MIC assays were performed according to methods published by Clinical and Laboratory Standards Institute (CLSI Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically Approved StandardmdashEighth Edition 2009 CLSI document M07-A8)

Immunocompetent lung infection model Female BALBc mice weighing 18 to 20 grams were infected with ~2 x 107 CFUmouse of P aeruginosa PA1145 a cystic fibrosis isolate from Childrenrsquos Hospital Boston via intranasal administration of 005 ml of cell suspension under light anesthesia One group did not receive drug treatment and lungs were harvested at 2 hour post-infection At 2 and 12 hours post-infection mice were treated with TP-433 tigecycline or amikacin intravenously or tobramycin intranasally Mice (n=6 per group) were treated with each drug concentration Twenty-four hours post initiation of treatment mice were euthanized by CO2 inhalation The lungs of the mice were aseptically removed weighed homogenized serially diluted and plated on MacConkey medium The plates were incubated overnight at 37 C in 5 CO2 CFU per gram of lung was calculated by enumerating the plated colonies then adjusting for serial dilutions and the weight of the lung Individual animal CFUgram lung data was plotted using GraphPad Prism Mean and standard deviations are show per dose group and statistical significance of dose group vs T=0 hr (2 hr post-challenge) or T=24 hr (24 hrs post-challenge) controls was determined by non-parametric Mann-Whitney analysis using GraphPad Prism

Immunocompetent thigh infection model Groups of 5 female specific-pathogen-free CD-1 mice weighing 22 plusmn 2 g were used On Day 0 animals were inoculated intramuscularly (01 mlthigh) with 3-5 x 106 CFUmouse of P aeruginosa PA694 a recent clinical isolate into the right thigh Two groups did not receive drug treatment and thighs were harvested at 2 hour post-infection Remaining mice were administered either vehicle TP-433 or meropenem at 2 and 12 hours post infection The muscle of the right thigh of each animal was harvested at 24 hr post-infection Harvested thigh tissues were homogenized in 2 ml of PBS pH 74 with a Polytron tissue homogenizer The homogenates were serially diluted and plated on Brain Heart Infusion agar + 05 charcoal (wv) for CFU determination per gram of thigh Individual animal CFUgram thigh data was plotted using GraphPad Prism Mean and standard deviations are show per dose group and statistical significance of dose group vs T=0 hr or T=24 hr controls was determined by non-parametric Mann-Whitney analysis using GraphPad Prism

Pharmacokinetics CD-1 mice (n=24) were received from Charles River Laboratories and were allowed to acclimate for two days prior to dosing A single dose was administered to each mouse by intravenous injection via the tail vein Body weight was measured prior to dosing and mice were dosed at a dose volume of 5 mLkg based on individual body weight Study animals were not fasted Mice (n=3 per time point) were euthanized using CO2 and blood samples were collected at sacrifice by cardiac puncture at eight PK time points (2 min 30 min 1 hr 2 hr 4 hr 6 hr 12 hr 24 hr) All time points were collected within 5 of the target time Blood collection (approximately 250 microL at sacrifice) was added into pre-chilled (0 ndash 4C) heparin-containing blood collection tubes The collected plasma (approximately 100 microL) was placed in sample tubes snap frozen on dry ice and immediately stored at nominally -80C Frozen plasma samples and dosing solution retains were analyzed for TP-433 concentration by LCMSMS using an internal standard Quality control (QC) samples (low medium high minimum of 6 standards with LLOQ lt 3 ngmL) and standard curves (in duplicate) were included in the bioanalytical run WinNonLin was used to determine individual and mean PK parameters ( Cmax Tmax Tfrac12 CL Vss AUC(0-t) AUC(0-) and MRT)

P aeruginosa is an opportunistic pathogen causing difficult-to-treat infections in compromised individuals such as those afflicted with diabetes cystic fibrosis and hospitalized patients who are intubated and undergoing mechanical ventilation

In rare circumstances P aeruginosa causes infections such as burn wounds folliculitis skin and soft tissue infections and external otitis in otherwise healthy individuals

Intrinsic resistance due to low permeability of the outer membrane and multidrug efflux pumps the spread of acquired resistance mechanisms including drug-inactivating enzymes and the dissemination of isolates with target-based drug resistance mutations has greatly reduced the effectiveness of currently marketed antibiotics against P aerguinosa infections

Tetraphasersquos chemistry platform has enabled the optimization of fully synthetic novel tetracycline-class molecules with improved activity against P aeruginosa in vitro (see Poster 1448) and demonstrated efficacy in vivo (this poster and Poster 1425) aimed at addressing this growing medical need

Objective To determine the PK in mice and to examine the efficacy of TP-433 in mouse lung and infection models challenged with P aeruginosa

Methods Lung infection model Immunocompetent female BALBc mice (18-20 grams) were infected with cystic fibrosis isolate P aeruginosa PA1145 (TP-433 MIC = 4 mcgml) via intranasal (IN) administration At 2 and 12 hours (hr) post-infection mice (n = 6) were treated intravenously (IV) with TP-433 (5 15 or 40 mgkg) tigecycline (40 mgkg) or amikacin (40 mgkg) Mice were euthanized by

CO2 inhalation 24 hr post-initiation of treatment Lungs were removed weighed homogenized serially

diluted plated on MacConkey agar and colony forming units (CFUs) per gram of lung were calculated Thigh model Groups of 5 female CD-1 mice (18-20 grams) were made neutropenic by administration of cyclophosphamide on Days -4 and -1 On Day 0 mice were inoculated with P aeruginosa PA694 (TP-433 MIC = 4 mcgml) into the right thigh TP-433 tigecycline and meropenem were administered at 5 15 and 40 mgkg IV 2 and 12 hr post-infection At 24 hr post-infection the muscle of the right thigh of each mouse was harvested homogenized serially diluted and plated on Brain Heart Infusion agar + 05 charcoal for CFU determination PK evaluation (WinNonLin) of TP-433 and tigecycline were performed in CD-1 mice after 1 mgkg IV administration and collection of 8 plasma samples over 24 hr TP-433 levels were quantified by LCMSMS

Results TP-433 was efficacious in the mouse lung infection model providing a 1 2 or 3-log reduction in CFUs at 3 12 and 29 mgkg respectively relative to 24-hr controls Amikacin a positive control reduced the CFUs in the lung by 355 log CFUs while tigecycline reduced CFUs less than one log In the neutropenic thigh model TP-433 at 40 mgkg provided a 22 log reduction from the 24-hr control counts while meropenem reduced bacterial burden ge4 log CFUs at doses ge15 mgkg The PK of 1 mgkg IV in mice produced a tfrac12 AUC(0-t) and Cmax of approximately 3 hr 348 ngmiddothmL and 487 ngmL respectively Conclusions TP-433 was efficacious in both P aeruginosa infection models In the lung model TP-433 was equipotent to amikacin at 40 mgkg and more potent than tigecycline at all doses TP-433 protected less well than meropenem in a neutropenic thigh model The AUC of TP-433 in mice was 26-fold higher than that observed with 1 mgkg IV tigecycline

The Intravenous Pharmacokinetics (PK) and Efficacy of TP-433 in Murine Infection Models Challenged with Pseudomonas aeruginosa

Y Deng T Grossman R Clark X-Y Xiao J SutcliffeTetraphase Pharmaceuticals Watertown US22nd ECCMID

31 March ndash 3 April 2012London United Kingdom

P 1426

ContactLeland Webster

Tetraphase Pharmaceuticalslwebstertphasecom

Abstract

Introduction

Results

Methods

Conclusion

Time Kill Assays

Pharmacokinetics of TP-433 administered IV to BALBc mice

TP-433 is a novel anti-Pseudomonas tetracycline that shows good pharmacokinetic properties in mice and strong efficacy in P aeruginosa lung and thigh infections

TP-433 4Tigecycline 16Amikacin 4

Tobramycin 05

Compound PA1145 MIC (microgml)

TP-433 4Meropenem 05

Compound PA694 MIC (microgml)

AUC0-t (nghmL) AUCinf (nghmL) T12 (hr) C0 (ngmL) Vss (Lkg) CL (mLminkg) MRT (hr)348 357 255 487 66 47 237

PK Parameter 1 mgkg IV

cfu reduction

p001 vs untreated 0 hr control

p001 vs untreated 24 hr control2

4

6

8

10

n=5 mice all other groups n=6 mice

Dose Group

P aeruginosa ImmunocompetentMurine Lung Infection Model

Lo

g 10

CF

U p

er

gra

m l

un

g

24 h

r

2

4

6

8

10

Dose Group

cfu reduction

p001 vs untreated 0 hr control

p001 vs untreated 24 hr control

P aeruginosa ImmunocompetentMurine Thigh Model

Lo

g 10

CF

U p

er

gra

m t

hig

h

24 h

r

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