British Journal of Pharmacology (2003) 140, 123–132
`
`& 2003 Nature Publishing Group All rights reserved 0007 – 1188/03 $25.00
`www.nature.com/bjp
`
`GLP-1 derivative liraglutide in rats with b-cell deficiencies:
`influence of metabolic state on b-cell mass dynamics
`
`*,1Jeppe Sturis, 1Carsten F. Gotfredsen, 1John Rømer, 1Bidda Rolin, 1Ulla Ribel, 1Christian L.
`Brand, 1Michael Wilken, 1Karsten Wassermann, 2Carolyn F. Deacon, 1Richard D. Carr & 1Lotte
`Bjerre Knudsen
`
`1Discovery, Novo Nordisk A/S, Novo Alle´ , DK-2880 Bagsværd, Denmark and 2The Panum Institute, University of Copenhagen,
`Copenhagen, Denmark
`
`1 Liraglutide is a long-acting GLP-1 derivative, designed for once daily administration in type II
`diabetic patients. To investigate the effects of liraglutide on glycemic control and b-cell mass in rat
`models of b-cell deficiencies, studies were performed in male Zucker diabetic fatty (ZDF) rats and in
`60% pancreatectomized rats.
`2 When liraglutide was dosed s.c. at 150 mg kg1 b.i.d. for 6 weeks in ZDF rats 6 – 8 weeks of age at
`study start, diabetes development was markedly attenuated. Blood glucose was approximately 12 mm
`lower compared to vehicle (Po0.0002), and plasma insulin was 2 – 3-fold higher during a normal 24-h
`feeding period (Po0.001). Judged by pair feeding, approximately 53% of the antihyperglycemic effect
`observed on 24-h glucose profiles was mediated by a reduction in food intake, which persisted
`throughout the study and averaged 16% (Po0.02).
`3 Histological analyses revealed that b-cell mass and proliferation were significantly lower in
`prediabetic animals still normoglycemic after 2 weeks treatment compared to vehicle-treated animals
`that had begun to develop diabetes. When the treatment period was 6 weeks, the liraglutide-treated
`animals were no longer completely normoglycemic and the b-cell mass was significantly increased
`compared to overtly diabetic vehicle-treated animals, while b-cell proliferation was unaffected.
`In the experiments with 60% pancreatectomized rats, 8 days treatment with liraglutide resulted in a
`4
`significantly lower glucose excursion in response to oral glucose compared to vehicle treatment. Again,
`part of the antihyperglycemic effect was due to reduced food intake. No effect of liraglutide on b-cell
`mass was observed in these virtually normoglycemic animals.
`In conclusion, treatment with liraglutide has marked antihyperglycemic effects in rodent models of
`5
`b-cell deficiencies, and the in vivo effect of liraglutide on b-cell mass may in part depend on the
`metabolic state of the animals.
`British Journal of Pharmacology (2003) 140, 123–132. doi:10.1038/sj.bjp.0705397
`Keywords: Glucagon-like peptide-1; type II diabetes; Zucker diabetic fatty; 60% pancreatectomy
`
`Abbreviations: AUC, area under the curve; b.i.d., twice a day; BrdU, bromodeoxyuridine; DPPIV, dipeptidyl-peptidase IV;
`GLP-1, glucagon-like peptide-1; OGTT, oral glucose tolerance test; ZDF, Zucker diabetic fatty
`
`Introduction
`
`Glucagon-like peptide-1 (GLP-1) is secreted from the intestinal
`L-cells
`in response
`to ingested carbohydrates and fat
`(Kreymann et al., 1987; Ørskov, 1992; Holst, 1997). The
`molecule has a spectrum of physiological effects, including
`glucose-dependent stimulation of insulin secretion and inhibi-
`tion of glucagon secretion (Gromada et al., 1998), inhibition of
`small bowel motility (Tolessa et al., 1998) and gastric emptying
`(Nauck et al., 1997), and reduction of appetite (Flint et al.,
`1998; Toft-Nielsen et al., 1999). Moreover, recent evidence
`suggests that GLP-1 and analogs act as trophic agents in the
`pancreas, causing b-cell proliferation and neogenesis (Buteau
`et al., 1999; Edvell & Lindstro¨ m, 1999; Xu et al., 1999; Perfetti
`et al., 2000; Moldrup et al., 2001; Farilla et al., 2002; Rolin
`et al., 2002), and some studies indicate that it also inhibits
`
`*Author for correspondence; E-mail: jstu@novonordisk.com
`Advance online publication: 29 July 2003
`
`b-cell apoptosis (Bregenholt et al., 2001; Farilla et al., 2002; Li
`et al., 2003). Thus, GLP-1 may be an important physiological
`regulator of b-cell mass. Taken as a whole, these properties
`make GLP-1 ideally suited to be developed as an antidiabetic
`agent, particularly since many of its effects are glucose-
`dependent and therefore treatment would likely be associated
`with a very low risk of hypoglycemia. Consequently, many
`studies have been undertaken and shown GLP-1 to be highly
`effective in reducing blood glucose levels in patients with type
`II diabetes (Nauck et al., 1993; Gutniak et al., 1994; Rachman
`et al., 1997; Larsen et al., 2001a). Native GLP-1 is, however,
`rapidly degraded by dipeptidyl peptidase IV (DPPIV; Mentlein
`et al., 1993; Deacon et al., 1995) and cleared by the kidneys
`(Deacon et al., 1996), giving the biologically active peptide a
`half-life of less than 2 min after i.v. administration (Deacon
`et al., 1996) and 1 – 2 h after s.c. administration (Knudsen et al.,
`2000). Since the presence of an elevated GLP-1 concentration
`is necessary in type II diabetic patients in order to obtain a
`continuous effect, the pharmacokinetic properties of the native
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`124
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`J. Sturis et al
`
`Liraglutide in ZDF and 60% pancreatectomized rats
`
`peptide make it less than ideal for therapeutic use. Liraglutide
`(Arg34Lys26-(N-e-(g-Glu(N-a-hexadecanoyl)))-GLP-1(7-37)),
`also known as NN2211,
`is a novel
`long-acting analog,
`obtained by derivatizing GLP-1 with a fatty acid side chain,
`which promotes albumin binding and prevents degradation by
`DPPIV (unpublished observations). Liraglutide and GLP-1
`are equipotent in vitro (Knudsen et al., 2000). When injected
`s.c., these features, combined with a slow release from the
`injection site, result in a compound with a prolonged plasma
`half-life of 14 h in pigs (Knudsen et al., 2000) and 10 – 12 h in
`man (Agers et al., 2002; Juhl et al., 2002), which makes it
`suitable for once daily administration (Knudsen et al., 2000).
`The present study was undertaken to investigate the effects
`of liraglutide on glycemia and b-cell mass in two b-cell-
`deficient rat models: male Zucker diabetic fatty (ZDF) rats, a
`model of type II diabetes in which insulin resistance and b-cell
`defects are prominent features (Clark et al., 1983; Etgen &
`Oldham, 2000), and 60% pancreatectomized Sprague – Dawley
`rats, a model of b-cell deficiency (Liu et al., 2000).
`
`Methods
`
`Animals
`
`All studies were carried out with permits from the Animal
`Experiments Inspectorate, Ministry of Justice, Denmark. Male
`ZDF (fa/fa) rats (ZDF) and lean male ZDF (fa/ þ or þ / þ )
`rats
`(lean) were purchased from Genetic Models
`Inc.
`(Indianapolis, IN, U.S.A.) and housed two per cage (study
`1a and 1c) or individually (study 1b). Male Sprague – Dawley
`rats were obtained from M&B, Lille Skensved, Denmark, and
`were housed individually. Unless otherwise stated, animals had
`free access to food (Purina 5008) and drinking water.
`
`Experimental procedures
`
`Dosing
`In all studies, the animals were dosed with sub-
`cutaneous injections (1 ml kg1) of vehicle or liraglutide at the
`various doses indicated at approximately 0715 – 0800 and
`1400 – 1415 h. Twice daily dosing was used because the
`pharmacokinetic half-life of liraglutide is only approximately
`4 h in rats (Novo Nordisk, data on file).
`
`Blood sampling Blood for the determination of whole-
`blood glucose concentration and HbA1c was collected into
`heparinized 10 and 5 ml glass tubes, respectively, by puncture
`of the capillary vessels in the tail tip, and diluted in analysis
`buffer. Samples for insulin measurements were also collected
`from the tail into 100-ml heparin-coated glass tubes and were
`centrifuged at 41C for 10 min and plasma was separated and
`stored at 201C until assayed.
`
`Oral glucose tolerance test Oral glucose tolerance tests
`(OGTTs) were performed in fasted animals. For ZDF rats,
`1 g kg1 glucose solution and for 60% pancreatectomized rats,
`2 g kg1 glucose solution was administered by gavage. Com-
`pound or vehicle was administered at the regular time points.
`Samples for the measurement of blood glucose and, in some
`cases, plasma insulin were drawn immediately prior to glucose
`administration and at intervals thereafter.
`
`British Journal of Pharmacology vol 140 (1)
`
`Studies in ZDF rats
`
`These studies were performed to investigate the ability of
`liraglutide to influence diabetes development and b-cell
`proliferation and mass in the ZDF rat model.
`Study 1a Three groups of animals (n¼ 6; age 7 – 8 weeks at
`the start of the study) were housed two per cage and treated
`b.i.d. with either vehicle or liraglutide (low dose, 30 mg kg1;
`high dose, 150 mg kg1) for 6 weeks. Initial random glucose
`concentrations were not significantly different between the
`three groups (vehicle: 6.5570.33; low dose: 6.4070.22; high
`dose: 6.4270.20 mm). Blood samples for 24-h profiles of
`glucose were taken approximately every 4 h on day 9, OGTTs
`were performed on days 21 and 36, and a final 24-h profile of
`glucose and insulin was carried out on day 41. Food and water
`intake were monitored daily (at approx. 0800 h), and body
`weight was measured regularly.
`
`Study 1b Pair feeding was employed in order to investigate
`to what extent the blood glucose-lowering effect observed with
`150 mg kg1 liraglutide in study 1a could be ascribed to the
`reduction in food intake. Four groups of animals (age 6 weeks
`at the start of the study) were individually housed and treated
`b.i.d. with either vehicle (lean, n¼ 9; ZDF vehicle, n¼ 7; ZDF
`pair-fed, n ¼ 8) or liraglutide (150 mg kg1, n¼ 8) for 6 weeks.
`Pair-matching was based on the animals’ initial body weights.
`Daily food consumption was measured for each liraglutide-
`treated animal and this amount of food was then made
`available to the pair-matched vehicle-treated animal on the
`following day. Glucose and insulin profiles (24 h) were assessed
`on day 38. Food and water intake were measured daily and
`body weight was recorded twice weekly.
`Study 1c Two groups of animals (n¼ 9 – 10; age 8 weeks at
`the start of the study) received either vehicle or liraglutide
`(200 mg kg1) b.i.d. for 2 weeks. OGTTs were performed after
`the first dose and again after 13 days. Food and water
`consumption were recorded throughout the study period.
`Body weight and fructosamine were measured before and at
`the end of the study.
`
`Studies in 60% pancreatectomized rats
`
`These studies were performed to test the effect of short-term
`liraglutide treatment on glucose tolerance and b-cell mass in
`60% pancreatectomized rats, a nongenetic model of b-cell
`deficiency.
`
`Study 2a
`In all, 12 male Sprague – Dawley rats of 80 – 100 g
`(100 g1)
`(50 ml
`received presurgical streptocillin treatment
`i.m.) prior to isofluoran anesthesia and were subjected to
`60% pancreatectomy using the method previously described
`(Liu et al., 2000). Briefly, the portion of the pancreas bordered
`by the spleen and ventriculus extending to the duodenum was
`carefully removed by gentle abrasion with cotton applicators,
`leaving the ligament with arteries and veins intact. The
`remnant pancreatic tissue was anatomically defined as the
`duodenal segment. All animals received postsurgical pain relief
`Finadyne (20 ml (100 g1) i.m.) and were allowed to recuperate
`for 4 days postoperatively. Animals were allocated into two
`groups (n¼ 6), based on an OGTT on day 4, after which they
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`J. Sturis et al
`
`Liraglutide in ZDF and 60% pancreatectomized rats
`
`125
`
`received either vehicle or liraglutide (100 mg kg1) b.i.d. for 4
`days. A second OGTT was carried out on day 8.
`
`Study 2b
`In all, 24 male Sprague – Dawley rats were
`subjected to 60% pancreatectomy and allocated into three
`groups (n ¼ 8) after an OGTT on day 4. One group was
`thereafter treated with vehicle, a second vehicle-treated group
`was pair-fed relative to the liraglutide-treated group, and the
`third group received liraglutide (150 mg kg1) b.i.d., all for 4
`days. A second OGTT was carried out on day 8.
`
`Histology
`In all studies, 4 h prior to killing, animals were
`injected with bromodeoxyuridine (BrdU, 100 mg kg1 i.p.).
`The animals were anesthetized with CO2, decapitated, and
`approximately 5 ml whole blood for determination of fructo-
`samine (study 1a) and liraglutide concentrations (study 1b)
`was collected from the trunk into 10 ml tubes containing
`0.12 ml 15% K3 ETDA. The pancreata were immediately
`isolated for histological analysis, weighed,
`fixed in 4%
`paraformaldehyde overnight, cut in small pieces, fractionated
`according to the smooth fractionator principle (Bock et al.,
`1999), dehydrated, and embedded in paraffin. Paraffin sections
`(3 mm) were cut, deparaffinized, rehydrated, the endogenous
`peroxidase blocked by H2O2 in 96% ethanol, and blocked with
`avidin and then biotin, before use for immunohistochemical
`staining of islet cell markers, which was carried out with slides
`from all pancreata in parallel. For antigen retrieval, the
`sections in 0.01 m citrate buffer pH 6.0, preheated to 901C,
`were heated 3 5 min in a microwave oven, before blocking
`with avidin and biotin blocking solutions (DAKO) and 10%
`normal goat serum. BrdU was stained by monoclonal mouse
`anti-BrdU (M0744, DAKO) 1 : 50 in 7% goat serum 3% rat
`serum in TBS, biotinylated goat anti-mouse Ig (E0433,
`DAKO) 1 : 300, and streptavidin peroxidase (Vectastain,
`Vector). The peroxidase activity was developed 5 min with
`(DAB) þ 0.01% H2O2þ 2.5%
`0.066% diaminobenzidine
`NiSO4 to render BrdU containing nuclei black. Insulin was
`stained with guinea-pig antiinsulin (#651041, ICN) 1 : 400 in
`7% rabbit serum 3% rat serum in TBS-T, peroxidase-coupled
`rabbit anti-guinea-pig Ig (P141, DAKO) 1 : 100, and developed
`with DAB for brown cytoplasm or with Vector Nova Red
`(Vector) according to the manufacturer, to stain the b-cell
`cytoplasm reddish brown. Finally, the slides were lightly
`counterstained with Mayer’s hematoxylin. Neighboring sec-
`tions were stained for non-b-cells with a mixture of mono-
`clonal mouse antiglucagon (GLU-001, Novo Nordisk)
`1 : 800þ rabbit antisomatostatin (A566, DAKO) 1 : 600þ
`rabbit antipancreatic polypeptide (A619, DAKO) 1 : 1000 in
`4% swine serumþ 4% goat serum þ 3% rat serum in TBS-T,
`detected using biotinylated goat anti-mouse IgG (E0433,
`1 : 400 þ biotinylated
`DAKO)
`swine
`anti-rabbit
`(E353,
`DAKO) 1 : 400, streptavidin peroxidase, and developed with
`DAB and NiSO4, as above. In ZDF studies 1a and 1b, b-cells
`were subsequently stained for insulin as described above.
`Reagents were obtained from DAKO, Copenhagen, Denmark,
`if not otherwise stated. BrdU staining of cell nuclei was
`examined in 1000 – 1500 b-cells per section in an Olympus BX-
`50 microscope with a video camera and monitor (Olympus,
`Copenhagen, Denmark), at a total on-screen magnification of
` 1920. The sections were systematically scanned using a PC-
`controlled motorized stage and CAST-GRID software (Olym-
`pus). The volume fractions of b- and non-b-cells were
`
`estimated by point-counting stereological techniques, at a
`total on-screen magnification of  960, a grid of 6 64 points,
`and step lengths of 1200  1000 mm2. The sections were
`examined with their origin blinded to the observer. Two
`sections cut 250 mm apart were stained and analyzed for each
`set of estimations.
`
`Assays Glucose concentrations were measured by the
`immobilized glucose oxidase method using an EBIO Plus
`autoanalyser (Eppendorf, Germany). Plasma insulin concen-
`tration was measured with an in-house ELISA method
`(Johansen et al., 1999) using 15 ml samples. Rat insulin was
`used as standard. Per cent HbA1c (Roche A/S) was measured
`by the enzymatic calorimetric method (COBAS MIRA Plus
`Autoanalyser, Roche Diagnostic Systems, Basel, Switzerland).
`The concentration of liraglutide was measured by sandwich
`ELISA (Wilken et al., 2000).
`
`Statistics Paired and unpaired t-tests and one-way analysis
`of variance (ANOVA) with Duncan’s and Tukey’s post hoc
`tests for pairwise group comparisons were employed. Area
`under the curve (AUC) was calculated with the trapezoidal
`rule. Data are expressed as mean7s.e.m. Differences with
`Po0.05 were considered significant.
`
`Results
`
`ZDF studies: metabolic parameters
`
`Glucose tolerance tests Study 1a: Both the low and high
`doses of liraglutide significantly lowered glucose excursions
`(AUC) compared to vehicle treatment. Differences were seen
`both during the first OGTT (Figure 1; day 21; Po0.0005,
`ANOVA), and during the second OGTT (Figure 1; day 36;
`2250790 mm min
`AUCs:
`vehicle
`vs
`low
`dose
`14507180 mm min vs high dose 760740 mm min, Po0.0002,
`ANOVA, all groups different by post hoc testing), demonstrat-
`ing a continuing antihyperglycemic effect at both dose levels.
`Plasma insulin concentrations were also increased in a dose-
`dependent manner (AUCs: vehicle 3375 nm min and low dose
`59712 nm min vs high dose 11778 nm min, Po0.0002, ANO-
`VA, high dose different from low dose and vehicle) and the
`effect of the drug increased with time. Thus, in the high-dose
`group, the AUC of insulin was increased between the first and
`second OGTT (Po0.005, paired t-test), the increase averaging
`66%. In contrast, in the vehicle group, the AUC of insulin was
`decreased between the first and second OGTT (Po0.0005,
`paired t-test), the decrease averaging 40%. In the second
`OGTT, the effect on the second phase of insulin secretion
`appeared particularly pronounced in the high-dose group.
`Study 1c: In the shorter-term study, no effect of the first dose
`was observed on glucose or insulin excursion during the
`OGTT, while significant effects on both parameters were
`observed on day 13 (data not shown).
`
`Profiles (24 h), fructosamine and HbA1c Study 1a: After
`41 days treatment, the final 24-h glucose profile (Figure 2) was
`significantly lower in the high-dose liraglutide group, the
`difference to the vehicle-treated group averaged B12 mm
`(glucose AUCs: vehicle 3171 m min and low dose 2673 m min
`vs high dose 1472 m min, Po0.0002, ANOVA, high dose
`
`British Journal of Pharmacology vol 140 (1)
`
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`Vehicle
`30 µg kg-1 bid
`150 µg kg-1 bid
`
`25
`
`20
`
`15
`
`10
`
`05
`
`Blood glucose (mM)
`
`Vehicle
`30 µg kg-1 bid
`150 µg kg-1 bid
`
`0
`
`80
`40
`Time (min)
`
`120
`
`0
`
`80
`40
`Time (min)
`
`120
`
`1500
`
`1000
`
`500
`
`0
`
`Plasma insulin (pM)
`
`126
`
`25
`
`20
`
`15
`
`10
`
`05
`
`Blood glucose (mM)
`
`1500
`
`1000
`
`500
`
`0
`
`Plasma insulin (pM)
`
`0
`
`80
`40
`Time (min)
`
`120
`
`0
`
`80
`40
`Time (min)
`
`120
`
`Figure 1 ZDF study 1a. OGTT after 21 days (left) and 36 days
`liraglutide treatment (right). Glucose (1 g kg1) was administered by
`gavage at time 0 to overnight fasted animals and BG and plasma
`insulin were measured basally and after the glucose challenge. All
`the three groups differed significantly with respect to total AUC
`after the glucose challenge (Po0.0005 by ANOVA, day 21,
`Po0.0002 by ANOVA, day 36).
`
`different from low dose and vehicle). In contrast, the 24-h
`insulin profile demonstrated increased insulin levels in the
`high-dose group (insulin AUCs: vehicle 0.7470.12 mm min and
`low dose 0.9270.30 mm min vs high dose 2.35 þ 0.34 mm min,
`Po0.002, ANOVA, high dose different from low dose and
`vehicle). Fructosamine was significantly reduced in the high-
`dose group (vehicle 22977 mm and low dose 219711 mm vs
`high dose 17478 mm, Po0.002, ANOVA).
`Study 1b: After treatment for 38 days, the AUCs of the 24-h
`glucose profiles were significantly different between all four
`groups (Figure 3). Lean animals had the lowest and ZDF
`vehicle animals the highest glucose AUC. Both liraglutide and
`pair feeding reduced the glucose AUC compared to the ZDF
`vehicle group. The reduction achieved by pair feeding alone
`was 7.5 m min, that is, approximately 53% of the 14.2 m min
`reduction observed with liraglutide treatment
`(Table 1).
`Similarly, HbA1c was significantly reduced in both ZDF
`liraglutide and ZDF pair-fed animals compared to vehicle,
`but was not normal compared with lean vehicle (Table 1).
`AUC for plasma insulin did not differ between ZDF liraglutide
`and ZDF pair-fed, but both were higher than lean vehicle and
`ZDF vehicle, which were similar to each other (Table 1). The
`reproducibility of the glucose and insulin results compared
`with study 1a strongly suggests that any stress caused to the
`animals by the individual housing did not
`influence the
`development of diabetes.
`Study 1c: Fructosamine levels were decreased in the
`liraglutide group compared to vehicle.
`
`British Journal of Pharmacology vol 140 (1)
`
`J. Sturis et al
`
`Liraglutide in ZDF and 60% pancreatectomized rats
`
`Vehicle
`30 µg kg-1 bid
`150µg kg-1 bid
`
`0900 1300 1700
`
`0100 0500
`2100
`Clock time
`
`0900
`
`Vehicle
`30 µg kg-1 bid
`150µg kg-1 bid
`
`30
`
`25
`
`20
`
`15
`
`10
`
`05
`
`Blood glucose (mM)
`
`3000
`
`2000
`
`1000
`
`0
`
`Plasma insulin (pM)
`
`0900 1300 1700 2100 0100 0500 0900
`Clock time
`
`Figure 2 ZDF study 1a. Glucose and insulin profiles (24 h) after 41
`days
`liraglutide treatment. Animals had free access
`to food
`throughout. Judged by 24-h AUCs, BG was significantly decreased
`(Po0.0002 by ANOVA) and plasma insulin was significantly
`increased (Po0.002 by ANOVA) in the group receiving high-dose
`liraglutide compared to low dose and vehicle.
`
`Food and water consumption, body weight
`
`In all the three ZDF studies, liraglutide treatment had an
`immediate strong effect on lowering both food and water
`intake immediately after dosing was initiated. With continued
`dosing, a less pronounced, but significant effect on food and
`water consumption was observed. At the end of the study, the
`overtly hyperglycemic vehicle-treated ZDF rats consumed
`significantly more water than the remaining groups. The data
`from study 1b are representative and are summarized in
`Table 2. Hematocrit measured at the end of study 1a was not
`reduced by liraglutide treatment (data not shown), indicating
`that the reduced water consumption was not associated with
`dehydration. Within each of the three ZDF studies, initial
`body weight did not differ between ZDF groups and in the
`early treatment period, the reduced food intake in liraglutide-
`treated animals
`resulted in an attenuated weight gain.
`However, as the treatment duration increased, the difference
`in weight disappeared, reflecting a treatment-related reduced
`loss of calories due to glucosuria. Data on body weight from
`study 1b are representative and are illustrated in Figure 4.
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`127
`
`Table 2 ZDF, study 1b: food and water intake
`
`Lean vehicle
`ZDF vehicle
`
`ZDF pair-fed
`ZDF 150 µg kg-1 bid
`
`0800 1200 1600 2000 2400 0400 0800
`Clock time
`
`30
`
`20
`
`10
`
`0
`
`Blood glucose (mM)
`
`3000
`
`2000
`
`1000
`
`0
`
`Plasma insulin (pM)
`
`0800 1200 1600 2000 2400 0400 0800
`Clock time
`
`Figure 3 ZDF study 1b. Glucose and insulin profiles (24 h) after 38
`days
`liraglutide treatment. Animals had free access
`to food
`throughout. Judged by 24-h AUCs, BG was significantly decreased
`(Po0.0001 by ANOVA) and plasma insulin was significantly
`increased (Po0.0001 by ANOVA) in animals receiving liraglutide
`compared to vehicle (see Table 1 for detailed analysis).
`
`Table 1 ZDF, study 1b: AUC of glucose and insulin
`during 24-h profile and HbA1c after 38 days treatment
`
`AUC glucose
`(m min)
`
`AUC insulin
`(mm min)
`
`HbA1c
`(%)
`
`Lean vehicle
`ZDF vehicle
`ZDF pair-fed
`ZDF
`liraglutide
`ANOVA
`
`0.670.02
`8.370.1
`0.870.1
`30.470.6*
`2.070.3*w
`22.970.2*w
`2.570.4*z
`16.270.2z}8
`Po0.0001
`Po0.0001
`*Po0.001 vs lean vehicle; wPo0.01 vs ZDF vehicle;
`zPo0.001 vs ZDF vehicle; }Po0.01 vs lean vehicle; 8Po0.05
`vs ZDF pair-fed, Tukey’s post hoc test. Data are mean7s.e.m.
`
`3.970.04
`8.670.2*
`6.370.4*z
`5.570.4z}
`Po0.0001
`
`Average
`daily food
`intake (g)
`
`20.270.4
`30.470.7*
`23.270.3*#
`23.870.4*#
`
`Average daily
`food intake,
`final week (g)
`
`Average daily
`water intake,
`final week (g)
`
`20.070.6
`30.870.9*
`23.570.5}#
`25.170.6*#
`
`2971#
`94711
`5674}#
`3873#
`
`Lean vehicle
`ZDF vehicle
`ZDF pair-fed
`ZDF
`liraglutide
`ANOVA
`
`Po0.0001
`Po0.0001
`Po0.0001
`*Po0.001 vs lean vehicle; #Po0.001 vs ZDF vehicle;
`}Po0.01 vs lean vehicle. Data are mean7s.e.m.
`
`Initial body weight
`
`*
`
`*
`
`*
`
`200
`
`100
`
`Body weight(g)
`
`0
`
`Lean vehicle
`
`ZD F vehicle
`
`ZDF pair-fed
`
`Z D F liraglutide
`
`Day 10-Day 0
`
`*
`
`**
`*
`
`**
`*
`
`Lean vehicle
`
`ZDF vehicle
`
`ZD F pair-fed
`
`ZD Fliraglutide
`
`Day 42- Day 0
`
`*
`
`75
`
`50
`
`25
`
`0
`
`(g)
`
`Body weight increase
`
`200
`
`100
`
`(g)
`
`Body weight increase
`
`Plasma concentration liraglutide
`
`In study 1b,
`the total plasma concentration (albumin
`bound þ free) of liraglutide was 160712 nm (approximately
`5 – 7 h after the final dose). The high total concentration
`reflects the albumin-binding properties of liraglutide.
`
`0
`
`Lean vehicle
`
`ZD F vehicle
`
`ZD F pair-fed
`
`ZD F liraglutide
`
`Figure 4 ZDF study 1b. Initial body weight and body weight
`increase after 10 and 42 days liraglutide treatment. *Po0.01 vs lean
`vehicle; **Po0.001 vs ZDF vehicle.
`
`British Journal of Pharmacology vol 140 (1)
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1046, p. 5 of 10
`
`

`

`128
`
`J. Sturis et al
`
`Liraglutide in ZDF and 60% pancreatectomized rats
`
`Beta-cell
`proliferation
`study 1a
`
`Beta-cell
`mass
`study 1a
`
`2
`
`1
`
`0
`
`% beta cellvolume
`
`ZDF Vehicle
`
`ZDF 30 µg kg-1 bid
`
`ZDF 150 µg kg-1 bid
`
`study 1b
`
`study 1b
`
`3
`
`2
`
`1
`
`% beta cell volume
`
`Lean vehicle
`
`ZDF pair-fed
`
`0
`ZDF vehicle
`ZDF 150 µg kg-1 bid
`
`study 1c
`
`study 1c
`
`1.00
`
`0.75
`
`0.50
`
`0.25
`
`0.00
`
`% beta cellvolume
`
`1.00
`
`0.75
`
`0.50
`
`0.25
`
`0.00
`
`beta cells
`
`%BrdU positive
`
`0.75
`
`0.50
`
`0.25
`
`0.00
`
`0.5
`
`0.4
`
`0.3
`
`0.2
`
`0.1
`
`0.0
`
`beta cells
`
`%BrdU positive
`
`beta cells
`
`% BrdUpositive
`
`ZDF vehicle
`
`ZDF 200 µg kg-1 bid
`
`Figure 6 Quantitation of b-cell proliferation and mass (volume
`fraction) in ZDF experiments. Study 1a: after 6 weeks treatment, b-
`cell volume fraction was increased in both the low- and high-dose
`liraglutide groups, though only statistically significant for the low-
`dose group (Po0.05) There was a weak trend towards increased b-
`cell proliferation as estimated by the BrdU indices. Study 1b: b-cell
`volume fraction in liraglutide-treated and pair-fed groups were
`higher than in the lean control group, but not significantly higher
`than in the vehicle-treated ZDF rats. There was no difference
`between the b-cell proliferation rates of the three groups of ZDF
`rats. Study 1c: b-cell volume fraction was significantly lower in the
`animals treated with liraglutide (Po0.002), and this relative decrease
`occurred concomitantly with a markedly reduced b-cell proliferation
`(Po0.006).
`
`groups of ZDF rats and the vehicle- (study 1a and 1b) or
`vehicle plus pair feeding (study 1b)-treated groups (not
`shown).
`
`Studies in 60% pancreatectomized rats: metabolic
`parameters
`
`In study 2a, liraglutide (100 mg kg1 b.i.d.) treatment resulted
`in a tendency towards lower blood glucose during the OGTT
`compared to vehicle at day 8, but the difference in AUC for
`glucose was not statistically significant
`(Figure 8,
`top).
`However,
`in study 2b,
`the higher dose of
`liraglutide
`(150 mg kg1 b.i.d.) resulted in significantly lower AUC for
`blood glucose at day 8 compared with both the vehicle and
`
`Figure 5 BrdU incorporation in b- and non-b-cells of the pancreas
`of a 13-week-old vehicle-treated diabetic ZDF rat from ZDF study
`1a. Black nuclei ¼ BrdU incorporation, brown cytoplasm ¼ insulin
`in b-cells. Filled arrows show BrdU-positive b-cells, empty arrow
`shows a BrdU-positive non-b-cell (exocrine or centro-acinar duct
`cell?). Horizontal bar indicates 50 mm.
`
`ZDF studies: histology
`
`Proliferation of b-cells was demonstrated by the incorporation
`of BrdU into b-cell nuclei (Figure 5). It should be noted that
`the number of non-b-cells in islets, duct cells, and exocrine cells
`that incorporated BrdU exceeded that of b-cells. The b-cell
`mass was estimated by point-counting morphometry. In study
`1a, after 6 weeks treatment in the study, b-cell mass (volume
`fraction) was increased in both the low- and high-dose
`liraglutide groups, although only statistically significant for
`the low-dose group (Figure 6a), and there was a weak trend
`towards increased b-cell proliferation as estimated by the
`BrdU indices. In general, the islet morphology in all groups of
`ZDF rats was quite irregular and the b-cell insulin-staining
`intensity variable with no treatment-related differences in
`staining intensity (not shown). In study 1b, b-cell volume
`fractions in liraglutide-treated and pair-fed groups were higher
`than in the lean control group, but not higher than in the
`vehicle-treated ZDF rats (Figure 6b). When expressed in terms
`of total b-cell mass, the liraglutide-treated group of ZDF rats
`had a higher b-cell mass than the ZDF vehicle animals
`(20.972.6 vs 12.471.8 mg, Po0.03). There was no difference
`between the b-cell proliferation rates of the three groups of
`ZDF rats and no obvious differences between insulin-staining
`intensities.
`After only 2 weeks treatment in study 1c, b-cell mass
`(volume fraction) was significantly lower in the animals treated
`with liraglutide (0.6670.04 vs 0.9070.06%, Po0.002), and
`this relative decrease occurred concomitantly with a markedly
`reduced b-cell proliferation (0.1370.04 vs 0.4670.07%,
`Po0.006, Figure 6c). In contrast, the insulin-staining intensity
`of b-cells in the liraglutide-treated rats was markedly higher
`than in the vehicle group (Figure 7).
`Non-b-cells were demonstrated by a cocktail of antibodies
`against glucagon, somatostatin, and pancreatic polypeptide.
`Stereological measurements of the volume fractions of non-b-
`cells in the pancreata from these experiments showed no
`treatment-related differences between the liraglutide-treated
`
`British Journal of Pharmacology vol 140 (1)
`
` PFIZER, INC. v. NOVO NORDISK A/S - IPR2020-01252, Ex. 1046, p. 6 of 10
`
`

`

`J. Sturis et al
`
`Liraglutide in ZDF and 60% pancreatectomized rats
`
`129
`
`Figure 7 Increased insulin staining intensity after 2 weeks liraglutide treatment. Representative sections from pancreata of ZDF
`rats show higher and more regular insulin-staining intensity in the liraglutide-treated rat than in the vehicle-treated rat. Note the
`better staining pattern and intensity of this 10-week-old newly diabetic vehicle-treated rat than that of the 13-week-old diabetic rat
`from the experiment shown in Figure 5.
`
`-30
`
`0
`
`60
`30
`Time (min)
`
`Vehicle
`Liraglutide
`
`90
`
`120
`
`P<0.05
`P<0.001
`
`P<0.05
`
`Vehicle Liraglutide Pair-fed
`
`150
`
`100
`
`50
`
`0
`
`∆AUC0-120 (mM×min)
`
`-30
`
`0
`
`30
`
`60
`Time (min)
`
`Vehicle
`Vehicle (pair-fed)
`Liraglutide
`
`90
`
`120
`
`10
`
`8
`
`6
`
`4
`
`2
`
`0
`
`16
`
`14
`
`12
`
`10
`
`02468
`
`Glucose (mM)
`
`Glucose (mM)
`
`Figure 8 OGTTs in experiments performed in 60% pancreatecto-
`fast glucose (2 g kg1) was
`mized rats. After an overnight,
`administered by gavage at time 0 to overnight fasted animals and
`BG was measured basally and after the glucose challenge. Top:
`study 2a. After dosing vehicle or 100 mg kg1 liraglutide b.i.d. for 4
`days, a nonsignificant trend towards improved glucose tolerance was
`observed in the treated animals. Bottom: study 2b. After dosing
`vehicle or 150 mg kg1 liraglutide b.i.d. for 4 days, significantly lower
`AUC for BG was observed in both the vehicle and pair-fed groups.
`Furthermore, pair feeding itself resulted in slightly, but significantly,
`reduced delta AUC during the OGTT.
`
`British Journal of Pharmacology vol 140 (1)
`
`pair-fed groups. Furthermore, pair feeding itself resulted in
`slightly, but significantly, reduced delta AUC during the
`OGTT (Figure 8, bottom).
`
`Studies in 60% pancreatectomized rats: histology
`
`In both studies 2a and 2b, the distribution of islet sizes showed
`a trend towards an increase in the liraglutide-treated animals.
`However, point-counting morphometry at sections through
`the remnant pancreas from study 2b revealed no significant
`differences in the total b-cell volume fractions (1.2770.16% in
`the liraglutide group, 1.1570.15% in the vehicle group, and
`0.9570.14% in the pair-fed group).
`There was no visible difference between the vehicle- and
`liraglutide-treated groups in the size or number of islets
`located in the regenerated area. Immunostaining for BrdU
`revealed a marked proliferation mainly in exocrine cells
`located at the border of the regeneration zones in both vehicle-
`and liraglutide-treated animals. At this time point, there was
`no effect of liraglutide treatment on the proliferative index in
`islets or in any other areas of remnant or regenerating
`pancreas.
`
`Discussion and conclusions
`
`We have demonstrated in two different rat models of b-cell
`deficiency that
`liraglutide has marked antihyperglycemic
`effects. In male ZDF rats, treatment with liraglutide strongly
`attenuated diabetes development in prediabetic animals, while
`treatment in 60% pancreatectomized rats reduced glucose
`excursions after an OGTT. In both models, the effects were
`partly