Gli immunomodulanti: il meccanismo d’azione

Giovanni A. Rossi

U.O. di Pneumologia e Allergologia

I.R.C.C.S. G. Gaslini, Genova

giovannirossi@ospedale-gaslini.ge.it

Fondazione

Gerolamo

Gaslini

Istituto

Giannina

Gaslini

Biological Response Modifiers

• Prodotti dall’organismo: Lattoferrina

• Di derivazione vegetale: Echinacea, Propoli e

Resveratrolo

• Di derivazione batterica: Lisati batterici, Frazioni

antigeniche, Frazioni ribosomiali e Glicoproteine

• Di produzione sintetica: Levamisolo, Isoprinosina e

Pidotimod

Lattoferrina

• L'affinità dei suoi domini cationici (+) per la superficie cellulare batterica, che ha una carica

(-), determina la morte del microrganismo per

citolisi

• La sua attività antimicrobica è correlata alla affinità per il Fe3+: sottraendo Fe3+ presente nelle secrezioni mucose, impedisce la proliferazione di virus, batteri e miceti

• E’ una proteina presente nel latte, nel colostro, nelle lacrime e nella saliva ma anche nei granuli specifici dei neutrofili

Propoli

• E’ una sostanza resinosa che le api raccolgono dalle gemme e dalla corteccia delle piante ed elaborano aggiungendo pollini, cera ed enzimi prodotti dalle api stesse

• Contiene oltre 150 componenti tra le quali: a) Minerali: Mg, Ca, I, K, Na, Cu, Zn, Mn e Fe b) Vitamine: B1, B2, B6, C e E c) Enzimi: G6PD, fosfatasi acida d) Acidi: a. salicilico, a. caffeico, a. p-cumarico, a. grassi f) Alcoli: alcol benzilico, alcol cinnamilico. g) Flavonoidi e Terpeni

• La composizione della propoli è variabile a seconda della zona di origine, della stagione, dell’annata, etc …

Resveratrolo

• Rinvenuto nella buccia dell'acino d'uva, è una delle fitoalessine prodotte naturalmente da parecchie piante, in difesa da agenti patogeni quali batteri o funghi

• Nel corpo umano il resveratrolo viene eliminato in brevissimo tempo ma si ha una scarsa conoscenza degli effetti collaterali della somministrazione prolungata di tale sostanza …….

• Possiede proprietà: antiossidanti, antinfiammatorie e antiaggreganti, legate alla capacità di bloccare la produzione della cicloossigenasi–2 (COX-2)

Echinacea o “Cone flower”

• L‘Echinea è una pianta utilizzata anticamente dagli Indiani del Nord America per medicare molte malattie cutanee e le reazioni locali da morso di serpente

• La radice contiene numerose sostanze attive: a) poliacetileni e un glucoside (l’echinacoside), dotati di un marcato effetto antibiotico e fungicida b) oli essenziali ad azione immunostimolante

• Le indicazioni cliniche sono: a) uso topico nelle infiammazioni cutanee e nelle ferite torpide b) uso sistemico nella profilassi e il trattamento delle malattie da raffreddamento

Major immunostimulant categories commonly used in childhood recurrent respiratory infections

• Bacterial extracts

• Synthetic chemical compounds

Staphylococcus aureus, Streptococcus mitis, Streptococcus pyogenes, Streptococcus pneumoniae, Klebsiella pneumoniae, Moraxella catarrhalis, Haemophilus influenzae

Streptococcus pneumoniae, Streptococcus pyogenes, Klebsiella pneumoniae, Haemophilus influenzae

Oral bacterial extracts: mechanisms of action

Ribosomal extract

D53

Immucytal

RU41740 K. pneumoniae

Biostim

Glycoprotein

Increases Fc- receptor dependent phagocytosis Enhances the oxidative metabolism Inhibits of PMN migration

Increases complement- and Fc receptor-dependent phagocytosis Enhances the oxidative metabolism Does not affect neutrophil migration

Bacterial extracts LW50029

IRS-19

OM-85 BV Bronchovaxom

Lantigen B

Induces lymphocyte proliferation in MALT Increases IFN synthesis Enhances Th1 immune response Increases secretory IgA synthesis Activates macrophages and NK cells

Activity of the oral bacterial extracts

TLR

Bacterial extracts

Synthetic immunostimulants: mechanisms of action

An energy molecule, participating in O2 metabolism and protein synthesis

Stimulates macrophage activity Stimulates T- and B-cell proliferation and maturation

Isoprinosine A nucleoside

precursor to adenosine

Stimulates T-lymphocytes Restores the phagocitic activity of macrophages and PMNs

Levamisole A compound belonging

to imidazothiazole derivatives

Withdrawn from the market due to the risk of serious side effects

Synthetic dipeptide active on both the adaptive and the innate immune responses

Pidotimod 1,3-thiazolidine-4-

carboxylic acid

Biological response modifiers

Caravaggio. L'incredulità di San Tommaso

Immunostimulants for preventing respiratory tract infection in children

• Objectives. To determine the efficacy and safety of

immunostimulants in preventing acute respiratory tract

infections (ARTIs) in children

Del-Rio-Navarro BE, Espinosa-Rosales FJ, Flenady V, Sienra-Monge JJL The Cochrane Library 2008, Issue 4, 1-65

• Conclusions. This review showed that immunostimulants

reduce the incidence of ARTIs in children, by 40% on average

• The safety profile of immunostimulants appears to be good

• This positive result should be interpreted with caution due to

the heterogeneity and the poor quality of the trials

The synthetic route assures purity and reproducibility of production process

Pidotimod

The spectrum of the different batches looks always the same

Bacterial extracts

The same does not occur with bacterial extracts

N

H

C O

S

COOH

N

Pidotimod

Questions

• Is biodegradation occurring in

GE tract?

• Do we have convincing in vitro

and experimental animal studies

• Which new studies are needed?

N

H

C O

S

COOH

N

Pidotimod

The spectrum of the different batches looks

always the same

Mean plasma concentration–time curve of pidotimod in 20 male volunteers after a single

800 mg oral dose of pidotimod

Liquid chromatogram of plasma sample of a volunteer 0.5 h after oral administration of pidotimod 800 mg

Zhang Y. Journal of Chromatography 2009; 877: 2566–2570

3.0-8.0 mg/ml

HLA-DR

CD83

CD86

Human dendritic cell activation induced by exposure to Pidotimod (1 μg/ml)

Giagulli C. International Immunopharmacology 2009; 9: 1366–1373

A.P.C.

Human CD4 T-cell activation induced by exposure to Pidotimod (1 μg/ml)

Giagulli C. International Immunopharmacology 2009; 9: 1366–1373

T-cells

IFN-g

Intranasal immunization with Pidotimod increases IFN-g

production by splenocytes and bone marrow B-cells and OVA-specific IgG production

OVA 50μg/ dose

Pidotimod (100μg/ dose)

PBS

Days 0, 14 and 21

Giagulli C. International Immunopharmacology 2009; 9: 1366–1373

Splenocytes

IFN-g

Bone marrow cells

IFN-g

IgG

Critical role of the CD30 co-stimulatory molecule in the development of allergic asthma

B T

CD30 OVA

Polte T. JACI 2006;118:942-8

CD30

Pidotimod (10 μg/ml) decreases the in vitro the PHA-induced expression of CD30 in peripheral blood mononuclear cells of

atopic asthmatic and normal children

Gourgiotis D. J Asthma 2004; 41: 285-7.

Po

siti

ve c

ells

(%

)

12.5

10.0

7.5

5.0

2.5

0

HLA-DR CD-30

Control

Polimod 0.007

N.S.

A. HLA-DR+ & CD-30+ cells

CD

30

po

siti

ve c

ells

(%

)

12.5

10.0

7.5

5.0

2.5

0

Control Pidotimod 10 mg/ml

Normal

Atopic 0.012

0.02

B. CD-30+ cells

CD30

The enhanced IFN-g production induced by intranasal immunization with Pidotimod increases the cytotoxic

activity of splenocytes

OVA 50μg/ dose

Pidotimod (100μg/ dose)

PBS

Days 0, 14 and 21

Giagulli C. International Immunopharmacology 2009; 9: 1366–1373

Splenocytes

IFN-g

Cytotoxic activity of Splenocytes

Which kind of new information is needed ?

Intracellular pathways involved

Innate - Adaptive Immune System

Possible intracellular signaling pathways involved in pidotimod actions

Surface or Cytoplasmic Receptor

Nuclear Factor-kB activation

Cytokine/Chemokine production, Adhesion molecule expression, Reactive O2 species generation

Calcium (Ca2+) mobilization

Proliferation, Differentiation, Activation

& Reactive O2 species generation

MAPK extracellular signal-regulated kinase (ERK)-1/2 phosphorylation Cell proliferation,

differentiation & TJ protein disruption

TNF-a

ERK ½ - dependent T. J. disassembly

The first preliminary answer from HBECs ? IL-4, IFN-g, TNF-a

Pro-inflammatory cytokines disrupt airway epithelial tight junctions Occludine and ZO-1

Petecchia L. Laboratory Investigation 2012 (in press)

Cytokine-induced T. J. disruption is associated with reduction of airway epithelial barrier integrity

Petecchia L. Laboratory Investigation 2012 (in press)

Inhibition of ERK-1 phosphorilation prevents cytokine-induced T. J. disruption

Petecchia L. Laboratory Investigation 2012 (in press)

ZO-1

Occlud

Petecchia L. Laboratory Investigation 2012 (in press)

Inihibition of ERK-1 phosphorilation prevents cytokine-induced T. J. disruption

Pidotimod and “constitutive” ERK ½ phosphorilation in airway epithelial cells

P-ERK 1/2

Tot-ERK 1/2

42 kDa

42 kDa

P-E

RK

1/t

ota

l ER

K r

atio

1.5 -

-

1.0 -

-

0.5 -

-

0.0 -

Pidotimod and TNF-a-induced ERK ½ phosphorilation in airway epithelial cells

P-ERK 1/2

Tot-ERK 1/2

42 kDa

42 kDa

CTR 5’ 30’ 1h TNF-a 5’ 30’ 1h

Medium Pidot 100mg TNF-a Pidot 100mg +TNF-a

P-E

RK

1/t

ota

l ER

K r

atio

0.75 -

-

0.50 -

-

0.25 -

-

0.0 - CTR 5’ 30’ 1h TNF-a 5’ 30’ 1h Pidot 100mg Pidot 100mg+TNF-a

Grazie per l’attenzione e … non mancate il 21-23 Giugno!

Pidotimod and “constitutive” and TNF-a-induced ICAM-1 expression by airway epithelial cells

8000

6000

4000

2000

0

IC

AM

-1 e

xp

ressio

n (

MF

C)

CTR TNF-a 1 10 100

10 (ng/ml) Pidotimod (mg/ml)

IC

AM

-1 e

xp

ressio

n (

MF

C)

--- Pidotimod

TNF-a 10 (ng/ml)

8000

6000

4000

2000

0

1 (mg/ml

10 (mg/ml)

100 (mg/ml)

IFN-g

Pidotimod induces a time-dependent modulation of ERK1/2 phosphorylation

ERK ½ - dependent T.J. lesion

The first preliminary answer from HBECs ?

IFN-g Quantitative evaluation of the effects of Pidotimod on ERK1/2 phosphorylation

Pidotimod (400 mg/day x 60 days) or placebo in 101 children (4.7+2.1 years) with recurrent URTI

Burgio GR. Arzneim Forsch Drug-Res 1994; 44: 1525-29.

Pidotimod in the acute phase and in the follow-up of RTI severe enough to require antibiotic

treatment

Maintenance

period

• Pidotimod

400 mg o.d.

• Placebo

o.d.

60 days

Caramia G. Arzneim-Forsch/Drug Res.1994; 44: 1480-82

• Acute phase

of the URTI

• Pidotimod

400 mg b.i.d.

+ antibiotics

• Placebo b.i.d.

+ antibiotics

15 days

60 children

60 children

Evaluation

Evaluation

Pidotimod in the acute phase and in the follow-up of URTI severe enough to require antibiotic treatment

Caramia G. Arzneim-Forsch/Drug Res.1994; 44: 1480-82

Oral purified bacterial extract (OM-85 BV) in acute respiratory tract infections in childhood:

a systematic quantitative review

• Aim. To summarised the evidence on the effectiveness of the immunomodulator OM-85 BV in the prevention of ARTI in children

Steurer-Stey C. Eur J Pediatr 2007; 166: 365-76.

• RESULTS. 13 studies (2,721 patients) of low to moderate quality tested OM-85 BV, but patients and outcomes differed substantially, which impeded pooling results

• CONCLUSION. Evidence in favour of OM-85 BV in the prevention of

ARTI in children is weak

• There is a trend for fewer and shorter infections and a reduction of antibiotic use

Which kind of new information is needed ?

“Modern” clinical studies Intracellular pathways

involved

P-E

RK

1/t

ota

l ER

K r

atio

0.75 -

-

0.50 -

-

0.25 -

-

0.0 - CTR 5’ 30’ 1h TNF-a 5’ 30’ 1h Pidot 100mg Pidot 100mg+TNF-a

Pidotimod and TNF-a-induced ERK ½ phosphorilation in airway epithelial cells

P-ERK 1/2

Tot-ERK 1/2

42 kDa

42 kDa

CTR 5’ 30’ 1h TNF-a 5’ 30’ 1h

Medium Pidot 100mg TNF-a Pidot 100mg +TNF-a