Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
The protocol was approved by the Novartis Animal Care and Use Committee (NACUC) of Novartis Pharmaceuticals Corporation and the study was conducted in compliance with all regulatory and NACUC requirements. The NACUC Approved Generic Protocol number was TX 4055.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
no guideline followed
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
IGS Wistar Hannover Rat; Crl: WI(Glx/BRL/Han)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
40 males and 40 females

Room temperature: 68-76°F (target range)
Room relative humidity: 30-80% (target range)
Lighting cycle: Fluorescent light for an approximate 12-hour light/12-hour dark cycle.
Animal caging: In pairs (except during the urine collection period); stainless steel wire bottom caging.
Acclimation period: Approximately 1 week
Diet: Certified Rodent Diet 18% #5LG3 (PMI feeds, Richmond,Indiana) ad libitum, except for study-defined fasting procedures and during the urine collection period.
Water: Water from the animal drinking supply was available ad libitum except during the urine collection period.
Environmental Enrichment: 100% nylon bones/balls (e.g., nylabones) were provided to each animal.

There were no known contaminants in the food or water expected to interfere with the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% (w/v) sodium carboxymethylcellulose (CMC), type 7HF, aqueous solution
Duration and frequency of treatment / exposure:
Route and frequency: Orally (gavage), once daily
Duration of dosing: At least 2 weeks

Dose volume: 5 mL/kg
Administration method: Syringe attached to a rigid metal feeding tube. Dosing times were documented in the raw data file. The dosing
formulations were shaken first and then stirred (via a magnetic stir plate and stir bar) prior to and during dosing.
Unused portions of dosing formulations were discarded at the end of each dosing day, with the exception of day 4 dose formulations, which were sent to Analytical Chemistry, Shared Local Services for analysis in response to clumps noted in the high dose formulation upon completion of dosing.
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
No. of animals per sex per dose:
four groups of animals (n=10/sex/group) at doses of 0 (control), 10, 50 and 150 mg/kg/day AHU377 base (equivalent to 0, 10.46, 52.3 and 156.9 mg/kg/day AHU377 calcium salt, respectively) at a dose volume of 5 mL/kg. Animals were dosed once daily for at least 2 weeks.
Control animals:
yes, concurrent no treatment
Details on study design:
Major activities Date, duration or study week
Protocol signed 08-Apr-2004
Animal arrival 06-Apr-2004
Start of pretest 12-Apr-2004
First day of dosing phase 19-Apr-2004 (day 1 of week 1)
Dosing phase length At least 2 weeks
Clinical observations Daily unless otherwise noted below
Ophthalmoscopic examinations Pretest and week 2
Clinical pathology Week 2
Toxicokinetic blood sampling Days 1-2 and 15-16
Last day of dosing phase* 04-May-2004
End of dosing (EOD) necropsies 04- and 05-May-2004
*All EOD scheduled sacrifices were dosed through and including the day prior to sacrifice.
Details on dosing and sampling:
Blood was obtained from the study animals on study days 1/2 and 15/16. Two animals per sex
per group were bled at 0.5, 1, 2, 6, and 24 hours post dose. Approximately 0.5 mL of whole
blood was collected from the retro-orbital venous plexus of anesthetized animals into tubes
containing EDTA and approximately 10-20 mg of sodium fluoride (NaF) and placed on ice.
BAPK weighed out NaF into EDTA-containing tubes, and the amount of NaF added to each
tube was recorded in the raw data.
All blood samples were centrifuged at approximately 4oC within approximately 30 minutes
after collection, and plasma was collected and transferred to a tube containing 5 μL H3PO4
(85%) and frozen at approximately -20ºC before transfer to the Toxicokinetic investigator.
All samples were analyzed, when possible. Plasma concentrations of AHU377 and the active
metabolite LBQ657 were measured using an LC-MS/MS method and AUC, AUC/dose, Cmax,
Cmax/dose, and tmax were determined when possible.
Statistics:
Generally, means and standard deviations were calculated on time-point specific data sets
(body weights, food consumption, clinical chemistry, hematology) and statistical differences
were assessed by an Analysis of Variance (ANOVA) followed by Bartlett’s test for
homogeneity of variances. If the variances were homogeneous, Dunnett’s t-test was employed
to determine the statistical significance between control and treated groups. If the variances
were not homogeneous, a modified t-test was used to determine which groups were
statistically different from the controls. For quantitative urinalysis and organ weight data
means and standard deviations were calculated. Plasma concentration-time data were
analyzed using suitable pharmacokinetic methods and were performed by BAPK. Specific
methodologies were documented in the final toxicokinetics report.

Results and discussion

Preliminary studies:
rats [Study 0370107], in which rats were administered AHU377 at dose levels of 50, 250, and
1000 mg/kg/day (base). In this study, one male dosed at 1000 mg/kg/day was sacrificed
moribund on day 7 with clinical signs of decreased locomotor activity, dehydration, labored
respiration, and respiratory rales. Test article-related clinical signs were noted in both sexes
(males at doses ≥ 250 mg/kg/day and in females at 1000 mg/kg/day). Decreases in mean body
weight gain were present in both sexes at doses ≥ 50 mg/kg/day, with slight body weight loss
in males and females dosed at 1000 mg/kg/day. Food consumption was slightly decreased in
both sexes at 1000 mg/kg/day. Clinical chemistry findings included slight increases in mean
alanine aminotransferase and aspartate aminotransferase activities in males dosed at
1000 mg/kg/day. Thymus weights were decreased in males at doses ≥ 250 mg/kg/day and in
females at 1000 mg/kg/day and correlated microscopically with lymphoid depletion.
Test-article related microscopic changes were present in the thymus (increased apoptosis
accompanied by lymphoid depletion; both sexes; doses ≥ 50 mg/kg/day) and stomach
(hyperplasia of nonglandular epithelium and concomitant hyperkeratosis at doses
≥ 50 mg/kg/day; vacuolation of nonglandular epithelium at doses ≥ 250 mg/kg/day; erosion of
glandular stomach and ulceration of the nonglandular stomach, male at 1000 mg/kg/day).
Therefore, doses of 10, 50, and 150 mg/kg/day were chosen for this study.
Main ADME results
Type:
absorption
Results:
(Cmax) at 0.5 h (tmax) or 1 hour post dose

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Analysis of plasma samples from group 1 (vehicle control) showed no measurable
concentrations of AHU377 and LBQ657. Rats in all treated groups were exposed to
AHU3777 and LBQ657. However, for the 10 mg/kg/day and 50 mg/kg/day dose groups, the
measurable concentrations of AHU377 were observed only up to 1 hr except for one sample at
2 hr. For the 150 mg/kg/day dose group, measurable concentrations of AHU377 were
observed in all samples up to 6 h post dose.
AHU377 was rapidly absorbed, reaching peak plasma concentration (Cmax) at 0.5 h (tmax) or
1 hour post dose. AHU377 (prodrug) was rapidly converted to LBQ657 (active metabolite).
Measurable concentrations of LBQ657 were observed in all treated groups up to 6 hr post
dose or longer. The Cmax of LBQ657 was at least 16 times greater than that of AHU377.
There were not enough non-zero data points to calculate the AUC for AHU377 for the
10 mg/kg/day and 50 mg/kg/day dose groups. For the 150 mg/kg/day dose group, there was
an approximate 20-fold greater exposure (AUC) for LBQ657 compared to AHU377.

No consistent difference in AHU377 and LBQ657 exposure or plasma concentration were
observed between male and female rats. After single and multiple doses with AHU377,
exposure to LBQ657 for female rats increased generally proportionally with dose. There
appeared to be a trend toward over proportional exposure to LBQ657 for male rats at the
150 mg/kg/day dose. However, the limited amount of data does not allow a clear conclusion.
The mean plasma exposure for LBQ657 on day 1 was similar to the exposure on day 15
suggesting no accumulation, with the possible exception of the male group dosed at
50 mg/kg/day (~2 fold) and the females dosed at 150 mg/kg/day (~ 3 fold).
Details on distribution in tissues:
There are no biologically significant alterations in organ weights when comparing the means
for the various groups. Relatively few macroscopic lesions were recorded at necropsy; none
are considered to be an unequivocal effect of the test article. The microscopic lesions seen in
the control and high-dose animals are considered to be incidental or a result of experimental
manipulation. None are considered to be a test article effect.
Based on the data generated by this study, the test article produces no changes to specific
organ weights, and no macroscopic or microscopic lesions in the organs and tissues evaluated.
Toxicokinetic parametersopen allclose all
Key result
Test no.:
#1
Toxicokinetic parameters:
AUC: 1310 ± 339 and 2630 ± 896 ng·h/mL
Remarks:
in males and females
Key result
Test no.:
#2
Toxicokinetic parameters:
Cmax: 383 and 497 ng/mL
Remarks:
in males and females

Metabolite characterisation studies

Metabolites identified:
yes
Remarks:
LBQ657
Details on metabolites:
Chemical name
4-{[(1S,3R)-1-(biphenyl-4-ylmethyl)-4-ethoxy-3-methyl-4-oxobutyl]amino}-4-oxobutanoic acid
Salt form / Ratio salt / acid / Ratio salt /base:
None
Molecular formula / Molecular weight
C22H25NO5 / 383.45

After oral administration, AHU377 (prodrug) was rapidly absorbed and converted to its active
metabolite (LBQ657) as indicated by its short tmax values (tmax ≤ 1.0 hr). No difference in
exposure to AHU377 and LBQ657 was observed between male and female rats after single
and multiple doses. Exposure to LBQ657 increased approximately proportionally with dose.
The exposure to LBQ657 was about 20-fold greater than that to AHU377 for the
150 mg/kg/day dose group. No accumulation of LBQ657 occurred with repeated dose.

Applicant's summary and conclusion

Conclusions:
No mortality occurred during the study and there were no clinical signs, changes in mean body weight parameters, or food consumption changes attributable to treatment with AHU377 at doses of 10, 50, or 150 mg/kg/day (base).
There were no treatment-related changes in hematology or urinalysis, or clinical chemistry changes indicative of overt toxicity in animals administered AHU377 by oral gavage at 10, 50, and 150 mg/kg/day for 2 weeks. Potentially treatment-related mild decreases in triglyceride concentration were present for males at doses ≥ 50 mg/kg/day and in females at 150 mg/kg/day.
There were no organ weight changes, macroscopic, or microscopic findings attributable to treatment with AHU377.
After oral administration, AHU377 (prodrug) was rapidly absorbed and converted to its active metabolite (LBQ657) as indicated by its short tmax values. No difference in exposure to AHU377 and LBQ657 was observed between male and female rats after single and multiple doses. Exposure to LBQ657 increased approximately proportionally with dose. The exposure to LBQ657 was about 20-fold greater than that to AHU377 for the 150 mg/kg/day dose group.
No accumulation of LBQ657 occurred with repeated dose.
In conclusion, administration of AHU377 to rats via oral (gavage) for two weeks at doses of 10, 50 and 150 was well tolerated at all dose levels examined. Potentially treatment-related findings were limited to decreases in triglyceride concentrations in males at doses ≥ 50 mg/kg/day and in females at 150 mg/kg/day and were not considered to be of toxicologic importance. Therefore, the no-observable-adverse-effect level in males and females was determined to be 150 mg/kg/day. The no-effect-level was 10 mg/kg/day in males and 50 mg/kg/day in females.