1. No category

Helicobacter pylori–binding microspheres
to prevent gastric cancer
Inês C. Gonçalves
icastro@ineb.up.pt
16th April 2015
“Encontros com a Inovação em Saúde”
Helicobacter pylori infection
World population
7 billion people
50%
Wroblewski et al. Clin Microbiol Rev 2010
Infected with
H. pylori
3,5 billion people
1%
Wroblewski et al. Clin Microbiol Rev 2010; Peek et al J. Pathol. 2006
Develop
gastric cancer
Have recomendation
for
H. pylori treatment
35 million people
20%
symptomatic
Malfertheiner et al. Lancet 2011
700 million people
20%
H. pylori treatment is
inneficient
Vakil et al. Am J Gastroenterol 2006
140 million people
The technology
Chitosan microspheres that, after oral administration, will adhere to
gastric mucosa, bind H. pylori and eliminate it through the
gastrointestinal tract.
Patent WO2013164652-A2
The biomaterial
Chitosan
NH3+
NH3+
Mucoadhesive properties
• Natural polymer
• Source: Squid pen (Chitin)
Other sources:
Mean size (m)
Chitosan microspheres
pH7.4
Diameter of 170 µm
450
400
350
300
250
200
150
100
50
0
*
*
289
167
172
175
pH 7.4
pH 6.0
pH 4.0
pH6.0
pH4.0
pH 2.6
pH2.6
345
pH 1.2
pH1.2
Stable in gastric acidic pH
I.C. Gonçalves*, et al. Acta Biomaterialia 9 (2013) 9370–9378
Chitosan microspheres
0,2mg Ch Mic
MKN45 gastric cells
Gastric retention time of 2h
Not cytotoxic
I.C. Gonçalves, et al. Acta Biomaterialia 9 (2013) 9370–9378
M. Fernandes*, I.C. Gonçalves*, et al. Int. J. of Pharmaceutics 454 (2013) 116– 124
Chitosan microspheres adhesion to H. pylori
J99
BabA+/SabA+
17875/Leb
BabA+/SabA-
17875 babA1A2
BabA-/SabA+
097UK
BabA-/SabA-
pH 6.0
pH 2.6
I.C. Gonçalves, et al. Acta Biomaterialia 9 (2013) 9370–9378
Helicobacter pylori adhesion
to gastric mucosa
The adhesion of H. pylori to the gastric mucosa involves
specific interaction between bacteria adhesins and
glycosylated receptors of gastric mucins and gastric
epithelial cells.
BabA
SabA
(blood group antigen
binding adhesin)
(sialic acid binding
adhesin)
Adapted from Magalhães A et al, 2010
binds to inflammationassociated sialyl-Lewisa and
silayl-Lewis X structures
[Mahdavi et al, 2002]
binds to H-type 1 and
Lewis b structures
expressed in normal
gastric mucosa
[Ilver et al, 1998]
GlyR-Ch Mic
Ch
“Click Chemistry”
Ch_
Leb
sLex
WO2013164652-A2 (PCT/GB2013/051181). MICROSPHERES. M. Cristina L. Martins et al.
H. pylori adhesion to GlyR Ch Mic
Ch
Ch_
Leb
sLex
+
+
+
+
BabA+/SabA- H. pylori
BabA+/SabA- H. pylori
BabA+/SabA- H. pylori
BabA+/SabA- H. pylori
WO2013164652-A2 (PCT/GB2013/051181). MICROSPHERES. M. Cristina L. Martins et al.
In vitro gastric mucosa model
Sections from paraffin
embedded stomachs
160
140
120
***
***
100
80
46%
60
60%
40
20
0
Mic Ch
Mic Ch_
Mic Leb
Mic sLex
180
[% in relation to the control]
[% in relation to the control]
17875/Leb adhesion to
mice gastric mucosa
180
17875/Leb adhesion to human
gastric mucosa sections
200
200
***
160
140
120
100
43%
80
60
40
20
0
Mic Ch
Mic Ch_ Mic Leb
Mic sLex
Welch - ANOVA: Tamhane’s T2 post hoc test (***p<0.001; **p<0.01)
Ex vivo and in vivo mouse
gastric mucosa model
Ex vivo
In vivo
fresh stomachs
0,2 mg Ch Mic 1x/day for 15 days
*
*
0,4 mg Ch Mic 1x/day for 15 days
63% reduction of H. pylori infection
One-way ANOVA with LSD post hoc (p*<0.05)
Conclusions
Ch microspheres are stable in acidic pH (do not dissolve), are
non-cytotoxic and are retained in mice stomachs for 2h.
Ch microspheres are able to bind different H. pylori strains
(unspecifically).
Glycan-modified microspheres bind H. pylori specifically
through carbohydrate-adhesin binding and remove H. pylori
adhesion, competing with carbohydrates from the gastric
mucosa.
In this panorama, chitosan microspheres should be considered
as alternative therapy for H. pylori infection treatment.
Advantages
•
No problems associated
resistance, side effects,…);
•
Huge market as H. pylori infection treatment:
• All infected population (3.5 billion people)
• Patients who have recommendation for treatment (700
million)
• Patients that do not respond to antibiotherapy (140 million);
•
Chitosan microspheres may be used as unspecific treatment,
while glycan coated microspheres may provide a personalized
treatment for specific bacterial strains.
•
Could also be used as preventive treatment or in combination
with other treatments, namely as local drug delivery system;
with
antibiotics
use
(bacterial
Acknowledgements
Cristina Martins
Celso Reis
Ana Margarida Costa
Ana Patrícia Henriques
Inês Vigário Rodrigues
Mariana Fernandes
Zé Ricardo Oliveira
Bernardo Antunes
Vanessa Graça
Catarina Seabra
Frederico Nogueira
Paula Parreira
Ana Magalhães
Joana Gomes
Leonor David
Patrícia Castro
Mário Barbosa
Isabel Amaral
Stefania Nardecchia
Maria Oliveira
Maria Lazaro
João Cortez
Paula Gomes
Rui Fernandes
Thomas Bóren
Paula Sampaio
Sofia Lamas
Fátima Carneiro
José Manuel Lopes
António Gouveia
Eliette Touati
Paulo Costa
FCT: “Glycobacter”, “Pyloricidal” and
“SweetMic” projects and Post-Doc grant
ON2 for project Norte-07-0124-Feder-00005