The Toxicologist - Society of Toxicology

777 NONCLINICAL CARCINOGENICITY ASSESSMENT OF
PRASUGREL.
L. A. Buckley 2 , A. Sanbuissho 1 , J. J. Starling 2 , M. Knadler 2 and J. A.
Jakubowski 2 . 1 Daiichi Sankyo Co., Ltd., Tokyo, Japan and 2 Eli Lilly & Co.,
Indianapolis, IN.
Prasugrel, a thienopyridine ADP receptor antagonist, is an orally administered prodrug
requiring in vivo metabolism to form the active metabolite that irreversibly inhibits
platelet activation and aggregation mediated by the P2Y12 receptor. A comprehensive
nonclinical safety assessment including genotoxicity and carcinogenicity
studies supported the chronic use of prasugrel in patients with atherothrombotic
disease. A special nonclinical carcinogenic risk assessment was undertaken to address
FDA-specific concerns relating to the potential for increases in human cancers.
Prasugrel was negative in a battery of genotoxicity tests and was not oncogenic
in a 2-year rat carcinogenicity study. In a 2-year mouse study, an increase in hepatocellular
adenomas was observed; this effect was considered secondary to enzyme
induction and not relevant to human safety. Further, the absence of any increase in
common background tumors at any other organ site in either rodent study indicated
a lack of tumor promoting activity (apart from the CYP450 induction-related
increase in mouse liver tumors). Several in vitro and in vivo tumor progression
studies provided further support that prasugrel is not a tumor promoter.
Specifically, cell culture studies with 3 human tumor cell lines (lung, colon, and
prostate) demonstrated that exposure of serum-starved cells to prasugrel’s active and
major circulating human metabolites does not increase cell proliferation relative to
starved cells stimulated to proliferate by the addition of 10% FBS. In vivo studies
with nude mice implanted with these 3 human tumor cells also demonstrated that
daily dosing of prasugrel does not increase tumor growth relative to vehicle control
groups despite levels of active metabolite up to 34-fold higher than exposures
achieved at steady state in patients receiving 10 mg maintenance doses. In summary,
traditional genotoxicity and 2-year bioassay studies as well as specially designed
tumor promotion studies in mouse xenograft models clearly demonstrated
prasugrel’s lack of tumorigenic potential.
778 OFF-TARGET PLATELET ACTIVATION IN MACAQUES
BY A THERAPEUTIC MONOCLONAL ANTIBODY.
M. J. Santostefano 1 , N. E. Everds 2 , H. M. Vargas 3 , M. Fort 4 , P. Narayanan 4 , D.
Tran 2 , W. Pan 5 , J. Kirchner 6 , C. Vissinga 6 and G. S. Elliott 7 . 1 CBSS - Toxicology,
Amgen, Seattle, WA, 2 CBSS - Pathology, Amgen, Seattle, WA, 3 CBSS - Investigative
Toxicology, Amgen, Thousand Oaks, CA, 4 CBSS - Investigative Toxicology, Amgen,
Seattle, WA, 5 Pharmacokinetics and Drug Metabolism, Amgen, Seattle, WA,
6 Inflammation Research, Amgen, Seattle, WA and 7 Clinical Pathology, Charles River
Laboratories, Reno, NV.
AMGX is a fully human neutralizing IgG2 monoclonal antibody with clinical applications
in autoimmune and inflammatory diseases. Dosing with ≥100 mg/kg IV
in cynomolgus monkeys resulted in rapid profound thrombocytopenia with decreased
platelet granularity, lowered mean arterial pressure, and transient loss of
consciousness and flushing. In contrast, none of these effects were observed after
100 mg/kg IV treatment with any of three other neutralizing monoclonal antibodies
against the same pharmacological target, indicating that the in vivo findings
were specific to AMGX. In vitro, AMGX induced platelet activation in whole
blood and platelet-rich plasma from cynomolgus (M. fascicularis), pigtail (M.
nemestrina) or rhesus (M. mulatta) macaque species, but not from human (H. sapiens)
or baboon (P. c. anubis), as assessed by both aggregometry and surface expression
of CD62P and PAC-1. These results were also specific to AMGX since none of
the other 3 neutralizing monoclonal antibodies directed against the same target
were able to induce in vitro platelet activation in any of the species tested. Together,
these data demonstrate that AMGX mediated activation of platelets in vivo and in
vitro through an off-target mechanism in a manner that was specific to macaques.
779 CINACALCET HCL: 6-MONTH ORAL TOXICITY
STUDY IN JUVENILE BEAGLE DOGS.
K. Black 1 , C. Dean 1 , N. Everds 1 , E. Lesage 2 and M. Loomis 1 . 1 Amgen Inc.,
Thousand Oaks, CA and 2 Charles River Laboratories Preclinical Services, Senneville,
QC, Canada.
Cinacalcet HCl is a calcimimetic agent that is approved for the treatment of secondary
hyperparathyroidism in adult patients with chronic kidney disease receiving
dialysis. To support pediatric use, a 6-month oral toxicity study was conducted in
juvenile beagle dogs. Male and females (8/sex/group; 10 weeks of age) received
cinacalcet HCl by gavage at dose levels of 0, 10, 30 or 100 mg/kg/day for 6 months,
following which 4/sex/group were euthanized and necropsies conducted. The remaining
animals entered a 3-month treatment-free phase. Emesis, tremors and de-
168 SOT 2011 ANNUAL MEETING
creased activity and decreased food consumption occurred at all dose levels.
Decreased body weight gain occurred at ≥30 mg/kg. During week 10, dosing was
suspended for 7 days for some females that received ≥30 mg/kg to allow them to regain
appetite and body weight, and 1 of these animals from the 100-mg/kg group
was euthanized in week 13 due to failure to recover. Pharmacologically-mediated
decreases in serum ionized calcium occurred at all dose levels. Decreased red cell
mass and increased platelets occurred at ≥30 mg/kg. Nonadverse histologic findings
consisted of minimal growth plate thickening and several lymphoid changes in the
thoracic cavity (lymphoid hyperplasia and mononuclear cell infiltration in the
esophagus). There were no treatment-related histologic changes in the heart or reproductive
tract tissues. Slight decreases in bone densitometry and geometry parameters
occurred at ≥30 mg/kg. All of the changes were fully to partially reversible
during the treatment-free phase. Based on the clinical intolerance and growth effects,
eg, decreased body weight gain, noted at ≥30 mg/kg, the no-adverse-effectlevel
in this study was 10 mg/kg. Most of the changes observed in the study were either
relatively minor, consistent with the pharmacologic effects of cinacalcet,
secondary to emesis or effects on food consumption and body weight gain, and/or
observed previously in adult animals.
780 QUANTIFIED TRACKING AND MONITORING OF
DIAZEPAM TREATED SOCIALLY HOUSED
CYNOMOLGUS MONKEYS.
C. Rose 1 , J. E. van der Harst 2 , R. C. de Heer 2 , B. M. Spruijt 2 and S. H. Korte 1 .
1 Covance Laboratories GmbH, Muenster, Germany and 2 Delta Phenomics BV,
Utrecht, Netherlands. Sponsor: G. Weinbauer.
The objective of this study was to assess effects of diazepam using the automated
analysis of digitized video images (EthoVision XT, Noldus IT, The Netherlands) of
group housed cynomolgus monkeys (Macaca fascicularis). Males (n = 3) and females
(n = 3) were injected with Diazepam (1.0 mg/kg) and behavioural parameters
related to activity, locomotion and spatial movements were analysed. General parameters
such as distance moved and velocity did not reveal the known sedative effects
of Diazepam. However, inspection of the automatically generated track images
indicated that diazepam treated animals had a more a meandering walking pattern
suggesting that Diazepam induced a loss of balance which was regained by corrective
movements. Therefore, parameters revealing specific aspects of the movement
pattern such as velocity profiles and turn angles have been analyzed. Most prominent
effects are represented by changes in the shape of the tracks, shift of the velocity
profiles and increases in turn angles. An increase in the mean velocity can be observed
in males (+51.3%) and females (+126.5%). In males loss of balance is
evidenced by an increase in the circular deviation of the mean turn angle (+18.3%)
and a decrease in the duration of movement along a straight line (-34,8%). Females
were hardly active during the control situation, which confounded the comparison
of control situation versus Diazepam treatment. This study demonstrated that parameters
such as distance moved and velocity may be insufficient to distinguish
drug effects. The detection of the loss of balance and subsequent corrective movements
urged for subsequent adjustment of the analysis protocols. This study
demonstrates the indispensable value of automated computerized analysis of video
tracking for quantifying differences for different dose levels in pre-clinical safety assessment.
Expert-knowledge on the tracking and analysis methods are pivotal for
data-interpretation and an optimal use of the techniques used here.
781 28-DAY TOXICITY STUDY WITH THE NON-
TRADITIONAL VEHICLE SOLUTOL HS15 AND
POLYETHYLENE GLYCOL 400 IN FEMALE BEAGLE
DOGS.
D. Kemp, J. Hailey, H. Jordan, R. Brown and D. Bailey. GlaxoSmithKline,
Research Triangle Park, NC.
Conventional vehicles do not always provide adequate systemic exposure of the test
article formulation to assess toxicity, in these cases we will consider non-traditional
vehicles. The lack of toxicology information with these vehicles has presented a
challenge. This study determined the impact of various formulations of Solutol
HS15 (Solutol)/polyethylene glycol (PEG) 400 on Beagle dogs to determine the
tolerability and toxicity following co-administration in a 28-day, oral, repeat-dose
study in female beagle dogs. Five groups of dogs (3/group) were given either reverse
osmosis-treated water, 10% Solutol/90% PEG 400 (2 mL/kg/day), 30%
Solutol/70% PEG 400 (2 mL/kg/day), 10% Solutol/90% PEG 400 (5
mL/kg/day), or 30% Solutol/70% PEG 400 (5 mL/kg/day). All dogs tolerated the
administration of 10% Solutol/90% PEG 400 or 30% Solutol/70% PEG 400 at a
dose volume of 2 mL/kg/day. Loose/watery feces and minimal mucus-cell hyperplasia
of the ileum was present in all groups; however, a higher incidence of
loose/watery feces was consistently present in animals given the dose volume of 5
mL/kg/day. Total bilirubin was minimally increased at all doses. An increase in red