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Intrapulmonary drug distribution
& pharmacodynamic interactions
of antifungal drugs with host cells
Russell E. Lewis, Pharm.D., FCCP, BCPS
Associate Professor
University of Houston College of Pharmacy &
The University of Texas M.D. Anderson Cancer Center
Houston, Texas
UH Anti-Infective
Research Laboratories
Outline
How does the pathophysiology of invasive pulmonary
aspergillosis impair drug delivery to the site of
infection?
Are pharmacokinetic differences among antifungal
agents important for the treatment of fungal
pneumonia?
Pathology of acute mould infections
neutropenic hosts
Inhalation of conidia
Conidia reach
distal alveolar space
and begin to germinate
Mononuclear and
polymorphonuclear
cells clear germinating
conidia and hyphal forms
Angioinvasive growth
In tissue with hemorrhage,
thrombosis, hypoxia,
necrosis, and dissemination
Impaired by neutropenia, glucocorticoids,
metabolic abnormalities, concomitant infections
Administer drugs to slow fungal proliferation;
and reduce, angioinvasion, and dissemination
Figure: R. Lewis
Pathology of acute mould infections
corticosteroid and mixed immunosuppression?
Inhalation of conidia
Conidia reach
distal alveolar space
and begin to germinate
Mononuclear and
polymorphonuclear
cells clear germinating
conidia and hyphal forms
Dysregulated
inflammatory
response
Figure: R. Lewis
Angioinvasive growth
In tissue with hemorrhage,
thrombosis, hypoxia,
necrosis, and dissemination
Immunopathology
hemorrhage,
necrosis
Progressive thrombosis- inflammation-necrosis
impair drug activity in invasive aspergillosis
Discrete nodules (neutropenia)
Nodular consolidation (steroids)
Coagulation necrosis without
Inflammatory cells
Encompassing
hemorrhage
Defined nodules with
hemorrhage
Solid nodular consolidation
with necrotic center
Dense neutrophilic
infiltrate on periphery
∆ 20% in response ~10 days
% Cured
All treated
Voriconazole
Amphotericin B
Nodular Lesion with
Halo Sign
(N=143)
Greene et al. Clin Infect Dis 2007;44:373-9.
Shibuya et al. J Infect Chemother 2004;10:138-45.
Nodular lesion without
Halo Sign
(N=143)
Pathogen growth and inflammation in the lung
evoke neovascularization and angiogenesis
Lung epithelium
• How does the pathogen or
host immunosuppression
affect the angioplasticity of
the lung?
TNFα
IL-8
VEGF
HIF-1α
bFGF
Bone marrow
derived pro-angiogenic
precursors
(CD34+, CD 133+)
– Implications for disease
pathogenesis?
– Implications for drug
delivery?
Asosingh & Erzuram. Biochem S. Tran 2009;37:805-810.
Invasive pulmonary aspergillosis in neutropenic mice
is associated with rapid down-regulation of multiple
genes involved in angioneogenesis
A. fumigatus
cyclophosphamide +
1 dose cortisone
(neutropenic)
whole lung
RNA
x fold expression
corticosteroid
only
Relative expression by RT-PCR at 24 hr
uncoupling of inflammatory-angiogenic
response in neutropenic animals?
PCR arrays
84 genes
angiogenesis
pathway
controls: same immunosuppression, no infection
Ben-Ami et al. Blood ; Advanced access published 2009
In vivo assessment of the anti-angiogenic
effects of Aspergillus infection
myocutaneous model of aspergillosis
(neutropenic, non-lethal)
In vivo Matrigel
plug (500
µL)bFGF +
heparin
Will surrounding
Aspergillus growth
suppress angiogenesis?
D+5
endothelial cell migration
capillary tube formation
D+7
erythrocyte-filled
lacunae
Remove and
image
Ben-Ami et al. Blood ; Advanced access published 2009
a Passanti et al. Lab Invest 1992;67:518-528.
injected 10 mm from infection site
A. fumigatus suppress angiogenesis in an
in vivo cutaneous infection model
∆laeA
AF 293
Control
Day +7 Matrigel plugs
Ben-Ami et al. Blood; Advanced access published 2009
Masson trichrome stain
Are secondary metabolites responsible for
this anti-angiogenic effect?
In vitro Matrigel experiments (HUVEC)
Growth medium
Growth medium
A. fumigatus culture filtrate
∆laeA culture filtrate
∆laeA complemented culture filtrate
∆glip
Ben-Ami et al. Blood; Advanced access published 2009
Gliotoxin suppresses angioneogensis
in a dose-dependent fashion
In vitro Matrigel experiments (HUVEC)
gliotoxin 3000 ng/mL
gliotoxin 30 ng/mL
gliotoxin 300 ng/mL
control
*gliotoxin lung concentrations in experimental pulmonary aspergillosis:
1000-4000 ng/gram lung
Ben-Ami et al. Blood; Advanced access published 2009
*Lewis et al. Infect Immun 2005;73:635-637.
Pathological features affecting drug delivery
depend on host immunosuppression
Neutropenic patient
Non-neutropenic patient
Diminished
drug delivery/
efficacy?
Diminished
drug delivery/
efficacy?
immune
response
Aspergillus
inhibition of
lung angiogenesis
Aspergillus
immune
response
PMN-mediated
tissue damage*
Impact of changing immune function, co-pathogens?
*evidence that gliotoxin also exacerbates this
effect in corticosteroid-immunosuppressed mice
Effect of amphotericin B on the survival or
immunosuppressed mice with invasive
pulmonary aspergillosis
Corticosteroid
Balloy et al. Infect Immunity 2005;73:494-503.
Chemotherapy (neutropenic)
Immunomodulatory effects of
antifungals in the lung
Amphotericin B
Liposomal
amphotericin B
Echinocandins
Triazoles
Dectin-1
TLR2
Activation of TLR2
TLR4
Activation of TLR4
Exposure or immunogenic
epitopes in cell wall
Fungal sensitization for
phagocytosis
relative pro-inflammatory
potential in the lung?
Ben-Ami et al. Clin Infect Dis 2008;47:226-35.
Outline
How does the pathophysiology of invasive pulmonary
aspergillosis impair drug delivery to the site of
infection?
Are pharmacokinetic differences among antifungal
agents important for the treatment of fungal
pneumonia?
Patterns of drug distribution for currently
available antifungals
Liver/
Spleen
Kidneys
Gut/gall
bladder
Lungs
Brain/
CSF
Eyes
Bladder/
urine
AMB
+
+
+
+
–
–
–
5FC
+
+
+
+
+
+
+
FLU
+
+
+
+
+
+
+
ITR
+
+
+
+
–
–
–
VOR
+
+
+
+
+
+
–
POS*
+
+
+
+
–
–
–
Echino
+
+
+
+
–
–
–
+, ≥50% of serum concentrations.
–, <10% of serum concentrations.
*Predicted.
1. Dodds-Ashley ES, et al . Clin Infect Dis. 2006;43:S28-S39.
2. Groll AH, et al. Adv Pharmacol. 1998;44:343-500.
3. Eschenauer G, et al. Ther Clin Risk Manag. 2007;3:71-97.
Steady-state concentrations of voriconazole and
anidulafungin in plasma, epithelial lining fluid and
macrophages in healthy adults
Similar data for posaconazole (Conte et al. Antimicrobial Agent Chemother 2009;53:707-7);
micafungin (Nicasio et al. Antimicrob Agent Chemother 2009;53:1218-20);
itraconazole (Conte et al. Antimicrob Agent Chemother 2004;48:3823-7)
Crandon et al. Antimicrob Agent Chemother 2009;Online Publication Sept 21, 2009
Concentration of amphotericin B in lung tissue, ELF,
PAMs and PBMs of healthy rabbits after once-daily
dosing for 8 daysa
Mean concentration + SD in:
Drug
Dose
(mg/kg)
Lung tissue
(ug/g)
ELF
(µg/mL)
PAM
(µg/mL)
PBMs
(µg/mL)
Plasma
(µg/mL)
DAMB
1
2.71 + 1.22
0.44 + 0.13
8.92 + 2.94
1.20 + 0.83
0.37 + 0.07
ABCD
5
6.29 + 1.17
0.68 + 0.27
5.43 + 1.75
2.44 + 1.90
0.37 + 0.12
ABLC
5
16.26 + 1.62
0.90 + 0.28
89.1 + 37
0.74 + 0.42
0.24 + 0.08
LAMB
5
6.32 + 0.57
2.28 + 0.57
7.52 + 1.43
1.51 + 0.78
26.4 + 4.99
Twenty-four hours after doses of either DAMB, ABCD, ABLC, or LAMB. All values represent the means ± SD from three to seven rabbits in
each dosing group. Plasma, concurrent plasma concentrations. P values from comparisons across dosage groups by Kruskal-Wallis
nonparametric analysis of variance (ANOVA) were as follows: lung, P = 0.0029; ELF, P = 0.0070; PAMs, P = 0.0246; PBMs, P = 0.4640; and
plasma, P = 0.0146. Between-group comparisons using Dunn's correction for multiple comparisons revealed significant differences in lung
tissue concentrations between DAMB- and ABLC-treated animals (P < 0.01), in ELF concentrations between DAMB- and LAMB-treated
animals (P < 0.01), and in PAM concentrations between ABCD- and ABLC-treated animals (P < 0.05).
a
Groll et al. Antimicrob Agent Chemother 2006;50:3418-23.
Our lipid formulations are engineered to reduce
nephrotoxicity, not improve efficacy
AMB-d or ABCD
28 days L-AMB 3 mg/kg
Liver
100 µg/gram
Spleen
50 µg/gram
Kidney
15 µg/gram
5-10 µg/gram
0.67 µg/gram
0.1 µg/gram in tissue
histologically infected
with Aspergillus
Lung
1 µg/gram
CNS
Vogelsinger et al. J Antimicrobial Chemother 2006;57:1153-1160.
Paterson et al. J Antimicrob Chemother 2003;52:873-876.
“Corpora non agunt nisi fixata”
-Paul Erlich
The way forward?
• Earlier detection= wider window for drug delivery to
target tissue
• Better understanding of the pathobiology that limits
drug delivery
– Angioneogenesis, tissue repair
– Inflammation
– Can we engineer solutions?
• How can this knowledge be used to improve
therapeutic approaches, especially in chronicallyinfected patients?
– Aerosolized, intermittent or novel drug delivery approaches
Acknowledgements
• M.D. Anderson Cancer Center
–
–
–
–
Dimitrios P. Kontoyiannis M.D., Sc.D
Ronen Ben-Ami, M.D. (angioneogensis work)
Nathan Albert, Konstantinos Leventakos, M.D.
Gregory May, Ph.D. (∆glip strain)
• NP Keller, Wisconsin (∆laeA)
• Funding for angioneogensis studies:
– EN Cobb Scholar Award (DPK)
– NCI Core Center Grant (CA 16672)
– NIH (1RO3AI083733-01) Kontoyiannis