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Animal models in preclinicalAnimal models in preclinical trials of aspergillosis
K l V Cl Ph DKarl V. Clemons, Ph.D.CIMR, SCVMC, & Stanford Univ.
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Why Perform Animal Models of Aspergillosis?
Studies on therapy Monotherapy & Combination therapy:AmB preps, azoles, echinocandins, etc.AmB preps, azoles, echinocandins, etc. Compare efficacies at same time!Results similar among models and to clinicalImmunotherapy:Immunotherapy:Various cytokines (interferon-γ, TNF-α, GM-CSF, etc.), other immunomodulators,Ablation of specific cytokinesAblation of specific cytokines
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The Ideal Animal Model
• No “warm fuzzy test tubes”!
Should mimic clinical disease
• Should be standardized reproducible &• Should be standardized, reproducible & affordable!
• Parameters of survival and infectious burden controllable: define the model first!controllable: define the model first!
• Wait until infection is established!
• Should reflect drug efficacy, toxicity and drug interactions similar to observed clinicallyy
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Animal Models – Control of VariablesAnimals – preclinical trial Humans – clinical trial
Syngeneic & many strains --Age & sex match --
C t l i l & t
No waySort ofNControl inocula & route --
Choice of inoculum strain --Control disease severity
No wayNo waySort of but really no wayControl disease severity --
Control exp. duration –Survival & CFU organs
Sort of, but really no wayYes – if they show upSurvival yes CFU noSurvival & CFU organs --
Route & duration of dosing --Control immunosuppression --
Survival – yes, CFU - noYes – to some degreeYes – to some degreeControl immunosuppression
Large #’s done at same time --Statistical analyses --
Yes to some degreeLimited # pts. & timeYes – more difficulty
Trials are prospective -- Some are - some are not
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Animal Models of AspergillosisAnimal Models of Aspergillosis
Primary model systems:• Murine – systemic, pulmonary, CNS, oculary p y• Rabbit – systemic, pulmonary, ocular• Guinea pig – systemic pulmonary• Guinea pig – systemic, pulmonary• Rat - systemic, pulmonary• Bird – pulmonary
Normal or immunosuppressed - depends on model
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Animal Models of AspergillusAnimal Models of Aspergillus
A. fumigatus is the most frequently usedg q y
Can test different Aspergillus species causingCan test different Aspergillus species causing clinically relevant disease.
Clinically:Clinically:A. terreus increase frequency & AMB resistantModels in rabbits and mice:
resistant to AMBs – like clinicalSaper, ICZ and POS effective
Dannaoui , et al. 2000 JMM; Walsh, et al. 2003, JID
Hanson, et al. 1995 JMVM; Steinbach, et al. 2004 AAC;
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Models of Aspergillosisode s o spe g os s
• Need to be cautious interpreting results!• Need to be cautious interpreting results!
What looks good in one model may not
look good in another model!
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Models of AspergillosisModels of Aspergillosis
• Systemic model – synergy between Mica and Nik Zy gy
• Pulmonary modelNO b t Mi d Nik Z– NO synergy between Mica and Nik Z
Different models using same drugs t i ltmay not give same result
Capilla Luque, et al, ’03. AAC. Clemons et al. 2006. Med Mycol
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Models of AspergillosisModels of Aspergillosis
Use models for drug efficacy comparisonsUse models for drug efficacy comparisons not likely to be done in a clinical trial
For example – is one lipid formulation of o e a p e s o e p d o u at o oAmB superior for treatment of
aspergillosis?aspergillosis?
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Summary - All Lipid AmB comparison Systemic Aspergillosis
1. All formulations prolonged survival, 2 AmBi nearly equivalent to dAmB; ABCD and2. AmBi nearly equivalent to dAmB; ABCD and
ABLC were less effective or equivalent depending on the severitydepending on the severity
3. No survivors cured in both kidneys and brain4. Each formulation efficacious, esp. in kidneys. 5. No lipid-formulation consistently superior to p y p
Fungizone even though higher doses given 6 All were equivalent to each other6. All were equivalent to each other
Clemons and Stevens. AAC, 2004
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Aspergillus Animal ModelsAspergillus Animal Models
Use models to look at new indications not possible in a clinical trial
Test efficacy of new and “old” drugsAl th• Alone – monotherapy
• Combination
Example CNS aspergillosisExample – CNS aspergillosis
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CNS Aspergillosis Monotherapy Studies
100 POS 5, 25
ving
100
80POS 100
POS 5, 25
ce S
urvi
v
60AmB 3
rcen
t Mic
40
20
CAS 5
Per 20
0
Therapy HPβCDICZ 50
D5W
0 5 10 15
Day Postinfection
Imai, et al. 2004, AAC
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Monotherapy against CNS Aspergillosis
Amphotericin B
• dAmB, iv partially effective
• AmBisome or ABLC iv effective not curative• AmBisome or ABLC iv effective not curative
Azoles
• ICZ and VCZ PO partially effective
• POS very effective
EchinocandinsEchinocandins
• Mica or CAS equally effective
NONE CURATIVE!
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Aspergillus Animal ModelsAspergillus Animal Models
Use to examine combination therapiesUse to try regimens to achieve cure
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CNS aspergillosisCNS aspergillosis
Clinically, CNS disease a common site of dissemination. Poor response to p
treatments and high mortality.
CNS Model Objectives• Examine combinations of antifungal drugs
( AmBi ABLC dAmB MICA VCZ ICZ( AmBi, ABLC, dAmB, MICA, VCZ, ICZ, CAS) or escalating dosages
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Combinations against CNS aspergillosisg p g• AmBi + echinocandins –improved survival, but no
significant enhancementsignificant enhancement• AmBi + VCZ – *significant enhancement*• Sequential AmBi + VCZ ca. as effective as
combination• dAmB + CAS – indifferent• Only combination regimens reduced CFUOnly combination regimens reduced CFU• Only AmBi +VCZ caused significant reduction in
brain and kidneybrain and kidney• No total cures were obtained
Imai, et al. 2005, JAC; Clemons, et al. 2005 AAC
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Combinations and CNS aspergillosisp g
• ABLC at 4 mg/kg = to higher dosagesg g g g– more not better
• ABLC + echinocandins – improved survival, but ns.ABLC echinocandins improved survival, but ns.– (lower doses of combination equal to higher doses!)
• ABLC + ICZ – indifferent• ABLC + ICZ – indifferent• ABLC + VCZ – *significant enhancement*• AmB + ICZ – slight enhancement?• AmB + CAS – indifferentAmB CAS indifferent
Similar to results with AmBisome (Clemons et al. AAC, 2005)Clemons et al. JAC, 2006
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Combination Therapy Pulmonary Aspergillosis
I t id d DBA/2 l d lIn steroid-suppressed DBA/2 pulmonary model
-AmBi + VCZ, CAS, or Mica-dAmB + Micad ca-Mica + NikZ-ICZ + Mica-ICZ + Mica
*N b tt th th **None better than monotherapy*
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Aspergillus Animal ModelsAspergillus Animal Models
Cure “The Holy Grail” of clinical medicineCure, The Holy Grail of clinical medicine
C hiCan you achieve cure Using high doses?
Using combination therapy?
Is more better?At what cost to tissues?
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Achieving cure systemic AspergillusAchieving cure- systemic Aspergillus
Test AmBi, CAS, VCZ• Dose-escalation• Loading doses AmBi• Combination
AmBi + CAS or VCZ
N i t i f f CURES!No improvement in frequency of CURES!Clemons, et al. AAC, 2005
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Achieving cure for CNS Aspergillosis by dose-escalation
100A Bi 15vi
ng
60
80AmBi 10
AmBi 15
A Bi 20ce s
urvi
40
60 AmBi 20AmBi 25
cent
mic
0 5 10 150
20D5WPe
rc
O C S & O S O
0 5 10 150
Day postinfection
NO CURES & MORE IS NOT BETTER!Clemons, et al. AAC, 2005
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Achieving cure-S t i P l CNS A illSystemic, Pulmonary or CNS Aspergillus
• More isn’t better and may be bad! – Increased dosages did not improve efficacy.g p y– Dose-escalation of AmBi or ABLC may be deleterious– AmBi loading doses no improvementAmBi loading doses no improvement
• MICA or CAS – flat dose-response – 1 =10 mg/kg• Suboptimal combo ≅ high dose AmBi or ABLC• Sequential therapy not effective for cureSeque a e apy o e ec e o cu e• No improvement in cure rate by any regimen
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What about other filamentous fungi?What about other filamentous fungi?
Increased frequency of zygomycosis
Treatments are poorly effective.
What works?What works?
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LAmB and echinocandin combination therapy against experimental mucormycosis
Ibrahim et al. 08 AAC:52:1556-8
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LAmB +Mica or LAmB +Anidula: DKA mouse model
3.0
neys
)
2.5
U/g
of K
idn
* LAmB + Anidula
1 5
2.0
Log
CFU *
Enhanced survivalLess for CFU
1.5Placebo LAmB5 Mica 1 LAmB +
Mica2.7
3.1
Kid
ney)
†
2.3
CFU
/g o
f K *LAmB + Mica Enhanced survival & CFU
1.5
1.9Lo
g10C
Placebo LAmB5 Anidula10 LAmB +Anidula
Ibrahim et al. 08 AAC:52:1556-8
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LAmB +Anidula: Neutropenic mouse model
100%Placebo
80%al
LAmB 3
Anidula 10
40%
60%
urvi
va
LAmB3 + Anidula 10
20%
40%Su *
0%0 7 14 21
Days post infection
Ibrahim et al. 08 AAC:52:1556-8
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What’s on the horizon for antifungals ?Azoles
• Isavuconazole (BAL-4815; Basilea)
– Prodrug, active against Candida and Aspergillus• Albaconazole (UR9825; Uriach)
– broad spectrumbroad spectrum• Ravuconazole (BMS207147 or ER30346: Eisai)
b d t– broad spectrum• ITF2534 (Italfarmaco)( )
– broad spectrum, in vivo Candida, Aspergillus CryptococcusCryptococcus
Pasqualotto and Denning, JAC, 2008; Clemons, et al ICAAC, 2008
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What’s on the horizon for antifungals ?Polyenes
• Patricin A – (SPA-753)( )– Candida & Cryptococcus in vivo, – Aspergillus in vitro (Rimaroli & Bruzzese, Chemother, 2000)
N f l ti f h t i i B• New formulations of amphotericin B
EchinocandinsEchinocandins• Aminocandin (HMR-3270; Novexel)
– Candida and Aspergillusp g
New classes• Arylamidine (T-2307)
• Candida, Aspergillus, & Cryptococcus (Mitsuyama et al AAC 2008))
• Sordarins??
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Summary of animal models in preclinical trials aspergillosis
• Results provide a basis for running clinical trials of antifungal therapy for aspergillosis
• Have been the basis for treatment with AmBs various azoles and echinocandinsAmBs, various azoles and echinocandins now approved for therapyPro ide the rationale for potential clinical trial• Provide the rationale for potential clinical trial of various combination therapies– (e.g., liposomal AmB + VCZ, or CAS or MICA)
• Will be necessary to bring new drugs to trialy g g
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Animal Models of Fungal InfectionsAnimal Models of Fungal Infections
• Show differences in susceptibility• Show effects of particular genes onShow effects of particular genes on
virulence (quantitative genetics!)Sh l ti d ifi it f h t• Show evolution and specificity of host-response
• Show correlation with therapeutic efficacyAllo s to ask q estions not ans erable• Allow us to ask questions not answerable in vitro and address the biology of host-parasite interactions