Comparative Pharmacodynamics of Amphotericin B Lipid Complex ABLC and Liposomal Amphotericin B LAMB

1
Comparative Pharmacodynamics of Amphotericin B
Lipid Complex (ABLC) and Liposomal Amphotericin B
(L-AMB) in a Murine Model of Acute Invasive
Pulmonary Zygomycosis
Contact Information Russell Lewis University
of Houston/ Texas Medical Center 1441 Moursund St
., 424 Houston, Texas 77030 713-795-8326
rlewis_at_uh.edu
R.E. Lewis,1,2 N.D. Albert,2 J. Hou,1 R.A.
Prince,1,2 and D.P. Kontoyiannis1,2
The University of Houston College of Pharmacy1
The University of Texas M.D. Anderson Cancer
Center2 Houston, Texas
Abstract M-2139
Abstract
Results
Methods cont.
Results cont.
Background Lipid formulations of amphotericin B
(AMB) differ in their rate of distribution to the
lung, however the importance of these
pharmacokinetic differences for the treatment of
invasive pulmonary zygomycosis (IPZ) remains
poorly understood. We compared the kinetics of
AMB accumulation in lung tissue and rate of
fungal clearance for ABLC and L-AMB treatment
regimens in an murine model of IPZ. Methods
BALB/c mice were immunosuppressed with
cyclophosphamide and cortisone before intranasal
inoculation with 1 x106 Rhizopus oryzae (RO)
conidia AMB MIC/MFC 0.5/8 µg/mL. ABLC, L-AMB,
or 5 dextrose water (control) was then
administered in daily intravenous doses (1, 5, or
10 mg/kg) in 40 mice starting 12 h after
infection and continued until day 5. At
predetermined times (0, 24, 72, and 120 h), 10
mice were euthanized, and lungs were harvested
for determination of lung fungal burden (conidial
equivalent (CE) Rhizopus DNA) by quantitative
PCR. Total AMB lung tissue concentrations were
determined by LC/MS. Results Both formulations
were ineffective at the 1 mg/kg/d dose. At 5
mg/kg/d RO lung fungal burden was significantly
lower in ABLC-treated animals vs. L-AMB (Plt0.05)
correlating with significantly higher AMB
concentrations in the lungs of ABLC-treated mice
during the first 72 hours of daily therapy. ABLC
and L-AMB were equally effective at 10 mg/kg.
Conclusion These data suggest that at doses lt
10 mg/kg/d, ABLC achieves effective
concentrations of AMB in tissue lung more rapidly
than L-AMB, resulting in improved control of
Rhizopus in an experimental model of IPZ.
Pharmacodynamic differences between the two
formulations were less evident at daily doses of
10 mg/kg/d.
Figure 1. Murine model of invasive pulmonary
zygomycosis
Figure 5. Higher daily doses are required for
L-AMB, but not ABLC, to reduce R. oryzae lung
fungal burden in experimental IPZ
Figure 2. L-AMB and ABLC have different patterns
of lung distribution and clearance of R. oryzae
in experimental IPZ
Each graph compares the relationship of A.
fumigatus and R. oryzae lung fungal burden for
L-AMB or ABLC therapy after 5 days of intravenous
dosing. Each datum point represents the mean /-
SD of the fungal-specific conidial equivalent
(C.E.) DNA in the lung determined by RT-qPCR for
5 mice (A. fumigatus)2 or 10 mice (R. oryzae).
Immunosuppression and infection protocols were
identical for the two datasets. Dotted line
represent median fungal burden associated with
mortality in R. oryzae model.
Conclusions

• Antifungal treatment
• Liposomal amphotericin B (L-AMB Ambisome, Gilead
  Sciences, Inc., Foster City, CA) Amphotericin B
  lipid complex (ABLC Abelcet, Enzon Inc.,
  Bridgewater, NJ) were reconstituted in D5W at the
  appropriate concentration for a 200 µL injection
• Mice received daily intravenous injections of
  each antifungals via the lateral tail vein
• L-AMB 1, 5, or 10 mg/kg/day
• ABLC 1, 5, or 10 mg/kg/day
• Normal saline, 200 µL (control)

• Although both lipid formulations of amphotericin
  B are effective for the treatment of pulmonary
  zygomycosis, significant differences were noted
  in the rate of drug distribution to the lung and
  dose-response curves in experimentally-induced R.
  oryzae pneumonia in neutropenic mice
• At 5 mg/kg/day, intravenous ABLC achieved higher
  AMB lung tissue concentrations compared L-AMB,
  resulting in significantly better clearance of R.
  oryzae from infected lung
• At 10 mg/kg/day, both lipid formulations achieved
  AMB concentrations in lung tissue during the
  first 72 hours that exceeded the MFC of the R.
  oryzae isolate, resulting in equivalent rates of
  fungal clearance
• Higher AMB tissue concentrations of amphotericin
  B were required to inhibit proliferation and
  invasion of R. oryzae versus A. fumigatus
  observed in a similar infection model
• Dosing strategies that achieve higher
  concentrations of AMB in the lung (use of 5 mg/kg
  ABLC or 10 mg/kg L-AMB) should be considered in
  the initial treatment phase of IPZ, as delay in
  the institution of effective therapy increases
  opportunities for angioinvasion and
  dissemination, and is associated with increased
  mortality in patients with zygomycosis5

Top row of graphs show the relationship of
Rhizopus oryzae fungal burden in 10 mice versus
time. Each datum point represents the mean /- SD
of R. oryzae conidial equivalent (C.E.) DNA
determined from lungs of 10 mice by real-time
quantitative PCR (RT-qPCR). L-AMB, Liposomal
amphotericin B ABLC, amphotericin B lipid
complex. Bottom row of graphs show the
corresponding mean /- SD of total amphotericin B
concentrations determined HPLC analysis of lung
homogenates. Concentrations were corrected for
weight and volume of homogenate. Plt 0.05 by
ANOVA with Dunns correction or Mann Whitney U
test, where appropriate. Dotted lines represent
the MIC/MFC of the infecting R. oryzae isolate.

• Quantitative real-time quantitative PCR assay
  (RT-qPCR)
• Adaptation of protocol described by Ibrahim et
  al.4
• Lungs homogenized in sterile PBS containing
  non-murine plasmid internal standard (Eimeria
  tenella PKG cDNA (GenBank accession no. AF491161)
  to correct for differences in DNA
  recovery-amplified in seperate RT-qPCR reaction
• 90 µL sample lysed with proteinase K DNA
  extracted using Qiagen DNeasy Tissue Kit (Qiagen,
  Valencia, CA)
• RT-qPCR performed on 5 µL DNA sample using
  oligonucleotide amplification primers and a
  dual-labeled fluorogenic hybridization probe
  complementary to the R. oryzae 18S rRNA gene
  (GenBank accession no. AF113440) on an Applied
  Biosystems 7000 Sequence Detection System
• Standard curve generated by spiking naïve lungs
  with 10-fold dilutions of AF 293 conidia
  (102-107), extracting DNA, and analysis of CT
  threshold by quantitative real-time PCR

Methods
Figure 3. L-AMB and ABLC exhibit dose-dependent
differences in extent of R. oryzae hyphal
invasion in experimental IPZ

• Test Isolate
• Rhizopus oryzae clinical isolate from patient
  with pulmonary zygomycosis
• Sub-cultured onto sabouraund dextrose agar (SDA)
  slants and grown for 7 days
• Conidia were harvested in 0.85 PBS and 0.01
  Tween 80 and filtered twice through sterile gauze
  to remove hyphal fragments
• Inoculum concentration verified by hemocytometer
  and viability confirmed by serial plating onto
  SDA
• Susceptibility determined by CLSI M38-A2
  methodology in RPMI 5 glucose and by Etest
• MIC endpoint optically clear well (M38-A2) or
  growth ellipse (Etest)
• MFC determined by plating 20 µL from optically
  clear wells controls on SDA and incubating
  until growth observed in control wells1
• MFC defined as lowest drug concentration that
  showed either no growth or fewer than three
  colonies (99 to 99.5 killing)1

Hyphal morphology on day 5 in tissue after
treatment with L-AMB or ABLC (1,5 or 10 mg/kg)
was compared in 5 mice per treatment group. Lungs
were fixed in 10 (vol/vol) formaldehyde and were
processed and embedded in paraffin wax, and
matched sections were stained with Grocott's
methamine silver nitrate. Representative lung
sections are presented. A dose-dependent
reduction in number of hyphae and invasion is
evident for both formulations. 200x bar12 µm
References
Figure 4. Reductions in Rhizopus oryzae pulmonary
fungal burden may require higher tissue
concentrations of amphotericin B than Aspergillus
fumigatus

• Espinel-Ingroff A. Fothergill, J. Peter et al.
  (2002) Testing Conditions for Determination of
  Minimum Fungicidal Concentrations of New and
  Established Antifungal Agents for Aspergillus
  spp. NCCLS Collaborative Study. J Clin Microbiol
  40, 3204-8.
• Lewis R.E. Wiederhold N.P. (2005). Murine model
  of invasive aspergillosis. Methods Mol Med 118,
  129-142.
• Lewis R.E., G. Liao, J. Hou, et al. (2007). A
  Comparative Analysis of Amphotericin B Lipid
  Complex and Liposomal Amphotericin B Kinetics of
  Lung Accumulation and Fungal Clearance in a
  Murine Model of Acute Invasive Pulmonary
  Aspergillosis. Antimicrobial Agent Chemother 51,
  1253-1258.
• Ibrahim A.S., J.C. Bowman, V. Avanessian et al.
  (2005). Caspofungin Inhibits Rhizopus oryzae
  1,3-ß-D-Glucan Synthase, Lowers Burden in Brain
  Measured by Quantitative PCR, and Improves
  Survival at a Low but Not a High Dose during
  Murine Disseminated Zygomycosis. Antimicrobial
  Agent Chemother 51, 1253-1258.
• Chamilos G., R.E. Lewis, D.P. Kontoyiannis.
  (2008). Delaying Amphotericin BBased Frontline
  Therapy Significantly Increases Mortality among
  Patients with Hematologic Malignancy Who Have
  Zygomycosis. Clin Infect Dis 47, 503-509.

• Determination of amphotericin B lung
  concentrations
• Total amphotericin B concentrations determined in
  lung by liquid chromatography/ mass spectroscopy3
• Calibration curve was linear over a range of 0.5
  to 10 µg/gram in tissue
• Mean inter- and intra-assay CV was lt10 with a
  lower limit of accurately detectable AMB of 0.05
  µg/gram

• Murine model of invasive pulmonary zygomycosis
• Modification of standardized model previously
  described for aspergillosis2,3
• 18-22 gram female Balb/c, n40 per treatment
  group
• Immunosuppression
• cyclophosphamide (IP) 150 mg/kg days -4 and -1
  100 mg/kg on day 3
• cortisone acetate (sub-Q) 300 mg/kg day -1
• Mice anaesthetized with 10 isoflurane oxygen
  and inoculated intranasally (35 µL) with 5x106
  conidia

The relationship of lung fungal burden (conidial
equivalent DNA, C.E. DNA) was compared to
amphotericin B tissue concentrations for R.
oryzae and A. fumigatus 293 (Aspergillus data
generated from a prior study with identical
methodology).3 EC50 and slope was determined by
fitting a a four parameter logistic Emax model
(sigmoidal curve with variable slope) to fungal
burden data YYmin (Ymax-Ymin)/110(log
ED50-X)slope using Graphpad Prism 5 Software.
MIC/MFC for R. oryzae 0.5/8 µg/mL A. fumigatus
0.25/4 µg/mL.

• Statistical analysis
• Comparisons between groups were performed by
  analysis of variance (ANOVA) with Dunns
  correction for multiple comparisons or the
  Mann-Whitney U test, as appropriate
• All P values were two sided, and a P value of
  lt0.05 was considered significant

This study was funded by an unrestricted grant
from Enzon Inc. who was not involved in the
design and conduct of the study, analysis and
interpretation of the data, nor the preparation
of this poster. Animals were cared for in
accordance with the highest standards for humane
and ethical care as approved by the institutional
animal care and use committees.