www.postersession.com

Results

Effects of TOP1210, a narrow spectrum kinase inhibitor, and selective

kinase inhibitors on the intestinal pro-inflammatory immune response in

ulcerative colitis

Martyn R Foster‡, Paolo Biancheri†, Matthew CT Fyfe, Thomas T MacDonald †, Adele Rowley‡, Sameer Sirohi, Yemisi Solanke, Steve

Webber‡, Eleanor Wood♯ and Claire A Walshe.‡

‡Topivert Pharma Ltd., London, UK; †Centre for Immunobiology, Barts and the London School of Medicine and Dentistry, London, UK; ♯Academic Department of Medical and

Surgical Gastroenterology, Homerton University Hospital, London, UK.

Figure 3. TOP1210 inhibitory effects on pro-inflammatory cytokine release from UC biopsies.

Introductio

n

Geometric Mean IC50 value (nM)

p38α Src Syk

TOP1210 65 10 17

BIRB-796 13 >1895 >1895

Dasatinib 378 6 >2049

BAY-61-3606 >2562 >2562 136

Geometric Mean IC50 value (nM)

Innate response: Monocytes Adaptive response: T

cells Epithelium

PBMCs Primary macrophages PBMCs PBMCs HT29 cells

IL-8

release IL-8 release

TNF-

release

IL-2

release

IFN-

release

IL-8

release

TOP1210 1.9 2.2 3.3 37 2.1 1.8

BIRB-796 >1895 >250 >474 >1895 >1895 >1895

Dasatinib >2049 >250 52 2.1 4.0 >2049

BAY-61-

3606 607 >250 >640 291 247 >2561

Table 2 : Effect of TOP1210 and selective kinase inhibitors on a

range of innate, adaptive and epithelial cellular response assays.

Table 1: Inhibitory effects of selective kinase inhibitors and the

NSKI TOP1210 on kinase activity in a biochemical Z-lyte based

assay.

Table 3. Inhibitory effects of the

NSKI TOP1210 and the selective

kinase inhibitors on pro-

inflammatory cytokine release by

Ulcerative Colitis myofibroblasts.

Figure 1. Exemplar data from cellular assays. TOP1210 inhibits

IL-8 release by LPS stimulated PBMCs (Figure 1A) and

macrophages (Figure 1B), and also LPS stimulated TNFα release

by macrophages (Figure 1C), with greater potency and efficacy

than any of the selective kinases tested.

• TOP1210 potently inhibits all kinases tested, whilst BIRB-

796, dasatinib and BAY-61-3606 show selective profiles.

IL-6

(IC50 ng ml-1)

IL-8

(IC50 ng ml-1)

TOP1210 2.2 2.1

BIRB-796 >1000 >1000

Dasatinib >1000 >1000

BAY-61-

3606

315 630

• Intracellular kinase activation plays a key role in inflammation and kinase inhibitors have been proposed as potential therapies in chronic inflammatory disorders such as ulcerative colitis.

• Selective kinase inhibitors, however, have proved disappointing, particularly in the treatment of rheumatoid arthritis and inflammatory bowel disease (IBD).

• Multi-kinase inhibition has been investigated as a strategy to improve efficacy.

• The activity of a narrow spectrum kinase inhibitor (NSKI), TOP1210, has been compared to selective kinase inhibitors (BIRB-796, dasatinib and BAY-61-3606) in a range of innate and adaptive inflammatory cell assays and in inflamed biopsies from ulcerative colitis (UC) patients.

Methods • Inhibitory effects on recombinant p38, Src and Syk kinases was

assessed in an ATP dependent ZYLTE™ based assay.

• Cellular assays were performed after 2hr preincubation with compound or vehicle. Peripheral blood monocytes (PBMCs) were stimulated with either lipopolysaccharide (LPS) or anti-CD3/CD28, monocyte derived macrophages with LPS, HT29 epithelial cells with IL1β and myofibroblasts isolated from inflamed UC mucosa with TNFα.

• Biopsies from inflamed ulcerative colitis patients were incubated with compound (24hr) and spontaneous cytokine release measured.

• A range of inflammatory cytokines in cellular and biopsy supernatants were measured by ELISA. Data is expressed as means ±s.e.m.of at least 3 determinations.

Conclusions

• Multi-kinase inhibition with NSKIs like TOP1210 leads to a efficacious and broad inhibitory profile in UC tissues and across a range of cell types including epithelial cells, innate and adaptive immune cells. Thus, NSKIs provide significant advantages over existing selective kinase approaches, and potentially offer much improved therapeutic benefit in IBD.

Results Introduction

Figure 2. Effect of TOP1210,

BIRB796 (BIRB), dasatinib

(DAS), or BAY-61-3606 (BAY)

on the release of IL-6 and IL-8

by UC myofibroblasts

• TOP1210 is more potent than selective kinase inhibitors in

reducing cytokine release from TNFα-stimulated UC

mucosal myofibroblasts.

IL-1 IL-6 IL-8

0 1 0.1 0.01 0.001

TOP1210 (g/ml)

0 1 0.1 0.01 0.001

TOP1210 (g/ml)

0 1 0.1 0.01 0.001

TOP1210 (g/ml)

*** *** ** ***

***

***

***

% i

nh

ibit

ion

vs D

MS

O

% i

nh

ibit

ion

vs D

MS

O

% i

nh

ibit

ion

vs D

MS

O

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

• TOP1210 is a potent and efficacious inhibitor in down-

regulating pro-inflammatory cytokine release from Ulcerative

Colitis biopsies.

• TOP1210 is a potent and efficacious inhibitor of innate and

adaptive immune responses. Generally, compared to

TOP1210, the selective kinase inhibitors have weak potency

and efficacy