International Journal of Obesity (2006) 30, 68–72
& 2006 Nature Publishing Group All rights reserved 0307-0565/06 $30.00
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ORIGINAL ARTICLE
Lower mean weight after 14 days intravenous
administration peptide YY3–36 (PYY3–36) in rabbits
AP Sileno, GC Brandt, BM Spann and SC Quay
Nastech Pharmaceutical Company Inc., Bothell, WA, USA
Objective: Endogenous peptide YY3–36 (PYY3–36) is associated with postprandial regulation of appetite. We investigated the
safety and effectiveness of peripherally administered synthetic human PYY3–36 for 14 days in New Zealand white rabbits. Weight
gain and food consumption were assessed and pharmacokinetics and toxicity characterized.
Research methods and procedures: In all, 24 animals were randomized to one of four intravenous treatment groups – control
(0.9% saline) or PYY3–36 bolus at 4.1, 41.0, or 205 mg/kg/day. Body weight and consumption of fixed food allotment were
measured daily. Hematology and serum chemistries were profiled at baseline and Day 15, and pharmacokinetics measured
following dose 14. Histopathologic examination of designated tissues and organs in control and PYY3–36 205 mg/kg animals was
conducted. All animals were subject to clinical and macroscopic observation.
Results: The trend effect of higher dose PYY3–36 on lower average weight was significant (P ¼ 0.01; Day 14 compared to
baseline) and its effect on reduced food consumption was suggested (P ¼ 0.065; number of days p75% food eaten, compared
with control). Hematology and clinical chemistries were within normal limits pretest and at Day 15. No clinical, macroscopic,
histologic, or microscopic changes related to the test article were observed over the course of study.
Discussion: Lower average weight occurs in rabbits treated once daily with intravenous injection PYY3–36 (205 mg/kg/day) over
14 days. No clinical or histologic signs of toxicity were observed. Further research is warranted to describe alternate routes of
peripheral administration for optimizing weight control.
International Journal of Obesity (2006) 30, 68–72. doi:10.1038/sj.ijo.0803067; published online 20 September 2005
Keywords: peptide YY3–36; PYY3–36; gastrointestinal hormone; satiety; radioimmunoassay
Introduction
Peptide YY3–36 (PYY3–36) is an endogenous hormone, produced by L cells of the gastrointestinal tract and released
postprandially in proportion to caloric intake.1–7 As a
selective Y2 receptor agonist, PYY3–36 influences the hypothalamus, including the melanocortin and neuropeptide
Y (NPY) systems of the arcuate nucleus, in its regulation
of appetite.7–11 Intraperitoneal administration of PYY3–36
(single dose 0.3 mg/100 g body weight) has been shown in
24-h fasted rats to produce plasma concentrations of PYY3–36
within the range of normal postprandial concentrations.11
Further, significant reduction in cumulative food intake and
consequent decreased body weight gain have been reported
for rats treated chronically with intraperitoneal PYY3–36
(5.0 mg/100 g body weight, twice daily for 7 days) when
Correspondence: BM Spann, Nastech Pharmaceutical Company Inc., 3450
Monte Villa Parkway, Bothell, WA 98021, USA.
E-mail: [email protected]
Received 4 August 2004; revised 29 June 2005; accepted 17 July 2005;
published online 20 September 2005
compared to a control population.11 In humans, a significant inhibition of food intake for a period of 12 h and a
decrease in cumulative 24-h caloric intake by obese and
lean subjects has been reported following a single peripheral
infusion of PYY3–36 (total dose, 2 nmol/m2 body-surface
area).12 The present study was performed to characterize
the pharmacokinetics, and investigate toxicity, food consumption, and reduction in weight gain effects, of chronic
peripheral administration of synthetic human PYY3–36 at
three dose levels over 14 consecutive days in rabbits.
Methods
In all, 24 New Zealand White rabbits (12 each male and
female; Myrtles Rabbitry Inc.), ages 12–14 weeks and
weighing 2.5–2.8 kg, were used in this study. The animals
were housed individually in stainless-steel cages and environmental conditions were maintained according to published practices of good animal husbandry.13 The study was
conducted at Provident Preclinical Inc. (Doylestown, PA,
USA) in compliance with Good Laboratory Practice (GLP)
Lower average weight after 14 days PYY3–36
AP Sileno et al
69
regulations as set forth in the United States Code of Federal
Regulations (21 CFR 58). Contract study facilities were in
compliance with the Animal Welfare Act of 1966 and
subsequent amendments for the care of animals. All animals
were acclimated for 11–12 days prior to the start of the study
and underwent physical examination.
Rabbits were individually identified and randomized to
one of four intravenous treatment groups: normal saline
(0.9%) control (Group I), or synthetic human PYY3–36 at
4.1 mg/kg (Group II), 41.0 mg/kg (Group III), or 205 mg/kg per
day (Group IV). Margins of safety for tested PYY3–36 dosages
(as compared with the expected human daily intravenous
dose of 20 mg) were 3.3X, 33X, and 166X, respectively. Each
of the four treatment groups was comprised of three males
and three females. Animals within each treatment group
were assigned to one of two subgroups (Squad A or B) to pace
the volume of clinical pathology and toxicokinetics procedures conducted on Day 15. Day 1 treatment was initiated
for Squad A animals, 1 day prior to that for Squad B.
Assigned test drug and control articles were administered
as intravenous bolus injection (0.4 ml/kg) via the marginal
ear vein for 14 consecutive days. Animals were observed each
day for signs of toxicity at 15 and 30 min, and 1, 3, and 5 h
postinjection. At 2–4 h following injection, animals were
provided 150 g Hi Fiber Rabbit Diet 5325 (Purina Mills Inc.,
Richmond, IN, USA) per day. The diet contained 3.67 kcal/g
of gross energy and 1.99 kcal/g of metabolizable energy.
Daily food consumption was measured as the estimated
percentage of food eaten (0, 1–25, 26–50, 51–75, and 76–
100%) over each 24-h period. Water was available to animals
at all times. Measurement of body weight was carried out
prior to dose injection or food allotment on baseline Day 1
and daily thereafter.
Hematology and fasting serum chemistry profiles (Table 1)
were collected for all animals on Day 1 prior to the first
intravenous dose, and on Day 15. Venous blood samples for
pharmacokinetics were drawn on Day 14 from all animals
before, and 2, 5, 10, 15, 30, and 45 min after, intravenous
injection of the study agent. Venous access was via jugular
vein or the marginal vein of the ear contralateral to that
used for injection. Blood samples (2 ml) were transferred to
laboratory test tubes containing potassium EDTA (Becton,
Dickenson and Company, Franklin Lakes, NJ, USA) and
protease inhibitor, aprotinin (0.6 TIU/ml; 50 ml/ml whole
blood; MP Biomedicals, Irvine, CA, USA). Plasma was
separated and dispensed into aliquots following centrifugation at 2366 r.p.m. at 41C for approximately 15 min. Aliquots
were frozen at 701C.
Table 2
Group
Group
Group
Group
All animals were anesthetized on Day 15 by intramuscular
injection of ketamine hydrochloride (20 mg/kg) and xylazine
(10 mg/kg; Vedco, St Joseph, MO, USA), euthanized by
exsanguination, and examined. Macroscopic lesions identified on examination and designated tissues and organs
(Table 2) were excised and preserved in 10% neutral-buffered
formalin. Representative sections of designated tissues and
organs collected from all Group I and Group IV animals were
routinely processed, embedded in paraffin, sectioned, and
stained with hematoxylin and eosin for microscopic evaluation. Histopathology processing, preparation, and evaluation were performed in accordance with the laboratory’s
standard operating procedures (Research Pathology Services
Inc.) and audited in compliance with GLP.
Test and control articles
Test drug article was synthetic human PYY3–36 (Bachem
California, Torrance, CA, USA), formulated to concentrations
of 10.25, 102.5, and 512.5 mg/ml. Sterile normal saline
solution (0.9%) was used as the control article. PYY3–36
formulations were stored at 2–81C. Single lot numbers of test
and control articles were used for each study group
throughout the 14-dose regimen.
Radioimmunoassay of PYY3–36
Concentration of plasma synthetic human PYY3–36 was
determined using radioimmunoassay methods developed
and validated by our laboratory. Calibrators and controls
were constituted in matrices identical to experimental
samples, and stripped of endogenous PYY. Plasma samples
and constituted calibrators and controls were extracted in
alcohol, evaporated under vacuum, and resuspended in a
standard phosphate buffer with aprotinin. Each was then
Table 1
Serum chemistry profile on Day 1 and Day 15
Sodium
Potassium
Chloride
Glucose
Calcium
Total protein
Albumin
Globulin
Albumin/globulin ratio
Cholesterol
Triglycerides
Urea nitrogen
Creatinine
BUN/creatine ratio
Inorganic phosphorus
Total bilirubin
Direct bilirubin
Alanine aminotransferase (ALT)
Alkaline phosphatase
Gamma glutamyltranspeptidase
Aspartate aminotransferase (AST)
Pharmacokinetic parameters for intravenous synthetic human PYY3–36 in rabbits (mean7s.d.)
I (0.9% saline)
II (PYY3–36, 4.1 mg/kg)
III (PYY3–36, 41 mg/kg)
IV (PYY3–36, 205 mg/kg)
Cmax (mg/kg)
Tmax (min)
AUC0–t (min mg/ml)
T1/2 (min)
0.000270.0001
0.02670.009
0.41470.166
2.19270.808
20722
2
2
2
0.00470.0037
0.33570.094
4.4770.606
30.117711.079
33.5715.2
13.772.3
19.374.2
19.173.2
International Journal of Obesity
Lower average weight after 14 days PYY3–36
AP Sileno et al
70
10.000000
1.000000
Mean (µg/mL)
mixed with rabbit polyclonal anti-human PYY antiserum,
incubated, and mixed with iodinated human PYY. Agents for
immunoprecipitation were goat anti-rabbit IgG and normal
rabbit serum. Bound and free fractions were separated by
centrifugation and the radioactivity in precipitates quantified
by gamma counter. Assay limits of quantification for synthetic
human PYY3–36 range from 40 to 1280 pg/ml. No high-dose
hook effect is observed through 16 000 pg/ml. The assay
antibody does not crossreact with human amylin, glucagon,
insulin, NPY, substance P, or pancreatic polypeptide.
0.100000
0.001000
0.000100
0.000010
0.000001
0
5
10 15 20 25 30 35 40 45
Time (min)
Figure 1 Mean plasma levels (log-linear).
3.05
Control (0.9% Saline)
SE
4.1 µg/kg
SE
41 µg/kg
SE
205 µg/kg
SE
3.00
2.95
Weight (kg)
Data analysis
Maximum drug concentration (Cmax), area under the curve
(AUC0–t), and half-life (T1/2) were calculated by noncompartmental modeling with data uncorrected for baseline from
time prior to dosing to time of last measurable concentration, using the linear trapezoidal rule (WinNonLins, Pharsight Inc.). Weight data for animals on Day 1 and Day 14
were analyzed using the one-way analysis of covariance
model with Day 1 results as the adjustment covariate.
Cook’s D statistic was used to detect observations of
undue influence to examine their effect on the regression
model.14,15 Food consumption data were analyzed using the
Cochran–Armitage test for trend and the Kruskal–Wallis
nonparametric one-way analysis of variance model was used
to compare group means.
Group 1
SD
Group 2
SD
Group 3
SD
Group 4
SD
0.010000
2.90
2.85
2.80
2.75
2.70
2.65
2.60
Results
0
2
4
6
8
10
12
14
Day
Pharmacokinetics
Peak mean plasma concentration (Cmax) of synthetic human
PYY3–36 in Group II, III, and IV animals were 0.02670.009,
0.41470.166, and 2.19270.808 mg/ml, respectively (Table 2).
Maximum concentration was reached at 2 min (Tmax), the
first time point assayed, for all groups administered active
agent. The mean T1/2 for PYY3–36 in Group III and Group IV
were 19.374.2 and 19.173.2 min. The mean T1/2 for lowdose PYY3–36 Group II was 13.772.3 min. AUC0–t was
0.33570.094, 4.47170.606, and 30.117711.079 min mg/ml
for Groups II, III, and IV, respectively (Figure 1).
Weight gain and food consumption
A significant trend of lower average weight for animals was
associated with higher dose PYY3–36 at Day 14, compared to
Day 1, adjusted for baseline weight, sex, and assigned squad
(P ¼ 0.01) (Figure 2). When animal 919 (Group IV) was
removed due to a large value for Cook’s D (influential
observation), the trend effect of the higher dose PYY3–36 on
lower average weight was highly significant (P ¼ 0.0001).
A trend for reduced food consumption was suggested for
higher doses PYY3–36 when dose groups were compared with
the number of days that animals ate p75% food (P ¼ 0.065)
or with the proportion of animals that ate p75% food for a
minimum of 1 day (P ¼ 0.14).
International Journal of Obesity
Figure 2 Daily mean weights (s.e.) by dose group.
Normative data for food consumption is 50 g/kg/day and
110 cal/kg/day for basal metabolism for New Zealand White
rabbits. All the animals weighed approximately 2.7–2.8 kg
(0.1) on Day 1. Using the Kleiber equation for maintenance
requirements (70 Wkg0.75), for a 2.7–2.8 kg rabbit, the
average energy maintenance requirements would be
150 kcal/day for all animals per group.
The ratio of food consumed to basal metabolism and
maintenance is independent of body weight and therefore
the calories of tissue formed per 100 calories of food
consumed are about the same for all the rabbits in the study.
The rate of gain is proportional to metabolic size and is
approximately the same for all rabbits in the study at a
weight of 2.7–2.8 kg on Day 1 using the equation Wkg0.75.
The rabbits were fed 150 g of a diet containing 3.67 kcal/g of
gross energy and 1.99 kcal/g of metabolizable energy. Therefore, a rabbit that ate 150 g of diet would have an available
298.5 kcal of metabolizable energy per day or energy intake
efficiency for maintenance and weight gain. Since the
animals weighed approximately the same (2.7270.1) when
entering the study, their food consumption, basal metabolism, and energy maintenance needs should be the same for
all groups and should not affect group comparisons on body
weight gain and food consumption.
Lower average weight after 14 days PYY3–36
AP Sileno et al
71
Table 3
Tissues and organs designated for histopathology in Group I (control) and Group IV (PYY3–36, 205 mg/kg)
No microscopic observations Histomorphologic observations
Group I
Group IV
Study number, male(m)/female(f)
Study number, male(m)/female(f)
901m 902m 903m
Adrenal
glands
Aorta
Cecum
Pancreas
Brain
Parathyroids Heart
Pituitary
Jejunum
Cervix
Prostate
Colon
Rectum
Kidneys
Duodenum Sciatic nerve
Epididymides Seminal
Liver
vesicles
Esophagus
Eyes
Femur
Skeletal
muscle
Spinal cord
Spleen
Lung
Gall Bladder Sternum
(with marrow
and bone)
Ileum
Stomach
Lacrimal
glands
Mammary
glands
Optic nerve
Ovaries
Urinary
bladder
Uterus
905f
Lymph node,
mandibular
906f
919m 920m 921m
Dilatation, ventricular
Infiltration, heterophilic
Macrophages, Peyer’s
patches
Dilatation, tubular,
multifocal
Infiltration, mononuclear
cell, focal
Cyst(s), medulla
Vacuolation,
hepatocellular, multifocal;
infiltration, mononuclear
cell, periportal
Minimal hyperplasia,
lymphoid,
Perivascular/peribronchial
Inflammation, interstitial,
multifocal
Microgranuloma
923f
924f
1
1
1
1
P
1
1
1
1
1
1
1
1
2
2
1
1
2
1
1
1
1
1
1
2
Congestion
2
1
2
1
2
Hyperplasia, lymphoid
Infiltration, heterophilic
Lymph node, Hyperplasia, lymphoid
mesenteric
Salivary glands Hyperplasia, ductular
Testes
Degeneration, diffuse,
unilateral
Thymus
Macrophages, pigmented
Thyroid
Cysts
Trachea
Inflammation, chronic,
focal/multifocal
Vagina
Congestion, acute
922f
2
Macrophages, pigmented,
multifocal
Emphysema, focal
Thymus
Tongue
904f
1
2
2
3
2
2
3
2
3
2
P
P
P
1
1
2
1 ¼ Minimal degree or amount of indicated change or lesion; 2 ¼ mild; 3 ¼ moderate; P ¼ indicated change or lesion present.
Energy expenditure and body composition at necropsy
was not evaluated.
Since food consumption was based on an estimation key
and not weight, we found eight instances in Group IV where
25% of the food was remaining and only one instance in the
control. Therefore, the changes in body weight across groups
are most likely due to food consumption between groups.
Safety and tolerance
Two animals (one female, one male) in Group III developed
tachypnea and bilateral reddening of ears following intravenous dose of PYY3–36 at 41.0 mg/kg, subsiding within
30 min and with no observed sequelae. All other animals
were observed to be free of abnormal or treatment-related
signs of toxicity up to 5 h postinjection. All animals survived
to study completion and necropsy.
Hematologic parameters were within normal limits on Day
1 and Day 15 for all animals in all treatment groups. Serum
chemistry results for all animals were within normal limits at
pretest and following the 14th consecutive days treatment.
Minimal to mild microscopic changes were observed in the
intravenous injection sites and surrounding perivascular
tissues. Observed changes occurred at similar incidence and
severity in both control Group I and test article Group IV
(PYY3–36 at 205 mg/kg). The most common changes in both
groups were perivascular mixed inflammatory cell (mononuclear and heterophilic) infiltrations and perivascular
International Journal of Obesity
Lower average weight after 14 days PYY3–36
AP Sileno et al
72
hemorrhage, edema, and fibroplasias. In control Group I
animals, venous thrombus and focal necrosis were observed
in one animal and focal perivascular necrosis in a second.
Histomorphologic observations are summarized in Table 3.
Microscopic changes, where observed in designated organs
and tissues, were graded as mild to moderate severity, and
were considered not related to administration of the test
article. No treatment-related differences for individual organ
weights (gross, or as a percentage of body weight and brain
weight) were identified.
Pulmonary tissues in 11 of 12 animals (Groups I and IV
combined) exhibited minimal to mild microscopic changes
including multiple foci of interstitial inflammation, hyperplasia of peribronchial and perivascular lymphoid tissues,
focal emphysema, foci of pigmented macrophages, and
occasional microgranuloma. Interstitial inflammation and
microgranulomas of pulmonary tissues are reported to be
incidental findings in laboratory animals enrolled in intravenous injection studies.16
Discussion
This study demonstrates that lower average weight occurs in
rabbits treated once daily with intravenous injection PYY3–36
(205 mg/kg daily dose) administered each of 14 consecutive
days when compared with saline controls. A suggested
trend in reduced food consumption was observed in rabbits
provided with a fixed daily food allotment. The diet was
appropriate for normal rabbit maintenance and weight gain.
Since all the animals entered the study at approximately the
same body weight, food consumption, basal metabolism,
energy expenditure, and maintenance should be the same
for all groups and should not affect group comparisons.
Energy expenditure is represented by basal metabolic rate.
There is a possibility that PYY3–36 may also raise basal
metabolic rates, thus resulting in weight loss. Lower average
weight gain and reduced food consumption have been
previously reported for freely feeding rodents treated twice
daily with intraperitoneal injection PYY3–36 (100 mg/kg daily
dose) over 7 days.11 Dose-dependent and significant inhibition of food consumption has also been demonstrated in
mice acclimated to laboratory handling for 1 week prior to
receiving a single intraperitoneal dose PYY3–36 (3.0, 30, and
100 mg/kg).17
No abnormal clinical or macroscopic laboratory findings
were attributed in this study to PYY3–36 at any of three tested
dose levels (4.1, 41, or 205 mg/kg daily dose). Observations
made at histopathology were considered to be incidental or
spontaneous in origin and independent of control or active
agent. Microscopic changes observed in intravenous injection site and in pulmonary tissues were similar in incidence
and severity for both Group I (control) and Group IV
animals. All hematology and clinical chemistry values were
within normal limits at Day 15 and did not change
significantly from those of Day 1 baseline.
International Journal of Obesity
Chronic peripheral administration of an active agent to
control food consumption and weight may require ondemand dosing for an as-yet-to-be determined regimen. This
study suggests peripheral administration of synthetic human
PYY3–36 is associated with no clinical or histologic signs
of toxicity after 14 days treatment. Further research is
warranted to describe alternate routes of peripheral administration, including intranasal route, with predicted peak
concentration and T1/2 values within the normal physiological range.
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