of an LC-MS/MS Method to Quantify the New TRPC6 Inhibitor SH045 (Larixyl N-methylcarbamate) and Its Application in an Exploratory Pharmacokinetic Study in Mice

Xiao-Ning Chai  1   2 , Friedrich-Alexander Ludwig  3 , Anne Müglitz  1 , Michael Schaefer  1 , Hai-Yan Yin  2 , Peter Brust  3 , Ralf Regenthal  4 , Ute Krügel  1
Affiliations

Validation of an LC-MS/MS Method to Quantify the New TRPC6 Inhibitor SH045 (Larixyl N-methylcarbamate) and Its Application in an Exploratory Pharmacokinetic Study in Mice

Xiao-Ning Chai et al. Pharmaceuticals (Basel). .

Abstract

TRPC6 (transient receptor potential cation channels; canonical subfamily C, member 6) is widespread localized in mammalian tissues like kidney and lung and associated with progressive proteinuria and pathophysiological pulmonary alterations, e.g., reperfusion edema or lung fibrosis. However, the understanding of TRPC6 channelopathies is still at the beginning stages. Recently, by chemical diversification of (+)-larixol originating from Larix decidua resin traditionally used for inhalation, its methylcarbamate congener, named SH045, was obtained and identified in functional assays as a highly potent, subtype-selective inhibitor of TRPC6. To pave the way for use of SH045 in animal disease models, this study aimed at developing a capable bioanalytical method and to provide exploratory pharmacokinetic data for this promising derivative. According to international guidelines, a robust and selective LC-MS/MS method based on MRM detection in positive ion mode was established and validated for quantification of SH045 in mice plasma, whereby linearity and accuracy were demonstrated for the range of 2-1600 ng/mL. Applying this method, the plasma concentration time course of SH045 following single intraperitoneal administration (20 mg/kg body weight) revealed a short half-life of 1.3 h. However, the pharmacological profile of SH045 is promising, as five hours after administration, plasma levels still remained sufficiently higher than published low nanomolar IC50 values. Summarizing, the LC-MS/MS method and exploratory pharmacokinetic data provide essential prerequisites for experimental pharmacological TRPC6 modulation and translational treatment of TRPC6 channelopathies.

Keywords: LC-MS/MS; SH045; TRP channels; TRPC6 inhibitor; channelopathies; labdane; larixol; mice; pharmacokinetics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
MS/MS of SH045 and (+)-larixol: (a) EPI spectrum of SH045 (product of m/z 364.3), (b) EPI spectrum of (+)-larixol (product of m/z 307.2), (c) chemical structures of SH045 and (+)-larixol and (d) explanation for fragmentation pattern in EPI spectrum of SH045.
Figure 2
Figure 2
Representative MRM chromatograms of (a) SH045 and (b) (+)-larixol as IS in mouse blank plasma (left panel), blank plasma spiked with 100 ng/mL of SH045 and 5 µg/mL IS (middle panel) and in a mouse plasma sample obtained 3 h following administration of SH045 (20 mg/kg, i.p., right panel).
Figure 3
Figure 3
Plasma kinetics of SH045 in mice after intraperitoneal (i.p.) administration (20 mg/kg body weight). (a) Mean plasma concentration-time course of SH045, (b) semilogarithmic plot indicative for first-order elimination kinetics (Pearson’s r = −0.991).
See this image and copyright information in PMC

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