Post on 22-Jan-2018
transcript
Human Babesiosis: The development of a new line of
drugs
Azan Virji
STARS Research Fellow 2015
PI: Professor Ben Mamoun Choukri
Mentor: Lauren Lawres
Department of Infectious Diseases, Yale School of Medicine
What is Babesiosis?
• Babesia microti
• Northeast & Northern
Midwestern States
• Spread by ticks
http://www.cdc.gov/parasites/images/babesiosis/map_babesiosis_by_county_2013.jpg
Babesiosis is commonly
misdiagnosed as malaria
https://classconnection.s3.amazonaws.com/915/flashcards/2974915/png/babesia1326269959514-14663CD9F46086C79A4.pnghttp://blogs.cdc.gov/global/2014/02/24/dpdx-15-years-of-strengthening-laboratory-capacity-for-parasitic-disease-
diagnosis/b_microti_vs_p_falciparum-2/
Babesia microti Plasmodium falciparum
Current therapies against Babesiosis
Vannier, E. et al. (2012, New England Journal of Medicine)
Recrudescence:
Resistance against current therapies
Treatment
Pereira, M. E. S. et al. (1996, Memórias do Instituto Oswaldo Cruz)
Addressing the problem
with current therapies
Current therapies are less effective than the new drug Endochin-like Quinolones
(ELQ)
Why are current therapies ineffective?
DNA Sequencing
In vivo study
SCID Mice10 mg/kg by oral gavage
Light Microscope counting
Day 45PCR- Cytochrome b gene
Yale Keck Sequencing Facility
Three of the four current therapies
are ineffective
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30
% P
aras
item
ia
Days post infection
Giemsa Counts
CONTROL
AZITHROMYCIN
QUININE
CLINDAMYCIN
Treatment
Atavaquone delays recrudescence
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30 35 40 45 50
% P
aras
item
ia
Days post infection
Giemsa Counts
CONTROL
ATAVAQUONE
Treatment
Endochin-like Quinolones also
delay recrudescence
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30 35 40 45 50
% P
aras
item
ia
Days post infection
Giemsa Counts
CONTROL
ELQ
Treatment
ATV + ELQ eliminates
recrudescence
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30 35 40 45 50
% P
aras
item
ia
Days post infection
Giemsa Counts
CONTROL
ELQ + ATV
Treatment
ATV + ELQ eliminates
recrudescence
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0 5 10 15 20 25 30 35 40 45 50
% P
aras
item
ia
Days post infection
Giemsa Counts
ELQ + ATV
Treatment
Why does a combination of
ATV and ELQ work better?
CL1
CL2
CL3
CL1
CL2
EL
QV
Sarewicz, M. et al. (2015, jhjournal)
Atavaquone
ELQ
GCT GTTAlanine Valine
Implications of this research
• Synergy
• Probability that mutation arises is lowered
10-7
ATV/ELQ
10-14
ATV+ELQ
• 2 mutations may lower fitness• Next steps: Dosing, Clinical trials
Acknowledgements
• Laboratory Of Infectious Diseaseso PI: Professor Ben Mamoun Choukri
o Mentor: Lauren Lawres
o Isaline and Pierre
o Yale School of Medicine
• STARS Summer Research Programo Dr. Moreno, Dr. Nelson and Dr. P And the TAs
o Howard Hughes Medical Institute (HHMI)
o Yale College
A%
Pa
ra
sit
em
ia
Co
ntr
ol
Qu
inin
e
Clin
dam
ycin
Azit
hro
mycin
Ato
vo
qu
on
e
En
do
ch
in
Ar t
esu
nate
0
2
4
6
8
*
*
*
B
FS
H
3.04%Control
YOYO Quinine
Endochin
Atovoquone
Artesunate Azithromycin
3.99
Clindamycin
3.40
4.5
1.02
1.3
2.8
3.01
Control
FS
C
YOYO-1Figure 1
Figure 2
% P
ara
sit
em
ia
- V 1 1 0 1 1 0 1 1 0
0
1
2
3
4
5
E L Q 2 7 1 E L Q 3 0 0 E L Q 3 1 6
V
4.66%
ELQ-1
1.50%F
SC
YOYO-1
FS
C
YOYO-1
ELQ-2
1.52%
ELQ-3
1.28%
FS
C
YOYO-1
FS
C
YOYO-1
A
BELQ 1 ELQ 2 ELQ 3
Figure 3 A B
C D
ELQ 1 ELQ 2
ELQ 3 ELQ 4
0 .0 0
0 .0 5
0 .1 0
2 .5
5 .0
2 5
5 0
7 5
E L Q 2 7 1 (1 0 m g /K g X 7 )
m o n ito r e d b y S m e a r
D a y s p o s t in fe c t io n
% P
ara
sit
em
ia
M o u s e # 1 6
M o u s e # 1 7
E L Q 2 7 1
C trl (F )
M o u s e # 1
M o u s e # 2
M o u s e # 3
T r e a t m e n t
1 5 8 1 2 1 5 1 9 2 3
C o n tro l 3
0 .0 0
0 .0 5
0 .1 0
2 .5
5 .0
2 5
5 0
7 5
E L Q 3 0 0 (1 0 m g /K g X 7 )
m o n ito r e d b y S m e a r
D a y s p o s t in fe c t io n
% P
ara
sit
em
ia
M o u s e # 1 6
M o u s e # 1 7
E L Q 3 0 0
C trl (F )
T r e a t m e n t
M o u s e # 4
M o u s e # 5
M o u s e # 6
1 5 8 1 2 1 5 1 9
C o n tro l 3
0 .0 0
0 .0 5
0 .1 0
2 .5
5 .0
2 5
5 0
7 5
E L Q 3 1 6 (1 0 m g /K g X 7 )
m o n ito r e d b y S m e a r
D a y s p o s t in fe c t io n
% P
ara
sit
em
ia
M o u s e # 1 6
M o u s e # 1 7
E L Q 3 1 6
C trl (F )
T r e a t m e n t
M o u s e # 7
M o u s e # 9
M o u s e # 8
1 5 8 1 2 1 5 1 9 2 3
C o n tro l 3
0 .0 0
0 .0 5
0 .1 0
2 .5
5 .0
2 5
5 0
7 5
E L Q 4 0 0 (1 0 m g /K g X 7 )
m o n ito r e d b y S m e a r
D a y s p o s t in fe c t io n
% P
ara
sit
em
ia
E L Q 4 0 0
C trl (M )
T re a tm e n t
M o u s e # 1 0
M o u s e # 1 1
M o u s e # 1 2
1
M o u s e # 1 7
M o u s e # 1 6
5 8 1 2 1 5 1 9
C o n tro l 3
Figure 4
A C
B
LabS1-S
+
V
LabS1-S
+
ELQ-1
LabS1-S
+
ELQ-3
CL1
CL2
CL3
CL1
CL2
CL3
CL1
CL2
EL
Q-1
EL
Q-3
V
V2
71
-R
Pa
ra
sit
em
ia (
%)
26
DP
I
0 .0
0 .5
2 0
3 0
4 0
5 0
6 0
1
10
Pa
ra
sit
em
ia (
%)
39
DP
I
0 .0 0
0 .0 5
0 .1 0
2 0
3 0
4 0
5 0
6 0
10
LabS1-S
+
V
LabS1-S
+
ELQ-1
LabS1-S
+
ELQ-3
D
19
Figure 5
Vannier, E. et al. (2012, New England Journal of Medicine)
What is currently unknown?
• Effective treatment: Are Endochin-like
Quinolones the solution?
• Pathophysiology: why does recrudescence
occur?
How to address the problem
of current therapies
• Prove that current therapies are ineffective and provide
evidence that new drug is significantly better than current
therapies
• Find out why current therapies do not work
• ELQs were considered potent due to previous studies
Methodology of infecting mice
• In vivo study
• SCID Mice/Rag2 knockout mice: Mice with weaker
immune system
• Mice from Dr. Ruslan Medzhitov’s Lab
• 107 parasites
• 10mg/ kg by oral gavage 4 to 11 days post infection (dpi)
• Control mice given vehicle with no drug
Methodology to test efficacy
• Blood collected from tail every 4 days
• Giesma Staining and Counting
• Flow cytometry to check counts
• DNA sequencing to identify mutations