Validation and Utility of Human ips-vascular Smooth Muscle Cells as an in vitro Vascular Model

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1 Validation and Utility of Human ips-vascular Smooth Muscle Cells as an in vitro Vascular Model Caroline Archer, Senior Scientist (Drug Safety and Metabolism) 30 th November 2017

2 Background Drug-induced changes in blood pressure (BP) are a common and undesirable side-effect in drug development 2 Source: Laverty H, Benson C, Cartwright E, et al. British Journal of Pharmacology. 2011;163(4):

arteries (mesenteric arteries) Vasoconstrictive or vasodilatory effects may translate into blood pressure changes Limitations Low

3 Typical Vascular Safety Approach In vitro screens Molecular: secp linked to haemodynamics In vitro assays Wire-myography In vivo studies Rat telemetry Regulatory studies Dog telemetry; MIBP Wire Myography Tests the effect of compounds on the tone of resistance arteries (mesenteric arteries) Vasoconstrictive or vasodilatory effects may translate into blood pressure changes Limitations Low throughput Requires up to 3 rats per compound Bespoke basis flag required from secp screen Some vessel to vessel variability Species translatability 3

In vitro Vascular Model A cellular in vitro model is required, to allow detection and mitigation of these changes prior to in vivo studies Assess value of ips-derived VSMC model; developed by Ncardia

4 In vitro Vascular Model A cellular in vitro model is required, to allow detection and mitigation of these changes prior to in vivo studies Assess value of ips-derived VSMC model; developed by Ncardia Healthy human fibroblasts à ipsc à vascular smooth muscle cells An alternative to primary human aortic smooth muscle cells Cellular in vitro Model 4 Image Source: Zhao, Y., Vanhoutte, PM. and Leung, SWS. Journal of Pharmacological Sciences. 2015; 129(2):83-94.

5 hips-vsmc Versus Primary Human VSMC Feature hips-vsmc Primary VSMC Low Donor variability (Genetic and Environmental) ü û Genetic manipulation ü û Phenotype switching required û ü Availability of cells Unlimited Limited Screening suitability ü û 5

6 Aims and Objectives Develop and validate hips-vsmcs for potential future use as a 2D vascular model suitable for detection of vasoactive compounds Recapitulate data from Ncardia Key vascular smooth muscle cell markers (IF) Response to tool compounds (Ca 2+ flux) Expand the set of vasoactive and nonvasoactive validation compounds Assess gene expression of key ion channels and receptors Develop a human 2D/3D co-culture with endothelial cells (HUVEC) Compare alongside a primary human/rat model system 6

Expression of general VSMC surface markers on

Positive expression of smooth muscle alpha-actin

RFP that was expressed following differentiation

actin; a constituent of contractile apparatus

protein, negative regulator of contraction SM22;

7 Expression of general VSMC surface markers on hips-vsmcs SM α-actin Calponin Merge TagRGF Positive expression of smooth muscle alpha-actin and calponin Cells could also be identified using RFP that was expressed following differentiation under the SM22 promoter 7 Smooth Muscle alpha actin; a constituent of contractile apparatus Calponin; Ca2+ binding filament-associated protein, negative regulator of contraction SM22; earliest marker of differentiated smooth muscle cells, also present in proliferating SMC

Technology Platforms: Vascular Contractility

Throughput Co-culture feasibility ü IonOptix

8 Technology Platforms: Vascular Contractility Assay FLIPR Tetra (Molecular Devices) Calcium High Throughput Co-culture feasibility ü XCELLigence Cardio ECR (ACEA) Impedence Med Throughput Co-culture feasibility ü IonOptix (IonOptix) Calcium/Contractility Low Throughput Co-culture feasibility û 8

9 Response to tool compounds TagRGF FLIPR Tetra used to dose and measure Ca 2+ flux in hips-vsmc Ca 2+ flux induced in hips-vsmc by the tool compounds Ang II and ET-1 9 Initial characterisation confirmed the phenotype and pharmacological responses to tool compounds

10 Vasoactivity wider validation compound set Vasocontriction/Vasodilation validation compounds were selected based: Clinical compounds Profile of known blood pressure change/vasoactivity in vivo Non-Vasoactive compounds: Clinical compounds No associated blood pressure change/vasoactivity in vivo 26 clinical compounds run through FLIPR Calcium assay 15 Vasoconstrictors 5 Vasodilators 6 Non-Vasoactive 10

11 Vasoactivity Results 11 Sensitivity Vasoconstrictors, 47 % Vasodilators, 40 % Specificity: Vasoconstrictors, 53 % Vasodilators, 60 % In vivo effect Active Inactive Vasoconstrictors 7/15 8/15 Vasodilators 2/5 3/5 Negative control 0/6 6/6 Compound In vivo Effect Result at Top Dose AZ1 Vasoconstrictor Inactive AZ2 Vasoconstrictor Active AZ3 Vasoconstrictor Inactive AZ4 Vasoconstrictor Inactive Angiotensin II Vasoconstrictor Active Endothelin-1 Vasoconstrictor Active AZ5 Vasoconstrictor Inactive AZ6 Vasoconstrictor Active AZ7 Vasoconstrictor Inactive AZ8 Vasoconstrictor Active Ionomycin Vasoconstrictor Active AZ9 Vasoconstrictor Inactive AZ10 Vasoconstrictor Active AZ11 Vasoconstrictor Inactive AZ12 Vasoconstrictor Inactive AZ13 Vasodilator Inactive AZ14 Vasodilator Active AZ15 Vasodilator Inactive AZ16 Vasodilator Inactive AZ17 Vasodilator Active AZ18 Negative Control Inactive AZ19 Negative Control Inactive AZ20 Negative Control Inactive AZ21 Negative Control Inactive AZ22 Negative Control Inactive AZ23 Negative Control Inactive

12 Gene expression Analysis Ion Channels A range of ion channels were expressed: Channel Gene Expression Relative Expression Level L-Type, Cav1.2 CACNA1C Low hao>hvsmc>hips T-Type, Cav3.1 CACNA1G Low hao>hips>hvsmc K ATP KCNJ11 Low hao>hips>hvsmc KCa1.1 KCNMA1 High hao>hips>hvsmc Nav1.2 KCN2A Medium hao>hvsmc>hips 12

13 Gene expression Analysis Cell surface receptors A range of cell surface receptors with a known role in vasoconstriction or vasodilation were expressed: Receptor Gene Expression Relative Expression Level ET A receptor EDNRA High hao>hips>hvsmc Thromboxane A2 receptor TBXA2R Medium hao>hvsmc>hips Alpha- adrenoceptor ADRA2A Low hao>hvsmc>hips Difference in magnitude of expression observed relevance unclear (the right control?) 13

14 Summary: 1. Gene expression analysis & pharmacological validation 2. Need to consider the other cell types in the vasculature; co-culture 14

15 VSMC Co-culture Can sensitivity and specificity of vascular model be improved? Increase physiological relevance by incorporating endothelial cells and/or ECM into the model 1. 2D multicellular 2. 3D ECM 3. Transwell 15

16 Transwell co-culture: hips-vsmc & HUVEC Treatment to Insert hips-vsmc assay plate ( 7 days) Primary HUVEC on insert transwell (24 hours) hips-vsmc loaded with Fluo4 HUVEC hips-vsmc Automated dosing 16

17 Transwell co-culture: hips-vsmc & HUVEC hips-vsmc hips-vsmc + HUVEC Delayed Calcium flux Vehicle 1 µm ET-1 Vehicle 1 µm ET-1 HUVEC barrier/diffusion 17

18 Current challenges Validation of hips-vsmc: Are they characteristic of a particular vascular bed? Can the phenotype be matured i.e. expression of receptors? Heterogenous population/purity to be determined Development of in vitro vascular assay: Comparison with Primary human aortic smooth muscle cell model/rodent species Ca 2+ transients as a surrogate for vasoactivity high throughput assay for SMC contraction? Improve physiological relevance through development of a co-culture model 18

19 Acknowledgements Amy Pointon Kelly Gray Milka Budnik-Zawilska Andreas Ehlich Matthias Gossmann Tristan Pritchard-Meaker Cardiovascular in vitro Safety Team 19

20 Confidentiality Notice This file is private and may contain confidential and proprietary information. If you have received this file in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this file is not permitted and may be unlawful. AstraZeneca PLC, 1 Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0AA, UK, T: +44(0) , 20

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