Invitro Activity of

PPIs on mouse macrophage

RAW264.7 cell line

Lansoprazole ,Omeprazol,Esmoprazol

In Vivo Activity of

Omeprazol on carrageenan-induced rat paw edema

LPS and H.pylori induced TNF-αand IL-1βproductionco-incubated with human neutrophils and human umbilical cord endothelial cells

inhibit the H. pylori extract induced expression of CD11b and CD18 on neutrophils and

neutrophil-dependent adhesion to endothelial

Anti-oxidant at and anti-inflammatory

efficacy at an intraperitoneal dose of

2.5 mg/kg

INTRODUCTION PPIs omeprazole,

lansoprazole, pantoprazole, esomeprazole, rabeprazole were purchased Swapnroop

chemicals, Aurangabad, India. Lambda carrageenan (22049 Sigma) and Thiazolyl Blue Tetrazolium Blue – MTT (M 2128 Sigma), Diclofenac sodium was generously supplied by Lord Venky Pharma Private Limited, Yanam, India. ELISA kits for determination of mouse TNF-α, IL-6 and IL-1β were purchased from eBioscience (San Diego, USA). Macrophage cell line, RAW 264.7 (ATCC TIB-71), was obtained from the American Type Culture Collection, Manassas, VA, USA.

MATERIAL EXPERIMENTAL PROTOCOL

Invitro Technique Invivo Technique

Experimental Animl group

Adult spargue Dawley Male

Rat ,11 treatment groups containing 6 rats each. The

acute in vivo antiphlogistic

activity of PPIs was tested at 15, 30

and 60 mg/kg oral doses.Orally

administered diclofenac sodium (5 mg/kg) served

as a standard drug.

Nitric Oxide

Measurmement

Carrageenan Induced paw inflammation

and Mechanical

Hyper AlgesiaRESULT

10 20 50 100 200 300 4000102030405060708090

100110

Effect of different PPIs on % survival of RAW 264.7 cells

OPZ LPZ PPZ RBZ EMZ

Conc of drug in uM

% S

urvi

val

Name of drug Omeprazole Lansoprazole Pantoprazole Rabeprazole Esomeprazole

IC 50 341.3 uM 149 uM 498 uM 146 uM 420 uM

% survival at 50 uM

100 80 82 85 99

% Survival at 400 uM

39 14 40 15 51

PPIs inhibit the LPS-induced cytokine release from RAW-264.7 cells

% survival at 50 uM

100 80 82 85 99

% Survival at 400 uM

39 14 40 15 51

 PPIs inhibit carrageenan-induced acute paw inflammation in rats

1 hr 4 hr 1 hr 4 hr 1 hr 4 hr 1 hr 4 hr0

5

10

15

20

25

30

35

40

45OPZ LPZ PPZ EPZ RPZ

15 mg/kg 30 mg/kg 60 mg/kg

Normal Carrageenan Standard

@

aa

b bb

b bb

c

bb

c

c cb

***

***

***

**

***

** c ***

***

******

***

***

***

***

**

@

% R

ise

in p

aw

vo

lum

e

 Figure 1: PPIs inhibit carrageenan induced acute paw inflammation in rats

15 mg/kg 30 mg/kg 60 mg.kg05

10152025303540455055

Normal Carrageenan Standard OPZ LPZ PPZ EPZ RPZ

@

a a a

a aa a a a

b b c

b b bc

Treatment

Pa

w w

ith

dra

wa

l th

resh

old

(g

)

1 hr 4 hr 1 hr 4 hr 1 hr 4 hr 1 hr 4 hr0

5

10

15

20

25

30

35

40

45OPZ LPZ PPZ EPZ RPZ

15 mg/kg 30 mg/kg 60 mg/kg

Normal Carrageenan Standard

@

aa

b bb

b bb

c

bb

c

c cb

***

***

***

**

***

** c ***

***

******

******

***

***

**

@

% R

ise

in p

aw

vo

lum

e

Effects of PPIs on the viability (% Survival) of RAW264.7 murine macrophages. Cells were incubated with various concentrations of PPIsfor 24 hr, and cell viability was determined by MTT assay.The results are expressed as means±SD. P<0.0001.

0

20

40

60

80

100

120

Treatment

Tnf-

leve

l ( p

g/m

g of

pro

tein

)

Effect of PPIs on TNF-α level in Paw tissue supernatant of carrageenan injected animal.

0

5000

10000

15000

20000

25000

a* a*a*b*

a*a* a*b*

Treatment

IL-6

leve

l (pg

/mg

of p

rote

in)

Effect of PPIs on IL-6 level in Paw tissue supernatant of carrageenan injected animal.

0

5000

10000

15000

20000

25000

a*a*

a*b*a*

a* a*

Treatment

IL-1

leve

l (pg

/mg

of p

rote

in)

  Effect of PPIs on IL-1β level in Paw tissue supernatant of carrageenan injected animal

DISCUSSION AND CONCLUSION Both In-vivo and In-vitro anti-inflammatory activity of Proton Pump Inhibitor was investigated. There were

three major findings in our study. First, PPIs reduced the rise in paw volume, by dose dependent manner and also reduced the pain in inflamed condition. Second, the suppression of NO production at the initial stage of

inflammation and third, in RAW 264.7 cell line PPIs exert anti-inflammatory effects through inhibition of the production of pro-inflammatory cytokine TNF-α, IL-1β and IL-6 from LPS stimulated macrophages

In conclusion, the overall results of this study clearly indicate that the Proton pump inhibitor had higher anti-inflammatory and cytokine inhibitory action at increasing dose. Although the exact mechanism(s) of Proton pump inhibitors on suppressing the cytokine were not investigated in the present study, this could constitute an area of

future studies

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In-vitro and In-vivo anti-inflammatory activity of proton pump inhibitors

Department of Pharmacology,(contact no.-9158830005, email adress-sanketgandhi111@gmail.com)R. C. Patel Institute of Pharmaceutical Education &

Research, Shirpur- 425405, Maharashtra, IndiaSanket Gandhi, Jayesh Patil, Umesh B. Mahajan, Sameer N. Goyal, Chandragouda R. Patil*

Effect of PPIs on viability of RAW 264.7 cell

By MTT

Assay

Effect of PPIs on Cytokinin release from

RAW 264.7 cell

cytokines levels TNF-a, IL1 and

IL6 levels by ELISA