RV521A clinical stage fusion inhibitor of Respiratory Syncytial Virus

Stuart Cockerill.

Respidart 2018

Thursday 29th November

Outline

• ReViral introduction

• RSV disease/Fusion mechanism

• Hit ID

• LO Progression to RV521

• Clinical update

ReViral Ltd.• Founded in 2011, ex Arrow Therapeutics

• Fusion Project initiated as founding project (H2L phase)

• Wellcome Trust £3.4M award to initiate LO phase

• Angel funding allows progression into pre-clinical development

• VC funding ($21M) and RV521 moves into P1 clinical studies

• PoC in phase 2a. Further VC funding ($55M)

Aug. 2011

Aug. 2013

Sep. 2015

Dec. 2017

Respiratory Syncytial Virus

• RSV is a leading cause of lower respiratory tract infection manifesting as bronchiolitis or pneumonia in infants, elderly and immunocompromised adults.

• RSV infection resulted in 3.2 million hospital admissions and an estimated 118,200 deaths in children

Fusion Inhibition

Battles et al., Nature, Chem. Biol.,2016, 12, 87-93

Post-fusion

•Inhibitor binds at the centre of the pre-hairpin structure•prevents formation of hairpin structure

Target Cell

MembranePre-hairpin Hairpin

Fusion

Viral Membrane

the virus approaches the

target cell

fusion protein activated to a

trimeric pre-hairpin intermediate

•Pre-hairpin intermediate rearranges

to form hairpin•allows membrane

fusion to occur •invokes a large conformational rearrangement

Design

Make

Test

Synthesis

Fusion IC50

Plaque IC50

CytotoxCC50

PhyschemProfiling

In vitro ADME

In vivo ADME

HAE modelBalbC mouse

Time of Addition

Mutant Generation

Mutant Sequencing

Subtype testing

Toxicity hERG, Ames

Pre-clinical and clinical evaluation

Project Screening Cascade

Lead Generation

7

Edwards, MP, Price DA, Ann. Reports in medicinal Chemistry, 45, 381, 2010Leeson PD, Springthorne B, Nat. Rev. in Drug Discovery, 6, 881, 2007

Set upStructural and Physicochemical analysis• Novel structural type:

• spirocyclic ring systems• A developable F inhibitor series

• target specific physicochemical space• Solubility, ppb, toxicology benefit• Specific logP range, 1

Stage 1• 30 compounds prepared• Potency demonstrated in a specific subset• Low oral PK demonstrated for a lead

compound

Spirocyclic bicyclics

N

N

NH2

A

PRA IC50 580nM, CC50 9,940nMclogP/D7.4 4.5/2.64

PRA IC50 240nM, CC50 13,750nMclogP/D7.4 4.06/2.2

PRA IC50 31nM, CC50 15,290nMclogP/D7.4 3.62/1.75

PRA IC50 310nM, CC50 23,110nMclogP/D7.4 3.29/1.42

PRA IC50 14,000nM, CC50 11,540nMclogP/D7.4 4.95/3.09

PRA IC50 3,400nM, CC50 33,400nMclogP/D7.4 4.06/2.2

PRA IC50 1,700nM, CC50 22,700nMclogP/D7.4 3.36/2.2

RV049

RV049 Pharmacokinetic Data

9

Cmax 95 / >100 / 5

clogP/D 3.6 / 1.75

Permeability MDCK(10-6 cm/s)

12

Kinetic Solubility(µM)

155

Protein Binding% Free ( r / d / h )

10/10/7

Metabolite ID Study

N

N

N

O

NH2

Hydroxyl incorporation as major metabolites in rat and human microsomes

Double hydroxylation incorporation in human microsomes only

Oxidation to carboxylic acid, a minor metabolite

Hydroxylation, a minor metabolite

Subst Posn.RSV FusionIC50 (nM)

RSV A2 PRA IC50(nM)

CC50 (mM)

4-Cl 11.1 180.7 5.2

5-F 0.7 4.2 7.6

5-Cl 4.5 25.4 0.71

6-F 0.467 0.75 3.0

6-Cl 1.2 6.7 4.5

7-Cl 46.7 2384.3 21.7

7-F 1.3 40.8 31.1

• Rat and human microsomal incubation • oxindole hydroxylation major human route

of metabolism in this system

Fluoro Series PK/Physicochemical Data

Solubility(uM)

Ppb (rdh. Fu %)

Clearence(microsomal, rdh,

ul/min/kg)

Perm (10-6 cm/s)

In vitro Clearence.(rat, hepatocyte, ul/min/106

cells)

F (%) rats (10mg/kg, po. 1mg/kg

iv)

6-F 176.3 33.3/15.1/18.4 45/6.23/50.7 0.75 43.1 25

7-F 1,230 37.8/2.29/6.96 37.8/2.29/6.96 0.185 ND 8

7-Fluoro (21)• Poor permeability • Reduced ppb Fu• Great solubility6-fluoro• Clearance high, also in hepatocytes• Better Fu, more permeable

solubility ok, potent

Chain Modifications

R 6/7-FRSV

fusionIC50 (nM)

RSV A2 PRA

IC50 (nM)DClogD7.4 PSA (A

2)

6 2.6 9.6 -2.87 98.29

7 2.6 2.4 -0.74 64.15

6 1.5 3.6 -1.23 84.38

6 0.91 1.3 -0.5 64.15

6 1.6 3.9 -1.2 84.38

6 1.2 2.4 -1.65 73.38

Objectives• Chain polarity search

• logP reduction• DlogP rel to isopentyl chain

• Retain lower PSANote:• HepG2 cytotoxicity >10mM• CF3-Butyl >25mM

Initial RV521 in vitro/in vivo mouse PK

13

Parameter Value

Solubility (mg/ml) 0.417

Permeability (10-6 cm/s)

0.5

Plasma protein binding. Fu (%, rat, dog, human)

52, 27, 67

Microsomal T1/2(mins) rat, dog, human

80, 532, 32

Hepatocyte T1/2(mins) rat, dog, human

93, 580, 211

F (%) 46%

Cl (ml/min/kg) 48

Vss (L/kg) 17.5

T 1/2 (iv, hrs) 4.2

Cmax (po ng/ml) 224

Profile suitable for progression into an in vivo model

paIC90

Balb-C Mouse Efficacy

14Mohapatra et al, Virol. J., 2005,2:3

• Infect intranasally with RSV at t = 0 hours

• Treat with RSV521 at t = 24 hours

• Harvest tissues at t = 4 days, peak viral load

• Assay RSV titres to measure inhibition

• Pre-treat with RSV521 at t = -2 hours

Pharmacokinetics in mouse :• Good bioavailability• Coverage above the protein-adjusted IC90 for

8hrs

• in vitro system that displays the morphologic and phenotypic characteristics of the in vivo human airway epithelium (in the absence of an immune system)

• cells from lung explants are placed in an air-liquid interface resulting in the formation of a pseudostratified, mucociliary airway epithelium

• compound added to basolateral surface with virus dosed and sampled from apical surface to replicate infection and systemic treatment

Apical surface exposed to airApical surface exposed to air

Human Airway Epithelial Model

• RV521 tested at 10nM, 100nM and 1000nM, +ve control: BMS-433771 (@ 1000nM)

• Initial dose t=- 2hours, subsequently dosed daily prior to infection on the apical side with virus (lucRSV)

• Virus determined @ d7, d8

RV521 RSV Spectrum and Selectivity

16

Cytotoxicity

Cell Type BJ H4-II-E-C3 HEK-293 HepG2 HK2 HUV-EC-C

IC50(μM)

16 14 30 31 >50 13

Cell Type Jurkat MRC-5 Neuro-2a NIH/3T3 NRK-52E SH-SY5Y SK-N-SH

IC50(μM)

30 13 15 19 19 30 26

EC50 (nM)

A5 6440

A5 5470

A2 Memphis37b

Birm1734/90

ATCC2012-10

BA Brasil

B9320

B Bb2

BA WV14617/85

A B

• Greater than a 1000 fold window• A and B subtype activity observed, IC50s

17

i.v. 1 mg/kg p.o. 10 mg/kg

p.o. 50 mg/kg

T1/2 (hrs) 1.78 9.92 12.0

Cl (ml/min/kg) 131.8

Vdss (L/kg) 21.97

Tmax 2.69 5.9

Cmax (ng/ml) 74.2 474.5

F (%) 41.8 131

RV521 rat/dog in vivo PK

i.v. 0.3 mg/kg

p.o. 3mg/kg

T1/2 (hrs) 12.5 7.4

Cl (ml/min/kg) 19.1

Vdss (L/kg) 18.8

Tmax 4.0

Cmax (ng/ml) 82.4

F (%) 62.6

Rat Dog

in vitro resistance generation

• RSV A strain Memphis 37b passaged in increasing concentrations of RV521 in HepG2 cells over 10 passages

• Single mutation D489Y identified in RSV F protein for RV521 resistant virus

• RV521 mutant shows cross resistance with GS-5806

• GS-5806 showed a larger EC50 shift than RV521 against all resistant variants (484x for GS5806, 76x for RV521)

• Similarly JNJ678

• The RV521 mutant remains sensitive to replication inhibitors (RSV604, ALS8176)

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Asp486Thr397

Asp487

Putative binding mode showing the orientation of the aminomethylene into a hydrophilic region characterized by the side chains of Aspartic acids 486 and 489 and Threonine 397 backbone carbony/, region and labels are shown in red

Thr397

Asp489

Asp486 Phe

488

Phe488

Ser398

Phe140

Phe488

Met396

Phe140

RV521 Synthesis

i). nBuLi, diisopropylamine, -70oC, then 1,2-dibromoethane,. ii). K2CO3, AcCN. iii). HCl,

DCM

RV521 pre-clinical/clinical evaluation

• Progressed through Pre-clinical GLP safety studies• No effect in Ames/Mutagenicity, Cardiac liability• Cyp 3A4 isoform responsible for metabolism• Broad Pharmacological profiling (5HT6 2.2mM)• Phase I in healthy adult volunteers evaluating safety and PK completed

• Target exposure of 3xEC90 achieved or exceeded with 200 mg and 350mg RV521

• Pre-clinical toxicology package complete, including juvenile studies.• Confirmed good safety profile for RV521

• Randomised, double blind phase 2a clinical trail in healthy adult volunteers inoculated with RSV

• Nasal inoculation with RSV A Memphis 37 – low passage clinical isolate

RSV Challenge - Study design

22

1 2 3 4 5 6 7 8 9 1011

12

RSV inoculation

Test 2x daily nasal wash for RSV • start dosing 12hr post positive• subjects negative for RSV on day 5

commence dosing (↑safety database)

First day dosing can commence

Final day dosing can commence

Last day of dosing

Discharge

Participants received RV521 (200mg or 350mg) or placebo twice daily for 5 days following confirmation of RSV infection or on the fifth day after inoculation if RSV was not detected

▪ Dosing started 12 hours following RSV positive nasal sample

▪ Viral load, disease severity, PK and safety were measured throughout the 12 day study quarantine period

Pre-defined efficacy endpoints▪ Primary : AUC RSV RT-qPCR viral load from twice daily nasal wash

▪ Secondary : viral load determined by cell-based infectivity assay from twice daily nasal wash, symptom scores (3x daily), and daily weight of nasal mucus produced.

11

0 1 2 3 4 5 6 70

1

2

3

Cell-Based Infectivity Assay Viral Load

Days since First dose

0 1 2 3 4 5 6 70

1

2

3

4

5

RT-qPCR Viral Load

Days since First dose

Me

an

Vir

al L

oa

d (

log

10

PF

Ue

/ml)

±S

E

RV521 Significantly Reduces RSV Viral Load

RV521 350mg RV521 200mg Placebo

Dosing Period

RV521 350mg RV521 200mg Placebo

Dosing Period

Me

an

Vir

al L

oa

d (

log

10

PF

U/m

l) ±

SE

23

Treatment group (ITT-I)

RV521 350mg (n=16) RV521 200mg (n=18) Placebo (n=19)

AUC RT-qPCR viral load (hours x log10 PFUe/ml) 185.26 ± 31.17 224.35 ± 37.60 501.39 ± 86.57

% Reduction relative to placebo; p value 63.05; p=0.002* 55.25; p=0.007*

AUC Plaque Assay viral load (hours x log10 PFU/ml) 38.29 ± 13.36 50.98 ± 14.89 162.35 ± 37.77

% Reduction relative to placebo; p value 76.42; p=0.012∞ 68.60; p=0.027∞

Plus-minus values are means ±SE of data from 1st dose to day 12. *Satterthwaite test. ∞ Wilcoxon rank-sum test.

0 1 2 3 4 5 6 7

0

2

4

6

8

1 0

D a ily N a s a l M u c u s W e ig h t

D a y s s in c e F irs t d o s e

Me

an

Na

sa

l M

uc

us

We

igh

t (g

)

SE

0 1 2 3 4 5 6 7

0

1

2

3

4

5

T o ta l S y m p to m S c o re

D a y s s in c e F irs t d o s e

Me

an

To

tal

sy

mp

tom

sc

ore

(

SE

)

RV521 350mg RV521 200mg Placebo

RV521 Significantly Reduces RSV Clinical Symptoms

RV521 350mg RV521 200mg Placebo

Dosing Period Dosing Period

24

Treatment group (ITT-I)

RV521 350mg (n=16) RV521 200mg (n=18) Placebo (n=19)

AUC total symptom score (hours x score) 82.41 ± 24.45 111.35 ± 33.88 381.82 ± 111.59

Reduction relative to placebo (%); p value∞ 78.42; p=0.002 70.84; p=0.009

Daily Nasal Mucus weight LS mean (g)& 0.27 0.33 0.61

Reduction relative to placebo (%); p value& 56.6; p=0.010 47.0; p=0.038

Plus-minus values are means ±SE of data from 1st dose to day12. ∞ Wilcoxon rank-sum test. fThe Least Square (LS) mean is calculated from a Mixed Model with Repeated Measures, adjusted for baseline mucus weight and treatment group as covariates, and subject as a random effect. The p value represents the LS mean difference between treatment groups.

Summary

• Potent inhibitor of RSV fusion process

• Good solubility, good pk in mice rats, dogs

• Good safety, exposure profile in phase 1 studies

• Therapeutic administration of RV521 in human challenge study significantly reduced RSV viral load and disease severity

• Only a single RSV F genetic variant detected following RV521 treatment which showed no evidence of clinical resistance or viral load rebound

25

Acknowledgements

• ReViral Ltd:• Ken Powell, Neil Mathews, Ian Fraser, Rachel Harland, Edward Littler, Elaine

Thomas, Alexandre Bedernjak, Khatareh Ahmadi

• ReViral Ltd Founders:• Richard Angell, James Lumley, Chris Pilkington, Keith Spencer, Joanne

Chapman, Spencer Campbell, Leena Sangar

• Contractors:• University of Queensland: Paul Young, Dan Watterson. Pirbright: Geraldine

Taylor. Ohio State: Mark Peeples. University of Surrey: Michael Paradowski, Simon Ward. Cyprotex, Onyx-Scientific, Pharmidex