Activity of PNU 100480 and its major metabolite in whole blood and broth culture models of TB Paul Converse 1, Jin Lee 1, Kathy Williams 1, Opokua Amoabeng 1, Kim Dionne 1, Nicole Parish 1, Robert Wallis 2, Eric Nuermberger 1 1 Center for Tuberculosis Research, Johns Hopkins University, Baltimore, MD 2 Pfizer Inc., Groton, CT
PNU 100480 Thiomorpholinyl derivative of linezolid In vitro: MIC = 0.25 μg/ml vs. M. tb H37Rv In vivo: Rapidly metabolized to cmpds with ~ same MIC as parent
Dose ranging activity of PNU, LZD in mice Lung log10 CFU count 8 7 6 5 4 3 2 1 0 0 25 50 100 130 260 Dose (mg/kg) D0 count = 7.49 INH PNU-100480 Linezolid Parameter Regimen MIC (mg/l) C max (mg/l) AUC 0-24 (mg-h/l) PNU-100480 + metab. 0.25 21.7 117 100 mg/kg a LZD 130 mg/kg b 0.25 58.4 379 a Compiled (i.e., sum of the parent + metab) concentration-time profile used for PK calcs. b For comparison, steady state values in humans are: C max, 18.3-18.8 mg/l; AUC 0-24, 215-294 mg-h/l Williams et al, AAC (2009);53:1314
PNU contributes sterilizing activity to RHZ and novel combinations Proportion (%) of mice with relapse after treatment for: Regimen* 3 months 4 months 5 months 6 months 2RHZ + 4RH 18 of 20 (90%) 1 of 20 (5%) 0 of 20 (0%) 2RHZU + 2 RHU 9 of 20 (45%) 1 of 20 (5%)** 2RHZL + 2 RHL 20 of 20 (100%) *R = rifampin, H = isoniazid, Z = pyrazinamide, U= PNU-100480 (sutezolid), L = linezolid **p< 0.005 vs. RHZ/RH control % (proportion) of mice with relapse after treatment for: Regimen* 2 months 3 months 4 months 2RHZ/4RH 100% (15/15) 64% (9/14) JCPaU 93% (14/15) 13% (2/15)** 7% (1/15)* JCPa 100% (15/15) 60% (9 /15) 33% (5/15) *R = rifampin, H = isoniazid, Z = pyrazinamide, U= PNU-100480 (sutezolid), J = bedaquiline, C = clofazimine, Pa = PA-824 **p< 0.05 vs. RHZ/RH control Williams et al, Am J Respir Crit Care Med 2009 Williams et al, Antimicrob Agent Chemother 2012
Objectives of study To gain insight into why PNU is more bactericidal than LZD despite achieving lower exposures in mice by: 1. Comparing the concentration response profile for LZD and PNU in several in vitro models of: extracellular infection (broth, plasma), and intracellular infection (WBA, J774 macrophages) 2. Determining the respective contributions of PNU 100480 (the parent) and PNU 101603 (the principal metabolite) in the same assays
Time to positivity in WB cultures
Whole blood activity by calculated log kill
WBA of PNU 100480 and PNU 101603
Oxie activity in spiked WB or plasma from healthy volunteers
Time kill study in complete 7H9 broth
Time kill study in J774 macrophages
Modeling the respective contributions of PNU 480 and 603 in humans Developed dose response curve for each in vitro model Used plasma PK profiles from Ph I SAD study to estimate the log CFU ct at each point of sampling Using control CFU cts (Time 0 or untreated), calculated the area under the 24 hr killing (or inhibition) curve for a single 1 g daily dose for each cmpd Assumed additivity (based on in vitro checkerboard assay) and calculated the contribution of each cmpd to the overall activity of the combination
Cumulative activity over 24 hrs by model
Conclusions In vivo activity of PNU 100480 likely derives from the combined activity of the parent and the sulfoxide metabolite Though the metabolite comprises ~85% of total serum AUC, the parent drives the activity against intracellular bacilli (e.g., WBA, J774, mice?) due to its striking potency advantage On the other hand, activity vs. extracellular M.tb may be driven by the more abundant metabolite Modeling cell kill based on actual CFU from WB or plasma culture gives results more like human EBA The potency advantage of PNU 100480 over linezolid will likely be smaller against extracellular as opposed to intracellular bacilli
Next steps Similar analysis for linezolid Repeat WBA CFU cts using additional volunteers More sophisticated models of additive oxie effects, using steady state PK data from humans and mice Confirm with one or more additional strains
Acknowledgements The work Kathy Williams Paul Converse Opokua Amoabeng Intellectual support Rokeya Tasneen Jin Lee Tong Zhu Ken Stover Bob Wallis Steve Brickner Mark Mitton-Fry Colleagues at the TB Alliance Funding support Pfizer, NIH (R01-AI-090820)