Transcript (PK) and Efficacy of TP-433 in Murine Infection Models Challenged

P 1426
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. Sutcliffe*
Tetraphase Pharmaceuticals, Watertown, US
Abstract
T1/2 (hr)
2.55
C0 (ng/mL)
487
10
6

4
cfu reduction:
m
g/
kg
IV
g/
kg
40
m
40
cl
in
e
ec
y
tig
to
br
am
g/
kg
m
40
3
43
TP
-
yc
in
IV
IV
*
g/
kg
TP
-4
3
TP
-4
3
3
5
re
a
un
t
3
PA1145 MIC
(µg/ml)
4
16
4
0.5
Compound
TP-433
Tigecycline
Amikacin
Tobramycin
P. aeruginosa Immunocompetent
Murine Thigh Model
Log10 CFU per gram thigh @ 24 hr
10

8

6


4
cfu reduction:
Time Kill
Assays
 , p0.01 vs. untreated 0 hr control
, p0.01 vs. untreated 24 hr control
40
m
TP
-4
33
33
-4
TP
m
er
g/
op
kg
en
IV
em
m
5
er
m
op
g/
kg
en
em
IV
15
m
er
m
op
g/
kg
en
em
IV
40
m
g/
kg
IV
g/
kg
15
m
5
33
-4
TP
IV
IV
m
g/
kg
te
d
re
a
re
a
te
d
24
0
hr
hr
2
un
t
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 mL/kg 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 µL at sacrifice) was added into pre-chilled (0 – 4C) heparincontaining blood collection tubes. The collected plasma (approximately 100 µL) was placed in sample tubes,
snap frozen on dry ice and immediately stored at nominally -80C. Frozen plasma samples and dosing
solution retains were analyzed for TP-433 concentration by LC/MS/MS using an internal standard. Quality
control (QC) samples (low, medium, high; minimum of 6 standards with LLOQ < 3 ng/mL) and standard
curves (in duplicate) were included in the bioanalytical run. WinNonLin was used to determine individual and
mean PK parameters ( Cmax, Tmax, T½, CL, Vss, AUC(0-t), AUC(0-), and MRT).
15
m
te
d
m
g/
kg
24
IV
*
hr
*
hr
0
te
d
re
a
un
t
Dose Group
un
t
Immunocompetent thigh infection model. Groups of 5 female specific-pathogen-free CD-1 mice weighing
22 ± 2 g were used. On Day 0, animals were inoculated intramuscularly (0.1 ml/thigh) with 3-5 x 106
CFU/mouse 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
7.4 with a Polytron tissue homogenizer. The homogenates were serially diluted and plated on Brain Heart
Infusion agar + 0.5% charcoal (w/v) for CFU determination per gram of thigh. Individual animal CFU/gram
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..
IN
 , p0.01 vs. untreated 0 hr control
, p0.01 vs. untreated 24 hr control
*n=5 mice; all other groups n=6 mice
 Tetraphase’s 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.
Immunocompetent lung infection model. Female BALB/c mice weighing 18 to 20 grams were infected with
~2 x 107 CFU/mouse of P. aeruginosa PA1145, a cystic fibrosis isolate from Children’s Hospital, Boston, via
intranasal administration of 0.05 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 CFU/gram 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 MannWhitney analysis using GraphPad Prism.


 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.
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
Standard—Eighth Edition, 2009. CLSI document M07-A8).
MRT (hr)
2.37


8
 In rare circumstances P. aeruginosa causes infections such as burn
wounds, folliculitis, skin and soft tissue infections, and external otitis in
otherwise healthy individuals.
Methods
CL (mL/min/kg)
47
P. aeruginosa Immunocompetent
Murine Lung Infection Model
2
 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.
Vss (L/kg)
6.6
40
Introduction
AUCinf (ng·h/mL)
357
in
Conclusions: TP-433 was efficacious in both P. aeruginosa infection models. In the lung model TP433 was equipotent to amikacin at 40 mg/kg, 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
2.6-fold higher than that observed with 1 mg/kg IV tigecycline.
AUC0-t (ng·h/mL)
348
IV
*
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 mg/kg, respectively, relative to 24-hr controls. Amikacin, a positive
control, reduced the CFUs in the lung by 3.55 log CFUs while tigecycline reduced CFUs less than one
log. In the neutropenic thigh model, TP-433 at 40 mg/kg provided a 2.2 log reduction from the 24-hr
control counts while meropenem reduced bacterial burden ≥4 log CFUs at doses ≥15 mg/kg. The PK
of 1 mg/kg IV in mice produced a t½, AUC(0-t), and Cmax of approximately 3 hr, 348 ng·h/mL, and 487
ng/mL, respectively.
PK Parameter @ 1 mg/kg IV
g/
kg
Methods: Lung infection model: Immunocompetent female BALB/c mice (18-20 grams) were infected
with cystic fibrosis isolate P. aeruginosa PA1145 (TP-433 MIC = 4 mcg/ml) 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 mg/kg), tigecycline (40 mg/kg), or amikacin (40 mg/kg). 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 mcg/ml) into the right thigh. TP-433, tigecycline, and meropenem were administered
at 5, 15, and 40 mg/kg 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 + 0.5% charcoal for CFU determination. PK evaluation (WinNonLin) of TP-433 and tigecycline
were performed in CD-1 mice after 1 mg/kg IV administration and collection of 8 plasma samples over
24 hr. TP-433 levels were quantified by LC/MS/MS.
Pharmacokinetics of TP-433 administered IV to BALB/c mice
Log10 CFU per gram lung @ 24 hr
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.
Results
m
ECCMID
31 March – 3 April 2012
London, United Kingdom
Contact:
Leland Webster
Tetraphase Pharmaceuticals
[email protected]
am
ik
ac
22nd
Dose Group
Compound
TP-433
Meropenem
PA694 MIC
(µg/ml)
4
0.5
Conclusion
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