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Description of key information

In a 52-week repeated dose oral toxicity study in marmosets treated with AHU377, a NOAEL of 25 mg/kg/day has been reported based on an increase in incidence and frequency of emesis. This is the lowest NOAEL observed for all repeated dose toxicity studies performed by oral route.


In other repeated dose oral toxicity studies performed in rats (13-weeks and 26-weeks in duration ), NOAELs of 400 mg/kg/day and 600 mg/kg/day were reported . Furthermore, 13-week repeated dose oral toxicity study in mice via diet revealed a NOAEL of 1000 mg/kg/day based on effects on serum chemistry. 


Finally, in a 104-week study, no effects were observed after oral administration of AHU 377 by gavage on rats at doses 50, 150 and 400 mg/kg/day. The NOAEL from the chronic MArmoset study has been identified as the point of departure for repeated dose toxicity.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 7 November 2005 to 4 January 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
yes
Remarks:
Animal used: non-rodent (marmoset).
Principles of method if other than guideline:
The purpose of this nonclinical laboratory study was to establish the toxicologic effects of AHU377, a neutral endopeptidase inhibitor, when administered to marmosets for at least 52 weeks. The study was designed to establish a no observable adverse effect level (NOAEL), to estimate the toxicokinetic profile of AHU377 and its metabolite LBQ657, and to estimate the potential for reversal of any toxic effects within a 4-week recovery phase.

AAHU377 (Batch nos. 0451002 and 0522003) was administered orally by gavage in 0.5% (w/v) sodium carboxymethylcellulose (CMC), type 7HF, aqueous solution to 4 groups (5/sex/group) of male and female marmosets (Callithrix jacchus) at doses of 0, 25, 100 and 200 mg/kg/day (base) for at least 52 weeks. Additional animals (2-4/sex) were included in the control and high dose groups and served as recovery animals for at least a 4-week recovery period upon the completion of at least 52 weeks of dosing. The dosing volume for all animals was 10 mL/kg. The animals were obtained from the Novartis stock colony. At the initiation of dosing, animals were approximately 13 months to 6 years of age and weighed 258.7 to 484.6 g for males and 249.8 to 468.9 g for females. Beginning 20-Jun-2006 (day 204) animals were supplemented with multivitamins, calcium, and vitamin D3 which were admixed into the food. Clinical observations, body weight, estimated food consumption determinations, ophthalmoscopic examinations and electrocardiographic evaluations were performed on all groups. Clinical pathology assessments were made on blood and urine samples collected pretest and during weeks 13, 25 (blood only), 52/53, and at the end of recovery. Clinical pathology assessments were also made on individual animals prior to early sacrifice and/or to monitor animal health. In addition, blood for special chemistries was also obtained from individual animals as part of a health check and from all surviving study animals during week 36 in order to monitor animal health specifically with respect to calcium metabolism. Blood was collected from study animals for toxicokinetic evaluations during weeks 1, 8, 39/40 and 50 at approximately 1, 2, 6 and 24 hours postdose and when possible from animal(s) prior to their early sacrifice. Organ weights were collected from scheduled sacrifice animals.
Complete necropsies including macroscopic assessment were performed. Microscopic examinations were performed on all gross lesions, and on protocol specified tissues from all main study animals and for all unscheduled sacrifices. Thyroid gland and gross lesions were processed from the recovery animals
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Active ingredient: AHU 377 (also identified as AHU377 Calcium salt per certificates of analysis E-05-49055 and E-05-53979 and AHU377 CALCIUM MODB FINE per certificate of analysis
B-06-42792. Variant names included AHU377-BAA.001 per certificates of analysis E-05-49055 and E-05-53979 and AHU377-BAA.002 per certificate of analysis B-06-42792.
Batch nos.: 0451002, 0522003 (beginning week 40); See Table 3-1.
Salt/base ratio: 1.046 (all certificates of analysis)
Drug content: 96.8-99.1%.
Species:
other: Marmoset
Strain:
other: Callithrix jacchus
Sex:
male/female
Details on test animals or test system and environmental conditions:
3.2.1 Experimental animals
Animal species and strain: Marmoset (Callithrix jacchus)
Breeder/supplier: Novartis stock colony
Number of animals ordered: 33 males and 34 females. See section 3.3.5.
Number of animals assigned to the dosing phase: 26 males and 26 females. See section 3.3.5.
Age: Approximately 13 months to 6 years (at start of dosing).
Body weight range: 258.7 to 484.6 g for males and 249.8 to 468.9 g for females (at start of dosing)
Acceptability: Following acclimation, a staff veterinarian released healthy animals for study purposes. After the start of treatment, the extra animals were returned to the Laboratory Animal
Services (LAS) colony.

3.2.2 Animal quarters/husbandry
Building/Animal room: 406/rooms 454, 455, 457, 458 during the dosing period and rooms 454 and 455 from the final day of terminal necropsy (01-Dec-2006, day 368) through the recovery necropsy. Animals were housed separately by sex.
Room temperature: 76-84°F (target range).
Room relative humidity: 40-80% (target range).
Lighting cycle: Fluorescent light for an approximate (± 1 hour) 12-hour light/12-hour dark cycle.
Animal caging: Singly housed in modified primate socialization cages.
Animal no. 1506 (control, female) was repeatedly found outside of its cage. Caging was modified to prevent future occurrences. Animal no. 1008 (control, male) was found outside of its cage on several occasions. In each case, the animal was examined and found to be in good health,
although animal no. 1008 was observed with skin bruising following one incident. No impact on study.
Acclimation period: At least 3 weeks
Diet: Purina 5040 New World Primate biscuits soaked in honey water with sweet potato and either macaroni, eggs, green beans, whole wheat bagel, fruit or vegetables or other approved treats (e.g., cereal, marshmallows). Modifications to the diet/feeding schedule (e.g., rice added to diet, increased quantity of food) were made as necessary for individual animals and were documented in the raw data file.
Animals were supplemented with Rep-Cal Herptivite
Multivitamin and Rep-Cal Calcium with Vitamin D3 (or equivalent) as documented in the raw data file beginning 20-Jun-2006 (day 204). These supplements were admixed into the daily food rations and this procedure was initiated following the early sacrifice of a control animal with a
suspected case of rickets. Animal no. 3001 (male, 100 mg/kg/day) was fasted overnight
on 17-Jul-2006 (day 231) due to a veterinary procedure performed on 18-Jul-2006.
Water: Water from the animal drinking supply was available ad libitum, except during urine collection period, from automatic dispensers.
Environmental enrichment: Included wood sticks, perches, nesting boxes with bed-o-cobs and/or music.
There were no known contaminants in the food or water that interfered with the conduct of the study.
Route of administration:
oral: gavage
Details on route of administration:
Route and frequency: Orally via gavage once daily, unless noted otherwise

Justification for route: Intended route in humans
Dose volume: 10 mL/kg
Doses were calculated and administered to one (tenths)
decimal place. A 5 cc syringe was used for animals weighing
= 500 grams. For animals weighing > 500 grams, a 10 cc
syringe was used.
Administration method: Syringe attached to an infant feeding tube or oral gavage
dosing catheter
Dosing times were documented in the raw data. The dosing
formulations were shaken 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, except when noted otherwise in the
toxicology raw data file.
Immediate reflux (IR) was documented following dosing for
individual animals of the control and AHU377-treated dose
groups. These occurrences were documented in the raw data
file and determined to have no impact on the study.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% (w/v) sodium carboxymethylcellulose (CMC), type 7HF, aqueous solution
Details on oral exposure:
Administration method: Syringe attached to an infant feeding tube or oral gavage dosing catheter
Dosing times were documented in the raw data. The dosing formulations were shaken 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, except when noted otherwise in the toxicology raw data file.
Immediate reflux (IR) was documented following dosing for individual animals of the control and AHU377-treated dose groups. These occurrences were documented in the raw data file and determined to have no impact on the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
AHU377 suspensions in 0.5% CMC, 2.09 mg/mL to 31.38 mg/mL (as salt), were found to be
stable for at least 35 days when stored at 6°C and at least 4 hours stirring at room temperature.
The 0.5% CMC was found to be suitable for use for at least 4 hours stirring at room
temperature, at least 15 days when stored at room temperature and at least 40 days when
stored at 6°C. The uniformity of the suspensions was confirmed. The samples of the dose
formulations for weeks 1, 4, 13, 26, 39 and 52 were analyzed. The concentrations were 95%
to 107% of targets. Six control samples were analyzed, and no AHU377 was detected.
Duration of treatment / exposure:
52 weeks
Frequency of treatment:
daily
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
+ 2 males and 4 females recovery for control
+ 3 recovery for the highest dose
Control animals:
yes, concurrent no treatment
Details on study design:
Pretest period: 3 weeks
Duration of dosing: At least 52 weeks
Recovery period: At least 4 weeks

- Dose selection rationale: Doses for this study were based on the results of a 4-week oral (gavage) toxicity study in marmosets with a 4-week recovery period with AHU377 alone and in combination with valsartan and hydrochlorothiazide [study 0470051]. In that study, AHU377 alone was administered at doses of 10, 50 and 200 mg/kg/day (base) for 4 weeks. Test article-related clinical signs were limited to feces with apparent compound and emesis with or without feed and/or apparent compound in animals receiving 200 mg/kg/day. No test article-related effects were noted for mortality/moribundity, body weight, food consumption, ophthalmology, electrocardiography, hematology, biochemistry, urinalysis, organ weights or macroscopic/microscopic pathology. Since the emetic changes at 200 mg/kg/day were considered dose limiting, a no observable effect level (NOEL) for AHU377 alone was established at 50 mg/kg/day. Therefore, doses of 0, 25, 100 and 200 mg/kg/day (base) were selected for this study.

- Rationale for animal assignment (if not random): Animals placed on study were selected based on available results from pretest examinations (e.g., body weights, estimated food consumption, clinical signs, clinical pathology, EKG’s and eye examinations) and manually assigned to 4 dose groups from the selected population based on pretest cage position, animal room (when possible) and sex. The manual assignment/randomization was documented in the raw data file and subsequently entered into the Xybion computer system. Animals from each dose group were housed in each animal room, whenever possible. No animal reassignments were made during the pretest or dosing phases of the study.

- Fasting period before blood sampling for clinical biochemistry: 18 hours (overnight).
- Rationale for selecting satellite groups: not relevant
- Post-exposure recovery period in satellite groups: not relevant
Observations and examinations performed and frequency:
3.4.1 Mortality
Twice daily (AM and PM) on weekdays and at least once daily on weekends, holidays and plant emergencies/closings. Due to inclement weather and a site closing on 09-Dec-2005 (day 11), the p.m. mortality check was not performed.

3.4.2 Clinical signs
Pretest and recovery: At least once daily
Dosing period: At least twice daily (prior to dosing and at approximately 2 hours postdose, except when noted otherwise in the raw data file). Additional observations were obtained as necessary and documented in the raw data.

3.4.3 Body weight
Pretest period: At least once weekly
Dosing period: Days 1, 4, 8 and 15 and at least weekly thereafter. .
Recovery period: Once weekly
Additional body weights were obtained as part of a health check for individual animals and
documented/retained in the raw data file.
Fasted terminal body weights were collected by Toxicology personnel prior to transfer to
Pathology for all scheduled necropsies, for the calculation of relative organ to body weight
determinations.

3.4.4 Food consumption
Estimated daily, from the beginning of the pretest period to the end of the recovery period,
except after any overnight fast, unless noted otherwise.

3.4.5 Ophthalmology
Pretest: All animals
End of dosing: All surviving animals
Recovery week 4: Not needed as there were no test article-related findings observed during the treatment phase.
Animals were anesthetized per SOP.

3.4.6 Electrocardiography
Pretest: All animals
Weeks 14, 26, 39 and 51: All surviving animals at approximately 1.5-2.5 hours postdose
Recovery week 4: Not needed as there were no test article-related findings observed during the treatment phase.
Tracings deemed unacceptable for evaluation by the cardiologist, were re-collected.
Sacrifice and pathology:
3.5 Clinical pathology
Hematology, hemostasis/thrombosis and/or clinical chemistry assessments were conducted pretest and during weeks 13, 25, 52 and at the end of recovery. Additional blood samples were obtained upon authorization by the Study Director (e.g., prior to early sacrifice, animal health monitoring, etc) for individual animals during weeks 9, 14, 29, 34, 38, 39 and/or 43 as documented in the raw data file. Results from these “unscheduled” analyses are included in the final report. Blood specimens were divided into EDTA-containing tubes for hematology (approximately 0.5 mL), serum collection tubes for clinical chemistry (approximately 1 mL) and sodium citrate tubes for coagulation (approximately 0.9 mL). Urine analyses were conducted pretest and during weeks 13 and 52/53 and at the end of recovery. An additional urine sample was obtained upon authorization by the Study Director (animal health monitoring) for an individual animal during week 42 as documented in the raw data file.
Results from this “unscheduled” analysis are included in the final report. For the collection of urine specimens (approximately 1 mL), up to a 3-hour collection from metabolism cages was obtained unless noted otherwise in the raw data.
All samples submitted were analyzed, whenever possible. Samples not obtained or deemed unacceptable for analysis by Clinical Laboratory personnel, were re-collected at the discretion of the Study Director, taking into account Novartis blood volume limits. Bone marrow smears
were not evaluated.
The left leg was routinely used for sampling; when the right leg was utilized, a notation was made in the raw data. Once the sample was obtained, the vein (or artery) was held off for at least 2 minutes. Other information relative to blood collection procedures was recorded in the raw data file: i.e., if arterial sampling was suspected, animals requiring multiple needle sticks (three or more), and/or animals that struggled excessively or were difficult to bleed.
The following parameters were assessed:

3.5.1 Hematology parameters:
erythrocytes Wintrobe indices white blood cell count hematocrit red cell distribution width (RDW) white blood cell differential hemoglobin reticulocytes platelets

Hemostasis/thrombosis parameters:
prothrombin time (PT) activated partial thromboplastin time (aPTT) fibrinogen

3.5.2 Clinical chemistry parameters:
alanine aminotransferase glucose calcium alkaline phosphatase blood urea nitrogen inorganic phosphorus aspartate aminotransferase creatinine triglycerides total bilirubin creatine kinase (CK)* cholesterol total protein sodium A/G ratio albumin (A) potassium globulins (G) chloride
*Isoenzyme analysis was not performed as determined by the Clinical Pathologist.

3.5.3 Special chemistry parameters:
Blood specimens (approximately 3 mL whole blood) were obtained from selected control animals as part of a health check (animal nos. 1004, 1007 and 1008 on 15-Jun-2006, day 199) and from all surviving study animals during week 36 (days 246 and 247). Samples were transferred into serum collection tubes, frozen at approximately -70° C and transferred to Seena Polivy, AniLytics Inc., 200 Girard Street, Gaithersburg, MD 20877 for determination of the below referenced parameters. These determinations were made in order to monitor animal health specifically with respect to calcium metabolism.
total calcium 1, 25 (OH) 2 Vitamin D3 parathyroid hormone ionized free calcium 25 (OH) Vitamin D3 phosphorus

3.5.4 Urinalysis parameters:
specific gravity glucose* protein* bilirubin* ketones* urobilinogen* blood* pH*
*test strip determinations

3.6.1 Necropsy procedures
Animals were fasted overnight (approximately 18 hours) prior to terminal necropsy. A prosector order was prepared per SOP. Complete necropsies were performed on all animals with a recording of macroscopic abnormalities for all protocol tissues.

3.6.2 Euthanasia:
For transfer to necropsy, Toxicology personnel anesthetized each animal with an
intramuscular injection (left leg) of a mixture of ketamine HCL plus xylazine HCL. Animals
were euthanized by induction of deep anesthesia with a sodium pentobarbital based iv solution
followed by exsanguination.

3.6.3 Sampling, weighing and histological processing of tissues:
Representative samples of the protocol tissues listed were collected and those indicated were weighed. Organ weights were recorded only at scheduled sacrifices. Fixation and storage of specimens were in 10% neutral-buffered formalin, except for the initial fixation of testes and epididymides (Bouin’s) and eyes (3% Sorensen’s buffered glutaraldehyde). Animal identification (implanted microchip) was stored together with tissue samples. Paired organs were weighed together and the parathyroid gland was weighed with the thyroid gland. Bone marrow smears were collected/prepared on all animals sacrificed early and on all surviving animals at scheduled necropsy.
The tissues specified in the list were processed to hematoxylin and eosin-stained tissue sections from all main study animals and for all unscheduled sacrifices. Sternum and femur from animal no. 1506, thyroid gland from the recovery males and macroscopic lesions from all recovery animals were additionally processed for microscopic evaluation.
Protocol tissues that were lost or could not be processed or examined were noted in the pathology raw data and the anatomic pathology appendix of this final report. These occurrences did not interfere with or impact the histological assessment of the study.

3.6.4 Microscopic examination and peer review:
All tissue sections processed were assessed with all observations recorded in the raw data. All
assessments were peer-reviewed per SOP. Bone marrow smears were not evaluated.
Other examinations:
3.7 Genomics sampling for possible investigational analyses
Prior to necropsy, at least 2.5 mL of blood was collected, stored and/or transferred per SOP.
The left leg was routinely used for sampling; if the right leg was utilized, a notation was made
in the raw data. Once the sample was obtained, the vein (or artery) was held for at least
2 minutes. The following information relative to blood collection procedures was recorded in
the raw data file: arterial sampling if suspected, animal(s) requiring multiple needle sticks
(i.e., three or more), animal(s) that struggled excessively or were difficult to bleed.
Tissues identified for genomics were the first samples taken (when possible) at scheduled
necropsies immediately after obtaining organ weights. Samples were shipped on dry ice for
possible gene expression analysis.
These exploratory investigations are not considered part of this study and as such, results of
these analyses, if any, were not included in the final report. Retention and reporting of any
data generated from these evaluations is the responsibility of ITOX (Investigational
Toxicology).

Statistics:
Generally, means and standard deviations were calculated and an Analysis of Variance
(ANOVA) performed on time-point specific data sets (e.g., body weights, clinical chemistry,
hematology). The ANOVA was followed by a Bartlett’s test for homogeneity of variances.
When the variances were homogeneous, Dunnett’s t-test was employed to determine the
statistical significance between control and treated groups. When the variances were not
homogeneous, a modified t-test was used to determine which groups were statistically
different from the controls. For organ weight data, an automated program was used to decide
whether parametric or non-parametric group comparisons should be made. This program uses
Kolmogorov's test to examine the normality of the data and Bartlett's test to examine the
homogeneity of variances. Accordingly, either Dunnett’s test or Student's t-test for parametric
group comparisons or Dunn's test or Wilcoxon's test (U-test) for non-parametric group
comparisons were used. For quantitative urinalysis data, means and standard deviations were
calculated. Specific methodologies used by DMPK are documented in the final toxicokinetics
report
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs considered test article-related at 25 mg/kg/day in either sex.
Emesis with or without feed and apparent compound was observed in individual males
(animal no. 2001 on days 12, 14, 139 and 202; animal no. 2003 on days 40, 96, 152-153, 181,
191 and 193; animal no. 2005 on days 80, 228 and 308) and females (animal no. 2501 on days
326, 333, 345, 346; animal no. 2504 on day 69; animal no. 2505 on days 26, 39, 97, 104 and
199). Emesis with apparent blood was also noted in female animal no. 2505 on a single day
(day 39). Due to the isolated/sporadic nature of these signs, they are not considered
toxicologically significant.
Emesis with or without feed and apparent compound was considered test article-related at the
higher doses (100, 200 mg/kg/day) based on the increased number of animals affected and/or
the increased frequency of occurrence, generally throughout the dosing period. The frequency
of emetic occurrences was more variable and not clearly dose-dependent as some individual
animals had about 80-200 episodes of vomiting (animal nos. 3002, 3005, 3505, 4002, 4005,
4007, 4502, 4503, 4505, 4506) and others had much fewer, about 20-70 episodes (animal nos.
3004, 3504, 4003, 4008, 4501, 4504, 4507, 4508).

During the recovery phase, no episodes of emesis were noted in any of the recovery animals,
indicating reversibility.
Fecal changes (e.g., soft, diarrhea, mucoid) were observed during the pretest, treatment and
recovery phases of the study. There were no distinguishable differences in the incidence and
severity of the various fecal changes observed in the treated animals compared to controls; as
such, the fecal changes were not attributable to AHU377. The skin bruising noted across all
dose groups including controls was related to the blood collection procedures.
All other clinical signs observed during the study were considered incidental and unrelated to
treatment with AHU377 due to the nature of the sign, lack of a dose response, occurrence
during the pretest phase, and/or occurrence in the control group.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test article-related moribundity occurred during the study.
Six animals were sacrificed prior to study termination for humane reasons and/or due to
moribundity. See section 4.4 for pathology results.
Animal no. 1007 (control, 0 mg/kg/day) was sacrificed on day 185. Prior to sacrifice this
animal exhibited clinical signs of a thin appearance (days 180-185), decreased locomotor
activity (day 184), and severe (100%) diarrhea (days 180-181). Body weight loss was also
evident over the course of the treatment period with a 36% loss on day 183 compared to
baseline. Upon necropsy, this animal (assigned as a male) was determined to be a female.
The cause of moribundity in this animal could not be determined based upon anatomic
pathology evaluations.
Animal 2003 (male, 25 mg/kg/day) was sacrificed on day 199. Prior to sacrifice relevant
clinical signs included a thin appearance (days 184-199), dehydration (day 191) and decreased
locomotor activity (day 199). Body weight loss was also evident over the course of the treatment period with a 32% loss on day 197 compared to baseline. The cause of moribundity
of the animal could not be determined based upon anatomic pathological evaluations.
Animal 3001 (male, 100 mg/kg/day) was sacrificed on day 234. Prior to sacrifice, clinical
signs included sunken eyes and/or dilated pupils (days 231-234), lacrimation, labored
respiration, visible swellings/tissue masses (days 221-234), and reddened skin (days 227-234).
Moribundity was due to a wide-spread, metastatic malignant adrenal pheochromocytoma.
Animal 3002 (male, 100 mg/kg/day) was sacrificed on day 268. Relevant clinical signs
included thin appearance (days 232-268), emesis mostly with feed and/or compound (between
days 1-267), dehydration (day 249), hunched posture (243-245), unkempt appearance (255-
268), decreased locomotor activity (days 249, 263-268), sunken eyes (days 266-268), and
feces with apparent blood (day 266). The cause of death of the animal could not be
determined based upon anatomic pathological evaluations.
Animal 1505 (female, 0 mg/kg/day) was sacrificed on day 303. This animal was first
observed to be limping/non-weight bearing appendage on day 283. Following veterinary
examination which included an X-ray of the limb, a fracture was noted. Animal no. 1505 was
sacrificed as a result of apparent mechanical trauma resulting in a fractured tibia and distal
femur.
Animal 3502 (female, 100 mg/kg/day) was sacrificed on day 98. Following blood collection
procedures obtained as part of a health check on day 51, this animal developed bruising of the
skin, and a visible swelling of the left leg. Animal no. 3502 was sacrificed due to the
debilitating effects of a large hematoma involving the left thigh and leg, presumably following
a prior blood sampling procedure.
All remaining animals survived until their scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no test article-related effects on mean body weight and mean absolute body weight
gains in AHU377-treated males during the course of the study. Mean body weights of the
treated male dose groups displayed adequate body weight gain; whereas the controls struggled
to maintain their baseline body weights until day 288. One male at 100 mg/kg/day (animal no.
3005), displayed body weight losses of approximately 12% (at study termination) to 25% (on
day 309).
In females, there were no test article-related effects on mean body weight and mean absolute
body weight gains in AHU377-treated animals at doses = 100 mg/kg/day. However at
200 mg/kg/day, body weight losses were evident in individual animals, notably animal nos.
4503 and 4504. By the end of 52 weeks of treatment, treatment-related body weight losses of
about 35% and 11% were noted in animals 4503 and 4504, respectively.
The body weight decreases in these and other animals during the course of the study may be
secondary to the frequent episodes of postdose emesis with feed.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test article-related effects on estimated food consumption determinations as all
groups, including controls, generally consumed 50-75% of the food presented.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular abnormalities associated with the administration of AHU377.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no hematologic changes or changes in coagulation parameters associated with the
administration of AHU377.
Changes present including decreases in red blood cell mass (animal nos. 3005, 4503),
increased neutrophil count (animal nos. 2003, 3004), and increased platelet counts (animal no.
2003) were not considered treatment-related as similar changes were not present in animals in
higher dose groups. Decreases in red blood cell mass in animal nos. 3002 and 3502 were due
to trauma and hemorrhage. All other changes, including those which achieved statistical
significance were considered incidental.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related changes present in clinical chemistry or in special chemistry
analyses. Variability in serum aspartate aminotransferase activity and creatine kinase activity
is considered to be due to handling.
Also noteworthy, but not related to compound administration, was the presence of marked
variability in serum alkaline phosphatase (ALP) activity as well as parathyroid hormone
(PTH) and 25(OH) Vitamin D3 concentrations. On day 199 of the study, PTH concentrations
as well as ionized calcium, 1, 25 (OH) Vitamin D3 and phosphorus concentrations were
measured in three animals (animal nos. 1004, 1005, 1008). Increased PTH concentrations
were noted in two of these animals (animal nos. 1004 and 1008), suggesting increased calcium
mobilization from bone. Vitamin D supplementation was initiated in the diet. On days 246
and 247, calcium metabolism was reassessed. Increased PTH concentrations accompanied by
decreases in serum Vitamin D concentrations was apparent in animal nos. 1001, 1004, 1006,
1506, 3002, 3005, 3505 and 4503. PTH activity was normal in animal no. 1008.
All other changes, including those which achieved statistical significance were considered
incidental.
Endocrine findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test article-related changes.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was a decrease in mean absolute and relative (to brain) thyroid weights in the terminal
sacrifice males at doses >= 25 mg/kg/day and in the high-dose recovery males. These organ
weight decreases correlated with decreased amounts of intrafollicular colloid, considered to be
a compound-related effect. Paradoxically, group mean thyroid weights were increased in the
treated females but with no apparent histological correlate.

Mean pituitary weights (absolute and relative to brain weight) were also statistically
significant in males at 200 mg/kg/day, however, these decreases were not considered to be
biologically significant in the absence of correlative histopathology.
All other organ weights in treated animals were comparable to those observed in their
respective control groups or were within the range previously observed in control animals
from other studies.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no apparent macroscopic findings at necropsy that were considered to be related to
AHU377 administration.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The only microscopic finding worthy of mention occurred in the thyroid gland of one of five
control males (animal no. 1002) and in all treated males except for animal no. 2001 and in all
three high-dose recovery males. This change was characterized by a slight to moderate
reduction in the amount of intra-follicular colloid. The epithelium of the follicles in the
affected animals appeared morphologically intact, being low cuboidal to cuboidal in height as
compared to the more flattened epithelial lining of the follicular walls caused by the presence
of abundant colloid.
Additional microscopic findings worthy of interest but unrelated to compound administration
consisted of gross and histological evidence of fibrous osteodystrophy in bones from animal
nos. 1004, 1007 and 1506 and a metastatic malignant adrenal pheochromocytoma in animal
no. 3001. The bone changes in the effected animals were reminiscent of those associated with
a severe chronic nephropathy; however, as the kidneys in these animals appeared essentially
within normal range as far as histological findings, their presence can be correlated to the inlife and clinical pathology observations consistent with dietary rickets.
All other microscopic observations were considered to be spontaneous in nature and unrelated
to AHU377 administration.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The only microscopic finding worthy of mention occurred in the thyroid gland of one of five
control males (animal no. 1002) and in all treated males except for animal no. 2001 and in all
three high-dose recovery males. This change was characterized by a slight to moderate
reduction in the amount of intra-follicular colloid. The epithelium of the follicles in the
affected animals appeared morphologically intact, being low cuboidal to cuboidal in height as
compared to the more flattened epithelial lining of the follicular walls caused by the presence
of abundant colloid.
Additional microscopic findings worthy of interest but unrelated to compound administration
consisted of gross and histological evidence of fibrous osteodystrophy in bones from animal
nos. 1004, 1007 and 1506 and a metastatic malignant adrenal pheochromocytoma in animal
no. 3001. The bone changes in the effected animals were reminiscent of those associated with
a severe chronic nephropathy; however, as the kidneys in these animals appeared essentially
within normal range as far as histological findings, their presence can be correlated to the inlife and clinical pathology observations consistent with dietary rickets.
All other microscopic observations were considered to be spontaneous in nature and unrelated
to AHU377 administration.
Details on results:
Electrocardiography:
There were no electrocardiographic abnormalities associated with the administration of AHU377.

Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
presumably yes
Conclusions:
In conclusion, the oral administration of AHU377 to marmosets for at least 52 weeks was
generally tolerated as there were no adverse effects on clinical and anatomic pathology
parameters. However, based on the increased incidence and frequency of emetic changes at
doses >= 100 mg/kg/day, the no observed adverse effect level (NOAEL) was considered to be
25 mg/kg/day.
Executive summary:

This study was conducted at Novartis Pharmaceuticals Corporation, East Hanover, New Jersey, in the facilities of Safety Profiling and Assessment (SP&A) and was based on requirements of the Good Laboratory Practice Regulations, Fed. Reg., Vol. 43, 21 CFR Part 58, 22-Dec-1978 and all subsequent amendments. Additional clinical pathology determinations (special chemistries) were conducted by AniLytics Inc., Gaithersburg, MD.


 


The purpose of this nonclinical laboratory study was to establish the toxicologic effects of AHU377, a neutral endopeptidase inhibitor, when administered to marmosets for at least 52 weeks. The study was designed to establish a no observable adverse effect level (NOAEL), to estimate the toxicokinetic profile of AHU377 and its metabolite LBQ657, and to estimate the potential for reversal of any toxic effects within a 4-week recovery phase.


 


AHU377 (Batch nos. 0451002 and 0522003) was administered orally by gavage in 0.5% (w/v) sodium carboxymethylcellulose (CMC), type 7HF, aqueous solution to 4 groups (5/sex/group) of male and female marmosets (Callithrix jacchus) at doses of 0, 25, 100 and 200 mg/kg/day (base) for at least 52 weeks. Additional animals (2-4/sex) were included in the control and high dose groups and served as recovery animals for at least a 4-week recovery period upon the completion of at least 52 weeks of dosing. The dosing volume for all animals was 10 mL/kg. The animals were obtained from the Novartis stock colony. At the initiation of dosing, animals were approximately 13 months to 6 years of age and weighed 258.7 to 484.6 g for males and 249.8 to 468.9 g for females. Beginning 20-Jun-2006 (day 204) animals were supplemented with multivitamins, calcium, and vitamin D3 which were admixed into the ,food. Clinical observations, body weight, estimated food consumption determinations, ophthalmoscopic examinations and electrocardiographic evaluations were performed on all groups. Clinical pathology assessments were made on blood and urine samples collected pretest and during weeks 13, 25 (blood only), 52/53, and at the end of recovery. Clinical pathology assessments were also made on individual animals prior to early sacrifice and/or to monitor animal health. In addition, blood for special chemistries was also obtained from individual animals as part of a health check and from all surviving study animals during week 36 in order to monitor animal health specifically with respect to calcium metabolism. Blood was collected from study animals for toxicokinetic evaluations during weeks 1, 8, 39/40 and 50 at approximately 1, 2, 6 and 24 hours postdose and when possible from animal(s) prior to their early sacrifice. Organ weights were collected from scheduled sacrifice animals. Complete necropsies including macroscopic assessment were performed. Microscopic examinations were performed on all gross lesions, and on protocol specified tissues from all main study animals and for all unscheduled sacrifices. Thyroid gland and gross lesions were processed from the recovery animals.


 


No mortality was noted during the study and no moribundity occurred at the highest dose level evaluated in this study (200 mg/kg/day). However, there were two males (one at 25 mg/kg/day and one at 100 mg/kg/day) that were euthanized on days 199 or 268, respectively, whose cause of moribundity was not determined upon histopathological assessment. Based on the lack of a dose response and the lack of test article-related findings in these animals at necropsy, a relationship to treatment was not established. Prior to sacrifice one or both of these animals displayed a thin or unkempt appearance, dehydration, hunched posture, decreased locomotor activity, and excessive body weight loss (32% relative to baseline). The cause of moribundity was established for the four other animals euthanized prior to study termination and were also not attributable to the administration of AHU377.


Test article-related clinical signs consisted of emesis with or without feed and apparent compound at the higher doses (100, 200 mg/kg/day) based on the increased number of animals affected and/or the increased frequency of occurrence, generally throughout the dosing period.


The frequency of emetic occurrences was more variable and not clearly dose dependent as some individual animals had about 80-200 episodes of vomiting and others had much fewer.


Body weight losses were evident in individual animals. One male at 100 mg/kg/day had losses of approximately 12% (at study termination) to 25% (on day 309) and two females at 200 mg/kg/day displayed losses of about 11-35% compared to baseline values by the end of 52 weeks of treatment. The body weight decreases during the course of the study may be secondary to the frequent episodes of postdose emesis with feed.



There were no test article-related effects on estimated food consumption determinations as animals from all dose groups generally consumed 50-75% of the food presented.


There were no ocular or electrocardiographic abnormalities associated with the administration of AHU377.



There were no hematologic changes or changes in coagulation parameters associated with the administration of AHU377. There were no test article-related changes present in clinical chemistry, special chemistry or urine analyses.



Noteworthy, but not related to compound administration, was the presence of marked variability in serum Alkaline phosphatase (ALP) activity as well as variable parathyroid hormone (PTH) and 25(OH) Vitamin D3 concentrations. On day 199, increased PTH concentrations were noted in animals 1004 and 1008. This prompted dietary supplementation
to the animals for the remaining course of the study. On day 246/247 the calcium status of the animals was reassessed. Increased PTH concentrations accompanied by decreases in serum Vitamin D concentrations, with or without increased ALP activity was apparent in animal nos. 1001, 1004, 1006, 1506, 3002, 3005, 3505 and 4503. Concentrations were normal in animal no. 1008. Histologic and clinical evidence of osteodystrophy was noted in three animals (nos. 1004, 1007 and 1506). The histologic changes combined with the abnormal biochemical alterations (increased PTH, decreased 25(OH) Vitamin D3) are consistent with the development of clinical Vitamin D-dependent Rickets (nos. 1004 and 1506) and suggest the presence of subclinical disease in the other animals. The mechanism of the rickets in the marmoset is believed to be due to a low number of Vitamin D receptors in the intestine and poor intestinal uptake of Vitamin D, necessitating high levels of Vitamin D supplementation in the diet (Takahashi N (1985)). Vitamin D supplementation was initiated on day 204. Not all animals were assessed on day 199, therefore response to therapy is difficult to ascertain.


These changes, accompanied by three histologic cases of osteodystrophy, suggest the presence of clinical and subclinical rickets in several animals. The presence of rickets did not affect the interpretation of the data nor the validity of the study.



There was a decrease in mean absolute and relative (to brain) thyroid weights in the terminal sacrifice males at doses ≥ 25 mg/kg/day and in the high-dose recovery males correlating with a reduction in intrafollicular colloid. The only microscopic finding worthy of mention occurred in the thyroid gland of one of five control males, in most of the treated males and in the highdose recovery males i.e. it was non-reversible. This change was characterized by a slight to moderate reduction in the amount of intra-follicular colloid. Reduced follicular colloid in the one control male indicates that this phenomenon is within the range of normal histological variation for the marmoset. In fact, scattered instances of reduced thyroid follicular colloid were similarly observed in scattered control and treated males, though there were no definitive thyroid weight alterations as in the males. Moreover, there were no inflammatory and/or degenerative changes associated with this change, nor apparent treatment-related effects on growth and body weight parameters. While the incidence of reduced thyroid follicular colloid appeared to be increased in the AHU377-treated males, it is considered to be of little if any toxicological significance.



Toxicokinetic analyses revealed systemic concentrations of AHU377 and LBQ657 following the oral administration of AHU377 at doses up to 200 mg/kg/day. Tmax was reached at 1-2 hours postdose. Based on the AUC and Cmax values, the exposure of AHU377 and LBQ657 appeared to be approximately dose proportional to the dose in the range tested. There were no consistent differences in exposure to AHU377 and LBQ657 observed between male and female marmosets. Following daily oral dosing of AHU377 for up to 346 days, the mean plasma exposure to AHU377 and LBQ657 was generally similar to that observed on day 1. 


 


In conclusion, the oral administration of AHU377 to marmosets for at least 52 weeks was generally tolerated as there were no adverse effects on clinical and anatomic pathology parameters. However, based on the increased incidence and frequency of emetic changes at doses >= 100 mg/kg/day, the no observed adverse effect level (NOAEL) was considered to be 25 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
chronic
Species:
monkey
System:
gastrointestinal tract
Organ:
stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the lack of significant adverse effect observed in the available studies, according to the Regulation (EC) No 1272/2008, AHU 377 does not have to be classified and has no obligatory labelling requirement for STOT-RE.