ANIMAL MODELS FOR THE STUDY OF ANTIMYCOBACTERIAL DRUGS

Transcription

1 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

2 . MAJOR TOPICS Anti-TB drugs Comparative features of animal models Global discovery program for novel anti-tb drugs Importance of fluoroquinolones and other drugs Rapid methods to screen drugs in mice Mouse infections with NTM Drugs for latent TB Alternative drug delivery systems

3 Anti-TB drugs

4 FIRST-LINE DRUGS against TB Isoniazid (H) Rifampin (R) Pirazinamide (Z) Streptomycin (S) Ethambutol (E) bactericidal bactericidal bactericidal bactericidal bacteriostatic oral oral oral injectable oral WHO 6 mo daily treatment: 2 mo HRZ(S/E) + 4 mo RH

5 SECOND-LINE DRUGS against TB KM, CM, AK ETH CS, PAS, TC OFL, CIP, MOX GAT, LEV bactericidal bactericidal bacteriostatic bactericidal injectable oral oral oral

6 PROMISING NEW DRUGS against TB Spigelman & Gillespie, Lancet 2006; 367:947

7 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.

8 Comparative features of animal models of pulmonary TB

9 Humans Mouse Rabbit Guinea pig Mononuclear cell granulomas Caseation necrosis Liquification/cavitation Extrapulmonary dissemination BL-3 space requirements NA Maintenance costs NA (adapted from McMurray et al, Trends in Molecular Medicine, 2001;7:135)

10 The mouse has been estensively used for preclinical assessment of experimental drugs against TB Medina & North, Immunology 1998; 93:270

11 Dose and routes of mouse infection for anti-tb drug screening Low-dose, aerosol infection CFU High dose infection i.v., i.n., i.p CFU (Orme et al, Antimicrob Agents Chemother 2001;45:1943)

12 Global discovery program for novel anti-tb drugs

13 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) ( 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.

14 TAACF (1): in vitro screening MICs against drug susceptible and resistant strains Cytotoxicity in Vero cells Killing in mouse bone marrow Mφ 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 (

15 TAACF (2): standard mouse model low-dose aerosol infection in C57BL/6 mice INH 25 mg/kg/day as positive control active drug = 0.7 log10 CFU reduction Orme et al, Antimicrob Agents Chemother 2001;45:1943

16 TAACF (3): rapid mouse model low-dose aerosol infection in IFN-γ KO C57BL/6 (GKO) mice efficacy after 8 days, less labor intensive, less compound active drug = 0.3 log10 CFU reduction H 25 M 100 M 200 M 400 Lenaerts et al, Antimicrob Agents Chemother 2003;47:783

17 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) (

18 The importance of fluoroquinolones for TB in particular moxifloxacin, to shorten the duration of TB therapy, and for the treatment of MDR TB

19 MOXIFLOXACIN: a stable cure was achieved 2 months earlier by substituting MOX for INH for treatment of drug susceptible strains 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

20 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); M (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

21 Pharmacokinetics of RMP, RFP, MOXI Rosenthal et al, Am J Respir Crit Care Med 2005;172:1457; Nuermberger et al 2006;174:94;

22 MOXI (3) against a highly resistant MDR strain Fattorini et al, Antimicrob Agents Chemother 2003;47:360

23 MOXI (4): the activity of MOXI against RM22 was not enhanced by 2 nd line drugs and LNZ, with the exception of ETH * BALB/c, 5x10 5 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

24 Other drugs

25 RIFALAZIL (KRM 1648) Lenaerts et al, Antimicrob Agents Chemother 2000;44:3167

26 Laca mice (out-bred), H37Rv, 1.5x10 5 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

27 Rapid methods to screen drugs in mice

28 (1) aerosol infection in IFN-γ KO C57BL/6 (GKO) mice efficacy after 8 days, less labor intensive, less active drug = 0.3 log10 CFU reduction H 25 M 100 M 200 M 400 Lenaerts et al, Antimicrob Agents Chemother 2003;47:783

29 (2) Shoen et al, J Antimicrob Chemother 2004;53:641 Treatment began 1 d. post-infection (10 6 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.

30 Mouse infections with NTM

31 M. avium TR Smooth transparent Smooth opaque OP Fattorini et al, J Med Microbiol 1994;40:129

32 in the Mφ the Transparent variant induces very low IL-1β, IL-6, TNF-α, GM-CSF and G- CSF 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

33 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

34 M.avium (905, type 1, TR) Beige mice, MAC 905, 10 7 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

35 M.avium 905 Fattorini et al, Microbial Drug Resistance 1999;5:227

36 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

37 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

38 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, 10 7 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

39 Smooth opaque Smooth transparent M. celatum TR BALB/c mice TR OP OP Fattorini et al, Microbiology 2000;146:2733

40 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 CLA, AZI CLA, AZI, EMB, AMI AMI CLA, AZI, EMB Beige mice, 10 7 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

41 Drugs for latent TB

42 latent TB, 2 x 10 9 individuals active TB, 8 x 10 6 cases/year

43

44 The Wayne tube AER ANA 0,06 % 0 2 1% 0 2 Muttucumaru, Tuberculosis, 2004;84:239

45 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

46 Metronidazole (1) Wayne et al, Antimicrob Agents Chemother 1994;38:2054

47 Metronidazole (2) Brooks et al, Antimicrob Agents Chemother 1999;43:1285

48 Metronidazole (3) (R,H,HR) (RM,HM,HRM) Paramasivan et al, Indian J Med Res. 1998;108:115

49 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

50 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 mg/kg/day Nuermberger et al. Am J Respir Crit Care 2005;1452

51 Laca mice (oubred mice). H37Rv, 1.5x10 5 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

52 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.

53 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

54 Nanoparticles (2) Gelperina et al, Am Respir Crit Care Med 2005;172:1487

55 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.

56 COLLABORATORS Manuela Pardini ElisabettaIona Federico Giannoni Lara Brunori Carla Palma Yong-Jun Li Yuming Fan Dejiang Tan Xiao Yan Bo Li Graziella Orefici