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

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Abstract

Purpose

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.

Methods

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

Results

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.

Conclusions

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.

Abbreviations

A:B:

Ratio of diastereoisomer A to B

API:

Active pharmaceutical agent

CLint:

Intrinsic clearance

D20:

Deuterated water

D6-DMSO:

Deuterated dimethyl sulfoxide

DMSO:

Dimethyl sulfoxide

d.r.:

Diastereoisomeric ratio

EGFR:

Epidermal growth factor receptor

EMA:

European Medicines Agency

FDA:

Food and Drug Agency

H/D:

Hydrogen/Deuterium

HPLC-MS:

High performance liquid chromatography coupled mass spectrometer

HPLC-UV:

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

IS:

Analytical internal standard

i.v.:

Intravenous

K3-EDTA:

Tripotassium ethylenediaminetetraacetic acid

KD:

Equilibrium dissociation constant

LC-MS/MS:

Liquid chromatography coupled tandem mass spectrometer

LLE:

Liquid liquid extraction

LLOQ:

Lower limit of quantitation

min:

Minutes

MRM:

Multiple reaction monitoring

NMR:

Nuclear magnetic resonance

NOE:

Nuclear Overhauser Effect

PK:

Pharmacokinetic

p.o. :

Per os, oral

PP:

Protein precipitation

PPM:

Parts per million

QC:

Quality control

RT:

Room temperature

s:

Second

TKI:

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Ethics declarations

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). https://doi.org/10.1007/s11095-022-03234-w

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  • DOI: https://doi.org/10.1007/s11095-022-03234-w

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