# In vitro-in vivo discord: A preclinical study of AZD2716 and its racemate with comparison to varespladib for the development of snake venom sPLA2 inhibitors

## TL;DR

This study compares sPLA2 inhibitors for snakebite treatment, finding that in vitro effectiveness does not always match in vivo results in mice.

## Contribution

Highlights the importance of preclinical testing by showing in vitro potency does not always translate to in vivo efficacy in drug development.

## Key findings

- AZD2716 and its racemate showed potent in vitro inhibition of sPLA2 across 26 snake venoms.
- In vivo mouse studies showed varespladib had better survival outcomes than AZD2716 enantiomers and racemate.
- In vitro efficacy did not predict in vivo performance, emphasizing the need for comprehensive preclinical testing.

## Abstract

We evaluated a family of repurposed sPLA2 inhibitors as novel candidate snakebite envenoming therapeutics. Stereospecific (R)-7 AZD2716 and its racemic mixture were compared to varespladib in an in vitro sPLA2 assay against a sample of 26 venoms from medically important snake species from five continents. All compounds demonstrated potent nano-to picomolar IC50 values, comparable to the benchmark inhibitory profile of varespladib. Surprisingly, however, this in vitro efficacy did not translate to survival in an in vivo mouse model under GLP standard conditions at an independent third party laboratory. In animal rescue studies evaluating both oral and IV dosing against the same four high sPLA2 venoms, varespladib demonstrated more consistent survival duration versus the chirally separated AZD2716 enantiomer and racemate following single-dose intravenous or two-dose oral drug administration. Additionally, the stereospecific AZD2716 did not provide the same survival advantage as the racemic mixture and neither molecule resulted in the same survival advantage as varespladib in vivo (p < 0.05), despite similar in vitro potency. These findings highlight the importance of following in vitro inhibition assays with preclinical studies in drug candidate selection for lead compounds and advancement to clinical development.

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•This work identifies a new family of potentially repurposed sPLA2 inhibitors with potent in vitro inhibition across a diverse cohort of snake venoms.•Surprisingly, these inhibitors showed inconsistent in vivo efficacy in a mouse model conducted under GLP reporting standards by an independent, third party, contract laboratory.•These differences highlight the importance of preclinical testing as in vitro potency may not translate to in vivo efficacy.

This work identifies a new family of potentially repurposed sPLA2 inhibitors with potent in vitro inhibition across a diverse cohort of snake venoms.

Surprisingly, these inhibitors showed inconsistent in vivo efficacy in a mouse model conducted under GLP reporting standards by an independent, third party, contract laboratory.

These differences highlight the importance of preclinical testing as in vitro potency may not translate to in vivo efficacy.