Pharvaris N : Bradykinin Challenge Model in Humanized Bradykinin B2 receptor Transgenic Rat

Bradykinin Challenge Model

in Humanized Bradykinin B2 receptor Transgenic Rat

Jolanta Skarbaliene, Michiel Joost van Esdonk, Juan Bravo, Christoph

Gibson, Jochen Knolle, Anne Lesage

2024 Bradykinin Symposium

Berlin, Germany; 5-6 September 2024

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This presentation includes data for an investigational product not yet approved by regulatory authorities.

Conflicts of interest disclosures

J. Skarbaliene employee of Pharvaris, holds stocks/stock options in Pharvaris. M. van Esdonk employee of Pharvaris, holds stocks/stock options in Pharvaris. J. Bravo employee of Pharvaris, holds stocks/stock options in Pharvaris.

C. Gibson employee of AnalytiCon Discovery, holds stocks/stock options in Pharvaris.

J. Knolle employee of JCK Consult and consultant to Pharvaris, holds stocks/stock options in Pharvaris.

A. Lesage employee of GrayMatters Consulting and consultant to Pharvaris, holds stocks/stock options in Pharvaris; advisor to Kosa Pharma.

This presentation includes data for an investigational product not yet approved by regulatory authorities.

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Why is a rat BK challenge model needed?

Established BK challenge models in non-human primates (NHP) and humans were effectively used to determine pharmacokinetic and pharmacodynamic (PK/PD) relationships of bradykinin B2 receptor antagonists

Advantages of a rat BK challenge model

• Enables early-stagePK/PD characterization
• Enables the refinement of human dose predictions
• Practical and scalable for broad research applications
• More cost-effective alternative to existing models
• Can be applied to genetically modified animals to address specific research questions

Lesage ASJ et al. AAAAI 2020.

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Development of a BK challenge model in a humanized bradykinin

B2 receptor transgenic rat line

Background

Bradykinin induces a transient hypotensive reaction following intra-arterial administration in rats, compared to intravenous administration.

Aim of the study

To develop the BK challenge model using transgenic humanized bradykinin B2 receptor Sprague Dawley male rats for evaluation of species selective and non-selective bradykinin B2 receptor antagonists

1. Identify the effective dose of BK for inducing hemodynamic effects.
2. Assess desensitization of hemodynamic effects after repeated BK administration.
3. Validate the model using icatibant as reference bradykinin B2 receptor antagonist

Adapted from Blais et al. 2005. Blood pressure modulator effect of BK administered via intravenous (i.v.) and intra- arterial (i.a.) routes in rats n = 5-9.***p < 0.001 vs. BK i.v.

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A humanized bradykinin B2 receptor transgenic rat model was

established and validated

Antagonist potency at recombinant bradykinin B2 receptors in

HEK293

• The recombinant humanized bradykinin B2 receptor was expressed in HEK293 cells and the effect of the mutation on antagonist potency of deucrictibant and icatibant was investigated.
• A transgenic humanized rat line was generated on a Sprague Dawley background using CRISPR/Cas9- mediated gene editing. This rat line showed no adverse phenotypes and appeared a healthy strain.

		Kb in nM (Ca2+ mobilisation)
	WT	Humanized	Ratio Kb values
	rat B2	rat B2	WT vs humanized B2
Deucrictibant	61.0 ± 23.0	0.45 ± 0.10	136
Icatibant	0.59 ± 0.02	0.53 ± 0.10	1

Antagonist affinity for endogenous bradykinin B2 receptor in

uterus of WT or transgenic rat

		Ki in nM (3H-BK)
	WT	Transgenic	Ratio Ki values
	rat uterus	rat uterus	WT vs transgenic rat
Deucrictibant	14.9 ± 4.1	0.55 ± 0.14	27
Icatibant	0.34 ± 0.10	0.14 ± 0.05	2

• Humanization of the rat bradykinin B2 receptor increases the antagonist potency and affinity of deucrictibant, a selective
  antagonist of the human bradykinin B2 receptor.
• Icatibant's antagonist potency and affinity for the recombinant bradykinin B2 receptor are not affected by humanization,

confirming its equipotent antagonism of the human and rat bradykinin B2 receptors.

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Development and validation of a BK challenge model in a humanized bradykinin B2 receptor transgenic rat line - Study design

Icatibant	BK challenge
iv injection	intra-arterial
via the lateral	injection of BK via
tail vein	the left common
	carotid artery
Anesthetized male rats
	Blood pressure (BP)
	measurement via the
	right common carotid
	artery and recorded
	using a BP transducer

Representative tracings depicting hypotensive effects of

BK following intra-arterial injection

Dose-response of BK

Desensitization testing

Icatibant testing

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Development and validation of a BK challenge model in a humanized bradykinin B2 receptor transgenic rat line - Study results

1. Evaluation of bradykinin-dose response (incremental BK doses every 30 min)

2. Evaluation of repeated administration of 0.8 nmol/kg BK (every 45 min)

3. Evaluation of icatibant effects on response to 0.8 nmol/kg BK (every 45 min)

BK-induced changes in BP (mmHg)

0

-20

-40

-60

BK (nmol/kg)

0.01 0.1 1 10 100

BK-induced changes in BP (mmHg)

0

-20

-40

-60

	BK (0.8 nmol/kg)
0	45	90	135	180	Time (min)

BK-induced changes in BP (mmHg)

0

-20

-40

-60

Icatibant 1 mg/kg

Icatibant 0.5 mg/kg

Icatibant 0.05 mg/kg

Icatibant 0.005 mg/kg

BK	BK	BK	BK	BK	(0.8 nmol/kg)
0	45	90	135	180	Time (min)
Icatibant
				Mean ±SEM, N=3-8

• Dose-dependentlowering of blood pressure demonstrated with BK (ED80 was estimated at approximately 1 nmol/kg)
• No apparent BK-mediated desensitization
• Icatibant at all tested doses (0.005 to 1 mg/kg, i.v.) inhibited the BK-induced hemodynamic response

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Summary

• We successfully developed a BK challenge model in humanized bradykinin B2 receptor transgenic rats that are pharmacologically responsive to bradykinin B2 receptor antagonists.
• The BK challenge model in humanized bradykinin B2 receptor transgenic rats offers a valuable, easy to manage, and cost-effective tool for efficacy studies compared to those involving non-human primates.

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Don't miss additional presentations for more insights!

Oral Presentation. Session VII Hereditary angioedema on Friday, 6th September, from 15:05 to 15:15

• Prophylactic Treatment With Deucrictibant Improves HAE Disease Control and HRQoL (Markus Magerl et al.)

Posters Poster session on Friday, 6th September, from 15:35 to 16:15

• The bradykinin challenge model translates across rat, monkey and human (Juan Bravo et al.)
• Deucrictibant inhibits carrageenan-induced edema in bradykinin B2 receptor transgenic rat (Anne Lesage et al.)
• Cardiovascular safety of repeated oral administration of the B2-receptor antagonist deucrictibant (Nieves Crespo et al.)
• A novel kinin biomarker assay for characterisation of bradykinin-mediated disorders (Evangelia Pardali et al.)
• A HMWK capillary immunoblotting assay to characterise bradykinin-mediated disorders (Evangelia Pardali et al.)
• Clinical trials conformity with AURORA COS: a systematic literature review (Remy S. Petersen et al.)
• Deucrictibant vs. Standard of Care in HAE: Propensity Score-Matched Analysis (Marc A. Riedl et al.)
• Treatment of HAE Attacks With Oral Deucrictibant: RAPIDe-2 Extension Results (Emel Aygören-Pürsün et al.)
• Long-TermSafety and Efficacy of Oral Deucrictibant for HAE Prophylaxis (Marc A. Riedl et al.)
• Prophylaxis of Hereditary Angioedema Attacks With Oral Deucrictibant: CHAPTER-1 Results (Emel Aygören-Pürsün et al.)

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