Evaluation of Tissue Distribution of TR-700 in Healthy Volunteers Using Microdialysis Martina Sahre1, Sreedharan Sabarinath1, PhD Maria Grant2, MD, Christoph Seubert3, MD, PhD, Carisa deAnda4, PharmD Hartmut Derendorf1, PhD University of Florida, College of Pharmacy, Departments of Pharmaceutics1, Therapeutics and Pharmacology2, Anesthesiology3, Trius Therapeutics4, San Diego, California

Overview Introduction Study Design Results Conclusion TR-700 / TR-701 Rationale Microdialysis Study Design Results Conclusion

TR-700 Oxazolidinone class Phosphate ester prodrug (TR-701) Limited water solubility Phosphate ester prodrug (TR-701) Rapid conversion to active drug

TR-700 Oxazolidinone class Indication complicated skin and skin structure infections (cSSSI) In vitro activity against: S. aureus (MS and MR), Linezolid-resistant MRSA carrying binding-site mutations, cfr gene S. pneumoniae (PS, PR), S. pyogenes, S. agalactiae, Enterococci (inc.. VRE) Rennie et al. (2003), Im et al. (2007), Jones et al. (2009), Shaw et al. (2007), Schaadt et al. (2009)

Rationale Plasma concentrations linked to PK/PD indices Free drug in tissue more relevant Drug molecules need to reach tissue Factors: pH of tissues, pKa of drug, density of tissue, blood-tissue barrier, lipophilicity. FDA and EMEA suggest to measure target tissue drug levels for dose-response studies

Mean Ceftobiprole Concentrations After Single 500 mg i.v. Dose Mean Linezolid Concentrations After Single 600 mg i.v. Dose C(tissue) < fc(plasma) C(tissue) > fc(plasma) Total Plasma,  Muscle Tissue,  Adipose Tissue, --- Free Plasma Barbour et al. (2009), Deghanyar et al. (2005)

Rationale Biopsy, skin blisters, imaging techniques AUC of Moxifloxacin after 400 mg p.o single dose: Measure unbound PK parameter in tissue using microdialysis Plasma Muscle Tissue Subcutaneous Tissue Saliva Capillary Blood Blister AUC [µg*h/mL] 19.8 8.5 8.0 17.6 18.7 12.3 Factors: pH of tissues, pKa of drug, density of tissue, blood-tissue barrier, lipophilicity. FDA and EMEA suggest to measure target tissue drug levels for dose-response studies Mueller et al. (1999)

Microdialysis Principle Diffusion through a semipermeable membrane “Mimics” blood vessel Sampling tool Assess free drug directly No volume added or taken from tissue. Only analyte taken or delivered. Courtesy of CMA

Recovery Probe calibration → recovery factor Probe is continuously perfused → no equilibrium cdialysate < cmedium Recovery defined as ratio cdialysate/cmedium Dialysate: liquid recovered from probe Medium: tissue or solution containing compound of interest Factors influencing recovery: Temperature, Membrane length, Flow-rate, Molecular size, Membrane properties, Tissue properties Probe calibration → recovery factor

Retrodialysis Perfusate contains known concentration of compound Dialysate concentration measured Calculated recovery used to adjust drug concentrations from experiment Courtesy of CMA

Study Design TR-700 measured in plasma, s. c. adipose and muscle tissue 15 healthy volunteers Pilot (n = 3) and Main Study (n = 12) Two microdialysis probes implanted Retrodialysis to calibrate probe Washout period Main Study Single dose of study drug, 600 mg p.o. Dialysate samples every 20 minutes for 12 h, blood samples for 24 h after dosing Protein binding samples 0, 2, 12h post dose CMA 60 probe, Courtesy of CMA, Sweden)

Pilot Study Results Recovery > 90% Washout period 4 h

Main Study: Results Protein Binding 87 % (1.3%) PB samples taken from each subject at 0, 2 and 12 h post dose and analysed using ultrafiltration. Each individuals plasma c(total) were adj. with their respective protein binding.

Mean PK Parameters (SD) Unit Plasma fPlasma Adipose Tissue Muscle Tissue Cmax (µg/mL) 5.37 (1.51) 0.70 (0.27) 0.66 (0.16) 0.74 (0.15) Tmax§ (hr) 2 (1-4) 3.33 (2.33-10.3) 3.67 (1.67-7) AUC0-12 (µg*h/mL) 38.8 (7.55) 4.96 (1.09) 5.27 (1.28) 5.95 (1.11) AUC0-24 57.1 (14.7) 7.28 (1.91) - AUC∞ 70.0 (24.8) 8.89 (3.06) 16.6 (15.8) 14.6 (10.5) t½§ 8.13 (5.94-12.8) 9.22 (5.98-85.9) 9.59 (6.15-48.2) CL/F (L/hr) 9.46 (2.91) * Vz/F (L) 113 (19.3) fAUC0-12, tissue/ fAUC0-12, plasma  1.08 (0.22) 1.22 (0.18) Protein Binding % 87.2 (1.3) * Not applicable; § Median (Range); λz: terminal elimination rate constant Cmax similar, T max 1 to 1.5 h later, AUC0-12 similar, t1/2 about 1 h longer in tissue, and free AUC0-12 ratios 1.1 and 1.2 show that free drug has no major barriers to distributing into tissues (observed only).

Conclusion TR-700 conc. in observed tissues similar to unbound plasma concentrations No significant difference between AUC0-12 for unbound plasma, adipose and muscle tissues Drug and microdialysis well tolerated Degree of tissue distribution corresponds with free drug available in plasma which is corroborated by the AUC0-12 being not stat. sign. Diff.

Acknowledgements Prof. Hartmut Derendorf Maria Grant, MD and Christoph Seubert, MD, PhD Sreedharan Sabarinath, PhD General Clinical Research Center at the University of Florida Philippe Prokocimer, MD Trius Therapeutics, Inc.