1. Animal Efficacy Studies for Antitubercular Agents V. Balasubramanian ASTRAZENECA R&D BANGALORE, INDIA

2.  Do we need animal model(s) for determining antitubercular efficacy?  Is the determination of blood levels sufficient to predict the outcome against infection? Some questions…...

3. Drug ClassIn vitro modein vivo efficacy of killingparameter  -lactamsTime dependentT > MIC AminoglycosidesConcentration dependentAUC/AUIC ratio FluoroquinolonesConcentration dependentAUC/AUIC ratio MacrolidesTime dependentAUC [half-lives and PAE high] T > MIC [half lives and PAE low] Some generalizations…..

4. Drug [mg/kg]MICCmaxFoldT 1/2 T > MIC [ug/ml][ug/ml] [h][h] Isoniazid [25]0.027350218 Rifampicin [25]0.1101003.528 Rifabutin [25]0.05102004510 d Rifapentine [25]0.021575013.25 d Pyrazinamide [100]63051036 Ethambutol [100]24236 Pharmocokinetic Properties of Anti-mycobacterial Drugs

5. Drug [mg/kg]MICCmaxFoldT 1/2 T > MIC [ug/ml][ug/ml] [h][h] Ethambutol [100]24236 Ethionamide [100]12224 Streptomycin [200]0.53628 Amikacin [200]13030212 Kanamycin [200]630526 Sparfloxacin [50]0.122.85.6518 Ofloxacin [200]1.01020735 Linezolid [50]17.71.4 PNU-100480 [50]70.7 Pharmocokinetic Properties of Anti-mycobacterial Drugs

6. Effect of Chemotherapy on Survival - Kradolfer et al Design Intravenous infection with M. bovis Ravenal strain Oral administration of drugs - day 11 & 12 post infection (Sm - s.c.) Minimal effect: Prolonging the survival of 50% mice in the treated groups - ED 50 HREAS Data from: Kradolfer, F. 1970. Antibiotica et Chemotherapia. 16:352-360 ED 50 [mg/kg] Isoniazid (H)2.7 - 3.4 Rifampicin (R)4.5 - 6.5 Ethambutol (E)35 - 55 Ethionamide (A)75 - 107 Streptomycin (S)75 - 108

7. Bactericidal Effect of Chemotherapy - Kradolfer et al Design Intravenous infection with M. bovis Ravenal strain Bactericidal effect: Culture negativity after prolonged oral treatment Ref: Kradolfer, F & Schnell, R. 1971. Chemotherapy. 16:173-182

8. Bactericidal Effect: An Initial Measure for Comparison Iv infection (~10 7 cfu); treatment by gavage started 1 wk pi., for 4 wks. Cynamon, M. H., Klemens, S. P., Sharpe, C. A., Chase, S. 1999. A. A. C. T. 43:1189-1191 Klemens, S. P., Grossi, M. A., Cynamon, M. H. 1994. A. A. C. T. 38: 2245-2248 Isoniazid, RifamycinsOxazolidinones

9. Baohong Ji, Lounis, N., Truffot-Pernot C., Grosset, J. 1995. A. A. C. T. 39: 1341 - 1344 Lounis, N., Baohong Ji, Truffot-Pernot C., Grosset, J. 1997. A. A. C. T. 41: 607 - 610 Aminoglycosides & Quinolones Iv infection (~10 7 cfu); treatment by gavage started 1 day pi., for 4 wks. Bactericidal Effect: An Initial Measure for Comparison

10. Bactericidal Effect: Infection Dose As a Variable Treatment by gavage started 1 day pi., for 4 wks. iv infection (~10 5 cfu)iv infection (~10 7 cfu) Miyazaki, E., R. E., Bishai, W. R. et al. 1999. A. A. C. T. 43: 85-89

11. PK Parameters Significant bactericidal activity in mice H >>> Z > Emb > Eth [Cmax, T > MIC] H > Z > Emb > Eth Rfp = Rlz = Rbu > Rif [Cmax, T > MIC] Rlz = Rfp > Rbu > Rif Ami > Sm = Kan [Cmax] Ami > Sm = Kan Spar > Lev = Ofla [Cmax] Spar > Lev = Ofla H > Linezolid > PNU100480 [?] H > PNU100480 > Linezolid No single parameter can independently Rlz = Rfp > Rbu > Rif = Spar > H predict across drug classes H > PNU100480> Lin > Z Z = Ofla > Emb > Eth

12. Clinical Efficacy in Tuberculosis Early Bactericidal Activity Sputum Conversion Emergence of resistance Relapse Rates MarkersFactors Combination Regimen Duration of Rx Frequency of Dosing

13. Inh, Rif, Emb Pza Rif [Early Bactericidal Activity] [Sterilizing Activity] 10 8 10 7 10 6 10 5 10 4 10 3 10 2 246 Cfu/ml in sputum Time (months) Why combination is needed and why is it at least six months long?

14. Drug Isoniazid++++ Rifampicin+++++ Pyrazinamide++++ Ethambutol+- Streptomycin+- Early Bactericidal Activity [EBA] Sterilizing Activity Measured by Cfu from sputum for 1 st 2 days after onset of treatment Relapse rates, 30 mo. after onset of treatment ImportanceCommunityIndividual

15. Model for the Initial and Continuation Phases of Rx Initial Phase Log 10 cfu /spleenSpontaneous Relapse [once daily for 2 months] (6 mo. post) Untreated06.65 ± 0.19 Inh+Rif25 + 103.00  0.54 Rif+PZA10 + 1500.86  0.44 *Inh+Rif+PZA25 + 10+ 1502.79  0.73 Continuation Phase % mice with [once daily for 4 months] +ve spleen cultures *+* 25+10+150035% *+Inh+Rif 25+10038% *+Rif+PZA 10+15009% Ref: J. Grosset, Truffot-Pernot, C., Lacroix, C., Ji. B. 1992. A.A.C.T. 36: 548-551 iv infection (10 7 cfu/animal)Rx: Oral gavage started 14 days pi.

16. Shortened Course - Daily Treatment with Rifapentine SpleenLungs Inh+Rif Inh+Rif+Pza Inh+Pza+Rfp 7/7 6/7 0/8 7/7 3/8 7/7 6/7 0/8 7/7 3/8 Ref: Cynamon, M. H. et al. 1999. A. A. C. T. 43: 2356-2360

17. Intermittent treatment with Rifapentine Rx 12 weeks iv. infection ~10 7 cfu / animal Cynamon, M. H. et al. 1999. A. A. C. T. 43: 2356-2360 Rx 8 weeks 47% Rif r mice 61% Grosset J. et al. 1998. Am J Respir Crit Care Med. 157:1436-1440

18. 0 BCGInfection 28 Dosing 44100128 8 wk 12 wk Prophylaxis Ref: Jabes, D., Bruna, C. D., Rossi, R., Olliaro, P. 1994. A. A. C. T. 38:2346-2350 *

19. Cornell Model for Effect on Reactivation Disease Inh+Pza 7 wks Test Rx 6 wks Infect iv 8.8x10 5 Cort 3 wks Sac Cort 3 wks Sac 291523263235Wks. Question: After the initial phase, what confers sterilization? Test regimens were R [15mg/kg]; RH [H25]; RZ [Z150]; RHZ % positive organs : R 81; RH 63; RZ 65; RHZ 71 [p = 0.3] However, trend chi-square suggested than addition of H or Z improved the sterilization effect of R Answer: At least in the Cornell model, none of the existing regimens confer complete sterilization Ref: Dhillon, J., Dickinson, J. M., Sole, K., Mitchison, D. A. 1996. A. A. C. T. 40: 552-555

20. Cornell Model for Effect on Reactivation Disease Inh+Pza 12 wks No Rx Test Rx 18 wks No Rx No Rx Cort Infect iv 3.95x10 5 Sac 0121634 Culture -ve organs 414448 TreatmentDosing% PositiveLog10cfuLog10cfu organsspleenlungs Placebo1003.24  2.25.77  0.11 Inh 25daily501.93  2.110.71  1.34 Rif 10twice weekly1004.11  0.405.39  0.49 Rpt 10once weekly1003.63  1.184.67  2.35 Inh+Rptonce weekly37.51.46  2.041.78  2.49 Ref: Miyazaki, E., Chaisson, R. E., Bishai, W.R. 1999. A. A. C. T. 43:2126-2130

21. Rifapentine Animal Data Daily dosing with Rfp offers the possibility of reduced Rx. However, due to long half life, possibility of accumulation Based on animal studies, twice weekly better than once weekly Based on daily dosing in animals, HZRfp > HZRif (relapse rates) However, relapse rates not determined in the case of intermittent Rx!) Intermittent Rfp approved by FDA in 1988 (1st in >15 yrs for TB) Clinical Trial data (surprises!) 2HRZ/4H 2 R 2 better than 2HRpZ/H 1 Rp 1, based on relapse data 4/5 HIV+ patients in 2HRpZ/H 1 Rp 1 developed Rif res Ref: Vernon, A. et al. 1998. Am. J. Resp. Crit. Care. Med. 157: A467

22. ROUTE OF INFECTION AS A VARIABLE Infection RouteIntravenousRespiratory Infection Dose10 6 cfu/animal10 cfu/animal Quantitationcfu / spleencfu / lungs InfrastructureSimpleSpecialized Tuberculosis Genitourinary 16.0% Peritoneal 3.7% Meningeal 4.2% Bone/Joint 8.5% Other 9.3% Miliary 9.8% Pleural 21.5% Lymphatic 27.0% Pulmonary 84.5% 14.6%

24. Model for Evaluating Primary Efficacy Time (days) 84 Respiratory Challenge (10 cfu / animal) Infect with M. tuberculosis Intravenous Challenge (10 6 cfu / animal) Log 10 cfu / lungs 14

25. 35 Log 10 cfu / organ Days Resp infection (100 cfu / animal) Once a week oral Rx Brooks, J & Orme, I. 1998. A. A. C. T. 42: 3047-3048 1/wk; 5 wks (resp) vs 6/wk;12 wks (iv) Similar results Combination Rx in the Respiratory Infection Model

26. Infect with M. tuberculosis RZ:8wk Test 3wk. 1wk Model for Evaluating Sterilization [20 wk. assay]

27. Bactericidal Effect Determined in Guinea Pigs Ref: Smith, D. W., Balasubramanian, V., Wiegeshaus, E. H. 1991. Tubercle. 72:223-2310

30. Culture negativity Limitation: poor thera. Index Not predictable from PK/PD Relapse rates 2HRZ/4RZ > 6HRZ > 2HRZ/4HR [8%] [34%] [38%] Towards clinical use... No drug given singly HRZ > HR > RZ > HZ RE, RS, HE, not effective In humans 2HRZ/4HR = 6HRZ [~8%]. 2HRZ/4RZ not given due to Z toxicity Rlz = Rfp > Rbu > Rif = Spar > H H > PNU100480 > Lin > Z = Ofla = Lev Z > Emb > Eth All significantly better than untreated control Compounds exhibiting statistically significant reduction in cfu PK predictive of efficacy within but not across Clinical Trial?

31. Clues from Animal Efficacy for Clinical Efficacy EBA Sputum conversion Drug resistance Relapse rates @ 30 mo. Duration of Rx Frequency of Rx Humans ?? Rate of culture negativity Resistance of survivors Relapse rates @ 6 mo. Time to culture negativity + relapse rates @ 6 mo. PK profiles such as T 1/2 Expt. animals

32. Efficacy Models During the Course of Development Effect (singly or combination) during early phase of infection *Survival *Bacterial counts Spontaneous Relapse after initial and continuation phase Reactivation following immunosuppression - Cornell Prophylaxis Effect on immunocompromised hosts All of the above in animals infected via the airway will be more efficient and relevant

33. VariablesBactericidal effectSpontaneous relapse* Vaccination-- Vaccination-infection interval-- Route of infectioni.v. or respi.v. or (resp) Infection inoculum~10 7 cfu or 100 cfu~10 7 cfu or (100 cfu) Infection-Drug interval2-4 weeks2-4 weeks Duration of initial treatment8 wks (iv); 2 wks (resp)8 wks; (4 wks) Duration of continuation Rx -16 wks; (8 wks) Duration of immunosuppression-- Post treatment interval1 day1 day, 3 & 6 months Measure of efficacyCfu / spleen or lungsCfu / spleen or lungs % mice with +ve organ cultures *conditions shown in parentheses not yet established Summary of important variables for the different efficacy models

34. VariablesSterilization*Prophylaxis Vaccination-BCG Vaccination-infection interval-4 weeks Route of infectioni.v. or (resp)i.v. or resp Infection inoculum~10 7 cfu or (100 cfu)~10 7 cfu or 100 cfu Infection-Drug interval2-4 weeks2-4 weeks Duration of initial treatment12 weeks8-12 weeks Duration of continuation Rx 12 weeks- Duration of immunosuppression3 weeks- Post treatment interval1 day, 2-4 weeks1 day Measure of efficacyCfu / spleen or lungsCfu / spleen or lungs % mice with +ve organ cultures *conditions shown in parentheses not yet established Summary of important variables for the different efficacy models