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Administrative data

Description of key information

In repeated dose oral toxicity studies in rats (up to 13-weeks and 26-weeks in duration ) and marmosets (up to 52-weeks in duration), NOAELs of 400 mg/kg/day, 600 mg/kg/day were reported in rats and 25 mg/kg/day reported in marmosets. Thirteen (13) week repeated dose oral toxicity studies in mice via gavage and in the diet revealed NOAELs of 1200 mg/kg/day and 1000 mg/kg/day, respectively. AHU377 has been associated with fetotoxicity and embryo-fetal toxicity in rabbits; the embryo-fetal NOAEL is rabbits was 200 mg/kg/day. In a fertility and early embryonic development study in rats, AHU377 was shown to have no effect on fertility up to 750 mg/kg/day. The no-observed-adverse-effect level (NOAEL) for paternal toxicity was considered to be 250 mg/kg/day and for maternal toxicity, 750 mg/kg/day. AHU377 had no effects on embryo-fetal toxicity in rats and was not teratogenic in rats and rabbits. AHU377did not affect survival, physical development, behavior or reproductive performance when administered during gestation, parturition and lactation to rats in a Pre and Postnatal Development study and effects were limited to transient effects on body weights between days 7 and 21 post partum at 750 mg/kg/day. The NOAEL for effects on the maternal (F0) generation was 750 mg/kg/day and the NOAEL for the development of offspring (the F1 generation) was 250 mg/kg/day. AHU377 was not genotoxic both in vitro and in vivo and was not carcinogenic in mice and rats with NOAELs of 1200 mg/kg/day and 400 mg/kg/day reported, respectively.

Key value for chemical safety assessment

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:
July - March 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
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 rats for 13 weeks. The
study was designed to establish a no observable adverse effect level (NOAEL) and a
maximum tolerated dose (MTD), to estimate the toxicokinetic profile, and to provide data for
dose selection for a future carcinogenicity study in rats.
Pretest period: Approximately 1 week
Duration of dosing: At least 13 weeks

Animal Numbers Dose (mg/kg/day)* Concentration (mg/mL)
Group Number/sex males females Base/Salt** Salt**
1 10 1001-10 1501-10 0 0
Control
2 10 2001-10 2501-10 50/52.3 5.2
Low
3 10 3001-10 3501-10 100/104.6 10.5
Mid
4 10 4001-10 4501-10 200/209.2 20.9
Mid-High
5 10 5001-10 5501-10 400/418.4 20.9
High
*Doses are not corrected for active moiety.
**Salt/base ratio for AHU377 is 1.046.

Route and frequency: Orally (gavage), once daily
Justification for route: Intended route in humans
Dose volume: For groups 1 and 5: 20 mL/kg
For groups 2-4: 10 mL/kg
Doses administered were calculated to tenths place.
Administration method: Syringe attached to a rigid metal feeding tube
Dosing times were documented in the raw data. 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.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
IGS Wistar Hannover Rat; Crl: WI (Glx/BRL/Han) IGS BR
Breeder/supplier: Charles River Laboratories, Raleigh, North Carolina
Number of animals
ordered: 56 males and 56 females
Sex:
male/female
Details on test animals and environmental conditions:
Animal species and strain: IGS Wistar Hannover Rat; Crl: WI (Glx/BRL/Han) IGS BR
Breeder/supplier: Charles River Laboratories, Raleigh, North Carolina
Number of animals
ordered: 56 males and 56 females
Novartis Confidential Page 13
Study no. 0570207 Toxicology report body AHU377
Number of animals
assigned to the dosing
phase: 50 males and 50 females
Age: Approximately 8 weeks (at start of dosing)
Body weight range: 210.8 to 258.6 g for males, and 163.0 to 198.6 g for females
(at start of dosing)
Acceptability: Following acclimation, a staff veterinarian released healthy
animals for study purposes. After the start of treatment,
extra/unused animals were transferred to Laboratory Animal
Services (LAS).
Animal quarters/husbandry
Building/Animal room: 406/360
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 (unless otherwise noted in the raw data), stainless
steel wire bottom caging
Acclimation period: Approximately 1 week
Diet: Certified Rodent Diet 18% #5LG3 pellets (PMI Feeds,
Richmond, Indiana), ad libitum, except for study-defined
fasting procedures
Water: Water from the animal drinking supply was available
ad libitum from automatic dispensers.
Environmental enrichment: 100% nylon bones/balls (e.g., nylabones) were provided to
each animal.
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:
Orally (gavage), once daily
Syringe attached to a rigid metal feeding tube
Dosing times were documented in the raw data. 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.
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
Route and frequency: Orally (gavage), once daily
Dose volume: For groups 1 and 5: 20 mL/kg
For groups 2-4: 10 mL/kg
Doses administered were calculated to tenths place.
Administration method: Syringe attached to a rigid metal feeding tube
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.
Dosage form

Administration form: AHU377 in 0.5% CMC Preparation for administration. Storage conditions: 2-8°C, protected from light
Stability and uniformity: Determined by Analytical Chemistry. Concentration (actual vs.nominal) under conditions
of administration: Samples of the test and control formulations prepared for
dosing in weeks 1, 5/6 and 13 were sent to Analytical
Chemistry and analyzed.
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, were found to be stable
for at least 35 days stored at 6°C and at least 4 hours stirring at room temperature. Aqueous
0.5% CMC was found to be suitable for use for at least 40 days stored at 6°C, at least 15 days
stored at room temperature and at least 4 hours stirring at room temperature. The uniformity
of the suspensions was confirmed. Samples of the formulations prepared for weeks 1, 5/6 and
13 were analyzed. Sample concentrations were 98% to 107% of target concentrations. Three
control samples were analyzed, and no AHU377 was detected.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
50, 100, 200,400 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
(CMC), type 7HF, aqueous solution was administered orally via gavage to IGS Wistar
Hannover [Crl: WI(Glx/BRL/Han)IGS BR] rats (10/sex/group) at doses of 50, 100, 200 and
400 mg/kg/day as base (or 52.3, 104.6, 209.2 and 418.4 mg/kg/day as salt). Ten additional
animals per sex received 0.5% (w/v) sodium carboxymethylcellulose (CMC), type 7HF,
aqueous solution and served as the controls. All groups were dosed for at least 13 weeks and
the dose volumes were 10 mL/kg for groups 2-4 and 20 mL/kg for groups 1 and 5. The
animals were received from Charles River Laboratories, Raleigh, North Carolina. At the
initiation of dosing, the animals were approximately 8 weeks of age and weighed 210.8 to
258.6 g for males, and 163.0 to 198.6 g for females. Clinical signs, body weight and food
consumption determinations, ophthalmoscopic examination and clinical pathology evaluations
(hematology, clinical chemistry and urinalysis) were performed. Complete necropsies
including macroscopic assessments were performed on all animals. Protocol-specified organs
were weighed and protocol-specified tissues were processed and microscopically examined.
Protocol-specified genomic tissues were collected at term necropsy. Blood samples for
toxicokinetic analysis of AHU377 and LBQ657 (an active metabolite of AHU377) were
collected from the nonrecovery study animals on study days 1/2 and in week 11 (day 76).
Observations and examinations performed and frequency:
In-life examinations
Data were collected on an individual animal basis with the exception of cage pan observations and food consumption.
Mortality
Twice daily (AM and PM) on weekdays and at least once daily on weekends and holidays
Clinical signs
Pretest: At least once daily
Dosing period: At least twice daily (predose and approximately 2 hours postdose)
Body weight
Pretest period: Once
Dosing period: Once weekly
Food consumption
Pretest: None
Dosing period: Once weekly
Ophthalmology
Pretest: All animals
Week 13: All surviving control and high-dose animals
Clinical pathology
Clinical pathology assessments were conducted on all surviving study animals in weeks 5 and

Blood specimens were collected from the sublingual vein from isoflurane/O2-anesthetized
animals into EDTA-containing tubes for hematology (approximately 0.5 mL) and serum
collection tubes for clinical chemistry (approximately 1 mL). For the collection of urine
specimens (approximately 1 mL), individual animals were placed in urine collection cages for
up to 5 hours. The following parameters were assessed.
Hematology parameters
erythrocytes Wintrobe indices white blood cell count
hematocrit red cell distribution width (RDW)
white blood cell differential
hemoglobin reticulocytes platelets
Clinical chemistry parameters
alanine aminotransferase globulins (G) chloride
alkaline phosphatase glucose calcium
aspartate aminotransferase Blood urea nitrogen inorganic phosphorus
total bilirubin creatinine triglycerides
total protein sodium cholesterol
albumin (A) potassium A/G ratio
Urinalysis parameters
specific gravity glucose* protein*
bilirubin* ketones* urobilinogen*
blood* pH*
*test strip determinations
Sacrifice and pathology:
Pathology
Necropsy procedures
Animals were fasted overnight (approximately 18 hours) prior to terminal necropsy. Fasted terminal body weights were collected by Pathology personnel prior to scheduled necropsies for the calculation of relative organ to body weight determinations. A prosector order was
prepared per Pathology SOPs. Complete necropsies were performed with a recording of macroscopic abnormalities for all protocol tissues.

Euthanasia
Scheduled sacrifices were exsanguinated while under isoflurane/O2 anesthesia.
Approximately 2.5 mL of blood was collected from isoflurane/O2-anesthetized animals at
scheduled sacrifices for possible investigational gene expression by Pathology personnel.
Once the animal was anesthetized, as determined by the loss of pedal and corneal reflexes,
anesthesia was maintained using a nose cone. While anesthetized, an incision was made in
the abdominal wall to expose the vena cava and abdominal aorta. After the appropriate
amount of blood was collected via needle and syringe, the abdominal aorta/vena cava was cut,
and the animal was exsanguinated.
Statistics:
Generally, means and standard deviations were calculated and an Analysis of Variance
(ANOVA) performed on time-point specific data sets (i.e., body weights, food consumption,
clinical chemistry and hematology). The ANOVA was followed by a 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 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, only means
and standard deviations were calculated.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant test article-related clinical signs noted in treated groups. Only
minor clinical sign, staining of fur, was noted primarily in males at 400 mg/kg/day.
All other clinical signs noted are considered unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no test article-related mortality or moribundity during the study. Two animals
(nos. 1006 and 5504) were found dead on days 91 and 76, respectively, following blood
collection. Both had discolored (red/dark) lungs at necropsy and histologically this correlated
with congestion. For animal no. 1006, there was additional pulmonary hemorrhage, which
together with the congestion was considered the cause of death and suggestive of a misgavage.
The pulmonary congestion was less severe for animal no. 5504 and with no other significant
findings, the cause of death was not determined.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no significant test article-related changes noted in any treated groups during the
study. Some minimal changes on body weight parameters noted in treated groups were not
considered toxicologically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test article-related changes noted in mean food consumption data from treated groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examinations performed during the study did not reveal any ocular changes
attributable to treatment with AHU377.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematologic changes associated with the administration of AHU377 were minimal. Changes
were seen only in males dosed at doses ≥ 200 mg/kg/day when compared to concurrent
controls. A slight, dose-dependent increase in the absolute neutrophil count was present on
day 30. All other values were within expected limits.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Changes in clinical chemistry were also very mild and none are considered of significance. A
minimal increase in serum alanine aminotransferase activity was apparent on both days 30 and
90 in males dosed at 400 mg/kg/day, but this increase was less than 70% over the concurrent
control values and is therefore not considered significant. Other changes included a minimal
decrease in glucose concentration in males dosed at 200 mg/kg/day on day 90 and an increase
in urea concentrations in males receiving 50 mg/kg/day on day 90. As these changes were
seen only in these groups, and not in animals receiving higher doses, they are not considered
to be test article-related. All other values were within expected limits or deemed insignificant.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test-article related changes in the urinalysis.
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):
A minimal test article-related decrease in mean thyroid gland weights (absolute, and relative
to body and brain) occurred in female animals dosed at doses ≥ 100 mg/kg/day and male
animals dosed at 400 mg/kg/day. In the absence of correlative histopathology, the
toxicological significance of this change is unclear. All other organ weight changes were
considered to be within the accepted normal range for the age, sex and strain of these animals,
and not related to administration of AHU377.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related macroscopic observations. Of interest, calculi (stones) were
found within the urinary tract of one female control animal, one female dosed at 50 mg/kg/day
and one female dosed at 200 mg/kg/day. Although unusual, the distribution and frequency of
these findings between groups suggests they are incidental, spontaneous and not related to
administration of AHU377.

There were no test article-related microscopic observations. The spectrum of histological
observations recorded was considered to be within the accepted normal range for the age, sex
and strain of these animals, to be spontaneous and/or incidental, and not related to
administration of AHU377.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Analysis of plasma samples from the control group showed that 13 out of 40 control samples
had measurable concentrations of AHU377 and/or LBQ657. 4 out of 13 samples showed both
AHU377 and LBQ657, and the remaining samples showed only LBQ657. Five samples were
collected on day 1 and eight samples on day 76 at time-points between 1-6 hours postdose.
Repeat analysis of these samples confirmed the original findings. The source of the observed
concentrations in these control groups was unknown. It is considered unlikely that these
animals were dosed with AHU377 since the ratios of exposures for AHU377 and LBQ657,
approximately 2- to 5-fold, in the four animals (nos. 1505, 1506, 1004 and 1005) which had
both measurable concentrations of AHU377 and LBQ657 were well below those seen in the
treated groups, ranging from 21- to 151-fold. The concentrations of AHU377 observed in all
control samples were within 3-fold of the LLOQ (20.0 ng/mL), and concentrations of LBQ657
in 11 samples were below 7-fold of the LLOQ (20.0 ng/mL). Furthermore, all but two
samples for LBQ657 were well below concentrations observed at corresponding time-points
in the low dose group. Therefore, these results are considered to have no adverse impact on
the result interpretation.
AHU377 was rapidly absorbed, reaching a peak plasma concentration (Cmax) between 0.5 - 1 h
(tmax) post dose. AHU377 was rapidly converted to LBQ657 (active metabolite). Significant
peak concentrations of LBQ657 were observed in all treated groups as early as 0.5 hour and
for at least 6 hours post dose. For all treated groups, there was an approximately 21- to
151-fold greater exposure (AUC) to LBQ657 than to AHU377.
No apparent difference in AHU377 or LBQ657 exposure was observed between male and
female rats after single and multiple doses at each dose level. Following daily oral dosing of
AHU377 for 76 days, the mean plasma exposure for AHU377 or LBQ657 was similar to that
on day 1 in both male and female rats, suggesting no accumulation.
After single and multiple doses of AHU377, exposure to AHU377 for males and females
increased proportionally with dose from 100 mg/kg/day to 400 mg/kg/day. At 50 mg/kg/day,
the exposure to AHU377 was under-proportional relative to the other three doses. Following
single and multiple doses and considering the observed SE, the increase in exposure in male
and female rats for LBQ657 was proportional to the dose.
Details on results:
There was no test article-related mortality or moribundity during the study. There were no
significant test article-related clinical signs, changes in body weight parameters, food
consumption data or ophthalmoscopic examination in any treated groups.
The only change noted in clinical pathology data was a very mild, dose-dependent increase in
the absolute neutrophil count in males at doses ≥ 200 mg/kg/day.
A minimal test article-related decrease in thyroid gland weights occurred in females at doses
≥ 100 mg/kg/day and males at 400 mg/kg/day, with no histological correlate. There were no
test article-related microscopic or macroscopic observations.In conclusion, oral administration of AHU377 to rats by gavage for 13 weeks was well
tolerated. Mild changes in clinical pathology were noted in males at doses ≥ 200 mg/kg/day,
and minimal organ weight decrease in thyroid gland were noted in females at doses
≥ 100 mg/kg/day and in males at 400 mg/kg/day with no correlated histological changes. The
no observable adverse effect level (NOAEL) for AHU377 in this study is considered at
400 mg/kg/day. A maximum tolerated dose (MTD) for AHU377 in rats is considered above
400 mg/kg/day under the conditions of this study.
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
other: Mild changes in clinical pathology were noted in males at doses ≥ 200 mg/kg/day, and minimal organ weight decrease in thyroid gland were noted in females at doses ≥ 100 mg/kg/day and in males at 400 mg/kg/day with no correlated histological changes
Key result
Critical effects observed:
not specified
Conclusions:
In conclusion, oral administration of AHU377 to rats by gavage for 13 weeks was well
tolerated. Mild changes in clinical pathology were noted in males at doses ≥ 200 mg/kg/day,
and minimal organ weight decrease in thyroid gland were noted in females at doses
≥ 100 mg/kg/day and in males at 400 mg/kg/day with no correlated histological changes. The
no observable adverse effect level (NOAEL) for AHU377 in this study is considered at
400 mg/kg/day.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The key effect that is considered for the determination of the occupational health category for AHU377 gastritis characterized by microscpopic GI changes and emetic effects observed in marmosets.
System:
endocrine system
Organ:
thyroid gland

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

Mode of Action Analysis / Human Relevance Framework

Upon ingestion, LCZ696 delivers systemic exposure to AHU377, a neprilysin (neutral endopeptidase 24.11, NEP) inhibitor (NEPi) and valsartan, an angiotensin receptor blocker (ARB). AHU377 is further metabolized via esterases to the active NEPi, LBQ657. The effects of NEP inhibition are attributed to the enhanced effects of biologically active natriuretic peptides (NPs). ARB blockade is specific and competitive at the angiotensin type 1 (AT1) receptor that mediates the deleterious effects of angiotensin II on the cardiovascular system.

Lowest pharmacologically active dose / estimated therapeutic dose: 100 mg/day

Route of administration: Oral

Additional information

Human data

Pharmacokinetics:

Absorption and Distribution

Following oral administration to healthy subjects, LCZ696 is rapidly absorbed delivering systemic exposures of LBQ657 (derived from AHU377 by enzymatic cleavage) and valsartan. Absolute bioavailability was not assessed with LCZ696 due to limitations with development of the intravenous formulation. The estimated absolute bioavailability of AHU377 is ≥60% following oral administration of LCZ696.

Metabolism

AHU377 is readily converted to LBQ657 by ester hydrolysis following oral administration. LBQ657 is the only circulating metabolite of AHU377 in the plasma.

Elimination

Following oral administration of 200 mg 14C-LCZ696 (the AHU377 moiety of LCZ696 was radiolabelled), AHU377 and valsartan are cleared from plasma with an apparent clearance of 49.4 L/h and 4.22 L/h, respectively.

Following oral administration of 14C-LCZ696 approximately 52% - 68% of the AHU377 dose was recovered in urine (primarily as LBQ657) and ~37 - 48% recovered in the feces (predominantly LBQ657). Unchanged AHU377 accounted for 0.8% - 2.8% of the dose in urine and 0.3% - 0.9% in feces.

The oral bioavailability is estimated to be 60%.

Justification for classification or non-classification

Single dose oral toxicity studies in mice and rats revealed NOELs of 2000 mg/kg in both species. In single dose intraperitoneal studies in mice and rats, a NOEL of 300 mg/kg was reported in mice but a NOEL was not identified in rats. In repeated dose oral toxicity studies in rats (up to 13-weeks and 26-weeks in duration ) and marmosets (up to 52-weeks in duration), NOAELs of 400 mg/kg/day, 600 mg/kg/day were reported in rats and 25 mg/kg/day reported in marmosets. Thirteen (13) week repeated dose oral toxicity studies in mice via gavage and in the diet revealed NOAELs of 1200 mg/kg/day and 1000 mg/kg/day, respectively. AHU377 has been associated with fetotoxicity and embryo-fetal toxicity in rabbits; the embryo-fetal NOAEL is rabbits was 200 mg/kg/day. In a fertility and early embryonic development study in rats, AHU377 was shown to have no effect on fertility up to 750 mg/kg/day. The no-observed-adverse-effect level (NOAEL) for paternal toxicity was considered to be 250 mg/kg/day and for maternal toxicity, 750 mg/kg/day.