Novel EP4 antagonists as a new therapyfor autoimmune diseases

0 = no signs of damage

1 = redness and swelling of less than two fingers or local part of foot pad

2 = redness and swelling of more than two fingers or less than two fingers and local part of foot 　 pad or whole foot pad3 = redness and swelling of more than two fingers and local part of foot pad or less than two 　　　 fingers and whole foot pad

4 = redness and swelling of more than two fingers and whole foot pad

0 = no signs of damage

1 = moderate inflammatory

2 = severe inflammation

Compound

Human EP4

binding KiHuman EP4

functional pA2*

Human IL-23production (IC50)

Human IL-6production (IC50)

Rat EP4

functional pA2*

Mouse EP4

functional pA2*Mouse IL-23production (IC50)

Selectivity

RQ-7

13 nM

8.3

3.1 nM

75 nM

8.2

8.1

16 nM

>200-fold**

RQ-8

<10 nM

～ 9.0

5.0 nM

-

8.3

9.0

<10 nM

>1000-fold**

Arthritis score was determined as follows;

Colitis score was determined macroscopically as followings;

2493471*Takako Okumura, Kiyoshi Kanazawa, Nobuyuki Takahashi, Ayano Sakakibara and Yukinori TakeRaQualia Pharma Inc., Research & Development, 5-2 Taketoyo, Aichi, 470-2341, Japan

Helper T cell (Th) subsets, Th1 and Th17 cells, play an important role in the development of autoimmunity in human diseases.

Recently, prostaglandin E2-EP4 receptor signaling was demonstrated to induce Th1 cell differentiation and Th17 cell expansion1).

PGE2-EP4 receptor signaling was also reported to induce IL-23 production in dendritic cells1). IL-23 promotes the expansion and survival of Th17 and has a potential of inhibiting Treg frequency2).

There are robust evidences that EP4 receptor predominantly mediates autoimmune diseases and allergy in animal models such as collagen antibody-induced arthritis and allergic contact dermatitis3,4).

In human, increase of IL-23 level has been associated with Crohn’s disease, RA, and psoriasis5, 6 , 7 ). IL-12/23 antibody is used for the treatment of psoriasis patients.

We investigated the EP4 pharmacology using RQ-00000007 (RQ-7) and RQ-00000008 (RQ-8), potent, and selective small molecule EP4 antagonists with different chemical class.

In vitro PharmacologyProstanoid Receptor Binding AssaysReceptor binding assays for the human EP1, EP2, EP3, EP4, DP, FP and IP receptors were performed using membrane preparations from HEK293 cells stably-expressed either human receptors. Radiolabeled ligands were used for each receptor binding assay. EP4 Cell Function AssaysCell function assays for the human and rat EP4 receptors were performed on intracellular cAMP accumulation in HEK293 cells stably-expressed either EP4 receptors. The cAMP level in the cell lysate was measured by SPA screening assay system (Amersham) or ELISA with the cAMP screen kit (Applied Biosystems).Cell function assay for the mouse EP4 receotor was performed on intracellular cAMP accumulation in HEK293 cells which transiently express mouse EP4 receptor. The cAMP level in the cell lysate was measured with HTRF cAMP kit.IL-23 Production in Human and Mouse Dendritic CellsMouse dendritic cells from the spleens of C57BL/6J mice were purified by magnetic activated cell sorting. These cells were cultured with 100 nM PGE2 and 10 μg/mL antibody to CD40 in the presence of RQ-7 or RQ-8 for 36 hrs. IL-23 levels in the supernatant collected at the end of culture were measured by ELISA (R&D Systems). Human dendritic cells from peripheral blood mononuclear cells (PBMCs) were incubated with 100 nM PGE1-OH, 10 ng/mL LPS, and 2.5 μg/mL R-848 stimulation in the presence of RQ-7 or RQ-8 for 24 hrs. The IL-23 level in the supernatant was measured by ELISA kit (R&D Systems).IL-17 Production in Mouse Th17 Cells by the Stimulation of IL-23Mouse splenic naïve T cells were incubated with 0.1, 1, and 10 ng/mL IL-23 under 1 ng/mL TGF-b, 50 ng/mL IL-6, and 5 μg/mL anti-IL-4 antibody for 96 hrs. The IL-17 level in the supernatant was measured by ELISA kit (R&D Systems).IL-6 Production in Human PBMCsHuman PBMCs were stimulated with 100 nM PGE2 and 5 µg/mL Concanavalin A in the presence of RQ-7 for 24 hrs. Then the amount of IL-6 in the supernatant was measured by ELISA kit (Cytoscreen).

In vivo PharmacologyDextran Sulfate Sodium (DSS)-Induced Colitis in MiceBALB/c female mice (8 week-old) were received 2.5% DSS ad libitum for 8 days. RQ-8 or vehicle (0.5% methylcellulose, MC) was orally administrated daily for 7 days. On Day8, the mice were sacrificed and each colon was harvested for evaluations. On Day8. Distal colon was isolated at a 2 cm segment (apart from 0.7 to 2.7 cm of the anus) and weighed.

Picryl Chloride (PCl)-Induced Contact Hypersensitivity in MiceBALB/c male mice (8 week-old) were sensitized by the application of 0.1 mL of 7% PCl to the abdomen on Day0. Seven days later, the mice were challenged by painting with 0.02 mL of 1% PCl both sides of the ear. Ear thickness was measured before and 24 hrs after the challenge. RQ-8 or vehicle (0.5% MC) was administered orally either on sensitization (S) and elicitation (E)-phase (Day0 to 8) or E-phase (Day7 to 8). Prednisolone was dosed on E-phase (Day7).Adjuvant-Incued Arthritis in RatsLewis male rats, (6 week-old) except for the normal control group, were given a subcutaneous injection of 600 μg of M. tuberculosis H37 RA suspended in 100 μL liquid paraffin into the right hind footpad on Day0. RQ-7 or RQ-8 or vehicle (0.1% MC) was administered orally twice daily from Day0 to Day21 (for 22 days; prophylactic treatment) or from Day12 to Day22 (for 11 days; therapeutic treatment). Rats in the normal and disease control group were given vehicle (0.1% MC). Hind paw volume was measured using hydroplethysmometer on Day21 or 22. At the day of final drug administration, rats were sacrificed and amputated hind paws were fixed, decalcified, embedded, sectioned, and stained with haematoxylin and eosin(H&E) for histopathological evaluation.Collagen Antibody-Induced Arthritis in MiceMouse monoclonal anti-type II collagen 5 clone antibody cocktail kit (Chondrex) was used for the development of arthritis. DBA/1J male mice (8 week-old) were given a intraperitoneal injection of 1.5 mg of anti-collagen antibody on Day1, and 50 µg of LPS (E.Coli 0111:B4) on Day4. Arthritic score of each paw was intermittently evaluated during the studies.

In vitro, RQ-7 and RQ-8 suppressed PGE2-induced IL-23 production in human and mouse dendrit ic cells, and PGE2-induced IL-6 production in human PBMCs.

In vivo, RQ-7 and RQ-8 suppressed disease on DSS-induced colitis in mice, PCl-induced contact hypersensitivity in mice, adjuvant-incued arthritis in rats and collagen antibody-induced arthritis in mice.

RQ-7 and RQ-8, potent and selective small molecule EP4 antagonists with different chemical class, suppress IL-23 production and demonstrate inhibitory activity in variety of autoimmune disease models.

There results suggest that a selective EP4 receptor antagonist offers an attractive therapeutic approach for autoimmune diseases through the inhibition of IL-23 production and suppression of Th1 and Th17.

1. Yao C, Sakata D, Esaki Y, et al. (2009). Nat Med 15: 633-640.2. Izcue A, Hue S, Buonocore S, et al. (2008). Immunity 28: 559-570. 3. McCoy JM, Wicks JR, Audoly LP. (2002). J Clin Invest 110: 651-658.4. Kabashima K, Sakata D, Nagamachi M, et al. (2003). Nat Med 9: 744-749.5. Schmidt C, Giese T, et al. (2005). Inflammatory Bowel Diseases 11(1): 16-23. 6. Kim HR, et al. (2007). Rheumatology 46(1): 57-64. 7. Lee E, Trepicchio WL, Oestreicher JL. (2004). J Exp Med 199: 125-130.

RQ-7 achieved proof of concept in osteoarthritis (OA) pain in two Phase 2a studies.RQ-8 is essentially ready for FIH, completed safety pharmacology, DMPK, and toxicology studies.* Inhibition of PGE2-induced cAMP elevation** Selectivity against over 100 receptors (include other prostanoid receptors), ion channels, transporters and enzymes

Human dendritic cells (>90% positive for CD11c and CD86, Lonza #CC-2701) were incubated with 100 nM PGE1-OH, 10 ng/mL LPS, and 2.5 μg/mL R-848 stimulation in the presence of RQ-7 or RQ-8 for 24 hrs. Data represent mean + SD (N = 2-3).

Background

Materials and Methods

ResultsTable 1

Figure 1

Profile of EP4 antagonists; RQ-7 and RQ-8

Effects of RQ-7 and RQ-8 on IL-23 Production in Human Dendritic Cells

Mouse splenic dendritic cells (>90% positive for CD11c) were incubated with 100 nM PGE2 and 10 μg/mL anti-CD40 stimulation in the presence of RQ-7 or RQ-8 for 36 hrs. Data represent mean + SD (N = 3).

Figure 2 Effects of RQ-7 and RQ-8 on IL-23 Production in Mouse Dendritic Cells

IL-23 (ng/mL)

A) Prophylactic Treatment, Paw Swelling on Day21

B) Therapeutic Treatment, Paw Swelling on Day22

C) Low-Magnification Photomicrographs of Representative Tarsal Joint Sections from Rats with Adjuvant-Induced 　 Arthritis

Rats were given a intrapiantar injection of 600 μg of M. tuberculosis H37 RA suspended in 100 μL liquid paraffin into the right hind footpad on Day0. RQ-7 or RQ-8 or vehicle (0.1% MC) was administered orally twice daily from Day0 to Day21 (for 22 days; prophylactic treatment) or from Day12 to Day22 (for 11 days; therapeutic treatment). Rats in the normal and disease control group were given vehicle (0.1% MC).

A, B) Paw Swelling at the Contralateral Hind Paw Hind paw volume was measured intermittently during test period. Represented data are expressed the paw volume of contralateral paw on Day22 or Day21 by prophylactic treatment (A), therapeutic treatment (B). Data represent mean + SEM (N = 8). **: p<0.01 versus disease control by One-way ANOVA, Dunnett post test###: p<0.001 versus disease control by t-test.

C) Photomicrographs of Representative Tarsal Joint Sections Photomicrographs of representative tarsal joint sections from contralateral foot stained with H&E (x 20).

Figure 7 Effects of RQ-7 and RQ-8 on Adjuvant-Induced Arthritis in Rats

Mouse splenic naïve T cells (>90% positive for CD4 and CD62L) were incubated with IL-23 under 1 ng/mL TGF-b, 50 ng/mL IL-6, and 5 μg/mL anti-IL-4 antibody under for 96 hrs. Data represent mean + SD (N = 3).

Figure 3 IL-17 Production in Mouse Th17 Cells Stimulated with IL-23

0 0.1 1 10

1000

2000

3000

4000

5000

0

PBMCs were incubated with 100 nM PGE2 and 5 µg/mL Concanavalin A stimulation in the presence of RQ-7 for 24 hrs. Data represent mean + SD (N = 3).

Figure 4 Effect of RQ-7 on IL-6 Production in Human PBMC Stimulated with PGE2

3000

2500

2000

1500

1000

500

0

Mice were given a intraperitoneal injection of mouse anti-collagen antibody cocktail on Day1, and LPS on Day4. RQ-7 was subcutaneously injected using osmotic pump (ALZET, #2001) at a injection dose of 400 µg/hr from Day0 to Day21. RQ-8 (3, 30 mg/kg) was orally administered, twice dailyfrom Day1 to Day21. Prednisolone (1 mg/kg) was orally dosed once a day from Day1 to Day21. Data represent mean +/- SEM (N = 4-8). ##: p<0.01 versus disease control group by t-test, *: p<0.05, **: p<0.01 versus disease control group by One-way ANOVA, Bonferroni test.

Figure 8 Effects of RQ-7 and RQ-8 on Collagen Antibody-Induced Arthritis in Mice

Mice were received 2.5% DSS ad libitum for 8 days. RQ-8 (3, 30 mg/kg) was administrated daily for 7 days. On Day8, the mice were sacrificed and each colon was harvested for evaluations. Colitis score was determined macroscopically. Distal colon was isolated at a 2 cm segment and weighed. *: p<0.05 versus disease control group by Mann-Whitney test (N = 6-10).

Figure 5 Effect of RQ-8 on DSS-Induced Colitis in Mice

DiseaseControl

Ear S

wel

ling

(mm

)

Figure 9 PGE2-EP4 Signaling and Immune Activation

Mice were sensitized by the application of 7% PCl to the abdomen on Day0. Then challenged by painting of 1% PCl both sides of the ear. Ear thickness was measured before and 24 hrs after the challenge, and the difference of ear thickness was used as on Day 7 a parameter of ear swelling. RQ-8 (3, 30 mg/kg) or vehicle (0.5% MC) was administered orally either on sansitization (S) and elicitation (E)-plase (Day0 to 8) or E-phase (Day7 to 8). Prednisolone (10mg/kg) was dosed on E-phase (Day7). Data represent mean + SEM (N = 10). **: p<0.01 versus disease control group by Dunnett's multiple comparison test following one-way ANOVA. ##: p<0.01 versus disease control group by unpaired t-test.

Figure 6 Effect of RQ-8 on PCl-Induced Contact Hypersensitivity in Mice

3 30

Prednisolone(mg/kg, PO, QD)

S+E phase E phase

**

****

**##

3 30 10

RQ-8(mg/kg, PO, BID)

0.00

0.05

0.10

0.15

0.20

0.25

Normal Control Disease Control Prophylactic TreatmentRQ-8 30 mg/kg, PO, BID

Therapeutic TreatmentRQ-8 30 mg/kg, PO, BID

Autoimmune Diseases

T reg

1)Th1 differentiation1)

2)Th17 expansion

1) Th17 expansion2) Inhibit Treg differentiation4)

Enhance IL-23 production

Dendritic cell

EP4

IL-6 IL-10

Conclusion

References

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Time after Antibody Injection (day)

Art

hriti

s Sc

ore

0

2

4

6

8

10

12

14

Time after Antibody Injection (day)

0 5 10 15 20 25 0 5 10 15 20 250

2

4

6

8

10

12

14

* * * *

** **

#### ##

## ## ## ## ##

Vehicle (0.9% NaCl, pH8.5, SC infusion) RQ-7 (400 µg/µl/hr, SC infusion)

Vehicle (0.5% MC, PO)RQ-8 (3 mg/kg, PO, BID) RQ-8 (30 mg/kg, PO, BID) Prednisolone (1 mg/kg, PO, QD)

Clinical candidates, RQ-7 and RQ-8, are potent, safe, and selective EP4 antagonists with different chemical class.

IL-1

7 Pr

oduc

tion

(pg/

mL)

IL-6

Pro

duct

ion

(pg/

mL)

RQ-7 (nM)

0

100

200

300

400

W/Ostim

Wstim

Wstim

Wstim

1000

RQ-7 (nM)

0

200

400

600

800

RQ-8 (nM)

IC50 = 3.1 nM

IL-2

3 Pr

oduc

tion

(pg/

mL)

IC50 = 5.0 nM

1 10 100 0.1 W/Ostim

1 10 100

ED70 = 0.9 mg/kg, PO, BID

****

** ** **###

1.6

1.8

2.0

2.2

2.4

2.6

2.8

Con

tral

ater

al P

aw V

olum

e (m

L)

Art

hriti

s Sc

ore

0.3 1 3 10 30DiseaseControl

NormalControl

RQ-8(mg/kg, PO, BID)

0 0.1 1 10 100 1000 W/Ostim

NormalControl

Colitis Score (Median)

0(Normal)

1(Moderate)

2(Severe)

Coliti

s Sc

ore

Distal Colon Weight/Length(Mean + SD)

20

25

30

Col

on W

eigh

t/Len

gth

(mg/

cm)

DiseaseControl

RQ-8(mg/kg, PO, QD)

30

*

DSS

3NormalControl

DiseaseControl

RQ-8(mg/kg, PO, QD)

3 30

*

DSS

** **

###

**

DiseaseControl 29 57 96 Normal

ControlRQ-7

(mg/kg, PO, BID)

1.61.82.02.22.42.62.83.03.2

Con

tral

ater

al P

aw V

olum

e (m

L)

ED70 = 57 mg/kg, PO, BID

**

** ** ** ** ###

1.6

1.8

2.0

2.2

2.4

2.6

2.8

0.3 1 3 10 30DiseaseControl

NormalControl

RQ-8(mg/kg, PO, BID)

Con

tral

ater

al P

aw V

olum

e (m

L)

ED70 = 0.3 mg/kg, PO, BID

EP4EP4

IL-17IFN-γ

T h1 T h17

PGE2

0

100

200

300

RQ-8 (nM)

1000 W/Ostim

RQ-7 (nM)

10 100 1000 10 100

IC50 = 16 nM IC50 = <10 nM

IL-2

3 Pr

oduc

tion

(pg/

mL)

IL-23