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Chemical, Analytical and Pharmacokinetic Characterisation of RO7304898, an API Consisting of Two Rapidly Interconverting Diastereoisomers

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Exploration of the chemical, analytical and pharmacokinetic properties of the API, RO7304898, an allosteric EGFR inhibitor, intended to be developed as a mixture of two rapidly interconverting diastereoisomers with composition ratio of approximately 1:1.


Assessment of diastereoisomer stereochemistry, interconversion rates, binding to EGFR protein, metabolic stability and in vivo PK in Wistar-Han rats was conducted.


The two diastereoisomers of the API undergo fast interconversion at physiologically relevant pH and direct EGFR binding studies revealed diastereoisomer B to be the active moiety. Pharmacokinetic studies in rat revealed a low-moderate total plasma clearance of the API along with similar plasma concentration-time profiles for diastereoisomers A and B, and the diastereoisomeric ratio reached stable equilibrium favoring formation of the potent diastereoisomer B. In in vitro incubations, the API was metabolically stable in plasma and hepatocyte suspension incubations in all species tested except that of rat hepatocytes. Additionally, only small species differences in the A:B composition were observed in vitro with the potent diastereoisomer B being the predominant form.


We demonstrated that the API, a mixture of two diastereoisomers; A (impotent) and B (potent), undergoes rapid interconversion which is faster than the apparent distribution and elimination rates of the individual diastereoisomers in vivo in rat, serving to diminish concerns that separate diastereoisomer effects may occur in subsequent pharmacologic and pivotal toxicological studies. Whilst vigilant monitoring of the diastereoisomeric ratio will need to be continued, this data adds confidence on the development pathway for this API to the clinic.

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Data Availability

All data generated or analysed during this study are included in this published article.



Ratio of diastereoisomer A to B


Active pharmaceutical agent


Intrinsic clearance


Deuterated water


Deuterated dimethyl sulfoxide


Dimethyl sulfoxide


Diastereoisomeric ratio


Epidermal growth factor receptor


European Medicines Agency


Food and Drug Agency




High performance liquid chromatography coupled mass spectrometer


High-performance liquid chromatography coupled ultra-violet (UV) spectroscopy


Analytical internal standard




Tripotassium ethylenediaminetetraacetic acid


Equilibrium dissociation constant


Liquid chromatography coupled tandem mass spectrometer


Liquid liquid extraction


Lower limit of quantitation




Multiple reaction monitoring


Nuclear magnetic resonance


Nuclear Overhauser Effect



p.o. :

Per os, oral


Protein precipitation


Parts per million


Quality control


Room temperature




Tyrosine kinase inhibitor


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Acknowledgements and Disclosures

Thomas Wirz, Monique Wittig-Kieffer, Fred Avenel and Pawel Dzygiel for bioanalytical method development and sample analysis. Annie Blondé, Claudia Senn and Veronique Dall’Asen for formulation and conduct of the rat in vivo single dose PK study and Laurent Gand for evaluation of the the rat pharmacokinetic parameters. Kenichi Umehara, Florian Klammers, Isabelle Walter and Vincent Monin for the conduct and evaluation of the in vitro plasma and hepatocyte studies. Dominique Burger expressed and purified the EGFR LR protein for the direct binding assay. Martin Binder for acquisition and interpretation of NMR data. Stefania Beato, Daniela Fraier, Luca Ferrari and Dietrich Türck for scientific discussions. The authors have no conflicts of interest to declare that are relevant to the content of this article.

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Authors and Affiliations



Conception and study design: Caroline Rynn, Katja Heinig, Filippo Sadojevich, Alfred Ross, Philipp Koldewey and Saša Miladinovic.

Acquisition and analysis of data: Caroline Rynn, Katja Heinig, Filippo Sadojevich, Alfred Ross, Philipp Koldewey and Saša Miladinovic.

Drafting and revision of the manuscript: Caroline Rynn, Katja Heinig, Filippo Sadojevich, Jeannine-Petrig Schaffland, Georg Jaeschke, Alfred Ross, Philipp Koldewey, Saša Miladinovic and Jin Wang.

Critical review of the manuscript: Daniela Fraier, Luca Ferrari and Stafania Beato.

Final review and approval: Caroline Rynn, Katja Heinig, Filippo Sadojevich, Jeannine Petrig Schaffland, Georg Jaeschke, Alfred Ross, Philipp Koldewey, Saša Miladinovic and Jin Wang.

Corresponding author

Correspondence to Caroline Rynn.

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Research Involving Animals

The protocol was reviewed and approved by the Institutional Animal Care and Use Committee of the Cantonal Veterinary Office Basel, Switzerland. The study was performed in accordance with the ‘Guide For The Care and Use of Laboratory Animals’ as adopted and promulgated by the U.S. National Institutes of Health.

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Heinig, K., Sladojevich, F., Petrig Schaffland, J. et al. Chemical, Analytical and Pharmacokinetic Characterisation of RO7304898, an API Consisting of Two Rapidly Interconverting Diastereoisomers. Pharm Res 39, 653–667 (2022).

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