Liraglutide precursor

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

june 2007 - July 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: European Parliament and Council Directive 2001/83/EC of 6 November 2001 of the Community Code Relating to Medicinal Products for Human Use, OJ L311/67-128, 28 November 2001 as amended Commission Directive 2003/63/EC, OJ L159, 27 June 2003.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Semaglutide

Cas Number:

910463-68-2

Molecular formula:

C187H291N45O59

IUPAC Name:

Semaglutide

Test material form:

solid: particulate/powder

Details on test material:

Molecular weight: 4113.58

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The active phamacautical ingredient Semaglutide, white powder, batch number: 412-G07011.
- Purity: NA
- Purity test date: NA

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Protected from light and moisture, and refrigerated (5-8°C).
- Stability under test conditions: NA
- Solubility and stability of the test substance in the solvent/vehicle: NA
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: NA


FORM AS APPLIED IN THE TEST (if different from that of starting material): Aerosolized for inhalation.

Test animals

Species:

monkey

Strain:

Macaca fascicularis

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: A total of 12 male and 12 female purpose bred cynomolgus monkeys (Macaca fascicularis) were
obtained from Vietnam via Belgrave Services.
- Age and weight at study initiation: The monkeys were approximately 29 to 35 months of age when treatment started and weighed 2.14
to 2.97 kg (males) and 1.71 to 2.32 kg (females).
- Fasting period before study: No
- Housing: The animals were housed in trios of the same sex/group per cage, to allow social interaction (ie
grooming, playing etc.), except for the period just before each administration until approximately 1
to 2 hours after dosing. Group housing of animals was carried out by removal of partitions between
adjacent cages.
- Diet (e.g. ad libitum): Each animal was offered 200 g of a standard dry diet (Old World Monkey Diet) daily. Each animal
was also offered two biscuit supplements (each weighing 25 g) daily. This basic ration was
supplemented on each day with fresh fruit produce, given in order to enrich the environment and
improve the interaction between the animals and their handlers. Each morning, the residue of the
food (not including any fruit or
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis were received routinely from the sawdust supplier. Each batch of diet was
analysed routinely by the supplier for various nutritional components and chemical and
microbiological contaminants. Supplier’s analytical certificates were scrutinised and approved
before any batch of sawdust or diet was released for use. The quality of the water supply is
governed by regulations published by the Department for Environment, Food and Rural Affairs.
Certificates of analysis were received routinely from the supplier. Since the results of these various
analyses did not provide evidence of contamination that might have prejudiced the study, they are
not presented.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 to 24 °C;
- Humidity (%): Not specified
- Air changes (per hr): Air extraction was via a balanced
system designed to provide approximately 12 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of light and 12 hours of dark per 24-hour.

IN-LIFE DATES: From: To: 20 march 2007 to September 2007

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

other: facemask

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.6 - <= 4.8 µm

Geometric standard deviation (GSD):

2.97

Remarks on MMAD:

The Mass Median Aerodynamic Diameters (MMAD) for Groups 3 and 4 are within the ideal range (1-4 μm) for a repeat dose inhalation study. From particle size analyses, the mean respirable fraction (% < 7 μm aerodynamic diameter) was greater than 81 % for these groups. The MMAD for Group 2 was slightly high, but still within an acceptable range. The percentage of material considered to be of a respirable size (<7 μm) was therefore low, at 60%. This resulted in the low achieved inhaled dose of 78% of target, despite the achieved chamber concentration being excellent at 98% of target.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
- Method of holding animals in test chamber:
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber:
- Air flow rate:
- Air change rate:
- Method of particle size determination:
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes/no

VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle:
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Overall mean chamber concentrations for the constant phase were 98, 88 and 88 % of the target concentrations for Groups 2, 3 and 4 respectively. For the rising dose phase mean chamber concentrations were 102% for Group 3 and 84 then 103% for Group 4.
The achieved inhaled doses were 78, 104 and 97% of the target doses for Groups 2, 3 and 4 respectively during the constant dose phase. For the rising dose phase achieved inhaled doses were 106% for Group 3 and 107 then 127% for Group 4.

Duration of treatment / exposure:

-Group 1(Control group) received air only for 2 weeks.
-Group 2 received sema at a dose of 0.5 mg/kg/day for 2 weeks in the constant dose phase.
-Group 3 received NNC 113-0217 at a dose of 0.5 mg/kg/day for one week in the rising dose phase, followed by a dose of 2.5 mg/kg/day for 2 weeks in the constant dose phase.
-Group 4 received a dose of 0.5 mg/kg/day for one week, followed by a dose of 2.5 mg/kg/day for one week in the rising dose phase, followed by a dose of 10 mg/kg/day for 2 weeks in the constant dose phase.

Frequency of treatment:

30 minutes per Day 7 Day per Week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50.7 mg/m³ air (analytical)

Remarks:

Group 2: Constant dose (males and females)

Doses / concentrations 2

Dose / conc.:

229 mg/m³ air (analytical)

Remarks:

Group 3: Constant dose (males and females)

Doses / concentrations 3

Dose / conc.:

53 mg/m³ air (analytical)

Remarks:

Group 3: Rising dose (males and females)

Doses / concentrations 4

Dose / conc.:

920 mg/m³ air (analytical)

Remarks:

Group 4: Constant dose (males and females)

Doses / concentrations 5

Dose / conc.:

43.7 mg/m³ air (analytical)

Remarks:

Group 4: Rising dose (week 1, males and females)

Doses / concentrations 6

Dose / conc.:

269 mg/m³ air (analytical)

Remarks:

Group 4: Rising dose (week 2, males and females)

No. of animals per sex per dose:

Three female and three male monkeys per sex per dose group (n=6/group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The target inhaled doses used in this study (0, 0.5, 2.5 and 10 mg/kg/day) were selected in conjunction with the Sponsor with reference to previous work with this compound performed in these laboratories (Huntingdon Life Sciences Report Number: JLY0073/070023). In that study doses of up to 0.626 mg/kg/day were clinically well tolerated with no evidence of overt toxicity.

- Rationale for animal assignment (if not random): After arrival the animals were allocated to groups employing a pseudo-random bodyweight stratification procedure to yield groups with approximately equal mean bodyweight (based upon the bodyweights provided by the animal unit). The animals were acclimatised to housing conditions in the primate building for at least four weeks before the start of treatment.

Positive control:

N/A

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Daily during the study, detailed observations were recorded at the following times in relation to dose administration:
During and immediately after dosing.
Between half an hour and two hours after completion of dosing.
As late as possible in the working day.

In addition, a more detailed weekly physical examination was performed on each animal to monitor general health. During the acclimatisation period, observations of the animals and their cages were recorded at least
once per day.


BODY WEIGHT: Yes
- Time schedule for examinations:
The weight of each monkey was recorded (prior to feeding) at least once per week during the acclimatisation period, on the day that treatment commenced, daily thereafter throughout the treatment period and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food (dry diet excluding biscuit supplements) supplied to each animal, that remaining and an estimate of any spilled were recorded daily during the last week of the acclimatisation period and throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and during Week 2 of the constant dose phase, the eyes of all animals allocated to the study were examined by means of a hand held direct ophthalmoscope. Animals
were examined without the use of anaesthesia or sedation.
Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic
solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil
dilated), lens, vitreous and fundus were examined.

- Dose groups that were examined: All animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Before treatment commenced and during Week 2 of the constant dose phase (before dosing), blood samples were obtained without overnight food deprivation from all animals using the femoral or
other suitable vein.
- Anaesthetic used for blood collection: NA.
- Animals fasted: Yes
- How many animals: All animals
- Parameters were examined: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV)
Total white cell count (WBC), Differential WBC count, Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before treatment commenced and during Week 2 of the constant dose phase (before dosing), blood samples were obtained without overnight food deprivation from all animals using the femoral or
other suitable vein
- Animals fasted: Yes.
- How many animals: All animals.
- Parameters were examined: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Total protein (Total Prot.), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos)

URINALYSIS: Yes
- Time schedule for collection of urine: Before treatment and during Week 2 of the constant dose phase, urine samples were collected from all animals during a 4 hour period in the afternoon.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters examined: pH, Specific gravity (SG), Protein (Prot), sodium (U-Na), potassium (U-K) and chloride (U-Cl)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: MORTALITY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Other examinations:

-Electrocardiography:Electrocardiograph tracings were recorded from all animals during the pre-treatment period for the three standard limb leads (I, II and III) and the three augmented limb leads (aVR, aVL and aVF). Further tracings were obtained during Week 2 of the constant dose phase, before dose and at approximately 2 hours after dose administration.

Statistics:

- Summary statistics (e.g. means and standard deviations) were calculated from computer-stored individual raw data.
- Pathological findings were analysed, where considered appropriate, using Fisher’s Exact test for each treated group versus the control.
- For the comparison of more than two treated groups against the control: Outliers were excluded and the reason was indicated. If Levene's test for variance homogeneity (Levene 1960) was not significant at the 1% level, then analysis of variance was performed. If the analysis of variance F-test was significant at the 5% level Dunnett's test (Dunnett 1955, 1964) was performed, otherwise a linear regression test was applied. If Levene’s test was significant at the 1% level, then a logarithmic transformation was tried. If Levene's test was still significant, Kruskal-Wallis test (Kruskal and Wallis 1952, 1953) was performed. If the Kruskal-Wallis test was significant at the 5% level Wilcoxon Rank Sum tests (Wilcoxon 1945) were performed, otherwise Jonckheere’s test (Jonckheere 1954) was applied.

Results and discussion

Results of examinations

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slightly low bodyweight loss (losses of between -0.04 and -0.22 kg), associated with slightly low food intake (reductions of 3 to 6% compared with Control), was apparent in all treated groups with no dose related trend apparent. However these findings are considered not adverse at the slight degree observed and because no evidence of poor condition was observed clinically.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Slightly low food intake (reductions of 3 to 6% compared with Control), was apparent in all treated groups with no dose related trend apparent.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

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):

Reductions in liver weight (16 to 28% reductions in absolute weights for males and 13 to 20% for females) and heart weight (22 to 24% reductions in absolute weights for males and 23 to 25% for females) were evident for all treated groups. However, the degree of effect was much reduced when differences in bodyweight were taken into account, and they were not associated with any changes in clinical pathology parameters or pathological findings. These differences are probably secondary to the bodyweight differences and are not considered to be indications of adverse effect on the organ itself.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

-Slightly low bodyweight gain, associated with slightly low food intake, was apparent in all treated groups with no dose related trend apparent. Reduction in bodyweight gain is a recognised pharmacological action of GLP-1 agonists, and is possibly related to appetite suppression. However the findings on this study are considered not adverse at the slight degree observed and because no evidence of poor condition was observed clinically.
-Lower absolute liver and heart weights were evident for all treated groups. However, the degree of effect was much reduced when differences in bodyweight were taken into account, and they were not associated with any changes in clinical pathology parameters or pathological findings. These differences are probably secondary to the bodyweight differences and are not considered to be indications of adverse effect on the organ itself.

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

920 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest concentration tested without causing adverse effects

Remarks on result:

other:

Remarks:

Highest concentration tested without causing adverse effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The local and systemic toxic potential and toxicokinetics of Semaglutide (a GLP-1 receptor agonist antidiabetic) to cynomolgus monkeys by inhalation administration was assessed over a period of up to 4 weeks. Based on the highest concentration tested which did not cause any adverse effects, the No Observed Adverse Effect Concentration (NOAEC) for Semaglutide was 920 mg/m3.

Executive summary:

 The local and systemic toxic potential and toxicokinetics of Semaglutide (a GLP-1 receptor agonist antidiabetic) to cynomolgus monkeys by inhalation administration (½ h per day) was assessed over a period of up to 4 weeks. Each group consisted of 3 male and 3 female monkeys.

-Group 1 was exposed to vehicle (air) for 2 weeks.

-Group 2 was exposed to Semaglutide at a conc level of 50.7 mg/m3 for 2 weeks in the constant dose phase

-Group 3 was exposed to Semaglutide at a conc level of 53 mg/m3 one week in the rising dose phase, followed by a conc level of 229 mg/kg for 2 weeks in the constant dose phase.

-Group 4 wass exposed to a conc level of 43.7 mg/m3 for one week, followed by a conc level 269 mg/m3 for one week in the rising dose phase, followed by a conc level 920 mg/m3 for 2 weeks in the constant dose phase.

There were no treatment-related effects on clinical observations, ophthalmic examination, haematology, blood chemistry, urinalysis, macropathology or histopathology.

 

There were no adverse effects of treatment. A slight bodyweight loss, associated with slightly low food intake, was apparent in all treated groups with no dose related trend apparent. These findings are likely a result of the pharmacological action of GLP-1 analogues, but the slight effects cannot be considered as adverse effects.

 

Reductions in liver and heart weight were evident for female and males in all treated groups. However, the degree of effect was much reduced when differences in bodyweight were taken into account, and they were not associated with any changes in clinical pathology parameters or pathological findings. These differences are probably secondary to the bodyweight differences and are not considered to be indications of adverse effect on the organ itself.

 

Based on the highest concentration tested which did not cause any adverse effects, the No Observed Adverse Effect Concentration (NOAEC) for Semaglutide was 920 mg/m3.