BAR 501, a Novel GPBAR1 Ligand, Reverses Intestinal and Liver Inflammatory Models Demonstrating That GPBAR1 Is an Essential Modulator of Innate Immunity in Entero-Hepatic Tissues

Barbara Renga*, Sabrina Cipriani #, Adriana Carino * , Silvia Marchianò * , Angela Zampella † and Stefano Fiorucci *

*Dipartimento di Scienze Chirurgiche e Biomediche, Università degli Studi di Perugia, Nuova Facoltà di Medicina e Chirurgia , Sant’Andrea delle Fratte,Perugia, Italy

#Dipartimento di Medicina, Università degli Studi di Perugia ,Nuova Facoltà di Medicina e Chirurgia, Perugia, Italy †Dipartimento di Farmacia, Università di Napoli Federico II, Napoli, Italy

Background. GPBAR1, also known as TGR5, is a bile acid receptor expressed in various cell types including monocyte/macrophages and Kupffer cells (the resident macrophages in the liver). GPBAR1 activation leads to reduction of cytokine release by macrophages and its deletion results in enhanced intestinal inflammation. BAR501 is recently described selective GPBAR1 agonist that activates the receptor with an EC50 OF 3 Μm (J Med Chem. 2014 Oct 23;57:8477-95). Aim of the study. To investigate whether BAR501 protects against development of intestinal and liver inflammation in rodent models of immune dysfunction.Material and methods. Hepatitis was induced by intravenous injection of ConA and colitis was induced by intra-rectal injection of TNBS. At day 0, male mice GPBAR1 wild type and GPBAR-/- were treated with a single dose of intrarectal TNBS (0,5 mg/mouse in 100μl solution 50% water and 50% ethanol). BAR501 was administered daily, at doses of 15 and 30 mg/kg. Weights and colitis score were evalueted daily, at day 7 mice were sacrificed and colon collected for length measurement, evaluation of macroscopic and histological scores (H/E) and cytokine assay. For the ConA model, female wild type and GPBAR-/- mice were pre-treated for 6 days with BAR501 (15 mg/kg). At day 6 ConA was administered at dose 10 mg/kg. Animals were sacrificed after 4, 8 and 24 hours post ConA injection and blood and liver collected for plasma AST and ALT measurement, liver histopathology analysis (H/E) and for cytokines assay (rt-PCR).

Results. Hepatitis induced by ConA, was severely exacerbated in GPBAR1-/-. BAR501 rescued from death and reduced AST and ALT levels by ~90%, IL-6, and TNFα by 60-70% and ameliorated the necrotic/inflammatory score in wild type mice but not in GPBAR1-/- mice. Colitis induced by TNBS was severely exacerbated in GPBAR-/- mice. BAR501 reduced mortality, ameliorates colitis symptoms (intestinal colitis scores and histological score) and reduced TNFα mRNA in wild type but not in GPBAR-/- mice. Conclusions. BAR501 exerts potent anti-inflammatory activity and modulates innate immunity

OHHOH

OH

BAR501

GPBAR1

% survival

1 2 3 4 5 6 70

25

50

75

100Gpbar+/+CTRL

Gpbar+/+ TNBS

Gpbar+/+TNBS+ +501(15mg/kg)

Gpbar+/+ TNBS+501 (30mg/kg)

Gpbar-/- CTRLGpbar-/- TNBSGpbar -/- TNBS+501(15mg/kg)

days

% s

urvi

val

delta weights

0 1 2 3 4 5 6 7-30

-25

-20

-15

-10

-5

0

5

10

Gpbar+/+CTRLGpbar+/+ TNBS

Gpbar+/+ TNBS +501(15mg/kg)

Gpbar+/+ TNBS+501 (30mg/kg)

Gpbar-/- CTRL

Gpbar-/- TNBS

Gpbar-/- TNBS+501(15)

days

%

-1 0 1 2 3 4 5 60.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Gpbar+/+ CTRL

Gpbar+/+ TNBS

Gpbar+/+ TNBS+501(15mg/kg)

Gpbar+/+ TNBS+501 (30mg/kg)

Gpbar-/- CTRL

Gpbar-/- TNBS

Gpbar-/- TNBS+501(15mg/kg)

TNBS

BAR501

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* TNBS +/+ vs ctrl +/+

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§ TNBS+ 501 -/- vs ctrl-/-

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TNBS+ 501+/+ vs TNBS +/+

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§

Time (Days)

Col

itis

scor

e

macroscopic colon score(thickness+ulceration)

CTRL

TNBS

TNBS+50

1(15)

TNBS+ 50

1 (30

)CTRL

TNBS

TNBS+50

1(15)

0.0

2.5

5.0

7.5

** *

**

Gpbar+/+

Gpbar-/-

colon lenght

CTRLTNBS

TNBS+501

(15)

TNBS+501

(30)

CTRLTNBS

TNBS+501

(15)

0.0

2.5

5.0

7.5

10.0

** *

Gpbar+/+

Gpbar-/-cm

GPBAR+/+

Gpbar1+/+ naive Gpbar1+/+ Con A Gpbar1+/+ BAR501

Gpbar1-/- naive Gpbar1-/- Con A Gpbar1-/- BAR501

0

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% a

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necr

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GPBAR -/-

ConA ConA

BAR 501

IL-

6 m

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5000

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25000Gpbar1-/- ConA+501Gpbar1-/- ConA

Gpbar1+/+ ConAGpbar1+/+ ConA+501

Time (hr)

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sma

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ls (U

I/L)

*

0 4 8 12 16 20 240

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Gpbar1+/+ ConA+501 Gpbar1-/- ConA+501

Gpbar1-/- ConA

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ConA ConA

BAR 501

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GPBAR-/- CTRL GPBAR-/-TNBS + 501 (15mg/kg)GPBAR-/- TNBS

10x10x 10x

GPBAR+/+ CTRL GPBAR+/+ TNBS

10x 10x

GPBAR+/+ TNBS+501(15 mg/kg) GPBAR+/+ TNBS+501(30 mg/kg)

10x 10x

A B

C D

E F

GPBAR1 receptor structure and BAR501 molecular structure

Figure 1: Effects of BAR501 on mice treated with ConA. (A-B) Plasmatic levels of transaminases (AST and ALT), (C-D) relative hepatic mRNA expression of IL6 and TNFα assayed by rt-PCR in livers from mice after 8 hours ConA treatment. (E) Hematoxylin/ eosin staining of livers after 8 hours ConA treatment . (F) % of liver necrotic areas calculated with ImageJ software, (* p<0,05).

Figure 2: Effects of BAR501 on colitis induced in mice with TNBS. (A) % delta weights, (B) colitis score, (C) % survival , (D) colon length, (E) macroscopic colon score (thickness plus ulceration), (F) hematoxylin/ eosin staining of colon. ( #, § vs Gpbar -/- CTRL; * vs Gpbar+/+ CTRL; Φ vs Gpbar+/+ TNBS; p< 0,05).

A B C

D E F

D'Amore C, et al. Design, synthesis, and biological evaluation of potent dual agonists of nuclear and membrane bile acid receptors. J Med Chem. 2014 Feb 13;57(3):937-54.