Cancer Therapy: Preclinical

The Intestinotrophic Peptide, GLP-2, Counteracts Intestinal Atrophy in Mice Induced by the Epidermal Growth Factor Receptor Inhibitor, Gefitinib KristineJuul Hare,1Bolette Hartmann,2 Hannelouise Kissow,1Jens Juul Holst,2 and Steen Seier Poulsen1

Abstract Purpose: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been introduced as antitumor agents in the treatment of cancers overexpressing the receptor. The treatment has gastrointestinal side effects which may decrease patient compliance and limit the efficacy. Glucagon-like peptide-2 (GLP-2) is an intestinal hormone with potent intestinotrophic properties and therapeutic potential in disorders with compromised intestinal capacity. The growth stimulation is highly specific to the gastrointestinal tract, and no effects are observed elsewhere. The aim of this study was to examine whether the inhibition of the EGFRinduces intestinal atrophy and if this can be counteracted by treatment with GLP-2. Experimental Design: Mice were treated for 10 days with either gefitinib orally, GLP-2 as injections, or a combination of both. After sacrifice, the weight and length of the segments of the gastrointestinal tract were determined, and histologic sections were analyzed by morpho- metric methods. Results: A significant atrophy of the small-intestinal wall was observed after treatment with gefitinib because both intestinal weight and morphometrically estimated villus height and cross- sectional area were decreased. The same parameters were increased by GLP-2 treatment alone, and when GLP-2 was combined with the gefitinib treatment, the parameters remained unchanged. Conclusions: Treatment with an EGFRtyrosine kinase inhibitor in mice results in small-intestinal growth inhibition that can be completely prevented by simultaneous treatment with GLP-2. This suggests that the gastrointestinal side effects elicited by treatment with EGFRtyrosine kinase inhibitors can be circumvented by GLP-2 treatment.

The epidermal growth factor receptor (EGFR) is expressed by EGFR-TKIs, gefitinib (Iressa) and erlotinib (Tarceva), with high approximately one-third of all human epithelial cancers, selectivity due to minimal effect on other tyrosine or serine/ including non–small cell lung cancer (NSCLC), prostate, breast, threonine kinases, are under clinical development (7, 8). colorectal, head and neck, ovarian, gastric, and pancreatic Primarily, they have been tested in NSCLC, but also in cancers (1–4). The EGFR pathway contributes to a number metastatic colorectal cancers, recurrent head and neck cancers, of processes involved in tumor survival and growth, such as and gliomas (for a review, see ref. 9). The potential role of the cell proliferation, inhibition of apoptosis, angiogenesis, and EGFR-TKIs has not been fully determined, but large phase II metastasis, thus making it an attractive target for anticancer studies where gefitinib or erlotinib were given as second-line therapies (5, 6). therapy to NSCLC patients showed antitumor activity as EGFR tyrosine kinase inhibitors (EGFR-TKI) that block the determined by tumor shrinkage, stabilization of disease, and signal transduction pathways implicated in the proliferation relief of symptoms, especially following erlotinib. Both EGFR- and survival of cancer cells and other host-dependent processes TKIs are generally well tolerated: the most frequent adverse promoting cancer growth have recently been introduced in effects noted were skin rashes and gastrointestinal symptoms the treatment of cancers, especially as second- or third-line such as nausea, vomiting, and especially diarrhea. A combina- therapy or in combination with chemotherapy. Orally active tion of EGFR-TKI (erlotinib) and a standard chemotherapy regimen (FOLFORI) had to be terminated due to excessive toxicity, including grade 3 diarrhea and vomiting (10). The Authors’Affiliations: Departments of 1Anatomy and 2Physiology, Panum Institute, occurrence of gastrointestinal symptoms is in accordance with University of Copenhagen, Copenhagen, Denmark the supposed role of the EGF system in the regulation of growth Received 3/8/07; revised 5/25/07; accepted 6/4/07. The costs of publication of this article were defrayed in part by the payment of page and differentiation in the gastrointestinal tract (11–13), charges. This article must therefore be hereby marked advertisement in accordance although a direct effect on the gastrointestinal tract by the with 18 U.S.C. Section 1734 solely to indicate this fact. inhibition of the EGF system by the EGFR-TKIs has never been Requests for reprints: Steen Seier Poulsen, Anatomy Department B, Panum shown. Institute, University of Copenhagen, 3 Blegdamsvej, DK-2200 Copenhagen N, f Denmark. Phone: 45-35327253; E-mail: [email protected]. Gastrointestinal side effects were observed in 80% of F 2007 American Association for Cancer Research. patients following treatment with gefitinib, and this required doi:10.1158/1078-0432.CCR-07-0574 dose reduction and caused treatment delay in some patients

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(14–17). Both the occurrence and severity of diarrhea and skin by immersion in ice-cold, freshly prepared buffered 4% paraformalde- rashes were found to be dose dependent (15, 16, 18). Because hyde. The fixed tissue samples were then dehydrated, embedded in A treatment with EGFR-TKIs most probably is going to be paraffin, and cut perpendicularly to the axis of their length into 10- m prolonged—several cycles of 4-week treatment periods—it is sections. The sections were stained with PAS-HE and were examined using a Leitz Ortoplan microscope fitted with a cooled camera, desirable to avoid the gastrointestinal side effects which can Evolution MP (MediaCybernetics). The cross-sectional area of the influence the general well-being of the patients. mucosa and muscular layers in the small and large intestines as well as Glucagon-like peptide-2 (GLP-2) is a 33-amino acid peptide the PAS-positive (mucus-containing) area in the stomach (indicated derived from the posttranslational processing of proglucagon. as square micrometers per millimeter mucosa) and colon (indicated as It has highly specific intestinotrophic effects (19–22), and PAS-positive area in percent of the cross-sectional area) was measured. following treatment with exogenous peptide, a growth response The thickness of the stomach mucosa and height of the gastric pits, is seen only in the intestinal system. GLP-2 is supposed to play the villus height, and crypt depth were also measured using Image-Pro an important role in the regulation of the size and absorptive Plus 5.0. The examination and the computer analysis of the histologic capacity of the gut. GLP-2 treatment increases small bowel sections were done without the knowledge of the origin of tissue weight and mucosal thickness in mice (20) by inducing a lower samples. Statistical analyses. The results are shown as mean F SE. Statistical rate of apoptosis in the enterocytes and a higher rate of crypt- significance of the differences obtained between initial and final body cell proliferation (19). The growth factor system activated by weight for each of the four treatment groups were assessed by an GLP-2 stimulation has not yet been unambiguously identified, unpaired t test. Comparison between groups was done by one-way although it has been shown that insulin-like growth factor I ANOVA, followed by Tukey’s post hoc analysis. Probability values of (IGF-I; ref. 23) as well as the keratinocyte growth factor (24) P < 0.05 were considered significant. seem to be involved. We hypothesized that unless GLP-2 exerts its growth- promoting action via the EGF system, the inhibition of Results intestinal growth by EGFR-TKIs might be counteracted by the Body weight changes during the study. Initial and final body treatment with GLP-2. In the present study, we describe the weight for the four groups are summarized in Table 1. Mice direct effect of short-term gefitinib treatment in mice on from group a (PBS) and group c (gefitinib) had a statistically morphometric parameters in the various segments of the significant (P = 0.048 and P = 0.00016) lower final body gastrointestinal tract, and we investigate whether the possible weight compared with their initial body weight (t test). PBS- growth-inhibiting effects of gefitinib might be counteracted treated mice lost in average 1.1 g (from 20.8 to 19.7 g), and by the simultaneous administration of GLP-2. gefitinib-treated animals lost 1.2 g (from 20.9 to 19.7 g). GLP-2– and GLP-2 + gefitinib–treated mice had minor and Materials and Methods insignificant weight losses. Statistical analysis comparing the four treatment groups (ANOVA) showed a significant difference P Animals. The animal studies were approved by the Danish Ministry ( < 0.05) in final body weight between animals in group b of Justice, Animal Experiments Inspectorate. Female C57bl mice (M&B) (GLP-2) and group c (gefitinib). weighing f21 g were housed in plastic-bottomed wire-lidded cages. Effects of GLP-2 and gefitinibon the weight and length of They were maintained throughout the course of the experiment on gastrointestinal organs. There was no significant difference water and chow (no. 1314, Altromin) ad libitum in animal facilities between the four treatment groups in the weight of the stomach with temperature (21jC)– and humidity (55%)–controlled rooms and the length of the small intestine and the length of the colon with a light-dark cycle of 12 h each. All animals were acclimatized for at (data not shown). Significant differences between the groups least 1 week before the study started. were found in the weight of the small intestine and the colon Experimetal design. Human recombinant GLP-2 (a generous gift (Fig. 1). The weight of the small intestine (expressed in percent from L. Thim, Novo Nordisk A/S, Bagsværd, Denmark) was dissolved in of the body weight) was significantly (P < 0.001) reduced by PBS containing 3.5 mg/mL Hemaccel (Behringwerke AG), which was F also used for control injections. The injection volume was 100 AL 17% in gefitinib-treated animals (3.58 0.09%) compared containing 25 Ag GLP-2, given as s.c. injections twice daily every 12 h for 10 days. EGFR inhibitor, gefitinib (Iressa), kindly donated from AstraZeneca, was prepared as a suspension (2.5 mg/mL) in 1% aqueous Tween 80 by homogenization with glass beads for f18 h. The dose Table 1. Body weight (BW) of mice treated 10 d volume was 0.2 mL administered by oral gavages twice daily every 12 h with PBS, GLP-2, gefitinib, or gefitinib + GLP-2 for 10 days. The suspension was kept at room temperature, with stirring during the experiment. PBS GLP-2 Gefitinib Gefitinib Animals were weighed and randomly allocated to the following + GLP-2 a b A c d groups of 10: ( ) PBS, ( ) GLP-2 (25 g), ( ) gefitinib (0.5 mg), and ( ) F F F c F A BW initial (g) 20.8 0.3* 21.2 0.2 20.9 0.2 21.0 0.4 GLP-2 (25 g) + gefitinib (0.5 mg). Animals in groups a and b were, in BW final (g) 19.7 F 0.4b 21.0 F 0.2 19.7 F 0.2b 20.4 F 0.4 addition, p.o. dosed with 0.2 mL water containing 1% Tween 80. Animals in group c had control injections with PBS. All animals were F sacrificed after 10 days of treatment. After removal of mesenteric fat and NOTE: Results are mean SE. *P luminal contents of the stomach and gut, the weight and length of the < 0.05 compared with final body weight of PBS-treated mice (t test). small and large intestines and the weight of the stomach were recorded. cP < 0.0002 compared with final body weight of gefitinib-treated When measuring length, all intestinal segments were vertically mice (t test). suspended with a 1.5-g weight to provide uniform tension. bP < 0.05 compared with final body weight of GLP-2 – treated mice Histologic sections and morphometric analysis. Tissue samples from (ANOVA). the small intestine (proximal, middle, and distal) and colon were fixed

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mucosa was similar to that of the controls. Animals treated with GLP-2 and GLP-2 + gefitinib showed increases (not statistically significant) in both pit height and amount of mucin compared with animals treated with gefitinib alone and control animals. The GLP-2 + gefitinib–treated group was comparable to the GLP-2 group. In the small intestine, the effects of gefitinib and GLP-2 were mainly seen in the proximal part (Table 2). The cross- sectional area of the proximal part of the small intestine in gefitinib-treated mice (3.40 mm2) was similar to that of the controls (3.27 mm2), whereas the GLP-2–treated mice (4.63 mm2) had a significantly increased area compared with controls (P < 0.01) and compared with gefitinib (P < 0.01). The proximal cross-sectional area in the GLP-2 + gefitinib– treated (4.00 mm2) animals was increased compared with gefitinib-treated mice and compared with controls (not significant). The cross-sectional area of the middle part of the small intestine was increased in GLP-2–treated animals both compared with the controls and the gefitinib-treated groups (P < 0.05; Table 2). No statistically significant differences between the four treatment groups were detected regarding the cross-sectional areas of the distal part of the small intestine (Table 2). The villus height (and thereby the small-intestinal surface area) was the parameter exhibiting the most pronounced changes following the treatments, and gefitinib and GLP-2 clearly had opposite effects (Fig. 2). Again, changes were most pronounced in the proximal part of the small intestine (Fig. 3), where the villus height was significantly decreased by 28% Fig. 1. Small-intestinal weight (A) and colonic weight (B) expressed as percent of the body weight in PBS-, GLP-2 ^ , gefitinib ^ , and gefitinib + GLP-2 ^ treated (P < 0.001) following treatment with gefitinib (0.51 mm) mice. Results are mean + SE. a, P < 0.05; A, P < 0.01; and AA, P < 0.001, compared compared with controls (0.71 mm). In contrast, villus height with gefitinib-treated mice. BB, P < 0.001, compared with GLP-2 ^ treated mice. P C, P < 0.01, compared with gefitinib + GLP-2 ^ treated mice. was significantly increased ( < 0.05) in GLP-2–treated animals (0.84 mm) compared with controls and compared with the gefitinib-treated animals (P < 0.001). When GLP-2 was with controls (4.30 F 0.06%). In contrast, the small intestine in given in combination with gefitinib, the gefitinib-induced villus the GLP-2–treated mice showed a marked increase (P < 0.001) atrophy was completely counteracted. Villus height in GLP-2 + in small-intestinal weight (5.74 F 0.10% of body weight) gefitinib–treated mice (0.75 mm) was comparable to controls compared with controls. Animals treated with GLP-2 in but was significantly increased compared with the gefitinib- combination with gefitinib (group d) showed a significantly treated group (P < 0.001). increased small-intestinal weight (4.82 F 0.11%) compared In the middle part of the small intestine, changes were less with both gefitinib-treated mice (P < 0.001) and controls pronounced (Fig. 2). The villus height in the gefitinib-treated (P < 0.01). This increase was, however, significantly lower animals (0.39 mm) was comparable to controls (0.43 mm). compared with the weight increase induced by GLP-2 alone GLP-2–treated (0.54 mm) and GLP-2 + gefitinib (0.48 mm)– (P < 0.001; Fig. 1A). The colonic weight was also influenced by treated animals had significantly increased villus height gefitinib treatment because the weight of the colon (in percent compared with controls (P < 0.001 and P < 0.05, respectively) of the body weight) was significantly (P < 0.001) decreased by and compared with gefitinib-treated mice (P < 0.001). 14% in the gefitinib-treated animals (1.10 F 0.02%) compared In the distal part of the small intestine, treatment with GLP-2 with controls (1.28 F 0.05%). Also, when compared with resulted in increased villus height compared with controls GLP-2–treated animals (1.24 F 0.02%) and gefitinib + GLP-2– (P < 0.01), gefitinib (P < 0.001), and gefitinib + GLP-2 treated animals (1.23 F 0.01), the colon weight of the gefitinib- (P < 0.05; Fig. 2). The villus height in gefitinib- and gefitinib + treated mice was significantly decreased (P < 0.01 and P < 0.05, GLP-2–treated animals was comparable to controls. respectively). The colon weight in the GLP-2–treated and the The depth of the crypts in the small intestine was not GLP-2 + gefitinib–treated animals was comparable to the influenced by any of the treatments (Table 2). There was a controls. tendency toward an increased crypt depth in the proximal and Morphometric analysis. Only minor changes of the morpho- middle part in the GLP-2–treated animals; however, this was metric parameters of the stomach were found (Table 2). There not significant (Table 2). were no differences in the height of the mucosa between the With respect to the large intestine, the four treatment four groups. Gefitinib-treated animals showed a minor decrease groups were comparable because no significant differences in (not statistically significant) in the height of the gastric pits cross-sectional area, crypt depth, and mucin area were seen compared with controls, whereas the amount of mucin in the (Table 2).

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Table 2. Morphometric data from mice treated 10 days with PBS, GLP-2, gefitinib, or gefitinib + GLP-2

PBS GLP-2 Gefitinib Gefitinib + GLP-2

Gastric mucosa Mucosal height (mm) 1.32 F 0.09 1.24 F 0.07 1.27 F 0.10 1.16 F 0.05 Height of gastric pit (Am) 177 F 17 193 F 15 162 F 21 200 F 9 Mucin area per millimeter mucosa (103 Am2) 65.25 F 10.3 87.65 F 8.5 65.75 F 8.6 90.16 F 8.3 Small intestine Cross-sectional area (mm2) Proximal 3.27 F 0.20* 4.63 F 0.34 3.40 F 0.15* 4.00 F 0.21 Middle 2.50 F 0.22c 3.48 F 0.19 2.54 F 0.24c 2.83 F 0.22 Distal 1.79 F 0.14 1.95 F 0.09 1.74 F 0.10 1.80 F 0.10 Crypt depth (mm) Proximal 0.118 F 0.008 0.151 F 0.009 0.127 F 0.014 0.123 F 0.010 Middle 0.113 F 0.005 0.123 F 0.006 0.110 F 0.004 0.115 F 0.007 Distal 0.124 F 0.008 0.121 F 0.005 0.108 F 0.005 0.115 F 0.008 Colon Cross-sectional area (mm2)2.97F 0.21 3.66 F 0.22 3.45 F 0.20 2.86 F 0.22c Crypt depth (mm) 0.033 F 0.002 0.043 F 0.003 0.034 F 0.002 0.043 F 0.005 Mucin area, % of cross-sectional area 66 F 7.2 58 F 8.2 51 F 4.8 62 F 8.6

NOTE: Results are mean F SE. *P < 0.01 compared with GLP-2 – treated mice. cP < 0.05.

Discussion resulting in reduced mucosal growth. Other consequences from a lack of this growth factor system have been described by There is increasing evidence that EGFR-TKIs have a place in Troyer et al. in knock-out mice missing some of the EGFR the future treatment of different adenocarcinomas. However, ligands (amphiregulin, EGF, and transforming growth factor-a). the side effects of blocking the EGF system are undesirable, The duodenum of these animals is prone to spontaneous especially in cancer patients where the overall health may ulceration, and the ileal villus height is reduced when compared already be impaired. The EGFR is present on most epithelial with wild-type mice (41). Lesions in the duodenum have also and stromal cells as well as on some glial and smooth muscle been reported after treating mice with an EGFR inhibitor (42). cells and is essential for normal function and development in EGFR-/- mice were described by Miettinen et al. (25). These the gut, kidney, urogenital system, and skin (5, 12, 13, 25). mice only survive for up to 8 days after birth and suffer from EGFR-inhibiting agents have an advantage over conventional impaired epithelial development in several organs. The pups chemotherapeutic agents in that they selectively block specific suffer from dehydration and malnutrition and die severely deregulated pathways in tumor cells while having less effects on undernourished. Post mortem findings include hemorrhagic, normal cell function (5, 26). Thus, the EGFR is an obvious distended intestines with a reduced number of shortened villi, target in cancer therapy and has already been proven efficient in thereby resembling necrotizing enterocolitis (25). clinical studies. However, gastrointestinal side effects need to be considered. GLP-2 is an intestinal hormone that acts through not yet settled pathways to induce intestinal growth. Several studies have shown that the growth factor, EGF and GLP-2, have similar trophic properties in the gastrointestinal tract (27–36). IGF has recently been suggested to have a crucial role as mediator of GLP-2–induced intestinal growth (23), but the possible role of the EGF system has not been elucidated. Our hypothesis was that EGFR-TKIs cause atrophy of the gastrointestinal tract, and unless the EGF system is crucial for the GLP-2–mediated stimulation of intestinal growth, treat- ment with GLP-2 can prevent this atrophy. Our results show that 10 days of oral treatment with the EGFR inhibitor gefitinib leads to a decrease in the weight of the small and large intestines and a decreased absorptive surface area due to a pronounced atrophy of the villi. EGFR activity in the small intestinal surface epithelium is almost entirely restricted to the proliferative crypt region. The receptor is located on the basolateral surface of the epithelium, and the Fig. 2. Villus height in the proximal, middle, and distal part of the small intestine in role of this growth factor system is probably to stimulate repair PBS, GLP-2, gefitinib, and gefitinib + GLP-2 ^ treated mice. Results are mean + SE. AA, P < 0.001,compared with gefitinib-treated mice. b, P < 0.05; B, P < 0.01; and and maintenance of the gut (12, 37–40). The reported atrophy BB, P < 0.001, compared with GLP-2 ^ treated mice. c, P < 0.05, compared with may be due to the absence of these effects on the mucosa gefitinib + GLP-2 ^ treated mice.

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Fig. 3. Proximal small intestine stained with PAS-HE showing differences in villus height and crypt depth. A, PBS. B, GLP-2 ^ treated group. C, gefitinib-treated group. D, GLP-2 + gefitinib ^ treated group. Bar, 100 Am.

These studies underline the importance of the EGF system in was excellent, although GLP-2 was given twice daily as s.c. the development, maintenance, and function of the normal gut. injections for 35 days (43). A similar improvement of Following treatment with GLP-2, we found increased small- absorptive function might be observed in patients on gefitinib and large-intestinal growth seen as both increased organ weight treatment also given GLP-2. and increased villus height. Changes were most pronounced The intestinal growth caused by exogenous GLP-2 regresses in the proximal part of the small intestine. When GLP-2 was to normal after cessation of treatment, indicating that the given to the gefitinib-treated mice, we found that the gefitinib- epithelial proliferation is dependent on ongoing GLP-2 admin- induced atrophy could be completely prevented because all istration (19). No side effects have been reported after treatment morphometric parameters of the gut were comparable or even with GLP-2, which is also in agreement with the assumption enhanced compared with PBS-treated controls. that its only target is the gut (19). The mechanism of GLP-2–induced growth is still not fully Physiologically, GLP-2 delays gastric emptying (enterogas- understood, but the involvement of local growth factors is trone effect; refs. 44, 45), perhaps through inhibiting centrally probable (24). Recently, the presence of IGF-I was suggested to induced antral motility (46), thereby acting as one of the be necessary for the GLP-2 intestinal growth response (23). mediators of the so-called ileal brake. The overall combination From the results of the present study, it seems unlikely that of intestinotrophic effects, functional improvement (27–30, GLP-2 acts via the EGF receptor because we were able to elicit 33, 34), and an enterogastrone effect makes GLP-2 promising identical GLP-2–induced growth responses with or without as an agent for treatment of intestinal insufficiency. These concurrent inhibition of the EGFR. physiologic properties of GLP-2 would also be of benefit to The gastrointestinal side effects observed in patients treated cancer patients suffering from diarrhea and weight loss. with gefitinib may be caused by the reduction of the absorptive Although GLP-2 has proved to be safe in long-term trials surface area resulting from atrophy of the small intestine. In (19, 21), recent studies show that GLP-2 is able to accelerate patients with short-bowel syndrome (where the absorptive growth of chemically induced colonic neoplasms in mice (47). area of the gut is also decreased), GLP-2 injections resulted in This effect of GLP-2 is not surprising in view of its trophic increased intestinal absorptive function, delayed gastric empty- action on the colonic mucosa. On the other hand, GLP-2 alone ing, and a general increase in lean body mass (43). Further- has never been found to induce neoplasia. Still, it may be more, morphometric analysis showed an increase in both advisable to perform colonoscopy in patients receiving long- villus height and crypt depth. Compliance in these patients term treatment with GLP-2.

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www.aacrjournals.org 5175 Clin Cancer Res 2007;13(17) September 1,2007 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. The Intestinotrophic Peptide, GLP-2, Counteracts Intestinal Atrophy in Mice Induced by the Epidermal Growth Factor Receptor Inhibitor, Gefitinib

Kristine Juul Hare, Bolette Hartmann, Hannelouise Kissow, et al.

Clin Cancer Res 2007;13:5170-5175.

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