Carsten F. Gotfredsen, Anne-Marie Mølck, Inger Thorup, Niels C. Berg Nyborg, Zaki Salanti,Lotte Bjerre Knudsen, and Marianne O. Larsen

The Human GLP-1 AnalogsLiraglutide and Semaglutide:Absence of HistopathologicalEffects on the Pancreas inNonhuman PrimatesDiabetes 2014;63:2486–2497 | DOI: 10.2337/db13-1087

Increased pancreas mass and glucagon-positive ade-nomas have been suggested to be a risk associatedwith sitagliptin or exenatide therapy in humans. NovoNordisk has conducted extensive toxicology studies,including data on pancreas weight and histology, inCynomolgus monkeys dosed with two different humanglucagon-like peptide-1 (GLP-1) receptor agonists. In a52-week study with liraglutide, a dose-related increasein absolute pancreas weight was observed in femalemonkeys only. Such dose-related increase was notfound in studies of 4, 13, or 87 weeks’ duration. Notreatment-related histopathological abnormalities wereobserved in any of the studies. Quantitative histology ofthe pancreas from the 52-week study showed an in-crease in the exocrine cell mass in liraglutide-dosedanimals, with normal composition of endocrine and exo-crine cellular compartments. Proliferation rate of theexocrine tissue was low and comparable betweengroups. Endocrine cell mass and proliferation rates wereunaltered by liraglutide treatment. Semaglutide showedno increase in pancreas weight and no treatment-related histopathological findings in the pancreas after13 or 52 weeks’ dosing. Overall, results in 138 nonhumanprimates showed no histopathological changes in thepancreas associated with liraglutide or semaglutide,two structurally different GLP-1 receptor agonists.

Pancreas safety has become a subject of much debate con-cerning dipeptidylpeptidase-4 (DPP-4) inhibitors (DPP-4is)

and glucagon-like peptide-1 (GLP-1) receptor agonists(GLP-1RAs). This concern is partly based on the well-described effect of GLP-1 to induce growth of pancre-atic b-cells (1). Both drug classes increase effectiveGLP-1 levels, but to a different degree, and have differentmodes of action; thus, it is important to differentiatebetween them, particularly when considering mechanistichypotheses for potential safety concerns or signals. GLP-1RAs mediate their effects directly through the GLP-1R(2,3). Although increased levels of GLP-1 and glucose-dependent insulinotropic peptide (GIP) are consideredimportant parts of the mechanism of action of DPP-4is(4), DPP-4 is known to degrade many other hormones (5).DPP-4is have been shown to increase GLP-1, GIP, andpeptide YY (6,7). This complicates the understanding ofboth desired and potentially undesired effects of this classof compounds. Within GLP-1RAs, differences exist insafety-related parameters. One subgroup is the exendin-4–based drugs with exenatide and lixisenatide, which arestructurally distinct from human GLP-1. Owing to the lowamino acid homology to native human GLP-1, these med-ications are associated with an increased number of im-mune reactions that are, however, all of a relatively mildform, that is, mostly antibody development, injectionsite nodules, and loss of efficacy (8,9). The other sub-group is based on human GLP-1 and contains liraglutide,taspoglutide, and larger covalently conjugated moleculessuch as albiglutide and dulaglutide. Clinical development

Diabetes Research Unit, Novo Nordisk, Måløv, Denmark

Corresponding author: Lotte Bjerre Knudsen, lbkn@novonordisk.com.

Received 11 July 2013 and accepted 18 February 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1087/-/DC1.

© 2014 by the American Diabetes Association. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

See accompanying article, p. 2219.

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PHARMACOLOGYAND

THERAPEUTIC

S

of taspoglutide was stopped due to severe immune-relatedside effects, with cases of anaphylactic shock, possiblycaused by the formulation (8,10). No such findings havebeen reported with other GLP-1–based analogs.

Semaglutide is a once-weekly GLP-1 analog that is inphase 3 clinical development (11). Where liraglutide isacylated with a palmitic acid and has an extra aminoacid as a spacer between the palmitic acid and the Lys26,where the fatty acid is attached, semaglutide is acylatedwith a stearic diacid at Lys26 but has a much larger syn-thetic spacer and is, furthermore, modified for DPP-4 sta-bility in position 8, where the amino acid a-aminobutyricacid has been introduced.

In support of drug development and regulatory ap-proval for treatment of chronic diseases, repeat dosetoxicity and carcinogenicity studies are conducted. Thesestudies are performed at different dosing durations tosupport the different phases of clinical development andwith doses aiming to obtain exposure several multipleshigher than the clinically relevant exposure with the aimto identify potential drug-related organ toxicity andcarcinogenicity. Repeat dose toxicity studies are typicallyconducted in a rodent and a nonrodent species. Forliraglutide and semaglutide, Cynomolgus monkeys werechosen as the nonrodent species. Repeat dose toxicitystudies in nonhuman primates are designed to screenfor potential hazards and are not designed or statisticallypowered for identification of differences in the incidenceor severity of individual organ changes. The number ofnonhuman primates per group is limited to three to fivefor ethical reasons (12). Because of the statistical limita-tions, standardization of the examinations is critical: Ifpathological findings are identified at a frequency or se-verity exceeding those in the in-study control group, theyare often compared with historical control data to assistinterpreting the significance of the finding. This principlealso applies to organ weights. Histopathological findingsin the pancreas from the repeat-dose studies in rodentsand nonhuman primates and from carcinogenicity studiesin rodents with liraglutide have been published previously(13). Liraglutide was not found to have a causal relation-ship to any histopathological findings.

Some studies in rats and mice have shown an increasedpancreas weight induced by DPP-4is or GLP-1RAs (14,15),and a recent ex vivo study with human pancreata sug-gested an increase in glucagonomas as well as increasedpancreas weight (16). Here, pancreas weight in Cynomol-gus monkeys is reported for liraglutide in toxicology stud-ies with 4, 13, 52, and 87 weeks’ dosing, and forsemaglutide in toxicology studies with 13 and 52 weeks’dosing, as well as a full histopathological evaluation ofthese same studies, except the 87 weeks’ study, whichhas been reported previously (13). For liraglutide, a fullquantitative histological assessment of the endocrine aswell as the exocrine pancreas was also performed in the52 weeks’ study.

RESEARCH DESIGN AND METHODS

The research design and methods for liraglutide stud-ies in Cynomolgus monkeys have been described pre-viously (13). All animals were examined daily forclinical signs in the in-life phase. Studies with sema-glutide were generally performed similarly, and bothfollowed international guidelines provided by Interna-tional Conference of Harmonization. Dose levels, du-ration, and group sizes for all studies are described inFig. 1. Compounds were administered as subcutaneousinjections.

Body Weight and Pancreas WeightTerminal body weight was obtained in sedated animalsimmediately before they were killed. The entire pancreasof each animal was examined macroscopically for any ab-normalities, excised, cleaned of fat and connective tissue,and weighed.

Tissue PreparationA transverse section from the midpart of the pancreasfrom all animals was sampled, and this section and therest of the pancreata were fixed in 10% neutral bufferedformalin for at least 48 h. The sections were dehydratedand paraffin-embedded according to standard histopath-ological procedures. According to international standardpractices, one section per animal was cut at a nominalthickness of 4–5 mm and stained with hematoxylin andeosin (17). The slides were read unblinded because this isrecommended by toxicopathology experts as a way toincrease the chance of separating subtle changes fromnormal background changes (18). Further details ofthe methodology have been previously described (13).The pancreas specimens from animals killed at termina-tion of dosing in the 52-week liraglutide study wereevaluated by quantitative histology (all groups for a-cellsand Ki-67, and only vehicle and high-dose groups for b-,d-, pancreatic polypeptide [PP], ductal, and acinar cells).The cranial and caudal remnants were sectioned longi-tudinally, cut into ;40 pieces, and distributed to fourcapsules, with one-fourth in each, according to thesmooth fractionator principle (19,20). Processing andstaining for detection of b-, a-, d-, and PP cells wasdone as previously described (21). The reactivity of theprimary antibodies to insulin, glucagon, somatostatin,and PP in monkey pancreas had been tested indi-vidually to validate the method. Costaining for ductplus acinar cells using mouse anti–CK-7 (Dako, Glostrup,Denmark) and rabbit anti–a-amylase (Calbiochem, Darm-stadt, Germany) plus Ki-67 using polyclonal rabbitanti–Ki-67 (Novus Europe, Cambridge, U.K.) followedthe same principles as above. Stained slides werescanned in a Hamamatsu NanoZoomer 2.0-HT (Hamamatsu,Hamamatsu City, Japan). Images were subsequently ana-lyzed automatically in the Visiopharm Integrator System(Visiopharm A/S, Hørsholm, Denmark) image analysisprogram.

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Calculations

Historical Control DataData on pancreas weights were collected from thecontract laboratories where the study of interest wasconducted to ensure matching for origin, age range, andenvironmental conditions to the highest possible extent.Historical control data on pancreas weights from animalsused as vehicle controls in other studies of 4, 13, and 39–52 weeks’ duration was used for comparison with liraglutide:4 week (n = 29 males and 28 females as historical con-trols), 13 week (n = 19 males and 19 females as historiccontrols), and 52 week (n = 22 males and 17 females ashistorical controls). No historical control data were avail-able for comparison with the 87-week study because thisis not a standard-length toxicology study.

Quantitative HistologyMeasurements were performed on samples obtained bya systematic uniform random sampling technique. The

mean value for each animal was calculated relative to thetotal tissue area counted for each estimate. Volume frac-tions were measured in percentage of the total pancreasvolume. Total cell mass in milligrams was calculated bymultiplying the volume fraction with the total pancreasweight.

StatisticsThe liraglutide data on pancreas weight were, per studyprotocol, analyzed by ANCOVA on body weights andpancreas weights combining both sexes and using day0 body weight and terminal body weight, respectively, ascovariates. These prespecified models were slightly differ-ent between studies because they were data-driven. Onlythe 52-week study reported an increase in pancreasweight when this analysis was used (22). To obtain con-sistency across studies and thus enhance comparisons,post hoc modeling was performed using the same modelsin all studies. In the prespecified analyses, terminal body

Figure 1—Absolute pancreas weight (g) in male (M) and female (F) control and liraglutide (subcutaneous once daily) (A–D) or semaglutide(subcutaneous twice weekly) (E and F ) dosed nonhuman primates (horizontal line indicates group mean in each data set). Liraglutide dosingfor 4 weeks (A), 13 weeks (B), 52 weeks (C ), and 87 weeks (D). Semaglutide dosing for 13 weeks (E) and 52 weeks (F ). Con M and Con F arehistorical control animals in panel A–C (from other studies of the same duration, run in the same facility). 0M and 0F are vehicle-dosedanimals on all graphs. A–C: 1M and 1F were dosed with 0.05 mg/kg/day, 2M and 2F with 0.5 mg/kg/day, and 3M and 3F with 5.0 mg/kg/dayliraglutide. D: 1M and 1F were dosed with 0.25 mg/kg/day and 2M and 2F with 5.0 mg/kg/day liraglutide. §Animal killed at 63 weeks onhumane grounds. In the 13-week semaglutide study, the high dose was reduced from 0.98 to 0.47 mg/kg after 6 weeks due to severedehydration in 2 female animals indicating that 0.98 mg/kg was not a tolerated dose. These two animals were killed after 36 days and notincluded in the analysis of pancreas weight, leading to a group size of two. E: 1M and 1F were dosed with 0.004 mg/kg, 2M and 2F with0.086 mg/kg, and 3M and 3F with 0.47 mg/kg semaglutide twice weekly. F: 1M and 1F were dosed with 0.01 mg/kg, 2M and 2F with0.06 mg/kg, and 3M and 3F with 0.36 mg/kg semaglutide twice weekly. A: Males: P = 0.036 by ANOVA across M0, M1, M2, and M3; P =0.054 vs. 0M for 1M by posttest; P = 0.18 by ANOVA across all groups, including Con M. Females: P = 0.38 by ANOVA across F0, F1, F2, andF3; P = 0.10 by ANOVA across all groups. B: Males: P = 0.73 by Kruskal-Wallis test across M0, M1, M2, and M3; P = 0.66 by Kruskal-Wallistest across all groups. Females: P = 0.93 by ANOVA across F0, F1, F2, and F3; P = 0.92 by ANOVA across all groups. C: Males: P = 0.37 byANOVA across M0, M1, M2, and M3; P = 0.86 by ANOVA across all groups. Females: P = 0.76 and P = 0.07 vs. 0F for 1F and 2F by posttest;P = 0.005 by ANOVA across all groups; P = 0.29, P = 0.98, and P = 0.08 for F1, F2 and F3, respectively, compared with Con F by posttest.D: Males: P = 0.68 by ANOVA across all groups. Females: P = 0.70 by ANOVA across all groups. E: Males: P = 0.36 by Kruskal-Wallis testacross all groups. Females: P = 0.025 by ANOVA across all groups; P = 0.30, P = 0.09, and P = 0.34 vs. F0 for F1, F2, and F3 by posttest.F: Males: P = 0.10 by ANOVA across all groups. Females: P = 0.19 by ANOVA across all groups.

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weight was taken into consideration in the statisticalmodels. However, that is problematic because liraglutidelowers body weight. The post hoc analysis is thus a one-way ANOVA of the pancreas weight for each study andsex separately. In case of variance inhomogeneity, mea-sured by means of the Bartlett and Brown-Forsythetests, a Kruskal-Wallis test was performed to evaluateoverall group effects. Because statistically significanteffects of liraglutide were seen in some studies forsome of the doses, a one-way ANOVA was also per-formed for the relative pancreas weight to further exam-ine a possible effect. The Dunnett multiple comparisontest was used as posttest after ANOVA in cases wherethe overall group effect was significant. Parameters fromquantitative histology were analyzed by the Studentt test for each sex, except for a-cell mass, where one-way ANOVA was used. P , 0.05 was considered statis-tically significant. Data are presented as mean 6 SEMunless otherwise stated.

RESULTS

Liraglutide

Pancreas WeightComparison With In-Study Control Groups. Pancreaticweight data from liraglutide studies are shown in Fig. 1. Asignificant increase that was apparently not dose-relatedwas found in the 4-week study in male animals (P = 0.036by ANOVA), whereas no increase was seen in females inthe same study. No statistically significant differenceswere found for any dose level in male or female animalsafter 13 or 87 weeks of dosing. Similarly, in the 52-weekstudy, no statistically significant differences were foundfor the male animals but a significantly higher pancreasweight, apparently dose-related, was found in the femalehigh-dose group compared with the in-study controlgroup (P = 0.007 by ANOVA).

Comparison With Historical Control Data. This compar-ison did not show any statistically significant differencesin pancreas weight for any dose level in males or femalesfrom the 4- and 13-week studies or males in the 52-weekstudy. When compared against historical controls infemales from the 52-week study, a statistically signifi-cant difference across groups was found (P = 0.005 byANOVA), but posttest showed that only the in-study con-trols exhibited a statistically significant different (lower)pancreas weight than the historical controls (n = 4 and17, respectively; P = 0.04).

Comparison of Pancreas Weight Adjusted for BodyWeight. An ANOVA analysis of the relative pancreasweight showed a significant increase compared with in-study controls in the male mid- and high-dose groups andin the female high-dose group in the 52-week liraglutidestudy (data not shown). There were no statisticallysignificant differences in the 4-, 13-, or 87-week liraglu-tide studies.

Recovery Animals. Additional animals were included toexplore reversibility of potential findings (n = two malesand two females, in the control and high-dose groups inthe 13- and 52-week studies for liraglutide and semaglu-tide). These additional animals were dosed for the sameduration as all other animals in the study but were keptfor additional 4 weeks after end of treatment before beingkilled. In the 52 weeks’ study, there was no longer anydifference in pancreas weight versus controls after the4-week treatment-free period (data not shown).

Pancreas HistologyHistological examination of the pancreata from the 4-,13-, and 52-week studies is shown in Table 2. Represen-tative histological sections of the different compartmentsof pancreas after 52 or 87 weeks of dosing are illustratedin Figs. 2 and 3. The histological examination did notreveal treatment-related differences between dosed andcontrol animals at any time point. The endocrine pancreasrevealed well-demarcated islets with normal pale islet cellsafter 52 and 87 weeks of dosing (Fig. 2). In the exocrinepancreas, all ducts appeared normal, both the large mainduct with the high columnar epithelium and abundantsurrounding connective tissue (shown in Fig. 3, left sidepanels, and in higher magnification in SupplementaryFig. 1) and the medium-sized interlobular ducts with thelower cuboidal epithelium and less surrounding connectivetissue (Fig. 3, right side panels). The small intercalated ductswith flat epithelium and no or sparse surrounding connec-tive tissue also appeared normal (not shown in figures). Theacinar cell parenchyma consisted of normal pyramid-shapedcells where the apical part was filled with eosinophilic zymo-gen granules and the basophilic basal part contained thenucleus, as shown in Fig. 3 and in Supplementary Fig. 2.

Quantitative Histology of Pancreas in 52-WeekLiraglutide StudyA quantitative histological assessment of the pancreas inthe 52-week liraglutide study was conducted to evaluatewhether there were changes in the mass of pancreatic celltypes that could not be identified by the qualitativehistological analysis. Table 1 shows absolute mass of b-,a-, d-, PP, duct, and acinar cells. Liraglutide-dosed mon-keys showed no significant differences in any of thesemeasures, except for absolute duct cell and acinar cellmass, which was significantly increased in the femalehigh-dose group. When the proportion of cells per volumeof pancreas was evaluated, no changes were found forductal cell volume in males (liraglutide 6.14 6 0.59 vs.control 6.71 6 0.62%, P = 0.53) or females (7.40 6 0.63vs. 6.55 6 1.29%, P = 0.57) or for acinar cell volume inmales (91.4 6 0.9 vs. 88.7 6 0.8%, P = 0.06) or females(90.3 6 0.4 vs. 90.0 6 0.7%, P = 0.71) high-dose com-pared with vehicle. Thus, the increased pancreas weightwas a balanced increase of the exocrine pancreas, with noapparent change in the ratio of ductal to acinar tissue. Tofurther evaluate if a-cell mass specifically was changed byliraglutide, the low- and medium-dose liraglutide groups

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were also evaluated quantitatively. Liraglutide did notchange a-cell mass in males in the low- or middle-dosegroups (30.0 6 8.4 and 24.2 6 3.3, respectively, vs.29.9 6 3.6 mg for controls, from Table 1) or females(16.0 6 1.6 and 20.4 6 2.0, respectively, vs. 22.6 6 3.3mg for controls, from Table 1), with P = 0.74 and P = 0.22for males and females across groups by ANOVA.

Examples of b- and non–b-cell staining (Fig. 4) anda-cell and proliferation (Ki-67) (Fig. 5, and higher magni-fication in Supplementary Fig. 3) in representative sectionsof pancreata from males and females in the vehicle and theliraglutide high-dose group of the 52-week dosing study areshown. In control and high-dose animals, glucagon stainingshowed a high and variable number of a-cells in islets(typically ;50% of non–b-cells were a-cells). Small num-bers of single cells or small clusters of glucagon-positivecells associated with ducts were seen in the exocrine pan-creas (Fig. 5). Very few cells in the endocrine and exocrinepancreas were positive for Ki-67, and there was no appar-ent difference between the liraglutide and control groups(data not shown). As a positive control for proliferation,lymph nodes present in 15 of the total of 32 pancreatashowed strong labeling of many cells in germinal centersand also some single cells (shown as inserts in Fig. 5).

SemaglutidePancreas weights from semaglutide studies are shown inFig. 1. In the 13-week study, there was no statistically

significant treatment-related effect on pancreas weightin males, whereas a significant difference was seen acrossgroups in the females (P = 0.02 by ANOVA). However, byposttest, no significant difference was found betweentreated groups and the control group, and no apparentdose-related effects were seen. In the 52-week study, nostatistically significant differences across study groupswere observed for pancreas weight; the highest pancreasweights were found in the control groups.

Histological examination of the pancreas from the13- and 52-week studies revealed common backgroundfindings of minimal to mild severity and with a focaldistribution. There were no signs of treatment-relatedeffects. The data are reported in Table 2.

DISCUSSION

Reported here are further data from nonhuman primatestudies conducted with liraglutide as a supplement topreviously published data on pancreas histology in mice,rats, and nonhuman primates (13). An apparent dose-related increase in absolute pancreas weight was foundin females in one of four monkey studies with liraglutideand in none of two monkey studies with semaglutide, andan increase that did not appear to be dose-related was foundin a 4-week liraglutide study, in males only (Table 3). Acomparison with historical control data was made. This isa common way of setting toxicological data into perspective

Figure 2—Liraglutide studies in nonhuman primates (hematoxylin and eosin staining). Endocrine pancreatic islets from males from 52weeks’ study (left) and females from 87 weeks’ study (right) from control (upper row) or liraglutide high-dose group (lower row). Well-demarcated islets with normal-looking pale islet cells. Liraglutide-dosed animals look similar to what is seen in control animals. Originalmagnification 3200; bar indicates 100 mm.

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Figure 3—Liraglutide studies in nonhuman primates (hematoxylin and eosin staining). Top row: 52 weeks’ study control males. Secondrow: 87 weeks’ study control females. Third row: 52 weeks’ study high-dose males. Bottom row: 87 weeks’ study high-dose females. Left:Original magnification 3200; bar indicates 100 mm. Ductular part of the exocrine pancreas with presence of a large main duct withcolumnar epithelium. The duct is surrounded by connective tissue but is still located within the parenchyma. The duct from high-dosegroup looks similar to that of the control monkeys. Right: Original magnification 3400; bar indicates 50 mm. Exocrine acinar cell paren-chyma and ductular part of the exocrine pancreas with presence of medium-sized interlobular ducts with cuboidal epithelium. The ductepithelium is lower and the amount of the surrounding connective tissue is lesser than for the large ducts. The duct from the high-dosegroup looks similar to that of the control monkeys. The acinar secretory compartment consists of pyramid-shaped cells where the apicalpart is filled with eosinophilic zymogen granules and the basophilic basal part contains the nucleus. The secretory compartment from high-dose group looks similar to that of the control monkeys.

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and may be especially useful in nonhuman primate studieswhere the number of animals is low due to ethical reasons.The histological analysis of the pancreas from the liraglu-tide and semaglutide studies did not reveal any potentiallyadverse findings that could be related to treatment (e.g.,pancreatitis, inflammatory cell infiltrations, or hyperpla-sia). Overall, this led to the conclusion that an increasein pancreas weight cannot be ruled out, but no consis-tent dose-related increases in pancreas weight were seenacross the liraglutide and semaglutide studies in mon-keys. In combination with the lack of treatment-relatedhistopathological changes, the data showed no adverseeffects on the pancreas by liraglutide dosing of up to

60-times the clinically relevant exposure for up to 87 weeksor semaglutide dosing for up to 52 weeks in monkeys.

The studies reported here have a high relevance forhumans because the pancreata of nonhuman primates areclosely related to humans anatomically and physiologi-cally. Additionally, the morphology of nonhuman primateislets is like that seen in humans and different fromrodents (23). Quantitative histology was used to assesschanges in the mass of different pancreatic tissue compo-nents, taking the three-dimensional structure of the or-gan into consideration (24). An inherent weakness ofthese studies is the relatively limited number of non-human primates per group because number of animals is

Table 1—Absolute mass (mg) for endocrine cell types, duct, and acinar cells in pancreas from monkeys dosed with vehicle orliraglutide for 52 weeks

Group (n = 4) b-Cells (mg) a-Cells (mg) d-Cells (mg) PP cells (mg) Duct cells (mg) Acinar cells (mg)

MaleVehicle control 49.1 6 4.4 29.9 6 3.6 17.3 6 2.5 3.73 6 1.06 251 6 36 3,319 6 454Liraglutide 5 mg/kg/day 43.4 6 9.1 24.0 6 3.5 13.8 6 3.5 6.30 6 2.02 312 6 55 4,563 6 482P 0.59 0.28 0.45 0.30 0.39 0.11

FemaleVehicle control 45.4 6 10.3 22.6 6 3.3 10.1 6 3.1 3.32 6 2.26 199 6 53 2,694 6 450Liraglutide 5 mg/kg/day 51.8 6 10.1 24.0 6 3.3 12.3 6 0.9 6.00 6 1.50 439 6 64 5,325 6 469P 0.67 0.78 0.52 0.36 0.03 0.007

Data are shown as mean 6 SEM. P values compared with control group same sex.

Figure 4—Fifty-two weeks’ liraglutide study in nonhuman primates. Male (left) and female (right) from control (upper row) or liraglutide high-dose group (lower row). Double immunohistochemical staining for b-cells (reddish brown) and non–b-cells (the sum of glucagon, somato-statin, and PP, violet/black). Islet structure and distribution of b-, and non–b-cells from liraglutide-dosed animals look similar to what is seenin control animals. Original magnification 3400; bar indicates 100 mm.

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constrained for ethical reasons. However, this report isbased on data from 90 and 48 animals dosed with liraglu-tide and semaglutide, respectively. Another limitation isthat only one transverse section was examined per ani-mal. As a consequence thereof, the statistical power islow. Three of the four studies that have described adverseeffects of DPP-4is or GLP-1RAs on the pancreas wereperformed in rodents (15,25,26). Thus, at the currentpoint in time with the very few studies available in non-human primates, the cumulative data in these studieshave a strong relevance for the assessment of adversepancreas effects in humans, despite the relatively limitednumber of animals per group.

A recent study with human pancreata from patientspreviously treated with sitagliptin or exenatide (sevensitagliptin, one exenatide) reported an increased pancreasweight (16). A number of important considerations withthe study design may have affected the results: the groupswere unbalanced, with an 18-year age difference betweenthe groups, and there was no attempt to control for typeof diabetes, weight, age, or sex. Pancreas weight dependson body weight, stage of diabetes, age, and sex (27,28).These methodological problems are clearly elucidated intwo related commentaries/reviews (29,30).

The data presented here do not show an increase in cellreplication or number of a-cells caused by liraglutide

treatment. Quantitative histology of the pancreas fromthe 52-week study with liraglutide demonstrated an in-crease of exocrine pancreas tissue with an apparently un-changed ratio between acinar and ductal cells and withnormal tissue architecture. Cell proliferation in the pan-creas measured by Ki-67 appeared unchanged by liraglu-tide; very low proliferation rates were found in allanimals. There appeared to be no change of b-, d-, orPP cell mass by 52 weeks of liraglutide treatment in thehigh-dose group and there was apparently no change ina-cell mass or indication of proliferation of a-cells in anyof the three liraglutide-dosed groups in the 52-weekstudy. The islets from the control and the liraglutidegroups in the 52-week study had ;50% non–b-endocrinecells, with a substantial portion of those being a-cells (25–30% of endocrine cell mass), less d-cells (;15%), and onlya few PP cells (4–7%). The fraction of the four endocrinecell types in our study is in agreement with data fromhuman pancreata and a descriptive study in Cynomolgusmonkeys (31–34). The intraislet organization of the b-and non–b-cells was random, with no clear rodent-likemantle and core, but with a more complex subunit struc-ture of mantles and cores, as characteristic for nonhumanprimates and humans (23,35,36). In all groups, includingthe controls, the distribution of a-cells was identical inislets and islets-like structures of variable size. The

Figure 5—Fifty-two weeks’ liraglutide study in nonhuman primates. Double immunohistochemical staining for glucagon (pink) and Ki-67(black). Male (left) and female (right) from control (upper row) or liraglutide high-dose group (lower row). Glucagon staining shows a high andvariable number of a-cells in islets and a small number of single cells and small clusters of glucagon-positive cells in the acinar exocrinepancreas and associated with ducts. The inserts show Ki-67 labeling in lymph nodes in the same sections. In total, 15 of 32 monkeys hadlymph nodes in these double-stained sections but none of the high-dose treated male monkeys had lymph nodes in such sections. Originalmagnification 3200.

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control and liraglutide-dosed groups both showed a num-ber of single glucagon-positive cells in the exocrine area,single cells and small clusters of glucagon-positive cellsassociated to the epithelial lining of both main andsmaller ducts. This finding of small clusters and singleendocrine cells is normal in Cynomolgus monkeys (34). Asimilar pattern with single cells in the exocrine areas andassociated to ductal structures was observed for b-cellsand less frequently with d-cells and PP cells, with nodifferences between groups. An apparently dose-relatedincrease in absolute pancreas weight was found in oneof four studies, and only in one sex, in nonhuman primate

studies with liraglutide. Despite this apparently dose-related increase in pancreas weight, no histopathologywas associated thereto, and there were no pancreaticintraepithelial neoplasia lesions (13).

The four studies that have suggested adverse effect ofDPP-4is or GLP-1RAs on the pancreas have described ordiscussed increased risk for pancreatitis, metaplasia, orinflammation and pancreatic adenocarcinomas and gluca-gonomas (15,16,25,26). In contrast, hundreds of otherstudies have investigated effects of these drugs on thepancreas but have not reported adverse effects; a feware referenced here for liraglutide (37–39). A study,

Table 2—Summary of histopathological findings in the 4-, 13-, and 52-week liraglutide studies and in the 13- and 52-weeksemaglutide studies

Males Females

4-week liraglutide dose levels (mg/kg/day) 0 0.05 0.5 5.0 0 0.05 0.5 5.0Animals examined 3 3 3 3 3 3 3 3Endocrine pancreasNo abnormality detected 3 3 3 3 3 3 3 3

Exocrine pancreasNo abnormality detected 3 3 3 3 3 3 3 3

13-week liraglutide dose levels (mg/kg/day) 0 0.05 0.5 5.0 0 0.05 0.5 5.0Animals examined 4 4 4 4 4 4 4 4Endocrine pancreasNo abnormality detected 4 4 3 3 2 3 4 4Prominent islets 0 0 1 1 1 0 0 0Fat infiltration, minimal 0 0 0 0 1 1 0 0

Exocrine pancreasNo abnormality detected 4 4 4 4 3 4 4 4Inflammatory cell infiltration, minimal 0 0 0 0 1 0 0 0

52-week liraglutide dose levels (mg/kg/day) 0 0.05 0.50 5.0 0 0.05 0.5 5.0Animals examined 4 4 4 4 4 4 4 4Endocrine pancreasNo abnormality detected 4 4 4 4 4 4 4 4

Exocrine pancreasNo abnormality detected 4 3 4 3 4 3 3 4Fibrosis, focal, minimal 0 1 0 0 0 0 0 0Inflammatory cell infiltration, focal, minimal 0 0 0 1 0 1 0 0Ectopic splenic tissue 0 0 0 0 0 0 1 0

13-week semaglutide dose levels (mg/kg twice weekly) 0 0.004 0.086 0.47 0 0.004 0.086 0.47Animals examined 4 4 4 4 4 4 4 4*Endocrine pancreasNo abnormality detected 4 4 4 4 4 4 4 3Islet atrophy 0 0 0 0 0 0 0 1

Exocrine pancreasNo abnormality detected 4 4 4 3 3 1 4 3Chronic focal inflammation, mild 0 0 0 0 0 1 0 0Inflammatory cell foci, minimal 0 0 0 1 1 1 0 1Focal brown pigment, minimal 0 0 0 0 0 1 0 0Ectopic splenic tissue 0 0 0 0 0 1 0 0

52-week semaglutide dose levels (mg/kg twice weekly) 0 0.01 0.06 0.36 0 0.01 0.06 0.36Animals examined 4 4 4 4 4 4 4 4Endocrine pancreasNo abnormality detected 4 4 4 4 4 4 4 4

Exocrine pancreasNo abnormality detected 3 3 4 4 3 1 3 3Focal arteritis/periarteritis, minimal 0 0 0 0 0 1 0 0Focal interstitial inflammatory cell infiltration, minimal 1 1 0 0 0 2 1 1Focal periductal inflammatory cell infiltration, slight 0 0 0 0 1 0 0 0

*Two animals were killed after 36 days, see Fig. 1 legend for details.

2494 GLP-1 Receptor Agonists and the Pancreas Diabetes Volume 63, July 2014

performed after others had reported potential adversefindings, confirmed the absence of pathology in diabeticrats and also did not show any regional differences in thepancreas induced by liraglutide when the pancreas wasdivided into four regions and examined by stereology(40). A recent publication used a human islet amyloidpolypeptide transgenic model similar to one of the earlierstudies, just in mice instead of in rats, treats the animalsfor 1 year instead of 12 weeks, used 20–25 animals ineach group instead of 8, and found no pancreas pathologyassociated with sitagliptin (25,41). The U.S. Food andDrug Administration recently published that it reassesseddata from 50 GLP-1 based therapeutics and found nochanges indicating pancreatic injury (42). To understandwhether there are any potential adverse effects of GLP-1RAs on the human pancreas, the expression pattern ofGLP-1R may be important. It has recently been recog-nized that most studies measuring the GLP-1R may beinvalid because the antibodies used are not specific forGLP-1R (30,43,44). G-protein–coupled receptors are no-toriously known for this problem (45–47). Some scientificjournals have provided new guidance for validationexperiments that must be available for reliable documen-tation of expression of a G-protein–coupled receptor (48).However, valid studies are available documenting GLP-1Rexpression (49). These studies measure receptor expres-sion by ligand binding and show that pancreatic adeno-carcinomas do not express GLP-1R (49,50). Thus, froma molecular target point of view, it seems unlikely thatGLP-1R agonism should directly worsen or induce pancre-atic adenocarcinomas when such tumors do not expressGLP-1R.

On the basis of the totality of information available toNovo Nordisk, the available information is insufficient toconfirm or exclude an association between liraglutide andpancreatitis, and there is no evidence that it increases therisk of pancreatic cancer in patients with type 2 diabetes.The Liraglutide Effect and Action in Diabetes: Evaluationof Cardiovascular Outcome Results—A Long Term Evalu-ation (LEADER; NCT01179048) will prospectively evalu-ate the overall safety of liraglutide. The trial has enrolled9,340 patients with type 2 diabetes and a high cardiovascu-lar risk profile. Patients are randomized 1:1 in a double-blind

study design to liraglutide or placebo and will be followedup for a minimum of 42 months for the primary end pointof adjudicated macrovascular events, including nonfatalmyocardial infarction, stroke, or cardiovascular death. Ad-judication of all adverse reactions related to pancreatitisand any neoplasm is an integral part of the protocolthroughout the duration of the LEADER study. Random-ized, controlled, long-duration trials with independent ad-judication are the only way to evaluate rare side effects, asalso recently mentioned by Kahn (29) and Drucker (30).The LEADER study will report in 2016.

Acknowledgments. The authors thank Charles Pyke, Novo Nordisk, forcareful reading of the manuscript and interpretations of the methodology used.Duality of Interest. Novo Nordisk markets liraglutide for the treatment ofdiabetes and has semaglutide in phase 3 clinical development. All authors arefull-time employees of Novo Nordisk and hold minor share portions as part oftheir employment. No other potential conflicts of interest relevant to this articlewere reported.Author Contributions. C.F.G. performed the quantitative histology.A.-M.M. collected and reviewed parts of the data and took part in drawingconclusions from the liraglutide studies. I.T. reviewed the histopathological dataand selected the photographs. N.C.B.N. took part in drawing conclusions from theliraglutide studies. Z.S. designed the semaglutide studies and concluded theresults for those. L.B.K. took part in drawing conclusions from the results ofthe studies and wrote the major part of ABSTRACT, INTRODUCTION, and DISCUSSION

sections. M.O.L. wrote the major part of the RESEARCH DESIGN AND METHODS and RESULTS

sections and some parts of the ABSTRACT and DISCUSSION and performed the statis-tical analysis. All authors reviewed the manuscript. L.B.K. is the guarantor of thiswork and, as such, had full access to all the data in the study and takes re-sponsibility for the integrity of the data and the accuracy of the data analysis.

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Table 3—Summary of dose-related tendencies for absolute pancreas weight (g) across studies with liraglutide and semaglutide

Pancreas weight, tendency for dose-related change?

Males Females Consistent in both sexes

4-week liraglutide * No

13-week liraglutide Yes

52-week liraglutide ** No

87-week liraglutide Yes

13-week semaglutide Yes

52-week semaglutide Yes

*P , 0.05 for a statistically significant increase in absolute pancreas weight, but not apparently dose-related. **P , 0.01 for anapparently dose-related increase in absolute pancreas weight.

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