ANIMAL MODELS FOR THE STUDY OF
ANTIMYCOBACTERIAL DRUGS
Istituto Superiore di Sanità, Rome
Department of Infectious, Parasitic and
Immune-mediated Diseases
LANFRANCO FATTORINI
Villars-sur-Ollon, Switzerland, October 8-12, 2006
ISTITUTO SUPERIORE DI SANITA’
DIPARTIMENTO M.I.P.I.
MAJOR TOPICS
.
9 Anti-TB drugs
9 Comparative features of animal models
9 Global discovery program for novel anti-TB drugs
9 Importance of fluoroquinolones and other drugs
9 Rapid methods to screen drugs in mice
9 Mouse infections with NTM
9 Drugs for latent TB
9 Alternative drug delivery systems
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Anti-TB drugs
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FIRST-LINE DRUGS
against TB
Isoniazid (H)
Rifampin (R)
Pirazinamide (Z)
Streptomycin (S)
Ethambutol (E)
bactericidal
oral
bactericidal
oral
bactericidal
oral
bactericidal
injectable
bacteriostatic
oral
WHO 6 mo daily treatment:
2 mo HRZ(S/E) + 4 mo RH
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SECOND-LINE DRUGS
against TB
KM, CM, AK
bactericidal
injectable
ETH
bactericidal
oral
CS, PAS, TC
bacteriostatic oral
OFL, CIP, MOX bactericidal
GAT, LEV
oral
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PROMISING NEW DRUGS
against TB
Spigelman & Gillespie, Lancet 2006; 367:947
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TREATMENT OF LATENT TB
(CDC recommendations, 2003)
INH
9 months
RMP
4 months
.
for persons who are contacts of patients
infected with INH-R, RMP-S Mtb strains
.
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Comparative features of animal
models of pulmonary TB
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Humans Mouse Rabbit
Guinea
pig
Mononuclear cell granulomas
++
++
++
++
Caseation necrosis
++
-
++
++
Liquification/cavitation
++
-
++
-
Extrapulmonary dissemination
++
++
++
++
NA
+
++++
++
NA
+
++++
++
BL-3 space requirements
Maintenance costs
(adapted from McMurray et al, Trends in Molecular Medicine, 2001;7:135)
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The mouse has been estensively used for preclinical
assessment of experimental drugs against TB
Medina & North, Immunology 1998; 93:270
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Dose and routes of mouse infection
for anti-TB drug screening
Low-dose,
aerosol infection
50-100 CFU
High dose infection
i.v., i.n., i.p.
106 - 107 CFU
.
(Orme et al, Antimicrob Agents Chemother 2001;45:1943)
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Global discovery program
for novel anti-TB drugs
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Global discovery program for
novel anti-TB drugs
In 1994 the National Institute for Allergy and
Infectious Diseases (NIAID) established the TB drug
screening program “TB Antimicrobial Acquisition And
Coordinating Facility” (TAACF)
(www.taacf.org)
to screen compounds for anti-mycobacterial activity
in high quality in vitro and in vivo assays.
The TAACF is a no-cost service. Sending drugs is
encouraged.
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TAACF (1): in vitro screening
9 MICs against drug susceptible and resistant strains
9 Cytotoxicity in Vero cells
9 Killing in mouse bone marrow Mφ
9 Compounds that perform well in Mφ are tested in the
mouse model (C57BL/6) and/or IFN-γ KO C57BL/6
(GKO) for Mtb, and beige mice for M.avium
(www.taacf.org)
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TAACF (2): standard mouse model
9 low-dose aerosol infection in C57BL/6 mice
9 INH 25 mg/kg/day as positive control
9 active drug = 0.7 log10 CFU reduction
20
65
-0.7
Orme et al, Antimicrob Agents Chemother 2001;45:1943
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TAACF (3): rapid mouse model
9 low-dose aerosol infection in IFN-γ KO C57BL/6 (GKO) mice
9 efficacy after 8 days, less labor intensive, less compound
9 active drug = 0.3 log10 CFU reduction
27
19
H 25
M 100
M 200
M 400
Lenaerts et al, Antimicrob Agents Chemother 2003;47:783
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TAACF (4)
To date, over 75,000 compounds
have been tested by the TAACF
program. About 0.3% of the
compounds which perform well in
vitro and ex vivo assays are
tested in mice-infected with Mtb
(Lenaerts et al, Antimicrob Agents Chemother 2003;47:783)
(www.taacf.org)
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The importance of
fluoroquinolones for TB
in particular moxifloxacin, to
shorten the duration of TB therapy, and
for the treatment of MDR TB
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MOXIFLOXACIN:
a stable cure was achieved 2 months
earlier by substituting MOX for INH for treatment of drug susceptible
strains
+3
+3
+3
+3
BALB/c mice, H37Rv by aerosol, drugs daily by gavage, relapse checked after 3 mo
RIF, INH, PYZ, MXF: 10, 25, 150, 100 mg/kg/day
Nuermberger et al, Am J Respir Crit Care Med 2004;170:1131
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MOXI (2):
intermittent, twice-weekly regimens containing
Rifapentine + MOXI gave a stable cure 2 months earlier for drug
susceptible strains
(
)
BALB/c mice, H37Rv by aerosol, drugs by gavage, relapse checked after 3 mo. R10 (5/7); Z150 or 300 (5/7 or 2/7); H25
or 75 (5/7 or 2/7); M2 100 (2/d, 5/7 or 2/7); P10 or 15 mg/kg/day (5/7 or 1/7)
Rosenthal et al, Am J Respir Crit Care Med 2006;174:94
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Pharmacokinetics of RMP, RFP, MOXI
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Rosenthal et al, Am J Respir Crit Care Med 2005;172:1457; Nuermberger et al 2006;174:94;
MOXI (3)
against a highly resistant MDR strain
Fattorini et al, Antimicrob Agents Chemother 2003;47:360
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MOXI (4):
the activity of MOXI against RM22 was not enhanced
by 2nd line drugs and LNZ, with the exception of ETH
*
BALB/c, 5x105 i.v., drugs 5/7 (gavage) ETH 100; CS 300;TC 60; PAS 750; MXF 100, LZ 50
mg/kg/day
Fattorini et al, Antimicrob Agents Chemother 2003;47:360
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Other drugs
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RIFALAZIL
(KRM 1648)
Lenaerts et al, Antimicrob Agents Chemother 2000;44:3167
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Laca mice (out-bred), H37Rv, 1.5x105 iv, drugs daily by gavage, RMP 12, INH 10, PZA 25, EMB
16, Econazole 3.3 mg/kg/day
Ahmad et al. FEMS Microbiol Letters 2006;261:181
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Rapid methods to
screen drugs in mice
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(1)
¾ aerosol infection in IFN-γ KO C57BL/6 (GKO) mice
¾ efficacy after 8 days, less labor intensive, less
¾ active drug = 0.3 log10 CFU reduction
27
19
H 25
M 100
M 200
M 400
Lenaerts et al, Antimicrob Agents Chemother 2003;47:783
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(2)
Shoen et al, J Antimicrob Chemother 2004;53:641
Treatment began 1 d. post-infection (106 IN) and was administered
for 2 days. Mice were euthanized 3 days post-infection and
CFU in the lungs were determined: about 1 month saved.
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Mouse infections
with NTM
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M. avium
TR
Smooth
transparent
OP
Smooth
opaque
Fattorini et al, J Med Microbiol 1994;40:129
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in the Mφ the Transparent
variant induces very low IL-1β,
IL-6, TNF-α, GM-CSF and GCSF than the Opaque variant.
Low cytokine induction may be
a pathogenic mechanism of the
Transparent variant
OP
TR
Fattorini et al, J Med Microbiol 1994;40:129
The intracellular growth
of the Transparent
variant was associated
with induction of proteins
involved in cell-wall
synthesis (KasA), protein
synthesis (EF-Tu) and
fatty acid metabolism
(FadE2, FixA),
Brunori et al, Proteomics 2004;4:3078
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Recommended therapy against
M.avium infections
•
•
•
•
Clarithromycin (or azithromycin) +
Ethambutol
Rifabutin
Amikacin (given for the initial 2-3 months for
previously untreated cavitary disease)
Iseman, Clin Chest Med, 2002;23:633
Wagner & Young, Infection 2003; 31:257
Karakousis et al., Lancet Infectious Diseases, 2004; 4:557
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M.avium (905, type 1, TR)
Beige mice, MAC 905, 107 i.p., 91 days, drugs (5/7) by gavage
CLA 50; AMI, EMB 100; CIP 40; CLO 20; RFB 10 mg/kg/day
Fattorini et al, Microbial Drug Resistance 1999;5:227
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M.avium 905
Fattorini et al, Microbial Drug Resistance 1999;5:227
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M.avium 905:
the most active combination was
CLA-AMI-EMB, in keeping with recommended therapy
Fattorini et al, Microbial Drug Resistance 1999;5:227
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M.avium 905:
no mutants developed against CLA and
AK while several mutants developed against CLO.
Fattorini et al, Microbial Drug Resistance 1999;5:227
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M.avium 905: INH is not recommended against M.avium but
the combination CLA-AMI-INH is active in beige mice
Ctrl
INH
CLA-AMI
CLA / CLA-INH
AMI
INH-AMI
CLA+AMI-INH
Beige mice, MAC 905, 107 i.p., 91 days, drugs (5/7) by
gavage CLA 50; AMI, EMB 100; INH 50 mg/kg/day
Fattorini et al, Antimicrob Agents 1995; 39:680
Fattorini et al, Antimicrob Agents 1998; 42:712
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Smooth opaque
Smooth transparent
M.
celatum
TR
TR
BALB/c mice
OP
OP
Fattorini et al, Microbiology 2000;146:2733
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M.celatum:
in the beige mouse model, CLA and AZI are the most
effective and RFB and RFM the least effective, in keeping with clinical
observations
RMP, RFB
RMP, RFB
CIP
RMP, RFB
INH,
EMB, AMI
AMI
CLA, AZI,
EMB, AMI
CLA, AZI,
EMB
CLA, AZI
Beige mice, 107 i.p., 56 days, drugs (5/7) by gavage
CLA, AZI, AMI, EMB 100; RMP, RFB 10 mg/kg/day
Fattorini et al, Microbiology 2000;146:2733
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Drugs for latent TB
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active TB,
8 x 106 cases/year
latent TB,
2 x 109 individuals
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The Wayne tube
AER
ANA
0,06 % 02
1% 02
Muttucumaru, Tuberculosis, 2004;84:239
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Wayne et al, Antimicrob Agents Chemother 1994;38:2054 & 1996,64:2062
Heifets et al, Annals Clin Microbiol & Antimicrob 2005;4:6
Lenaerts et al, Antimicrob Agents Chemother 2005;49:2294
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Metronidazole (1)
Wayne et al, Antimicrob Agents Chemother 1994;38:2054
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Metronidazole (2)
Brooks et al, Antimicrob Agents Chemother 1999;43:1285
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Metronidazole (3)
(R,H,HR)
(RM,HM,HRM)
Paramasivan et al, Indian J Med Res. 1998;108:115
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First-line drugs plus PA-824
Swiss mice, H37Rv, aerosol, drugs 5/7 or 1/7 (gavage), relapse checked after 3 mo
R, 10; Z, 150; H, 25; PA-824, 100 mg/kg/day
Nuermberger et al, Antimicrob Agents Chemother 2006;50:2625
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MOXIFLOXACIN + PA-824
BALB/c BCG-vaccinated mice, H37Rv, aerosol, 6 mo, drugs 5/7 or 1/7 (gavage),
relapse checked after 3 mo
R10 (5/7); PZA, 150; EMB, 100; INH, 25 (5/7) or 75 (1/7); MXF, 100; PA-824 100 mg/kg/day
Nuermberger et al. Am J Respir Crit Care 2005;1452
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Laca mice (oubred mice). H37Rv, 1.5x105 iv, INH+PZA by gavage at 10
and 25 mg/kg for 12 wks. No CFU at this time. Econazole orally twice
daily for 6 weeks at 3.3 mg/kg/day. Dexamethasone for 2 days then
relapse checked after 6 wks
Ahmad et al. FEMS Microbiol Letters 2006;258,200
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Alternative drug
delivery systems
• Patient non-compliance is the major drawback
of long-duration chemotherapy of TB
• Adherence to treatment may be improved with
long-term duration drug formulations
releasing the drugs in a slow manner allowing
reduction in frequency and dosing numbers.
• The most studied delivery systems are
nanoparticles, for their high carrier
capacity, feasibility of administration
routes including inhalation and oral route.
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Nanoparticles (1)
.
Guinea pigs nebulized with PLG-NP
(poly-DL-lactide-co-glycolide) containing INH, RMP, PZA
Pandey et al, J Antimicrob Chemother 2003;52:981
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Nanoparticles (2)
Gelperina et al, Am Respir Crit Care Med 2005;172:1487
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CONCLUSIONS
• Experimental evaluation of anti-TB drugs in animals
is essential to screen new compounds and drug
combinations.
• The mouse, despite being an imperfect animal model
of human TB, is a cost-effective model extensively
used for preclinical assessment of experimental drugs
against TB.
• The guinea pig model is presently used mostly to test
alternative drug delivery systems.
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COLLABORATORS
• Manuela Pardini
• Elisabetta Iona
• Federico Giannoni
• Lara Brunori
• Carla Palma
• Yong-Jun Li
• Yuming Fan
• Dejiang Tan
• Graziella Orefici
• Xiao Yan
• Bo Li
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