[1,1'-Biphenyl]-4-pentanoic acid, .gamma.-[(3-carboxy-1-oxopropyl)amino]-.alpha.-methyl-, 4-ethyl ester, calcium salt (2:1), (.alpha.R,.gamma.S)-

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 Feb 2014 - 29 Aug 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S5 (R2) Guideline for Industry: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility

Deviations:

yes

Remarks:

Minor deviations (see report)

GLP compliance:

yes (incl. QA statement)

Justification for study design:

The Wistar Hannover rat was selected as the test animal because 1) it is a standard species accepted for use in embryo-fetal development studies; 2) this species and strain has been demonstrated to be sensitive to developmental toxicants; and 3) historical data and experience
exist at the test facility.

The requirement for, and use of animals in this research was the responsibility of the Sponsor, in that the research did not unnecessarily duplicate previous animal experiments. The research was conducted in the absence of acceptable non-animal alternatives to provide meaningful data and was designed such that it did not require an unnecessary number of animals to accomplish its objectives.

The study plan, study plan amendments and procedures involving the care and use of animals in this study were reviewed and approved by PCS-MTL Institutional Animal Care and Use Committee (IACUC) before conduct. During the study, the care and use of animals was conducted in accordance with the guidelines of the USA National Research Council and the Canadian Council on Animal Care (CCAC). This non-clinical health and environmental safety study was reviewed and approved by the East Hanover Novartis Animal Care and Use Committee (EH-NACUC).

Test material

Test material information

Constituent 1

Reference substance name:

calcium bis(3-{[(2S,4R)-1-{[1,1'-biphenyl]-4-yl}-5-ethoxy-4-methyl-5-oxopentan-2-yl]carbamoyl}propanoate)

EC Number:

935-847-3

Cas Number:

1369773-39-6

Molecular formula:

C48H56CaN2O10

IUPAC Name:

calcium bis(3-{[(2S,4R)-1-{[1,1'-biphenyl]-4-yl}-5-ethoxy-4-methyl-5-oxopentan-2-yl]carbamoyl}propanoate)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Active ingredient: AHU377 (AHU377 BAA.002, calcium salt form)
Batch no.: 0723009
Retest date: 31-Jul-2014
Supplier: Novartis Technical Research and Development (TRD)
Purity (by HPLC): 100.2% (purity assumed to be 100% for dose calculation purposes)

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rattus norvegicus/Wistar Hannover Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

No. of animals assigned to the dosing phase: 96/sex
Age: Males 12 weeks and females 9 weeks at start of dosing
Body weight range: 298-355 g (males) and 167-215 g (females), at start of dosing
Acceptability: All assigned animals were considered suitable for use on study

Room temperature: 19°C to 25°C
Room relative humidity: 30-70%
Lighting cycle: Fluorescent light for a 12-hour light/12-hour dark cycle
Animal caging: Throughout the study, animals were housed in solid-bottomed cages equipped with an automatic watering valve on corn-cob bedding. On arrival, animals were individually housed until randomization; following randomization, animals were group housed (up to 3 animals of the same sex and same dosing group together), unless otherwise recommended by the clinical veterinarian (i.e., for animal nos. 3018 and 3017 that were single housed from 28-Feb-2014 to 20-Mar-2014 to allow for healing of a skin lesion at the dorsal cervical lesion of animal no. 3018). During cohabitation, each female was paired with a male and animals were returned to group housing following confirmation of mating or at the end of the cohabitation period, as applicable.
Acclimation period: An acclimation period of two weeks was allowed between animal arrival and the start of treatment in order to accustom the animals to the laboratory environment.
Diet: All animals had free access to standard certified pelleted commercial laboratory diet (PMI Certified Rodent 5002: PMI Nutrition International Inc.) throughout the study, except during designated procedures.
The feed is analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are on file at the test facility.
Water: Municipal tap water softened, purified by reverse osmosis and exposed to ultraviolet light, was freely available (except during designated procedures).
Periodic analysis of the water is subcontracted to management authorized analytical laboratories. The analytical results are retained in the archives of PCS-MTL.
Animal enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with items such as hiding tubes and a chewing object, except during designated activities.
There were no known contaminants in the food or water that would interfere with the conduct
of the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) hydroxypropylcellulose

Details on exposure:

Route and frequency: Oral (gavage), daily
Justification for route: Intended route in humans

Males were given the test or reference item formulations once daily beginning 28 days before cohabitation, during cohabitation and continuing through the day before euthanasia, for a total of at least 56 days. Females were given the test item and/or the reference item formulations once daily beginning 14 days before cohabitation, during cohabitation and continuing until day 6 postcoitum (i.e., of gestation). Females not mated after completion of the 14-day cohabitation period were given the test item and/or the reference item formulations until the day before necropsy.
Animals were given a daily oral dose (gavage) of the dose formulation using a plastic gavage tube. The dose volume was adjusted for each animal based on the most recently recorded body weight and administered at approximately the same time each day (i.e., ±1 hour for each group). The dosing formulations were placed on a stir plate for at least 30 minutes prior to and constantly during dosing and were administered within a 4-hour period following transfer to room temperature. Reflux was observed once between study days 1 and 63 for a few animals (male nos. 1014, 3007, 3010, 4007, 4009 and female nos. 1512, 2516 and 4512) in each group.

Details on mating procedure:

Within each dosage group, a consecutive order was used to assign rats to cohabitation (i.e., pairing), one male to one female. The cohabitation period consisted of a maximum of 14 days. Females with spermatozoa observed in a smear of the vaginal lavage and/or a copulatory plug observed in situ were considered to have mated (day designated as day 0 postcoitum [i.e., of gestation]) and were returned to group housing. Females not mated after completion of the 14-day cohabitation period were returned to group housing and were euthanized 8 days after completion of the mating period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Based on stability data provided by the Sponsor, AHU377 suspensions in 0.5% (w/v) hydroxypropylcellulose (grade HF), NF (Klucel), at 1.0 to 125.5 mg/mL (salt) are chemically
stable for 12 days at 6°C, 24 hours at room temperature and 4 hours stirring at room temperature.

Homogeneity of the dose formulations was assessed at the dose concentrations intended for use on the study. It was assessed from the preparation vessel and the dose aliquot jars
as follows:
Following the first preparation of the suspension (including stirring) but prior to aliquoting into daily containers, two sets of samples (target volume of 1 mL), one in duplicate for analysis and the other in triplicate as back-up samples, were sampled from approximately the top, middle, and bottom of the group 2 and 4 test item formulations and from the middle of the vehicle control and group 3 test item formulation. An additional sample (10 mL) was taken from each formulation for density measurement. As suspensions were prepared on a weekly basis and stored in daily dosing containers, the homogeneity was also assessed following resuspension of the
dosing container for groups 2 and 4 on the first day of use.
Two sets of samples (target volume of 1 mL), one in duplicate for analysis and the other in triplicate as back-up samples, were sampled from approximately the top, middle, and bottom of the group 2 and 4 test item formulations and from the middle of the vehicle control and group 3 test item formulation. All samples were transferred to HDPE
containers.
Concentration of the dose formulations was also assessed at the dose concentrations intended for use on the study. It was assessed from the preparation vessel as follows:
Following week 5 and the last preparation of the suspension (including stirring) but prior to aliquoting into daily containers, two sets of samples (target volume of 1 mL), one in duplicate for analysis and the other in triplicate as back-up samples, were sampled from approximately the
middle of the test item formulations and the vehicle control.
For concentration, the criterion for acceptability was mean sample concentration results within or equal to ± 15% of theoretical concentration (individual sample concentration result within or equal to ± 20%). For homogeneity, the criterion for acceptability was a relative standard deviation
(RSD) of concentrations of 5% for each group.

Duration of treatment / exposure:

Males: 28 days before cohabitation, during cohabitation and continuing through the day before euthanasia, for a total of at least 56 days.

Females: 14 days before cohabitation, during cohabitation and continuing until day 6 postcoitum (i.e., of gestation)

Frequency of treatment:

once daily

Details on study schedule:

Major activities Date or study day no.
Study plan signed 03-Feb-2014
Experimental start date 04-Feb-2014
Animal arrival/Start of acclimation 04-Feb-2014 (males) 18-Feb-2014 (females)
First day of dosing 18-Feb-2014 (males) 04-Mar-2014 (females)
Last necropsy 25-Apr-2014
Experimental completion date 29-Aug-2014

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24 males and 24 females per goup

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses for this study were based on preliminary findings in the AHU377 26-week oral (gavage) toxicity study in rats conducted at doses of 50, 150 and 600 mg/kg/day [Novartis reference no. 1370484]; on the results of the AHU377 oral (gavage) embryo-fetal development study in rats conducted at doses of 75, 250 and 750 mg/kg/day [Novartis reference no. 0570301] and on the AHU377 oral (gavage) pre and postnatal study in rats conducted at doses of 50, 250 and 750 mg/kg/day [Novartis reference no. 1070349]. Based on results from those studies, doses of 75, 250 and 750 mg/kg/day were selected for the current study.

- Rationale for animal assignment (if not random): One week before initiation of treatment, animals were assigned to groups using a
computer-based randomization procedure. Males and females were randomized separately. No animals at the extremes of the weight range or in poor health were assigned to the study. Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. The disposition of all animals was documented in the study records. All animals remaining unassigned to groups were released from the study on study day 3 and their disposition documented.

- Fasting period before blood sampling for clinical biochemistry: not relevant.

Examinations

Parental animals: Observations and examinations:

3.5.1 Mortality
Twice daily (AM and PM) on weekdays and weekends; once daily on the day of animal arrival and on the last day of necropsy.

3.5.2 Clinical signs
Cageside observations: On non-dosing days, once daily starting on the day of randomization when no detailed examinations were scheduled; on days of dosing, predose (when no detailed
examinations were scheduled) and within 1 to 3 hours post dose.
Detailed examinations: On the days of body weight assessment

3.5.3 Body weight
Individual body weights were measured twice weekly commencing on the day of randomization and extending through the treatment period, including the day animals were placed for mating and, for males, the day of scheduled necropsy. Mated females were weighed on days 0, 3, 7, 10 and 13 postcoitum.

3.5.4 Food consumption
Food consumption (cage measurements) was quantitatively measured twice weekly commencing the day of randomization and until initiation of the mating period.
Food consumption for mated females was measured on days 0 to 3, 3 to 7, 7 to 10 and 10 to 13 post coitum.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage for 14 consecutive days beginning with the first day of dosage administration and then until spermatozoa were observed in a smear of the vaginal contents and/or a copulatory plug was observed in situ during the cohabitation period.

Postmortem examinations (parental animals):

3.6.3 Ovarian and uterine examinations
The reproductive tract was dissected from the abdominal cavity. The uterus was opened and the contents examined. The embryos were removed from the uterus.
The ovaries and uterus were examined for number and distribution of:
¿ Corpora lutea
¿ Implantation sites
¿ Placentae (size, color or shape)
¿ Early resorptions
¿ Live and dead embryos

Uteri of apparently nonpregnant females were stained with 10% aqueous (v/v) ammonium sulfide solution and examined for implantation sites (Salewski, 1964).

3.6.5 Necropsy
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Necropsy examinations were conducted under the supervision of a board-certified veterinary pathologist or other qualified personnel with appropriate training and experience in animal anatomy and gross pathology.

3.6.6 Organ weights
The organs identified for weighing in the Tissues collection and preservation table were weighed at necropsy for all animals. Paired organs were weighed individually, unless otherwise indicated.

3.6.7 Tissue collection and preservation
Representative samples of the tissues identified in the Tissue collection and preservation table were collected at scheduled euthanasia and were preserved in 10% neutral buffered formalin. Unless specifically cited below, all other tissues were discarded. A standard list of tissues were collected at scheduled necropsy from the first two gravid control females and the first two control males examined, as per PCS-MTL SOP, and were retained for possible future comparison. No histopathological evaluation was performed and all tissues have been discarded.

Statistics:

Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to
the matrix below when possible, but excluded semi-quantitative data, and any group with less
than 3 observations. Litter values were used where appropriate.

Reproductive indices:

3.5.6 Cohabitation/mating.
Within each dosage group, a consecutive order was used to assign rats to cohabitation (i.e., pairing), one male to one female. The cohabitation period consisted of a maximum of 14 days. Females with spermatozoa observed in a smear of the vaginal lavage and/or a copulatory plug observed in situ were considered to have mated (day designated as day 0 postcoitum [i.e., of gestation]) and were returned to group housing. Females not mated after completion of the 14-day cohabitation period were returned to group housing and were euthanized 8 days after completion of the mating period.

3.6.4 Male reproductive assessments
To assess the potential toxicity of the test item on the male reproductive system, the endpoints
listed below were evaluated for each main study male at scheduled euthanasia.

3.6.4.1 Sperm motility
Sperm motility was evaluated using computer-assisted sperm analysis (CASA). Motility was evaluated following dispersion, into an appropriate medium, of sperm from the left vas deferens.

3.6.4.2 Sperm concentration
A homogenate was prepared from the left cauda epididymis for evaluation to determine sperm concentration (sperm per gram of tissue weight; manual count using a hematocytometer). Sperm concentration (millions/gram of epididymis) was assessed from two counts of sperm obtained from the cauda epididymis.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation and/or wet fur on the lower jaw and/or muzzle were observed in a few males at
75 mg/kg/day and most males and females at 250 and 750 mg/kg/day during the treatment
period, including the gestation period. A few males at 250 and 750 mg/kg/day were noted to
be hypersensitive sporadically during the treatment period. These observations were
considered to be non-adverse.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There was no unscheduled mortality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For males at 250 and 750 mg/kg/day, significantly lower body weight gains, compared to
controls, were noted during the first week of the premating treatment period. At
750 mg/kg/day, this continued throughout the remaining 3 weeks of this period of the study.
As a result, the overall weight gains (study days 1 to 28) were significantly decreased at
250 and 750 mg/kg/day, being 12 and 35% lower than controls. The body weights at
750 mg/kg/day were significantly lower from study day 15 until the end of the study (-4 to
-6%). These lower body weights for males at 750 mg/kg/day continued during the mating and
post mating treatment periods. Body weight decreases in males at 750 mg/kg were of
sufficient magnitude to be considered adverse. An a few occasions, statistically significant
differences were noted for body weight gain values for males at 75 mg/kg/day but these were
considered to be the result of random variation and not test item related.
There were no AHU377-related effects upon body weights or body weight gains at any dose
level for the females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were slightly lower food intakes for males at 250 and 750 mg/kg/day during the
premating treatment period, particularly for the first week of treatment. Overall, all values
(study days 1 to 28) were reduced by 5% and 8% at 250 and 750 mg/kg/day, respectively,
compared to controls.
The food consumption of the males at 75 mg/kg/day and females at <= 750 mg/kg/day was
unaffected.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The estrous cycles of the AHU377-treated females were not affected.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no effect of AHU377 upon the day to mating, mating or fertility indices, or the conception rates.

Ovarian and uterine findings:
For AHU377-treated females, there were no effects upon the numbers of corpora lutea, implantations, live embryos, dead embryos, resorptions or the pre or post-implantation losses. The increased pre or post-implantation loss values noted for the treated groups were not considered related to the test item since there was no dose response noted and because values were within the historical control values for these parameters.

There were no toxicologically significant differences in mean for sperm parameters, including
sperm motility and concentration, between AHU377-treated animals and vehicle-treated control animals.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

paternal toxicity

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

2

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Food efficiency:

not specified

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

750 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, administration of AHU377 by daily oral gavage at dosages of 75, 250 and
750 mg/kg/day to males and females resulted in adverse effects on body weights and food
consumption in males, but not females, at 750 mg/kg/day. There was no evidence of effects
on male or female reproductive function or embryolethality at any dose. 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. The NOAEL for reproductive
function and early embryo-fetal development was considered to be 750 mg/kg/day, the highest
dose level administered.

Executive summary:

This study was conducted at Charles River Laboratories Preclinical Services Montreal, 22022 Transcanadienne, Senneville, Quebec, Canada, H9X 3R3 and was based on requirements of the OECD Principles of Good Laboratory Practice (GLP) and as accepted by Regulatory Authorities throughout the European Union, United States of America (FDA), Japan (MHLW), and other countries that are signatories to the OECD Mutual Acceptance of Data Agreement. Exceptions included: the test item characterization and stability testing performed by the Sponsor followed FDA Good Manufacturing Practice (GMP) regulations.

 

The design of this study was based on the study objective and the following study design guidelines: ICH Harmonised Tripartite Guideline S5 (R2) Guideline for Industry: Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility.
The purpose of this study was to determine the potential adverse effects of AHU377, a neutral endopeptidase inhibitor, on Wistar Hannover Crl:WI (Han) rats before cohabitation, through mating and implantation. This study evaluated ICH Harmonised Tripartite Guideline stages A and B of the reproductive process and was designed to detect effects on the estrous cycle, tubal transport, implantation, and development of preimplantation stages of the embryos of females and permit detection of functional effects (e.g., effects on libido or epididymal sperm maturation) that may not be detected by histological examinations of male reproductive organs.

 

AHU377 (batch no. 0723009) was administered by oral gavage at doses of 75, 250 and 750 mg/kg/day (base) to three groups (n = 24/sex) of male and female rats. An additional group of rats (n = 24/sex) received the vehicle, 0.5% (w/v) hydroxypropylcellulose (grade HF), NF (Klucel), aqueous solution, at an equivalent dose volume (10 mL/kg), and served as controls. Males were given the test or reference item (vehicle) formulations once daily beginning 28 days before cohabitation, during cohabitation and continuing through the day before euthanasia, for a total of at least 56 days. Females were given the test item and/or the reference item formulations once daily beginning 14 days before cohabitation, during cohabitation and continuing until day 6 postcoitum (i.e., of gestation).

 

Rats Wistar Han (Crl:WI[Han]) were obtained from Charles River Laboratories, Kingston, New York, USA. At the start of dosing, males were 12 weeks and weighed 298 to 355 g; females were 9 weeks of age and weighed 167 to 215 g at the onset of treatment. Cesarean sections were performed on day 13 post coitum (pc) for main study animals.

 

The following parameters and endpoints were evaluated: mortality and moribundity, clinical signs, body weights, food consumption, estrous cycles, reproductive performance, organ weights, ovarian and uterine examinations, sperm evaluations, and gross pathology.

 

There was no mortality. Signs of salivation/wet fur were observed primarily for males and females at 250 and 750 mg/kg/day during the study although salivation was also noted for few males at 75 mg/kg/day.

For males, overall weight gains (study day 1 to 28) were significantly decreased at 250 and 750 mg/kg/day. The male body weights at 750 mg/kg/day were significantly lower from study day 15 until the end of the study. There were lower food intakes for males at 250 and 750 mg/kg/day during the premating treatment period, with differences from controls ranging from 5% to 8%. There were no AHU377-related effects upon body weights or food intake at any dose level for the females.

 

The estrous cycles of females and the parental performance parameters (mean day to mating, mating and fertility indices and the conception rate) of the males and females were not affected by treatment with AHU377. There were no compound-related effects upon the ovarian and uterine parameters (numbers of corpora lutea, implantations, live embryos, dead embryos, resorptions or the pre or post-implantation losses).

 

No macroscopic observations attributed to the effect of treatment with AHU377 were observed. There were no differences in mean absolute or relative organ weights between

 

AHU377-treated animals and vehicle-treated control animals. Sperm parameters, including sperm motility and concentration, were unaffected by treatment with AHU377.

 

In conclusion, administration of AHU377 by daily oral gavage at dosages of 75, 250 and 750 mg/kg/day to males and females resulted in adverse effects on body weights and food consumption in males, but not females, at 750 mg/kg/day. There was no evidence of effects on male or female reproductive function or embryolethality at any dose. The NOAEL for paternal toxicity was considered to be 250 mg/kg/day and for maternal toxicity, 750 mg/kg/day. The no-observed-adverse-effect level (NOAEL) for reproductive function and early embryo-fetal development was considered to be 750 mg/kg/day, the highest dose
level administered.