Transcript MARAVIROC

Potenziale ruolo dell’antagonista
del CCR5, Maraviroc, nel
trattamento di patologie
non-AIDS correlate.
Daniela Francisci
Clinica di Malattie Infettive
Università di Perugia
BACKGROUND
• HIV infection appears to increase risk of cardiovascular
disease.
• Chronic inflammation may be one mechanism underlying
this increased risk.
• Atherosclerosis is actually recognized to be a chronic
INFLAMMATORY DISEASE, where an initially protective
response continues to excess and become detrimental
“ response-to-injury” (Ross R, NEJM 1999)
• Endothelial dysfunction increases vascular permeability,
allowing circulating monocytes and T cells to migrate
into the sub-endothelial space
The continued presence of immune cells togheter with migration and proliferation of
vascular smooth muscle cells serves to perpetuate the immune response and promote
atherosclerotic plaque formation.
CCR5 and Atherosclerosis
• The critical role of inflammation and immune cells
makes it unsurprising that many chemokines and
chemokine-receptors have been linked to
atherosclerosis
• Chemokines are proinflammatory cytokines that recruit
leukocytes from the blood into tissues, playing a
pivotal role in human inflammatory disease
• Chemokines exert their biological functions through
binding to transmembrane G-protein coupled receptors
Two of these,CCR2 and CCR5 bind MCP-1 and MIP1(α-β)
and RANTES
MIP 1 α
CCL3/macrophage
inflammatory protein- 1 α
MIP1 β
CCL4/macrophage
inflammatory protein- 1
β
RANTES
Regulated on Activation,
Normal T cell Expressed
and Secreted
CCR5 is expressed on
monocytes/macrophages
T cells, endothelial and
vascular smooth muscle
cells.
CCR5 signaling is involved in trafficking of T cells to inflamed tissues and may
increase T cell activation directly
CCR5 is particularly noteworthy given the availability of an
approved antagonist : MARAVIROC
CCR5 Delta 32
• CCR5Δ allele, which produces a prematurely
truncated form of CCR5,was recognized and found to
lead to near complete resistance to HIV-1 infection in
the homozygous state, and slower progression to
AIDS in heterozygotes
(Dean et al., 1996; Huang et al., 1996; Zimmerman et al.1997)
CCR5Δ32
• The CCR5delta32 allele has been linked with:
-
Reduced susceptibility to coronary artery disease
(Szalai et al, 2001, Afzal et al,2008)
- Reduced early onset of coronary heart disease in women
(Pai et al, 2006)
- Protection against early episode of myocardial infarction
(Gonzalez et al, 2001; Balistreri et al 2008, Mountinghe 2008)
- Lower risk for cardiovascular disease and mortality in
patients with chronic renal failure and reumatoid arthris
(Rodrìguez-Rodrìguez et al, 2011)
- Higher plasma high density lipoprotein (HDL) cholesterol
and lower plasma triglycerides
(Hyde et al,2010)
OUR PURPOSE
• By using a murine model of genetic dyslipidemia (ApoE -/- mice)
we investigated whether Maraviroc :
- counteracted the early, ritonavir-induced
progression to atherosclerosis in young mice
and
- interfered with spontaneous plaque
progression resulting from underlying
dyslipidemia in old mice
Model 1 EARLY ATHEROSCLEROSIS ( ApoE-/- 8 weeks old mice)
( treatment for 13 weeks).
Groups: ApoE-/- mice plus vehicle (n=8-10); ApoE-/- mice plus RTV
(5mg/kg ip),(n=8-10); ApoE-/- mice plus RTV (5mg/kg ip) plus MVR (50
mg/kg per os daily), (n=8-10).
Model 2 LATE ATHEROSCLEROSIS (ApoE-/- 39 weeks old mice)
( treatment for 13 weeks)
Groups: Apo E-/- + vehicle( n=13); Apo E-/- + MVR 50 mg/kg per os daily
(n=13).
We have previously shown that Ritonavir induces
inflammation in adipose tissue, increasing the levels of
proinflammatory cytokines and accelerates the
extension of atherosclerotic aortic plaques and CD36
over expression on circulating monocytes.
(Circulation. 2013;127:2114-2124.)
MVC attenuates atherosclerotic plaque progression in RTV-exposed ApoE−/− mice
Naive
RTV
RTV + MVC
*
*
lesion area
(pixel)
100000
40X
75000
50000
25000
0
Ritonavir
Naive
Alone
Maraviroc
10x
10x
10x
% area Mac3 staining on
plaques
ApoE -/75
*
*
50
25
0
Ritonavir
Naive
Alone
Maraviroc
ApoE-/-
10x
Model 1
10x
10x
*
1000
500
C
7500
5000
2500
Alone
Naive
Alone
Maraviroc
Ritonavir
Alone
Naive
ApoE
75
RANTES
(pg/ 500 g of protein)
*
20
10
*
50
25
-/-
30
IL-6
(pg/ 500 g of protein)
*
F
Maraviroc
Ritonavir
Ritonavir
E
MCP-1
(pg/ 500 g of protein)
50
ApoE -/-
D
20
10
0
0
Alone
Naive
0
Maraviroc
Alone
Ritonavir
ApoE
Naive
-/-
H
Ritonavir
*
5.0
2.5
Alone
Naive
30
20
10
ApoE -/-
Alone
Maraviroc
Ritonavir
Maraviroc
Ritonavir
ApoE -/-
0
0.0
Naive
40
INF
(pg/ 500 g of protein)
*
Alone
Maraviroc
ApoE -/-
7.5
IL-17A
(pg/ 500 g of protein)
100
Maraviroc
Naive
ApoE -/-
30
*
0
0
0
G
150
TNF
(pg/ 500 g of protein)
ICAM
(pg/ 500 g of protein)
*
*
B
*
10000
VCAM
(pg/ 500 g of protein)
A
1500
Naive
Maraviroc
Ritonavir
ApoE -/-
Maraviroc
counterregulation of
RTV-induced plaque
inflammation
52- week- old ApoE
mice naive
52- week -old ApoE
mice + maraviroc
MODEL 2
% lesion area (plaques)
Sudan IV staining
*
40
30
20
10
0
Naive
Maraviroc
ApoE-/-
% area Mac3 staining on
plaques
MAC-3 staining
75
50
*
25
0
CCR5 staining
Naive
Maraviroc
ApoE-/-
Maraviroc inhibits spontaneous
atherosclerosis progression
*
500
30000
20000
*
10000
-/-
ApoE
G
IL-17A
(pg/ 500 g of protein)
5000
200
TGF
(pg/ 500 g of protein)
RANTES
(pg/ 500 g of protein)
250
150
100
50
*
0
Naive
Maraviroc
4000
3000
2000
1000
*
5.0
2.5
0.0
Maraviroc
ApoE-/-
10
*
0
Naive Maraviroc
ApoE -/-
-/-
Naive Maraviroc
ApoE-/-
I
2
1
10
5
0
Naive
Maraviroc
-/-
Adhesion molecules and inflammatory
cytokines in atherosclerotic aortic plaques
7.5
5.0
*
2.5
0.0
Naive
10
15
ApoE
M
7.5
20
ApoE-/-
ApoE -/-
10.0
3
Naive Maraviroc
-/-
% of CD11b/CCR-5 cells
% of CD11b/CD36 cells
L
ApoE
0
0
Naive Maraviroc
ApoE
H
20
0
INF
(pg/ 500 g of protein)
ApoE-/-
F
50
E
Naive Maraviroc
Naive Maraviroc
Naive Maraviroc
100
0
0
0
D 30
150
IL-2
(pg/500 g of protein)
VCAM
(pg/ 500 g of protein)
ICAM
(pg/ 500 g of protein)
1000
C
MCP-1
(pg/ 500 g of protein)
B40000
1500
TNF
(pg/ 500 g of protein)
A
Naive
Maraviroc
ApoE-/-
MCV decreases blood monocyte expression
of CD36 and CCR5
We have shown that :
In a mouse model of genetic dyslipidemia, MVC
reduced the atherosclerotic progression by
interfering with inflammatory cell recruitment into
plaques.
In mice characterized by a general RTV-induced
inflammation, MVC reversed the pro-inflammatory
profile.
Properly designed clinical studies are
required to evaluate whether present
observations extend to clinical settings..
CCR5 and CANCERS
Inappropriate or prolonged expression of
chemokines and/or chemochine receptors results in
an excessive infiltration of leukocytes into inflammed
tissues :
- chronic inflammation
- autoimmune diseases
- tumor growth and tumor dissemination
Adenocarcinoma gastrico
• 4° neoplasia in ordine di frequenza
• 2° causa di morte neoplasia-correlata
• OS a 5 anni: 20-30% nei pazienti sottoposti a trattamento
chirurgico
Recidiva
dopo
intervento
Carcinosi
peritoneale
Ridotta
Chemiosensibilità
Sopravvivenza
media: 6-9 mesi
Sasako M, et al Lancet Oncol 2010
Incidenza in Umbria
L’incidenza in Umbria,
nell’area dell’alta valle
del Tevere, è di 60-70
casi ogni 100.000
abitanti
Sovrapponibile
all’incidenza in
Giappone
CCR5 and Gastric cancers
Previous studies have shown that :
• RANTES and CCR5 are highly expressed in gastric
cancers with lymph nodes matastatis
• RANTES and its receptor CCR5 were associated
with the metastatic potential of this tumor.
• Expression levels of RANTES in lymph nodes with
cancer invasion were substantially increased
(Lim HK, et al 2003; Cao Z et al,2011; Graziosi L, et al. 2013, Fukuda K et al.,2009)
CCR5/RANTES and Gastric cancer
Role of RANTES and Its Receptor in Gastric Cancer Metastasis
CAO Z., et al. J Huazhong Univ Sci Technol, 2011
CCR5 and Gastric cancer
Prognostic Significance of Expression of CCL5/RANTES
Receptors in Patients With Gastric Cancer
Survival curve in patients with positive expression of
CCR5 (solid line) or negative expression. (dashed line)
H. Sugasawa, Journal of Surgical Oncology, 2008
The potential relevance of MVC to gastric cancer therapy is
unknown.
We have designed a study to investigate the effect of this agent
in a rodent model of gastric cancer dissemination
Risultati in vitro (1)
Espressione di CCR5 nelle linee cellulari di cancro gastrico
Tutte e 3 le linee cellulari di cr
gastrico sono positive per CCR5,
con livelli maggiori nella forma
indifferenziata
L’espressione di CCR5 è
maggiore a livello
intracellulare
Risultati in vitro (2)
Espressione di CCR5 nelle cellule di cancro gastrico dopo
impianto nel cavo peritoneale
La percentuale di cellule positive per
CCR5 incrementa del 100%
Interazione con il microambiente
tumorale
Risultati in vitro (3)
Effetti dell’inibizione con Maraviroc di CCR5 sulla
proliferazione cellulare
Il trattamento con Maraviroc per 3 giorni riduce la proliferazione cellulare, con
effetti maggiori sulla linea meno differenziata
Risultati in vitro (4)
Effetti degli agonisti e antagonisti di CCR5 nella adesione delle
cellule neoplastiche
Gli agonisti di CCR5
incentivano l’adesione,
mentre la loro attività è
ridotta nel trattamento
combinato con Maraviroc
Risultati in vivo (1)
Effetti dell’azione di agonisti e antagonisti del CCR5 nel
modello di carcinosi peritoneale
Carcinosi
peritoneale:
Il trattamento con
Maraviroc per 7
giorni ha ridotto la
diffusione
peritoneale, con
riduzione del
numero e delle
dimensioni dei
noduli
Aumento della sopravvivenza dopo 7
giorni di trattamento con Maraviroc
Risultati in vivo (2)
Effetti del trattamento con Maraviroc nello xenograft
sottocutaneo
I topi trattati con
Maraviroc hanno
presentato una
significativa riduzione
del tasso di crescita
tumorale
Conclusioni:
 CCR5 è coinvolto nella proliferazione e nell’adesione
neoplastica.
 L’inibizione di CCR5 inibisce la crescita e la diffusione delle
cellule tumorali in modelli preclinici
Possibile
TARGET THERAPY per il trattamento e la prevenzione
della Carcinosi Peritoneale di origine Gastrica
GRAZIE PER L’ATTENZIONE
Acknowledgment
•
Dipartimento di Medicina Sperimentale e Scienze Biochimiche
Baldelli Franco
Francisci Daniela
Schiaroli Elisabetta
•
Dipartimento di Medicina Clinica e Sperimentale
Fiorucci Stefano
Renga Barbara
Cipriani Sabrina
Mencarelli Andrea
D’ Amore Claudio
•
Dipartimento Scienze Chirurgiche, Radiologiche e Odontostomatologiche
Donini Annibale
Graziosi Luigina
B
200
*
*
*
*
n° adherent monocytes
(cells/optical field)
n° adherent monocytes
(cells/optical field)
A
100
0
*
*
400
300
200
100
0
Naive
Ritonavir 500nM
Ritonavir 1 M
Maraviroc
Maraviroc
Naive
TNF
TNF
Maraviroc
+
MAraviroc
Maraviroc inhibits U937 (mature macrophages) cell adhesion to
RTV or TNF-α- activated endhotelial cells
Materiali e metodi (2)
 Valutazione in vitro:
3. Adesione al peritoneo parietale murino dopo trattamento delle
cellule con agonisti e antagonisti
Cellule coltivate con MIP1α, MIP-1β, RANTES ±
MARAVIROC
Sospensione cellulare
in peritoneo parietale
su piastra
Spettrofotometria a
fluorescenza
Materiali e Metodi (1)
 valutazione in vitro:
1. Espressione cellulare di membrana ed intracellulare di CCR5
su linee cellulari di cancro gastrico
-MKN74
-MKN45
-KATO III
2.
Citometria a flusso
Proliferazione cellulare dopo stimolazione con agonisti (MIP-1 α, MIP-1
β, RANTES) e antagonisti recettoriali ( MARAVIROC)
Cellule in coltura con :
- MIP-1 α, MIP-1 β, Rantes
- MARAVIROC
- medium di controllo
Conta cellulare
- EXPERIMENTAL PERITONEAL CARCINOMATOSIS
- XENOGRAPH MODEL
Gastric cancer human cell lines were injected
intraperitoneally or subcutaneously ,respectively,
in NOD-SCID mice.
Mice were randomized in two groups :
- Controls
- MVC treated at the dose of 50 mg/kg twice time day
Materiali e metodi (3)
 valutazione in vivo degli effetti del trattamento farmacologico
dopo induzione del tumore gastrico in 2 modelli murini
1. Carcinosi peritoneale:
Sospensione
Cellulare:
2. Xenograft sottocutaneo
Linea cellulare MKN74
MKN45
MKN45
Inoculo in cavità peritoneale
(topi NOD/SCID)
Veicolo inerte
Maraviroc
Volume noduli peritoneali e
mesenterici
Innesto sottocute
(topi NOD/SCID)
Veicolo inerte
Maraviroc
Volume noduli e %
inibizione maraviroc
A
B
surface expression
*
intracellular expression
*
*
100
CCR5 expression
(% of positive cells)
CCR5 expression
(% of positive cells)
20
10
*
75
50
25
D
MKN74
III
KA
TO
KN
CCR-5 Intracellular
KATO III
MKN45
G
H
MKN74 surface expression
MKN45 surface expression
20
15
10
0
pre-impantation
post-implantation
KATO III surface expression
20
CCR5 expression
(% of positive cells)
CCR5 expression
(% of positive cells)
CCR5 expression
(% of positive cells)
M
E
CCR-5 Surface
Autofluorescence
F
-4
4
-7
KN
M
C
5
III
0
KA
TO
5
-4
KN
M
M
KN
-7
4
0
10
5
0
10
0
pre-impantation
post-implantation
pre-impantation
post-implantation
Proliferation assay
A
B
MKN45
Kato III
MKN74
0
0
-10
-20
*
% of inhibition
(vs control)
0
% of inhibition
(vs control)
% of inhibition
(vs control)
C
-10
-20
*
-30
ctrl
maraviroc
-30
-10
-20
-30
-40
*
-50
ctrl
maraviroc
ctrl
maraviroc
Adhesion MNK-74
Adhesion assay
adhesion MNK45
% vs control group
50
25
0
50
25
-25
0
-50
Ctrl MIP-1
Ctrl MIP-1
MIP-1 Rantes alone MIP-1
MIP-1 Rantes alone MIP-1
MIP-1 Rantes
MIP-1 Rantes
Maraviroc
Maraviroc
Adhesion Kako III
C
*
*
50
% vs control group
% vs Control group
75
25
*
0
-25
-50
Ctrl MIP-1
MIP-1
Rantes alone MIP-1
MIP-1
Maraviroc
Rantes
SURVIVAL
Mesenteric nodules
Peritoneal
nodules
20
*
*
Number
Number
30
20
E
10
100
0
Ctrl
0
Maraviroc
Ctrl
Maraviroc
Percent survival
10
Maraviroc
(10 mg/kg,i.p.)
50
Control
Total volume
*
1000
0
750
500
250
-2.5
-5.0
-7.5
0
Ctrl
Maraviroc
0
1
2
3
4
5
6
Weeks
0.0
% vs day 0
3
Size (mm )
Weight
-10.0
Ctrl
Maraviroc
7
8
9
10
XENOGRAPH
MKN45 volume
750
0
vehicle
Maraviroc
start of
treatment
500
3
(mm )
volume of nodule
Day 30
B
250
0
0
10
20
% of inhibition
(vs control)
A
-20
-40
-60
30
Ctrl
Maraviroc
days
C
D
MKN74 volume
vehicle
Maraviroc
start of
treatment
900
600
300
% of inhibition
(vs control)
0
1200
volume of nodule
3
(mm )
Day 30
-20
-40
0
0
10
20
days
30
-60
Ctrl
Maraviroc
The histo-pathology analysis of xenograph nodules demonstrates that
the administration of MVC resulted in extensive intra-tumoral necrosis
B
*
20
% of CD11b/CCR5 cells
*
10
10.0
*
7.5
5.0
C
2.5
0.0
0
Alone
Naive
Maraviroc
Alone
Naive
Ritonavir
Maraviroc
75000000
Ritonavir
ApoE -/-
ApoE -/-
CRP
(pg/ml)
% of CD11b/CD36 cells
A
50000000
25000000
D
E
*
*
mRNA relative expression
Epidydimal fat ratio
0
IL-6
1.00
0.75
0.50
0.25
Alone
*
50
*
Naive
ApoE -/-
40
30
20
10
0
0.00
Alone
Naive
Ritonavir
ApoE -/-
Alone
Maraviroc
Naive
Maraviroc
Ritonavir
ApoE
-/-
Figure 4
Maraviroc
Ritonavir
A
B
150
750
600
INF
(pg/ml)
IL-17
(pg/ml)
125
100
75
450
300
50
150
25
0
0
naive ConA naive ConA
naive ConA naive ConA
Ctrl.
Ctrl.
Maraviroc
ApoE-/-
ApoE-/-
C
D
30
20
300
TGF
(pg/ml)
TNF
(pg/ml)
Maraviroc
10
200
100
0
naive ConA naive ConA
Ctrl.
Maraviroc
-/-
ApoE
0
naive ConA naive ConA
Ctrl.
Maraviroc
ApoE-/-
Supplemenntary Figure 1
IL10RB
mRNA relative
expression
1.5
Met
NME1
FAT1
1.5
2
1.5
LTB
1.5
*
1.0
*
1.0
1.0
1.0
*
*
0.5
0.5
0.0
0.5
0.0
NT
MARA
*
1
0.5
0
0.0
NT
MARA
*
NT
MARA
0.0
NT
MARA
NT
MARA
Figure 8
ApoE-/A
Ritonavir
B
Ritonavir
+ Maraviroc
C
*
*
100000
D
lesion area
(pixel)
Naive
75000
50000
25000
0
40X
Ritonavir
Naive
Alone
Maraviroc
ApoE -/-
10x
I
F
G
10x
10x
L
M
H
% area Mac3 staining on
plaques
E
75
*
*
50
25
0
Ritonavir
Naive
Alone
ApoE-/-
10x
10x
10x
Maraviroc
B
200
*
*
*
*
n° adherent monocytes
(cells/optical field)
n° adherent monocytes
(cells/optical field)
A
100
0
*
*
400
300
200
100
0
Naive
Ritonavir 500nM
Ritonavir 1 M
Maraviroc
Maraviroc
Naive
TNF
TNF
Maraviroc
+
MAraviroc
Maraviroc inhibits U937 cell adhesion to RTV or TNF-α- activated
endhotelial cells
The potential relevance of MVC to gastric cancer therapy
is unknown.
We have designed a study to investigate the effect of this
agent in a rodent model of gastric cancer dissemination
A
B
60
150
50
*
INF
(pg/ml)
IL-17A
(pg/ml)
40
100
30
20
50
#
10
#
0
0
naive ConA naive ConA naive ConA
Ctrl.
Alone
naive ConA naive ConA naive ConA
Ctrl.
Maraviroc
Alone
Ritonavir
ApoE
ApoE
C
Maraviroc
Ritonavir
-/-
-/-
D
40
#
3000
TGF
(pg/ml)
TNF
(pg/ml)
30
20
2000
1000
10
#
0
0
naive ConA naive ConA naive ConA
Ctrl.
Alone
Maraviroc
naive ConA naive ConA naive ConA
Ctrl.
Alone
Ritonavir
ApoE -/-
Maraviroc
Ritonavir
ApoE -/-
Figure 5
PURPOSE
• To verify in a early atherogenetic model the
extension of the RTV–induced pro-atherogenic
and pro-inflammatory profile and the efficacy of
MVC in reducing atherosclerotic plaque
evolution and local and systemic inflammation.
(ApoE-/- 8 weeks old mice )
• To investigate wheter MVC attenuated plaque
development in a late phase of spontaneous
atherogenesis when dyslipidemia plays a major
pathogenic role
(ApoE-/- 39 weeks old mice)
CONCLUSIONS
Properly designed clinical studies are
required to evaluate whether present
observations can be extended to
clinical settings…