Transcript Antifungal drugs

A novel approach for non-invasive diagnostic
imaging of invasive fungal infections
Fungal Infections
Invasive fungal infections (IFI) are a serious threat to human health
mostly due to high proliferation rate of fungal pathogens.
These infections are especially dangerous in case of immunocompromised
Individuals, whose number is growing due to:
General aging of the population.
Common use of immunosuppressive agents in patients who received
transplants, implants, intensive anticancer therapies, or suffer autoimmune
diseases, like rheumatoid arthritis or multiple sclerosis.
Spread of immunocompromising diseases (e.g. diabetes, AIDS)
www.gaffi.org
www.life-worldwide.org
The size of the fungal problem
Over 300 million people affected by
serious Fungal Infection worldwide
25 million - life-threatening and/or
hugely debilitating
www.fungalinfectiontrust.org
Fungal Infections cases
Skin infections
Respiratory infections
Eye infections
Lung infections
Annual estimated mortality from serious fungal infection worldwide
The treat of fungal infections is not yet widely recognized, even
though these infections are associated with high mortality rates.
Over 1,350,000 people are estimated to die annually
Fungal infection
Cryptococcal meningitis
Pneumocystis pneumonia
Case fatality rate
15-20% USA
>50% developing world
~15% in AIDS
~50% non-AIDS
Estimated
deaths
Comments
600,000
CDC estimate
>80,000
Most cases in Africa not
diagnosed and 100% mortality
Many missed diagnoses
globally
Invasive aspergillosis
~50% mortality in
developed world
>100,000
Candida bloodstream
infection
~40% mortality
>120,000
~15% mortality in
developed world
>450,000
Under-diagnosed and mistaken
for tuberculosis
<1% ?
~100,000 asthma
deaths - ~50%
related to SAFS
Uncertain
>1,350,000
Significant underestimate
Chronic pulmonary
aspergillosis
Severe asthma with fungal
sensitization (SAFS)
Total
Acute invasive fungal infections (IFIs) due to Candida
species have a high associated mortality
The most clinically significant fungal species are Candida
and Aspergillus
Candida spp. Crude Mortality
Wisplinghoff et al. 2004 CID 39(3):309-17
Challenges in IFI diagnostics
The diagnosis of IFI is complex since it requires a combination of
clinical observation, laboratory investigation, and radiological
or other diagnostic imaging methods
The clinical representation is often non-specific and can be caused
by a wide range of infectious organisms, underlying illness or
complications of treatment
Uncertainties and controversies in disease definition and in selecting
of the standardized methods for establishing the diagnosis
Existing diagnostic approaches for IFI
Available Tests
Limitations
Culture Test
Must be used in combination
with histopathologic or
cytopathologic examination
Antigen Detection
(commonly used for express
diagnosis)
PCR Assay
High incidence of falsepositive and false negative
results
Lack of standardization
X-ray Imaging,
Inherently non-specific.
Computed Tomography (CT) Must be used in combination
with clinical and
microbiological factors
X-ray image of fungal lung infection
This chest x-ray shows the affects of a
fungal infection, coccidioidomycosis.
In the middle of the left lung (seen on
the right side of the picture) there are
multiple, thin-walled cavities (seen as
light areas) with a diameter of 2 to 4
centimeters. To the side of these light
areas are patchy light areas with
irregular and poorly defined borders.
Other diseases that may explain these
x-ray findings include lung abscesses,
chronic pulmonary tuberculosis,
chronic pulmonary histoplasmosis,
and others
Missed diagnosis often leads to death, serious
chronic illness or blindness in, case of eye infections
Early diagnosis saves lives
Relationship between hospital mortality and the timing of
antifungal treatment.
Morell et al. AAC. 2005 49: 3640
Our goal
To save more lives by
Development of fungal-specific probes,
that could be used to reliably and promptly
image pathogens in human body
Our approach
Diagnostic probe
fluorophore
fluorophore
drug
drug
Incubation
Fungal cell
drug
drug
Detection
Target
Fungal
cell
Our approach takes advantage of highly specific and sensitive antifungal drugs
as targeting molecules
By coupling a fluorophore that can be visualized, the new probes would be able
to detect the presence of fungal pathogens
Antifungal drugs
Antifungal drugs work by exploiting the differences between mammalian
and fungal cells.
Unlike bacteria both fungi and humans are eukaryotes, and therefore
are more similar at the biological level. This makes it more difficult
to discover drugs that target fungi without affecting human cells.
As a consequence, many antifungal drugs cost severe side effects
Therefore, for development of the diagnostic probes the ideal would be
the drugs that do not have any interaction with host cells and tissues
and target exclusively fungal cells.
Also these drugs should have broad specificity targeting many
fungal species
Antifungal drugs chosen for diagnostic probes design
H2N
C H3
HO
C H3
O
Caspofungin
OH
O
OH
N H2
N
H
H
N
H3 C
O
NH
N
O
N
NH
HO
N
H
N
NH
OH
O
CH3
O
O HO
OH
OH
Binds with high affinity and specificity to its fungal target glucan synthase
O
Me
O
H
N
N
N
OH
N
F
Me
O
F
N
N
N
Posaconazole
N
Potent and selective inhibitor of fungal cytochrome p 450 lanosterol
14 demethylase
Both drugs have broad spectrum activity and are fungi-specific since they
target the enzymes absent in mammalian cells
The synthesis of fluorescently labeled caspofungin
Antifungal drugs should have suitable groups for the attachment of the label
H2N
C H3
HO
C H3
Caspofungin
O
OH
N H2
N
H
H
N
H3 C
O
NH
OH
O
N
O
N
NH
HO
NH
H
N
N
OH
O
CH3
N
C H3
O HO
BODIPY-Fl-NHS
H3 C
H3 C
O
F
B
N
F
O
O
OH
N H2
N
H
H
N
H3 C
O
NH
HO
C H3
Caspofungin-BODIPY
OH
NH
O
OH
OH
N
O
N
NH
HO
N
H
N
NH
OH
O
CH3
O
O HO
OH
OH
Modified caspofungin probe retains high
antifungal activity with C. albicans
Labeling should not affect targeting properties of the drugs.
H2N
C H3
HO
C H3
Caspofungin
O
OH
N H2
N
H
H
N
H3 C
O
NH
O
MIC 50 nM
N
OH
O
N
NH
HO
NH
H
N
N
OH
O
CH3
H3 C
O
O HO
OH
OH
N
H3 C
C H3
F
B
N
NH
O
F
HO
C H3
O
Caspofungin-BODIPY
O
OH
N H2
N
H
H
N
H3 C
O
NH
OH
N
O
N
NH
HO
N
H
N
NH
OH
MIC 80 nM
O
CH3
O
O HO
OH
OH
The synthesis of fluorescently labeled posaconazole
O
Me
H
N
O
N
N
N
OH
N
F
Me
O
N
F
Posaconazole(SCH65592)
N
Succinic anhydride
Me-Im, DMSO
N
O
Me
H
N
O
N
N
O
N
N
F
O
C
O
C
OH
Me
O
N
N
F
4-nitrophenol
DCC, THF
N
O
Me
H
N
O
N
N
N
O
N
F
O
O
C
C
O
Me
NO2
O
N
N
F
1,4-diaminobutane
N
O
Me
H
N
O
N
N
Fluorescent tag
O
C
O
N
F
O
N
C
NH –(CH2)4-NH2
Me
O
N
F
N
C H3
BODIPY-FL-NHS
N
O
Me
O
H
N
N
N
N
O
N
F
Me
N
C
O
F
C
NH –(CH2)4-NH
O
O
F
N
O
N
N
Pos-Fl
N
B
F
C H3
Imaging of C. albicans using fluorescent
caspofungin probe
Fluorescent
Light transmission
The fluorescence intensity was consistent with the existing data about
the distribution pattern of glucan synthase throughout the cell
features, which was indicative for the specificity of labeling
Fluorescent
Light transmission
The labeling is highly specific
Labeling was reduced with partially drug-resistant strain
No labeling occurred with the fluorophore, or drug alone
Pre-incubation with non-modified drug eliminated the labeling
Fluorescent imaging of Aspergillus fumigatus
by caspofungin fluorescent probe
Light transmission
Fluorescence
Next challenge

In vivo imaging
Fluorescence Molecular Tomography (FMT)
is powerful in vivo imaging approach
Imaging of fluorescent material (e.g. labeled microbial pathogens)
in body can be achieved through excitation by powerful light
source and detection of the induced fluorescence. Moving the
excitation source around a scanned object with simultaneous
signal acquisition allows 3-D imaging of the area of interest.
Diagnostic probe
fluorophore
drug
Target
fluorophore
drug
Target
Steps:
1. Probe injection
2. Clearance of unbound probe
3. Fluorescence imaging
4. Optical imaging
FMT requires near-infrared fluorescent labels
5. Superposition of
fluorescent and optical
images
Body tissues are transparent for near infrared light
630-760 nm
Transparency window
of Transparency
body tissues window
of body tissues
window
The most of the current Near-Infrared fluorescent
labels are bulky
DDAO
Cl
O
HO
N
Cy 7.5
Cy 5
Cl
Improving of the DDAO fluorescent properties
by 7-amino modification
DDAO
O
HO
pH-dependent
emission
Cl
Original
ionizable
group
N
Molar absorbance
Brightness
Emission maximum
48,000
3,600
660 nm
Cl
7-amino DDAO
Cl
O
R-HN
pH-independent
emission
Cl
N
Molar absorbance
Brightness
Emission maximum
53,000
6,000
680 nm
Developed
compound
Light absorption spectrum of body tissues and the emission
properties of the synthesized DDAO fluorophore
100000
80000
Cl
60000
O
R-HN
40000
N
Cl
0
7-amino DDAO
7-amino DDAO emission
is in the middle of tissue
transparency window
7-amino DDAO is chemically and
photochemically stable, which
reduces photo bleaching
7-aminoDDAO
emission
20000
640
680
Transparency window
of body tissues
window
720
The synthesis of DDAO-based reactive compounds
for design of diagnostic probes
Cl
S=C=N–(CH2)4-NH
O
Amine-reactive derivative
N
Cl
III
1. CSIm2
2. TFA
Cl
1,4-DAB-(AcOH)2
O
HO
N
Cl
H2N –(CH2)4-NH
Cl
O
N
DDAO
Inexpensive,
commercially available
AcBr-PNP
Cl
BrAc-NH–(CH2)4-NH
Cl
N
II
IV
Thiol-reactive derivative
3MI-Pr-OSu
O
O
N (CH2)2 C
O
Cl
NH –(CH2)4-NH
O
O
N
V
Thiol-reactive derivative
Cl
Cl
The synthesis of DDAO-labeled caspofungin
H2N
O
NH
OH
N H2
HO
C H3
C H3
N
H
H
N
H3 C
Caspofungin
OH
O
O
N
O
N
NH
HO
NH
H
N
N
OH
O
CH3
O
O HO
OH
OH
DDAO-NCS
H2N
O
NH
OH
S
C H3
N
H
H
N
H3 C
Caspofungin-DDAO
N
OH
O
O
O
N H – C – NH –(CH 2)4-NH
HO
C H3
Cl
N
DDAO
O
N
NH
HO
N
H
N
NH
OH
O
CH3
O
O HO
OH
OH
Cl
non-infected
infected
FMT detection of C. albicans in infected mice kidneys using
caspofungin-DDAO fluorescent probe
treated
non-treated
Tomographic detection of C. albicans in live mice
using fluorescent Casp-DDAO compound
Imaging of the infection progress
Time elapsed
after infection
Signal
intensity
Day 0
0.04
Day 1.5
0.32
Day 2.5
0.41
Conclusions
We developed fluorescently labeled fungi-specific
diagnostic probes that can be used for microscopic
and tomographic imaging of the pathogens.
Tomographic imaging will allow more precise and
targeted drug delivery, which could increase the
therapeutic efficiency and as such, restrict the
emergence of drug resistance
Our team
RHRI Center, Rutgers, the State University of New Jersey. USA
Arkady Mustaev Lab
David Perlin Lab
Synthesis and characterization
of the diagnostic probes
Validation of the probes using
Fluorescent microscopy and FMT
Arkady Mustaev
David Perlin
Laura Wirpsza
Min Hee Lee
Shyamala Pillai
Yanan Zhao
Steven Park
Posaconazole-DDAO can act as “signaling”
molecule, thereby providing high imaging contrast
fluorophore
Stacking
interactions
drug
drug
Target
Target
Unquenched
(Fluorescent)
fluorophore
Quenched
O
H
O
N
N
N
F
Quencher
O
F
Me O
O C
N
N
Me
C
Cl
O
NH –(CH2)4-NH
N
N N
N
DDAO
O
Pos-DDAO
Cl
fluoropohore
Incidence of Invasive Fungal Infections (IFI)
 Solid Organ Transplant
 Kidney
 Heart
 Heart-Lung/Lung
 Pancreas
 Liver
5 - 42%
5 – 14%
5 – 32%
15 – 36%
18 – 38%
7 – 42 %
 Bone Marrow Transplant
15 - 25%
 Intensive Care Unit
17%
• Singh, N. CID 2000; 31:545-53
• Vincent JL. Intensive Care Med 1998; 24: 206-216
Opportunistic Candida infections in the
immunocompromised are a major cause of clinical
morbidity and mortality, and are the 4th leading cause
of nosocomial bloodstream infections
Percentage of BSIs (rank)
BSIs per
10,000
admissions
Total
(n = 20,978)
CoNS
15.8
Staphylococcus aureus
Enterococcus species
Crude mortality, %
ICU
(n = 10,515)
Non-ICU
ward
(n = 10,442)
Total
ICU
NonICU
ward
31.3 (1)
35.9 (1)a
26.6 (1)
20.7
25.7
13.8
10.3
20.2 (2)
16.8 (2)a
23.7 (2)
25.4
34.4
18.9
4.8
9.4 (3)
9.8 (4)
9.0 (3)
33.9
43.0
24.0
Candida species
4.6
9.0 (4)
10.1 (3)
7.9 (4)
Escherichia coli
2.8
5.6 (5)
3.7 (8)a
7.6 (5)
22.4
33.9
16.9
Klebsiella species
2.4
4.8 (6)
4.0 (7)a
5.5 (6)
27.6
37.4
20.3
Pseudomonas
aeruginosa
2.1
4.3 (7)
4.7 (5)
3.8 (7)
38.7
47.9
27.6
Pathogen
39.2 47.1 29.0
Wisplinghoff et al. 2004 CID 39:309-17