2486 Diabetes Volume 63, July 2014 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 Analogs Liraglutide and Semaglutide: Absence of Histopathological Effects on the Pancreas in Nonhuman Primates Diabetes 2014;63:2486–2497 | DOI: 10.2337/db13-1087 Increased pancreas mass and glucagon-positive ade- and glucagon-like peptide-1 (GLP-1) receptor agonists nomas have been suggested to be a risk associated (GLP-1RAs). This concern is partly based on the well- with sitagliptin or exenatide therapy in humans. Novo described effect of GLP-1 to induce growth of pancre- Nordisk has conducted extensive toxicology studies, atic b-cells (1). Both drug classes increase effective including data on pancreas weight and histology, in GLP-1 levels, but to a different degree, and have different Cynomolgus monkeys dosed with two different human modes of action; thus, it is important to differentiate glucagon-like peptide-1 (GLP-1) receptor agonists. In a between them, particularly when considering mechanistic 52-week study with liraglutide, a dose-related increase hypotheses for potential safety concerns or signals. GLP- in absolute pancreas weight was observed in female 1RAs mediate their effects directly through the GLP-1R monkeys only. Such dose-related increase was not (2,3). Although increased levels of GLP-1 and glucose- found in studies of 4, 13, or 87 weeks’ duration. No dependent insulinotropic peptide (GIP) are considered treatment-related histopathological abnormalities were important parts of the mechanism of action of DPP-4is observed in any of the studies. Quantitative histology of (4), DPP-4 is known to degrade many other hormones (5). PHARMACOLOGY AND THERAPEUTICS the pancreas from the 52-week study showed an in- DPP-4is have been shown to increase GLP-1, GIP, and crease in the exocrine cell mass in liraglutide-dosed peptide YY (6,7). This complicates the understanding of animals, with normal composition of endocrine and exo- crine cellular compartments. Proliferation rate of the both desired and potentially undesired effects of this class exocrine tissue was low and comparable between of compounds. Within GLP-1RAs, differences exist in groups. Endocrine cell mass and proliferation rates were safety-related parameters. One subgroup is the exendin- – unaltered by liraglutide treatment. Semaglutide showed 4 based drugs with exenatide and lixisenatide, which are no increase in pancreas weight and no treatment- structurally distinct from human GLP-1. Owing to the low related histopathological findings in the pancreas after amino acid homology to native human GLP-1, these med- 13 or 52 weeks’ dosing. Overall, results in 138 nonhuman ications are associated with an increased number of im- primates showed no histopathological changes in the mune reactions that are, however, all of a relatively mild pancreas associated with liraglutide or semaglutide, form, that is, mostly antibody development, injection two structurally different GLP-1 receptor agonists. site nodules, and loss of efficacy (8,9). The other sub- group is based on human GLP-1 and contains liraglutide, Pancreas safety has become a subject of much debate con- taspoglutide, and larger covalently conjugated molecules cerning dipeptidylpeptidase-4 (DPP-4) inhibitors (DPP-4is) such as albiglutide and dulaglutide. Clinical development Diabetes Research Unit, Novo Nordisk, Måløv, Denmark © 2014 by the American Diabetes Association. See http://creativecommons.org Corresponding author: Lotte Bjerre Knudsen, [email protected]. /licenses/by-nc-nd/3.0/ for details. Received 11 July 2013 and accepted 18 February 2014. See accompanying article, p. 2219. This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1087/-/DC1. diabetes.diabetesjournals.org Gotfredsen and Associates 2487 of taspoglutide was stopped due to severe immune-related RESEARCH DESIGN AND METHODS side effects, with cases of anaphylactic shock, possibly The research design and methods for liraglutide stud- fi caused by the formulation (8,10). No such ndings have ies in Cynomolgus monkeys have been described pre- – been reported with other GLP-1 based analogs. viously (13). All animals were examined daily for Semaglutide is a once-weekly GLP-1 analog that is in clinical signs in the in-life phase. Studies with sema- phase 3 clinical development (11). Where liraglutide is glutide were generally performed similarly, and both acylated with a palmitic acid and has an extra amino followed international guidelines provided by Interna- acid as a spacer between the palmitic acid and the Lys26, tional Conference of Harmonization. Dose levels, du- where the fatty acid is attached, semaglutide is acylated ration, and group sizes for all studies are described in with a stearic diacid at Lys26 but has a much larger syn- Fig. 1. Compounds were administered as subcutaneous thetic spacer and is, furthermore, modified for DPP-4 sta- injections. bility in position 8, where the amino acid a-aminobutyric acid has been introduced. Body Weight and Pancreas Weight In support of drug development and regulatory ap- Terminal body weight was obtained in sedated animals proval for treatment of chronic diseases, repeat dose immediately before they were killed. The entire pancreas toxicity and carcinogenicity studies are conducted. These of each animal was examined macroscopically for any ab- studies are performed at different dosing durations to normalities, excised, cleaned of fat and connective tissue, support the different phases of clinical development and and weighed. with doses aiming to obtain exposure several multiples higher than the clinically relevant exposure with the aim Tissue Preparation to identify potential drug-related organ toxicity and A transverse section from themidpartofthepancreas carcinogenicity. Repeat dose toxicity studies are typically from all animals was sampled, and this section and the fi conducted in a rodent and a nonrodent species. For rest of the pancreata were xed in 10% neutral buffered liraglutide and semaglutide, Cynomolgus monkeys were formalin for at least 48 h. The sections were dehydrated fi chosen as the nonrodent species. Repeat dose toxicity and paraf n-embedded according to standard histopath- studies in nonhuman primates are designed to screen ological procedures. According to international standard for potential hazards and are not designed or statistically practices, one section per animal was cut at a nominal thickness of 4–5 mm and stained with hematoxylin and powered for identification of differences in the incidence eosin (17). The slides were read unblinded because this is or severity of individual organ changes. The number of recommended by toxicopathology experts as a way to nonhuman primates per group is limited to three to five increase the chance of separating subtle changes from for ethical reasons (12). Because of the statistical limita- normal background changes (18). Further details of tions, standardization of the examinations is critical: If the methodology have been previously described (13). pathological findings are identified at a frequency or se- The pancreas specimens from animals killed at termina- verity exceeding those in the in-study control group, they tion of dosing in the 52-week liraglutide study were are often compared with historical control data to assist a fi fi evaluated by quantitative histology (all groups for -cells interpreting the signi cance of the nding. This principle and Ki-67, and only vehicle and high-dose groups for b-, fi also applies to organ weights. Histopathological ndings d-, pancreatic polypeptide [PP], ductal, and acinar cells). in the pancreas from the repeat-dose studies in rodents The cranial and caudal remnants were sectioned longi- and nonhuman primates and from carcinogenicity studies tudinally, cut into ;40 pieces, and distributed to four in rodents with liraglutide have been published previously capsules, with one-fourth in each, according to the (13). Liraglutide was not found to have a causal relation- smooth fractionator principle (19,20). Processing and fi ship to any histopathological ndings. staining for detection of b-, a-, d-, and PP cells was Some studies in rats and mice have shown an increased done as previously described (21). The reactivity of the pancreas weight induced by DPP-4is or GLP-1RAs (14,15), primary antibodies to insulin, glucagon, somatostatin, and a recent ex vivo study with human pancreata sug- and PP in monkey pancreas had been tested indi- gested an increase in glucagonomas as well as increased vidually to validate the method. Costaining for duct pancreas weight (16). Here, pancreas weight in Cynomol- plus acinar cells using mouse anti–CK-7 (Dako, Glostrup, gus monkeys is reported for liraglutide in toxicology stud- Denmark) and rabbit anti–a-amylase (Calbiochem, Darm- ies with 4, 13, 52, and 87 weeks’ dosing, and for stadt, Germany) plus Ki-67 using polyclonal rabbit semaglutide in toxicology studies with 13 and 52 weeks’ anti–Ki-67 (Novus Europe, Cambridge, U.K.) followed dosing, as well as a full histopathological evaluation of thesameprinciplesasabove.Stainedslideswere these same studies, except the 87 weeks’ study, which scanned in a Hamamatsu NanoZoomer 2.0-HT (Hamamatsu, has been reported previously (13). For liraglutide, a full Hamamatsu City, Japan). Images were subsequently ana- quantitative histological assessment of the endocrine as lyzed automatically