ORIGINAL ARTICLE

The Intestinotrophic Peptide, GLP-2, Counteractsthe Gastrointestinal Atrophy in Mice Induced by the EpidermalGrowth Factor Receptor Inhibitor, Erlotinib, and Cisplatin

Andreas Rosen Rasmussen • Niels-Erik Viby •

Kristine Juul Hare • Bolette Hartmann •

Lars Thim • Jens Juul Holst • Steen Seier Poulsen

Received: 14 July 2009 / Accepted: 10 December 2009 / Published online: 29 January 2010

� Springer Science+Business Media, LLC 2010

Abstract

Purpose Erlotinib, an epidermal-growth-factor receptor

inhibitor, belongs to a new generation of targeted cancer

therapeutics. Gastrointestinal side-effects are common and

have been markedly aggravated when erlotinib is combined

with cytostatics. We examined the effects of erlotinib alone

and combined with the cytostatic, cisplatin, on the gastro-

intestinal tract and examined whether glucagon-like pep-

tide-2 (GLP-2), an intestinal hormone with potent

intestinotrophic properties, might counteract the possible

damaging effects of the treatments.

Experimental Design Groups of ten mice were treated for

10 days with increasing doses of erlotinib alone or in

combination with cisplatin and/or GLP-2. Weight and

length of the gastrointestinal organs were determined and

histological sections were analyzed with morphometric

methods as well as BrdU- and ApopTag-staining to

determine mitotic and apoptotic activity.

Results Erlotinib was found to induce small-intestinal

and colonic growth inhibition through an increased apop-

totic activity but had no effect on mitotic activity. The

combined treatment with cisplatin synergistically aggra-

vated the intestinal growth inhibition. Erlotinib, and espe-

cially the combination therapy, increased the weight of the

stomach contents considerably. Concomitant treatment

with GLP-2 counteracted the intestinal mucosal atrophy

induced both by erlotinib alone and combined with cis-

platin through a reduction of the apoptotic activity. There

was no influence on the mitotic activity.

Conclusions The findings demonstrate that the intestinal

mucosal damage induced by erlotinib alone and in com-

bination with cisplatin can be counteracted by GLP-2

treatment, which might suggest a role for GLP-2 in the

treatment of the gastrointestinal side-effects caused by

these cancer therapeutics.

Keywords GLP-2 � Erlotinib � Cisplatin �Chemotherapy-induced enteritis � Chemotherapy

Introduction

The tyrosine kinase inhibitor (TKI) erlotinib (Tarceva; OSI

Pharmaceuticals, Inc.) belongs to a new generation of tar-

geted cancer therapeutics. It inhibits the intracellular tyr-

osin kinase domain of the Epidermal Growth Factor

Receptor (EGFR) and thus a number of processes pro-

moting tumor survival and growth in which the EGFR

pathway is involved. These include cell proliferation,

inhibition of apoptosis, angiogenesis, and metastasis [1, 2].

The EGFR is expressed in many epithelial cancers and its

expression is associated with rapid disease progression,

poor survival, and poor response to chemotherapy [3, 4].

Erlotinib has been approved for second-line treatment of

non-small cell lung cancer (NSCLC) [5, 6], as well as first-

line treatment of locally advanced/metastatic pancreatic

cancer in combination with gemcitabine [6, 7]. Trials are

also ongoing concerning the use of erlotinib in several

other cancer forms [7].

Among the major side-effects of erlotinib are gastroin-

testinal symptoms as diarrhea, stomatitis, and anorexia [6].

A. R. Rasmussen � N.-E. Viby � K. J. Hare � B. Hartmann �J. J. Holst � S. S. Poulsen (&)

Department of Biomedical Sciences, The Panum Institute,

University of Copenhagen, 3 Blegdamsvej, 2200 Copenhagen N,

Denmark

e-mail: spoulsen@sund.ku.dk

L. Thim

Novo Nordisk A/S, Novo Nordisk Park, 2760 Maløv, Denmark

123

Dig Dis Sci (2010) 55:2785–2796

DOI 10.1007/s10620-009-1104-x

Besides being troublesome for the heavily burdened

patients, the gastrointestinal side-effects could make dose

reduction necessary and thus impair effectiveness or lead to

a halt of treatment.

The EGFR is supposed to play a role in the regulation of

growth and differentiation as well as healing in the gas-

trointestinal tract [8–10]. The receptor is located at the

basolateral membrane of the surface epithelium in the

small intestine and is activated primarily by epidermal

growth factor (EGF) or transforming growth factor alpha

(TGF-a) [9]. The EGFR pathway has been demonstrated to

stimulate proliferation of enterocytes in the gastrointestinal

tract [11–14]; to up-regulate electrolyte and nutrient

transport in the enterocyte [15], increase the expression of

brush border enzymes [16], and to induce epithelial resti-

tution [17–20]. An inhibition of these functions by erlotinib

could explain the gastrointestinal symptoms experienced

by the patients. The direct effects of erlotinib on the gas-

trointestinal tract, however, have not been characterized.

Oncological treatment often involves combination

therapy where several drugs are combined in order to target

different mechanisms of the malignant transformation, and

this strategy will probably aggravate the gastrointestinal

adverse effects associated with a TKI. Thus a combination

of erlotinib and the standard chemotherapy regime

FOLFIRI had to be terminated due to excessive toxicity

including grade 3 diarrhea and vomiting [21].

Glucagon-like peptide-2, GLP-2, would be an obvious

candidate for the treatment of any growth inhibition or

mucosal damage induced by erlotinib alone or combined

with cytostatics. It is a 33-amino-acid peptide derived from

the posttranslational processing of proglucagon in intesti-

nal L cells. It is central in the physiological regulation of

the size and absorptive capacity of the small intestine and

is secreted in response to intake of nutrients. In rodents,

GLP-2 has been shown to significantly increase small-

bowel weight, probably through an increase in crypt cell

proliferation and/or an inhibition of the apoptotic activity

resulting in lengthening of the intestinal villi and expansion

of the crypt compartment [22–25]. GLP-2 also increases

the nutrient absorption through increased production of

brush border enzymes [26]. In the colon, GLP-2 has been

shown to possess intestinotrophic capabilities in mice [27]

and pigs [28, 29].

The GLP-2 receptor (GLP-2R) has been localized to

subepithelial myofibroblasts, enteric neurons, and ente-

roendocrine cells [26], and the intestinotrophic effect of

GLP-2 is probably mediated by the release of one or more

local growth factors following receptor stimulation. Studies

have suggested keratinocyte growth factor (KGF) to be

involved in the colon [30], and insulin-like growth factor I

in both the small intestines and the colon [31]. In an earlier

study, the intestinotrophic effects of GLP-2 was not

impaired by the EGF TKI inhibitor, iressa. However, a

recent extensive study has reported that the pan-ErbB

inhibitor CI-1033 does inhibit the actions of GLP-2 and

that ligands to the EGF receptors are upregulated by GLP-2

[32]. Thus, the interaction with the EGF system by means

of tyrosinkinase inhibition does not seem to interfere with

the GLP-2 effects whereas these are blocked by complete

inhibition of the ErbB signaling network. The reason for

this difference remains to be elucidated. The GLP-2R has

not been localized in other organ systems and the growth

stimulating effect is confined to the intestinal system. This

lack of receptors in other tissues minimizes the risks of

inducing further neoplastic growth or other side-effects of

GLP-2 treatment.

The clinical implications of treatment with GLP-2/GLP-2

analogues are still under investigation, but clinical trials have

shown that teduglutide, a dipeptidyl peptidase IV-resistant

GLP-2 analogue, can improve the intestinal function of

patients with short bowel syndrome [33]. Other possible

future clinical applications of GLP-2 are IBD, intestinal

insufficiency caused by prolonged parenteral nutrition, and

chemotherapy-induced enteritis [34].

In the present study we examined the changes in mor-

phology and size as well as in the proliferatory and apop-

totic activity of the gastrointestinal organs in mice

following treatment with increasing doses of erlotinib

given alone or in combination with the cytostatic, cisplatin.

Furthermore, we investigated whether any possible intes-

tinal damage or atrophy might be counteracted by simul-

taneous administration of GLP-2.

Materials and Methods

Animals

Female NMRI mice weighing *30 g were housed in

plastic-bottomed wire-lidded cages. During the experi-

ments, the mice were provided with water and chow (no

1314, Altromin) ad libitum and kept in animal facilities

with temperature (21�C) and humidity (55%)-controlled

rooms with a light–dark cycle of 12 h each. Cages, bed-

ding, and water bottles were autoclaved before use and

changed every fifth day. All animals were allowed to

acclimatize for at least 1 week before the experiments were

started. The animal studies were approved by the Danish

Ministry of Justice, Animal Experiments Inspectorate.

Test Agents and Administration

A long-acting dipeptidylpeptidase IV (DPP-IV)-resistant

GLP-2 derivative (NNC 103-0066, Novo Nordisk A/S) was

dissolved in PBS to a stock solution of 1 mg/ml. A volume

2786 Dig Dis Sci (2010) 55:2785–2796

123

of 100 ll containing 25 lg GLP-2 was administrated as

s.c. injections twice daily every 12 h for 10 days. The

EGFR inhibitor erlotinib (Tarceva), kindly donated from

OSI Pharmaceuticals, was produced as a fine suspension

with methylcellulose (0.5% v/w) in demineralized water

and 0.5 ml of the various doses were prewarmed to 37�C

and administrated to the mice i.p. once daily for 10 days.

Cisplatin (Mayne Pharma) was provided in sterile saline

solution of 1 mg/ml and was diluted further with sterile

saline to 0.12 mg/ml. The dose of 2 mg/kg (50% of max-

imal tolerable dose—unpublished data) was administered

i.p. as 0.5 ml of the solution once a day for 10 days.

Experimental Design

Animals were weighed and randomly allocated to the fol-

lowing experimental groups of ten animals.

I. The dose-related effects of erlotinib on the gastroin-

testinal tract. The following groups were included: (1)

methyl-cellulose (placebo), (2) erlotinib 25 mg/kg, (3)

erlotinib 50 mg/kg, (4) erlotinib 100 mg/kg, and (5) erl-

otinib 200 mg/kg.

II. The ability of GLP-2 to counteract intestinal atrophy

induced by erlotinib and the effect on the mitotic and

apoptotic activity. (1) methylcellulose, (2) erlotinib

(100 mg/kg), (3) erlotinib (100 mg/kg) ? GLP-2 (25 lg).

III. The ability of GLP-2 to counteract intestinal atrophy

induced by erlotinib and cisplatin in combination. (1)

methylcellulose, (2) cisplatin (2 mg/kg), (3) GLP-2

(25 lg), (4) erlotinib (100 mg/kg) ? cisplatin (2 mg/kg),

(5) erlotinib (100 mg/kg) ? cisplatin (2 mg/kg) ? GLP-2

(25 lg). The dose of 100 mg/kg erlotinib and 2 mg/kg

cisplatin was in preliminary trials established to be the

maximally tolerable dose (MTD) (unpublished data).

All animals were killed after 10 days of treatment. They

were weighed, and the weight of the stomach and weight

and length of the small and large intestines were recorded

after removal of mesenteric fat and contents of the gut.

When measuring length, the intestinal segments were

vertically suspended with a 1.5-g weight to provide uni-

form tension [24]. In trial I, we observed that mice given

erlotinib had a large amount of gastric contents, which

added considerably to their total body weight. Therefore

the contents of the stomach were weighed in all animals in

trials II and III and subtracted from the bodyweight. In trial

II, all mice were injected i.p. with 50 mg/kg BrdU 2� h

before killing. The observers were blinded in each trial.

Histological and Morphometric Analysis

Tissue samples from the colon and the proximal, middle,

and distal part of the small intestine were fixed by

immersion in ice-cold, freshly prepared buffered 4%

paraformaldehyde. The samples were transferred to alcohol

(70%) within 12 h. The fixed tissue samples were then

dehydrated, embedded in paraffin, and cut perpendicularly

to the axis of their length into 10-lm sections. The sections

were stained with PAS-hematoxylin and examined using a

Leitz Ortoplan microscope fitted with a camera, Evolution

MP (MediaCypernetics). Morphometric analyses were

done by means of Image-Pro Plus 5.0 software.

Measurements included the cross-sectional area of the

mucosa and tunica muscularis of the small and large

intestines as well as the PAS-positive (mucus containing)

area (indicated as PAS-positive cross-sectional area in

percent of total cross-sectional area). The villus height,

crypt depth, number of crypts per millimeter circumference

and the length of the circumference were also measured. In

trial II, the number of mitoses in the crypts was counted in a

transverse histological section from each part of the intes-

tine and divided by the number of crypts in that section.

Bromodeoxyuridine (BrDU) was applied as a marker of

proliferation, and the BrDU-positive cells were visualized

by means of monoclonal mouse anti-BrdU as described in

[35]. The ApopTag Peroxidase in situ Detection Kit (Mil-

lipore, Billerica, US) was used as described by the manu-

facturer to detect DNA strand breaks as biochemical

markers of apoptosis. The number of apoptoses per section

on villi and in the crypts was counted separately. The

examination and computer analysis of the histological

sections were carried out blinded.

Statistical Analyses

The results are shown as mean ± SEM. The groups were

compared by one-way ANOVA followed by Tukey’s post

hoc analysis. Probability values of P \ 0.05 were consid-

ered significant.

Results

Trial I. The Dose-Related Effects of Erlotinib on the

Gastrointestinal Tract

The results of trial I are shown in Table 1 as well as Figs. 1

and 2. Doses of 25 and 50 mg/kg erlotinib resulted in

slight, non-significant reductions in the intestinal weights

and morphometric parameters. Following 100 mg/kg, there

was a statistically significant intestinal growth inhibition,

which was most pronounced in the distal part of the small

intestine. The weight of the small intestine and the colon as

well as the villus height in both the middle and distal part

of the small intestine were significantly reduced compared

to placebo. The dose of 200 mg/kg erlotinib resulted in the

death of several animals on day 4–5 of the trial, and the rest

Dig Dis Sci (2010) 55:2785–2796 2787

123

of the animals were euthanized due to their bad general

condition.

Trial II. The Ability of GLP-2 to Counteract Intestinal

Atrophy Induced by Erlotinib and the Effect on the

Mitotic and Apoptotic Activity

Trial II results are shown in Table 2 and Figs. 1 and 2.

Compared to the placebo group, treatment with erlotinib,

100 mg/kg, caused reduction of the body weight to

81 ± 2% (P \ 0.001) and the weight of the small intestine

to 79 ± 5% (P \ 0.01). The weight of the colon was not

affected. Moreover, the weight of the stomach contents was

increased to 236 ± 34% (P \ 0.01). The addition of GLP-

2 completely counteracted the reduction in the weight of

the intestine, which in spite of erlotinib-treatment,

increased to 117 ± 5% (P \ 0.05) of placebo values. The

increased weight of the stomach contents were partly

counteracted, being reduced to 153 ± 30% of placebo,

non-significant compared to erlotinib as well as placebo.

The loss of body weight was not affected by the addition of

GLP-2.

Morphometric Analysis

The growth-inhibiting effect of erlotinib was most pro-

nounced distally in the small intestine where the villus

height was reduced 66 ± 4% of placebo (P \ 0.05) and the

crypt depth was reduced to 81 ± 4% of placebo (P \ 0.05).

The cross-sectional area of the mucosa was slightly but non-

significantly reduced in the small intestine and significantly

reduced in the colon to 80 ± 7% of placebo (P \ 0.05). In

the colon, the area stained for mucin with PAS was reduced

significantly to 76 ± 5% (P [ 0.05).

GLP-2 completely counteracted the growth inhibition

induced by erlotinib and several parameters were even

above the levels of the placebo group. Thus, the cross-

sectional area of the mucosa was increased to 132 ± 9%

(P \ 0.05) and 138 ± 28% of placebo (P \ 0.05) in the

middle and distal part of the small intestine. Also in the

colon, the significant reduction of the cross-sectional area

of the mucosa was completely counteracted. In all three

small-intestinal segments, the villus height was increased

significantly compared to the erlotinib-treated group and

even compared to the placebo group, where there was

an increase to 121 ± 7% (P \ 0.05) and 144 ± 4%

(P \ 0.001) in the proximal and middle part of the small

intestine. The crypt depth was significantly increased in

the proximal part of the small intestine of the erlotinib ?

GLP-2- treated mice to 118 ± 4% (P \ 0.05) of placebo.

The addition of GLP-2 did not affect the area stained for

mucin with PAS which was 59 ± 10% (P \ 0.05) of pla-

cebo in the distal part of the small intestine and 69 ± 10%

of placebo (P \ 0.05) in the colon.

Table 1 Dose-related effects of erlotinib on the gastrointestinal tract

Gr.1 Methyl-

cellulose

Gr.2 Erlotinib

25 mg/kg

Gr.3 Erlotinib

50 mg/kg

Gr.4 Erlotinib

100 mg/kg

Body weight (g) 30.47 ± 0.07 29.82 ± 1.31 28.73 ± 0.77 29.23 ± 0.88

Small intestine

Length (cm) 60.25 ± 1.22 53.56 ± 1.20AA 58.67 ± 0.96b 63.17 ± 0.91BB,c

Weight (g) 1.15 ± 0.04 1.15 ± 0.06 1.11 ± 0.04 0.95 ± 0.06a,b

C.s.a. of mucosa (mm2)

Proximal part 3.02 ± 0.20 3.05 ± 0.17 3.01 ± 0.16 2.99 ± 0.24

Middle part 2.62 ± 0.14 2.49 ± 0.13 2.78 ± 0.37 2.22 ± 0.15

Distal part 1.70 ± 0.14 1.49 ± 0.18 1.76 ± 0.23 1.25 ± 0.14

Villus height (mm)

Proximal part 0.469 ± 0.031 0.483 ± 0.025 0.469 ± 0.025 0.425 ± 0.033

Middle part 0.280 ± 0.017 0.286 ± 0.015 0.229 ± 0.018 0.219 ± 0.016b

Distal part 0.215 ± 0.009 0.187 ± 0.010 0.177 ± 0.014 0.159 ± 0.010A

Colon

Length (cm) 11.43 ± 0.24 10.88 ± 0.39 12.32 ± 0.29b 10.46 ± 0.36C

Weight (g) 0.38 ± 0.01 0.42 ± 0.02 0.36 ± 0.01 0.31 ± 0.04b

Cross-sectional area of mucosa (mm2) 2.46 ± 0.10 2.42 ± 0.18 2.34 ± 0.17 2.27 ± 0.07

Cross-sectional area of muscularis (mm2), crypt depth, and mucin area are not included in the table, since no tendencies or significant differences

between the groups were observeda P \ 0.05 compared to gr. 1, b P \ 0.05 compared to gr. 2, c P \ 0.05 compared to gr. 3A P \ 0.01 compared to gr. 1, B P \ 0.01 compared to gr. 2, C P \ 0.01 compared to gr. 3AA P \ 0.001 compared to gr. 1, BB P \ 0.001 compared to gr. 2

2788 Dig Dis Sci (2010) 55:2785–2796

123

Apoptotic and Mitotic Activity

The results are apparent from Fig. 3. The number of

mitoses/crypt was not influenced in any part of the gut

neither by erlotinib alone nor the combination with GLP-2.

The number of apoptosis was, however, significantly

increased by erlotinib—most pronounced in the distal part

of the small intestine. The number counted in the crypts per

circumference was increased to 393 ± 120% (P \ 0.05)

and 318 ± 66% (P \ 0.001) of placebo in the middle and

distal part of the small intestine. The number of apoptosis

on the villi/circumference showed a similar but non-sig-

nificant pattern.

The addition of GLP-2 reduced the increase in apoptotic

activity induced by erlotinib. In the crypts the number of

apoptosis was reduced to 114 ± 40, 167 ± 34 and 117 ±

22% of placebo in the proximal, middle and distal part of

the small intestine, thus being almost comparable to pla-

cebo. The number of apoptosis on the villi was not sig-

nificantly influenced by the addition of GLP-2. These

observations indicate that an increased apoptotic activity in

the intestinal crypts is the fundamental cellular mechanism

behind the intestinal growth inhibition induced by the

EGFR-TKI erlotinib and that GLP-2 counteracts this

mechanism.

Trial III. The Ability of GLP-2 to Counteract Intestinal

Atrophy Induced by Erlotinib and Cisplatin

in Combination

The results are apparent from Table 3 as well as Figs. 1

and 2. The mice treated with a combination of erlotinib and

cisplatin had a significant loss of body weight to 65 ± 1%

of placebo (P \ 0.001). The weight of the small intestine

was reduced to 64.7 ± 3.5% and the colonic weight to

70 ± 3% (P \ 0.001) of placebo. The weight of stomach

contents was considerably increased to 478 ± 48% of

Fig. 1 Weight of the small intestine (a) and stomach contents (b) as

% of placebo in selected groups from trial I–III. * P \ 0.05 and

** P \ 0.001 compared to placebo. # P \ 0.001 compared to

100 mg/kg erlotinib. ¤ P \ 0.001 compared to erlotinib ? cisplatin

Fig. 2 Villus height in the

proximal, middle and distal part

of the small intestine as % of

placebo in selected groups from

trial I–III. * P \ 0.05 and

** P \ 0.001 compared to

placebo. # P \ 0.05 and## P \ 0.001 compared to

100 mg/kg erlotinib.¤ P \ 0.05

and ¤¤ P \ 0.001 compared to

erlotinib ? cisplatin

Dig Dis Sci (2010) 55:2785–2796 2789

123

placebo (P \ 0.001) or corresponding top 8% of the

bodyweight.

GLP-2 alone had the opposite effect. The weight of the

small intestine and the colon was increased to 144 ± 5%

(P \ 0.001) and 125 ± 6% (P \ 0.001) of placebo in

accordance with the intestinotrophic properties of GLP-2.

The total body weight was not influenced by GLP-2 alone.

When GLP-2 was given together with erlotinib and cis-

platin this resulted only in little and non-significant effects

on the weight parameters.

Morphometric Analysis

Cisplatin by itself had no influence on any of the mea-

sured parameters. However, when erlotinib and cisplatin

were given in combination, this resulted in changes far

more advanced than the ones resulting from erlotinib

alone, indicating a strong potentiating interaction of the

two drugs in combination The cross-sectional-area of the

mucosa was reduced to 70 ± 7, 62 ± 6, 61 ± 8% (all

P \ 0.05) and the villus height to 61 ± 2% (P \ 0.001),

Table 2 The ability of GLP-2 to counteract intestinal atrophy induced by erlotinib

Gr.1 Methyl-cellulose Gr.2 Erlotinib

100 mg/kg

Gr.3 Erlotinib

100 mg/kg ? GLP-2

Body weight (g) 29.37 ± 0.60 23.73 ± 0.71AA 24.34 ± 0.49AA

Stomach

Weight (g) 0.22 ± 0.01 0.27 ± 0.02 0.26 ± 0.02

Contents of stomach (g) 0.60 ± 0.06 1.42 ± 0.20A 1.02 ± 0.17

Small intestine

Length (cm) 49.33 ± 1.17 49.14 ± 0.89 49.04 ± 0.70

Weight (g) 1.39 ± 0.06 1.09 ± 0.07A 1.62 ± 0.07a,BB

Cross-sectional area of mucosa (mm2)

Proximal part 3.37 ± 0.21 3.36 ± 0.32 3.94 ± 0.22

Middle part 3.18 ± 0.29 2.66 ± 0.15 4.19 ± 0.30a,BB

Distal part 1.77 ± 0.09 1.37 ± 0.13 2.45 ± 0.53b

Villus height (mm)

Proximal part 0.570 ± 0.016 0.461 ± 0.040 0.691 ± 0.041a,BB

Middle part 0.360 ± 0.019 0.304 ± 0.018 0.517 ± 0.015AA,BB

Distal part 0.266 ± 0.011 0.189 ± 0.012a 0.293 ± 0.036B

Crypt depth (mm)

Proximal part 0.091 ± 0.004 0.093 ± 0.004 0.108 ± 0.004a

Middle part 0.097 ± 0.003 0.102 ± 0.004 0.110 ± 0.005

Distal part 0.109 ± 0.005 0.089 ± 0.004a 0.092 ± 0.005

Mucin area (% of cross-sectional area)

Proximal part 2.13 ± 0.33 1.38 ± 0.40 1.64 ± 0.29

Middle part 2.56 ± 0.3 1.57 ± 0.26 1.73 ± 0.33

Distal part 3.22 ± 0.40 2.67 ± 0.34 1.89 ± 0.33a

Colon

Lenght (cm) 9.92 ± 0.67 10.04 ± 0.50 11.25 ± 0.53

Weight (g) 0.30 ± 0.01 0.30 ± 0.01 0.34 ± 0.01

Cross-sectional area of mucosa (mm2) 2.48 ± 0.06 1.98 ± 0.18a 2.37 ± 0.12

Crypt depth (mm) 0.228 ± 0.016 0.191 ± 0.019 0.212 ± 0.015

Mucin area (% of cross-sectional area) 34.83 ± 3.20 24.92 ± 1.64a 24.39 ± 3.00a

Mitoses/crypt (nr.) 4.4 ± 0.6 5.3 ± 1.3 5.2 ± 1.4

Apoptoses/circumference (nr.) 2.6 ± 0.7 5.5 ± 1.6 5.9 ± 1.9

Cross-sectional area of muscularis, crypts/mm circumference, and the circumference are not included in the table, since no significant differences

between the groups were observeda P \ 0.05 compared to gr. 1, b P \ 0.05 compared to gr. 2A P \ 0.01 compared to gr. 1, B P \ 0.01 compared to gr. 2AA P \ 0.001 compared to gr. 1, BB P \ 0.001 compared to gr. 2

2790 Dig Dis Sci (2010) 55:2785–2796

123

73 ± 13% (n.s.) and 60 ± 5% (P \ 0.001) of placebo in

the proximal, middle, and distal part of the small intestine,

respectively.

The effect of GLP-2 administration per se was most

pronounced proximally in the small intestine. The cross-

sectional area of the mucosa was increased to 166 ± 9%

(P \ 0.001) and 140 ± 6% (P \ 0.05) and the villus

height to 139 ± 5% (P \ 0.001) and 162 ± 5%

(P \ 0.001) of placebo in the proximal and middle part of

the small intestine. The crypt depth did not change sig-

nificantly in any of the segments.

When GLP-2 treatment was added to the combination of

erlotinib and cisplatin, the pronounced growth inhibition

induced by this treatment was almost abolished. The

measurements were non-significant compared to the

methylcellulose-treated mice. Importantly, the villus height

increased significantly in all parts of the small intestine

(P \ 0.01 or 0.001) compared to the erlotinib ? cisplatin-

treated mice and was comparable to placebo. Thus GLP-2

was able to counteract the pronounced reduction of the

absorptive mucosal surface area of the gut induced by the

combination of cytostatics and EGFR-TKI.

Fig. 3 Apoptotic and Mitotic

activity. The number of

apoptoses in the crypts a and on

the villi b per circumference and

the number of mitoses per crypt

c in the proximal, middle and

distal part of the small intestine.

* P \ 0.05 and ** P \ 0.001

compared to placebo.# P \ 0.05 compared to

100 mg/kg erlotinib

Dig Dis Sci (2010) 55:2785–2796 2791

123

Finally, we observed a significantly (P \ 0.01 or 0.001)

increased area of mucus stained by PAS in the erloti-

nib ? cisplatin-treated mice (338, 244 and 239% of pla-

cebo) in the proximal, middle, and distal part of the small

intestine. The addition of GLP-2 had no significant effect

on this parameter.

Discussion

Erlotinib is an EGFR-TKI—a new group of targeted cancer

therapeutics. It has been approved for treatment in two

regimes and several new trials are ongoing. Among the

major side-effects are diarrhea and other gastrointestinal

symptoms.

The EGF-receptor is supposed to play a role in the

regulation of growth and differentiation as well as healing

in the gastrointestinal tract [8–10]. Therefore it is a likely

hypothesis that erlotinib causes intestinal growth inhibition

and that this might be part of the mechanism that causes the

gastrointestinal adverse effects of erlotinib. We investi-

gated this hypothesis in mice, and a growth inhibitory

effect was confirmed by our results in trial II. The maximal

tolerable dose of erlotinib resulted in significant reductions

Table 3 The ability of GLP-2 to counteract intestinal atrophy induced by erlotinib and cisplatin

Gr.1 Methyl-cellulose Gr.2 Cisplatin Gr.3 GLP-2 Gr.4

Erlotinib ? Cisplatin

Gr.5 Erlotinib

? Cisplatin ? GLP-2

Body weight (g) 28.60 ± 0.62 29.14 ± 0.98 29.43 ± 0.54 18.55 ± 0.43AA,BB,CC 20.47 ± 0.78AA,BB,CC

Stomach

Weight (g) 0.24 ± 0.02 0.26 ± 0.01 0.26 ± 0.02 0.25 ± 0.01 0.22 ± 0.01

Contents of stomach (g) 0.29 ± 0.06 0.52 ± 0.03 0.33 ± 0.04 1.39 ± 0.14AA,BB,CC 1.24 ± 0.09AA,BB,CC

Small intestine

Length (cm) 49.77 ± 0.59 50.52 ± 1.25 52.63 ± 0.38 49.15 ± 0.81c 48.94 ± 0.90c

Weight (g) 1.31 ± 0.06 1.46 ± 0.06 1.89 ± 0.07AA,BB 0.84 ± 0.04AA, BB,CC 0.97 ± 0.06A,BB,CC

C.s.a. of mucosa (mm2)

Proximal part 2.45 ± 0.07 2.81 ± 0.16 4.08 ± 0.22AA,BB 1.72 ± 0.18a,BB,CC 2.29 ± 0.24CC

Middle part 1.99 ± 0.20 2.33 ± 0.20 2.79 ± 0.11a 1.24 ± 0.12a,BB,CC 1.97 ± 0.15c,d

Distal part 1.30 ± 0.13 1.63 ± 0.07 1.59 ± 0.14 0.80 ± 0.1010a,BB,CC 1.18 ± 0.06b

Villus height (mm)

Proximal part 0.456 ± 0.022 0.454 ± 0.021 0.632 ± 0.021AA,BB 0.279 ± 0.011AA,BB,CC 0.401 ± 0.035CC,D

Middle part 0.281 ± 0.018 0.293 ± 0.017 0.452 ± 0.013AA,BB 0.206 ± 0.036CC 0.378 ± 0.021a,DD

Distal part 0.213 ± 0.017 0.256 ± 0.015 0.259 ± 0.016 0.130 ± 0.011A,BB,CC 0.217 ± 0.019D

Crypt depth (mm)

Proximal part 0.089 ± 0.004 0.097 ± 0.005 0.088 ± 0.005 0.097 ± 0.009 0.101 ± 0.005

Middle part 0.100 ± 0.007 0.106 ± 0.007 0.107 ± 0.005 0.080 ± 0.004b,c 0.105 ± 0.005

Distal part 0.096 ± 0.007 0.108 ± 0.006 0.104 ± 0.006 0.077 ± 0.003b 0.095 ± 0.004

Mucin area (% of c.s.a.)

Proximal part 0.49 ± 0.15 0.55 ± 0.09 0.42 ± 0.05 1.66 ± 0.24AA,B,CC 1.59 ± 0.40A,B,C

Middle part 0.86 ± 0.13 0.65 ± 0.14 0.65 ± 0.08 2.10 ± 0.41A,BB,CC 2.46 ± 0.26AA,BB,CC

Distal part 1.34 ± 0.24 1.21 ± 0.16 1.03 ± 0.19 3.26 ± 0.47A,BB,CC 2.87 ± 0.57a,b,C

Colon

Length (cm) 9.9 ± 0.41 9.02 ± 0.13 10.73 ± 0.32BB 9.15 ± 0.25CC 9.56 ± 0.24

Weight (g) 0.30 ± 0.01 0.31 ± 0.01 0.37 ± 0.02AA,B 0.21 ± 0.01AA,BB,CC 0.24 ± 0.01A,BB,C

C.s.a. of mucosa (mm2) 1.52 ± 0.08 1.51 ± 0.09 1.69 ± 0.09 1.29 ± 0.10 1.52 ± 0.18

Crypt depth (mm) 0.18 ± 0.01 0.19 ± 0.01 0.21 ± 0.01 0.19 ± 0.02 0.22 ± 0.01

Mucin area (% of c.s.a.) 16.87 ± 2.28 16.03 ± 1.57 19.50 ± 2.43 13.50 ± 2.31 15.56 ± 2.79

Cross-sectional area of muscularis, crypts/mm circumference and the circumference are not included in the table, since no significant differences

between the groups were observed

C.s.a cross-sectional areaa P \ 0.05 compared to gr. 1, b P \ 0.05 compared to gr. 2, c P \ 0.05 compared to gr. 3, d P \ 0.05 compared to gr. 4A P \ 0.01 compared to gr. 1, B P \ 0.01 compared to gr. 2, C P \ 0.01 compared to gr. 3, D P \ 0.01 compared to gr. 4AA P \ 0.001 compared to gr. 1, BB P \ 0.001 compared to gr. 2, CC P \ 0.001 compared to gr. 3, DD P \ 0.05 compared to gr. 4

2792 Dig Dis Sci (2010) 55:2785–2796

123

of the weight of both the small intestine and the colon and a

decreased absorptive surface area due to reductions of the

cross-sectional area of the mucosa, the villus height, and

crypt depth. These effects were most pronounced in the

middle and distal part of the small intestine as well as in the

colon.

No studies have so far been undertaken regarding the

effects of erlotinib on the gastrointestinal tract in animal

models. We have previously investigated the effect on the

GI tract of another EGFR-TKI, gefitinib, and found minor

but significant reductions in the small intestinal and colonic

weight and morphometric parameters (only in the proximal

small intestine) [36]. Only on dosage of gefitinib was

investigated and probably this dose was suboptimal.

In the present study we have made a dose-response trial

to define the maximal tolerable dose of the EGFR-TKI, and

found that this dose resulted in growth inhibition in all

parts of the intestinal system, most pronounced in the

middle and distal part of the small intestine.

It is likely that erlotinib in the future will be adminis-

tered in combination with other antineoplastic drugs and

this might potentiate the intestinal growth inhibition asso-

ciated with erlotinib. In the present study, we found that the

combination of erlotinib and cisplatin resulted in severely

aggravated intestinal atrophy in all investigated parameters

and in all parts of the gut. Since no growth-related changes

were observed in the group receiving cisplatin alone, it

must be concluded that erlotinib and cisplatin synergisti-

cally aggravate the intestinal atrophy. Cisplatin induces

apoptotic cell death through cross-linking of DNA. It is

thus possible that the synergy observed can be explained by

an increased vulnerability of the intestinal mucosal cells,

secondary to the erlotinib induced blockade of the apop-

totic modulating properties of the EGFR system. A similar

synergy could be expected when erlotinib is combined with

other cytostatics in future anti-neoplastic regimes.

The reduction of villus height and cross-sectional area of

the mucosa in the mice receiving erlotinib causes a

decrease in the total surface area of the small intestine. This

might impair the absorption of electrolytes and water and

might be instrumental in inducing the diarrhea observed in

patients receiving erlotinib. It is likely that the reductions

of the morphological parameters are also reflected on the

cellular level. The EGFR pathway has been shown to

regulate the electrolyte and nutrient transport in the en-

terocyte in vivo [15] and stimulate expression of brush

border enzymes [16]. Inhibition of these functions could

also contribute to reduced absorptive capacity and possible

osmotic diarrhea [37].

We observed a threefold, significant increase of the

amount of mucin localized to goblet cells in the small

intestine when the maximal dose of erlotinib and cisplatin

was given in combination. No effect was observed in the

colon. On the contrary, erlotinib alone induced a reduction

of the area of PAS stained mucus, especially in the colon,

whereas cisplatin only had little and non-significant influ-

ence on that parameter. This might reflect that erlotinib and

cisplatin in combination induces pathobiological changes

that are different from the changes elicited by each drug

alone. GLP-2 only had a small effect on this parameter.

We hypothesized that GLP-2 might be able to counteract

the intestinal atrophy induced by erlotinib alone and in

combination with cisplatin. GLP-2 is known to have highly

specific intestinotrophic effects in rodents [22–26] and has

been used with success in the treatment of patients with

short bowel syndrome in increasing the absorptive capacity

and lean body mass [38].

GLP-2 administered twice daily for 10 days resulted in a

significant increase in the weight of the small intestine and

the colon as well as morphometric parameters, being most

pronounced in the proximal part of the small intestine.

When GLP-2 was administered in the same dose together

with erlotinib it significantly and almost completely coun-

teracted the growth-inhibitory effects of erlotinib associ-

ated with morphometric parameters and small intestinal

weight. The body weight was not restored to normal by

GLP-2, but this probably reflects that erlotinib exerts its

effects in many organs throughout the body, in accordance

with the widespread distribution of the EGF receptor,

whereas the effect of GLP-2 is restricted to the gastroin-

testinal tract.

When GLP-2 was administered to mice given the

combination therapy, erlotinib and cisplatin, we found that

GLP-2 was able to completely counteract the reductions in

the absorptive mucosal capacity associated with the very

pronounced changes in morphometric parameters caused

by this treatment. Also in this group there was only a minor

effect of GLP-2 treatment on the body weight and the

intestinal weights, reflecting that GLP-2 primarily has its

receptors and exerts its effects in the mucosa of the

intestine.

Both erlotinib alone and the combination with cisplatin

caused a considerable increase in the weight of the stomach

contents, up to 8% of the total body weight. The small

intestine was observed to be almost empty. We have found

no previously published observations of this effect of a

tyrosine kinase inhibitor. A possible hypothesis could be

that erlotinib induces gastric retention. This might be part

of the cause of the nausea and anorexia that reduce food

intake and absorption of nutrients in patients treated with

erlotinib. We found that GLP-2 to some degree reduced the

increase in the weight of the stomach contents, especially

following erlotinib alone. This is in contrast to previous

findings in healthy animals and humans of delayed gastric

emptying following GLP-2 administration where this effect

of GLP-2 was thought to be part of the normal

Dig Dis Sci (2010) 55:2785–2796 2793

123

physiological regulation of digestion [39–41]. These find-

ings require further studies into the effects of erlotinib and

GLP-2 on gastric emptying.

It has been shown in vivo that EGF infusion increases

the mitotic activity in the intestinal crypts in rats [11, 12].

No former study has investigated the effect of antagonizing

the EGFR system on the apoptotic or mitotic activity in the

gastrointestinal tract. We observed that erlotinib had no

influence on the mitotic activity, but on the contrary on the

number of apoptosis, which was increased 3 to 4-fold in the

crypts of the middle and distal part of the small intestine.

Thus it seems that the intestinal atrophy caused by erlotinib

is mediated by means of an up-regulation of the apoptotic

activity rather than a down-regulation of the proliferative

activity.

When GLP-2 was given in combination with erlotinib,

the apoptotic activity in the crypts of the mucosa in the

middle and distal part of the small intestine was almost

completely normalized to the level of the placebo-treated

mice. The effects on the villi were less consistent and

non-significant. The finding that GLP-2 counteracts the

increased apoptotic activity in the intestinal crypts induced

by erlotinib is in agreement with earlier reports describing

an anti-apoptotic effect of GLP-2. Reduced apoptotic

activity has been reported both in normal mice [25] and in

mouse models of chemotherapy or indomethacin induced

enteritis [42, 43].

There has been conflicting results on the effects of

GLP-2 on the intestinal proliferative activity. In the

present study we found no effect of neither erlotinib nor

the addition of GLP-2 on the mitotic activity. In some

studies, GLP-2 was found in healthy mice to induce

increased proliferation in the small-intestinal crypts [22,

25, 31]. As in our study, others have found that GLP-2

exclusively affects the apoptotic activity and does not

increase mitotic activity [44]. The discrepancies might be

related to differences in the methods applied for detection

of mitosis. In our study and in the other study with

negative effect on the mitotic activity, BrDU was used as

a marker of proliferation. The studies finding increased

mitotic activity use Proliferative Cell Nuclear Antigen

(PCNA) [22] or Ki-67 [31], except for a recent study

using BrDU [32]. Furthermore, we studied the effects of

GLP-2 on the mitotic rate in animals that were treated

with erlotinib, whereas the other studies focused on the

effect of GLP-2 alone.

Since GLP-2 affects the apoptotic activity and induces

growth, it must be considered whether GLP-2 could act as a

promoter for tumors. It must be emphasized, however, that

the GLP-2-receptor is only localized to the gastrointestinal

tract and has never been found to be expressed by tumors

or cell lines outside the gastrointestinal tract. The intestinal

growth caused by exogenous GLP-2 regresses to normal

after cessation of treatment [25], and GLP-2 alone has

never been found to initiate tumor growth. Moreover,

chemotherapy-induced diarrhea (CID) would generally

only require short-term treatment. Since the GLP-2

receptor is localized also to the colon, GLP-2 might

accelerate the growth of colonic tumors and it should

therefore not be used in cases where chemotherapy is given

as a treatment of gastrointestinal cancers. The potential risk

of growth of gastrointestinal tumors has not been finally

clarified. We have previously shown that GLP-2 is able to

accelerate the growth of pre-existing chemically induced

colonic neoplasms in mice [45], but a recent study of APC

knockout-mice treated with GLP-2 did not confirm that risk

[46].

In conclusion, we have demonstrated that treatment with

the maximum tolerable dosage of the EGFR TKI, erlotinib,

induced growth inhibition throughout the intestinal tract

primarily by increasing the apoptotic activity. This growth

inhibition is highly potentiated when the EGFR TKI is

combined with a cytostatic, which might become a com-

mon clinical situation. These inhibitory effects can be

completely abolished when GLP-2 is added to the

treatments.

The side-effects associated with cancer therapy with

EGFR TKI and cytostatics are another of the clinical

entities that await further investigations and trials with

respect to the possible beneficial effects of treatment with

GLP-2 or GLP-2 analogues.

Acknowledgments Andreas Rosen Rasmussen is the recipient of a

scholarship from the Danish Agency for Science, Technology and

Innovation. The technical assistance of Heidi Paulsen and Jette

Schousboe is highly appreciated.

References

1. Woodburn JR. The epidermal growth factor receptor and its

inhibition in cancer therapy. Pharmacol Ther. 1999;82(2–3):

241–250.

2. Normanno N, De Luca A, Bianco C, et al. Epidermal growth

factor receptor (EGFR) signaling in cancer. Gene. 2006;366(1):

2–16.

3. Siegel-Lakhai WS, Beijnen JH, Schellens JH. Current knowledge

and future directions of the selective epidermal growth factor

receptor inhibitors erlotinib (Tarceva) and gefitinib (Iressa).

Oncologist. 2005;10(8):579–589.

4. Brabender J, Danenberg KD, Metzger R, et al. Epidermal growth

factor receptor and HER2-neu mRNA expression in non-small

cell lung cancer Is correlated with survival. Clin Cancer Res.

2001;7(7):1850–1855.

5. Cohen MH, Johnson JR, Chen YF, Sridhara R, Pazdur R. FDA

drug approval summary: erlotinib (Tarceva) tablets. Oncologist.2005;10(7):461–466.

6. European Agency for the Evaluation of Medicinal Products

(EMEA). Tarceva� [European product information]. Available at

http://www.emea.europa.eu/humandocs/PDFs/EPAR/tarceva/H-

618-PI-en.pdf. Accessed March 4, 2008.

2794 Dig Dis Sci (2010) 55:2785–2796

123

Ref Type: Generic

7. Bareschino MA, Schettino C, Troiani T, Martinelli E, Morgillo F,

Ciardiello F. Erlotinib in cancer treatment. Ann Oncol. 2007;18

(Suppl 6):vi35–vi41.

8. Barnard JA, Beauchamp RD, Russell WE, Dubois RN, Coffey RJ.

Epidermal growth factor-related peptides and their relevance to

gastrointestinal pathophysiology. Gastroenterology. 1995;108(2):

564–580.

9. Dignass AU, Sturm A. Peptide growth factors in the intestine.

Eur J Gastroenterol Hepatol. 2001;13(7):763–770.

10. Playford RJ. Peptides and gastrointestinal mucosal integrity. Gut.1995;37(5):595–597.

11. Berlanga-Acosta J, Playford RJ, Mandir N, Goodlad RA. Gas-

trointestinal cell proliferation and crypt fission are separate but

complementary means of increasing tissue mass following infu-

sion of epidermal growth factor in rats. Gut. 2001;48(6):803–807.

12. Kitchen PA, Goodlad RA, FitzGerald AJ, et al. Intestinal growth

in parenterally-fed rats induced by the combined effects of glu-

cagon-like peptide 2 and epidermal growth factor. JPEN J Par-enter Enteral Nutr. 2005;29(4):248–254.

13. Kurokowa M, Lynch K, Podolsky DK. Effects of growth factors

on an intestinal epithelial cell line: transforming growth factor

beta inhibits proliferation and stimulates differentiation. BiochemBiophys Res Commun. 1987;142(3):775–782.

14. Matsuda K, Sakamoto C, Konda Y, et al. Effects of growth

factors and gut hormones on proliferation of primary cultured

gastric mucous cells of guinea pig. J Gastroenterol. 1996;31(4):

498–504.

15. Opleta-Madsen K, Hardin J, Gall DG. Epidermal growth factor

upregulates intestinal electrolyte and nutrient transport. Am JPhysiol. 1991;260(Pt 1 6):G807–G814.

16. Goodlad RA, Raja KB, Peters TJ, Wright NA. Effects of uro-

gastrone-epidermal growth factor on intestinal brush border

enzymes and mitotic activity. Gut. 1991;32(9):994–998.

17. Skov OP. Role of epidermal growth factor in gastroduodenal

mucosal protection. J Clin Gastroenterol. 1988;10(Suppl 1):

S146–S151.

18. Itoh M, Imai S, Joh T, et al. Protection of gastric mucosa against

ethanol-induced injury by intragastric bolus administration of

epidermal growth factor combined with hydroxypropylcellulose.

J Clin Gastroenterol. 1992;14(Suppl 1):S127–S130.

19. Dignass AU, Podolsky DK. Cytokine modulation of intestinal

epithelial cell restitution: central role of transforming growth

factor beta. Gastroenterology. 1993;105(5):1323–1332.

20. Itoh M, Matsuo Y. Gastric ulcer treatment with intravenous

human epidermal growth factor: a double-blind controlled clini-

cal study. J Gastroenterol Hepatol. 1994;9(Suppl 1):S78–S83.

21. Messersmith WA, Laheru DA, Senzer NN, et al. Phase I trial of

irinotecan, infusional 5-fluorouracil, and leucovorin (FOLFIRI)

with erlotinib (OSI-774): early termination due to increased

toxicities. Clin Cancer Res. 2004;10(19):6522–6527.

22. Drucker DJ, Erlich P, Asa SL, Brubaker PL. Induction of intes-

tinal epithelial proliferation by glucagon-like peptide 2. Proc NatlAcad Sci U S A. 1996;93(15):7911–7916.

23. Drucker DJ, Shi Q, Crivici A, et al. Regulation of the biological

activity of glucagon-like peptide 2 in vivo by dipeptidyl peptidase

IV. Nat Biotechnol. 1997;15(7):673–677.

24. Hartmann B, Thulesen J, Kissow H, et al. Dipeptidyl peptidase IV

inhibition enhances the intestinotrophic effect of glucagon-like

peptide-2 in rats and mice. Endocrinology. 2000;141(11):4013–

4020.

25. Tsai CH, Hill M, Asa SL, Brubaker PL, Drucker DJ. Intestinal

growth-promoting properties of glucagon-like peptide-2 in mice.

Am J Physiol. 1997;273(1 Pt 1):E77–E84.

26. Dube PE, Brubaker PL. Frontiers in glucagon-like peptide-2:

multiple actions, multiple mediators. Am J Physiol EndocrinolMetab. 2007;293(2):E460–E465.

27. Dube PE, Forse CL, Bahrami J, Brubaker PL. The essential role

of insulin-like growth factor-1 in the intestinal tropic effects of

glucagon-like peptide-2 in mice. Gastroenterology. 2006;131(2):

589–605.

28. Drucker DJ, Yusta B, Boushey RP, DeForest L, Brubaker PL.Human [Gly2]GLP-2 reduces the severity of colonic injury in a

murine model of experimental colitis. Am J Physiol. 1999;276(1

Pt 1):G79–G91.

29. L’Heureux MC, Brubaker PL. Glucagon-like peptide-2 and

common therapeutics in a murine model of ulcerative colitis.

J Pharmacol Exp Ther. 2003;306(1):347–354.

30. Orskov C, Hartmann B, Poulsen SS, Thulesen J, Hare KJ, Holst

JJ. GLP-2 stimulates colonic growth via KGF, released by sub-

epithelial myofibroblasts with GLP-2 receptors. Regul Pept.2005;124(1–3):105–112.

31. Dube PE, Forse CL, Bahrami J, Brubaker PL. The essential role

of insulin-like growth factor-1 in the intestinal tropic effects of

glucagon-like peptide-2 in mice. Gastroenterology. 2006;131(2):

589–605.

32. Yusta B, Holland D, Koehler JA, et al. ErbB signaling is required

for the proliferative actions of GLP-2 in the murine gut. Gas-troenterology. 2009;137(3):986–996.

33. Jeppesen PB, Sanguinetti EL, Buchman A, et al. Teduglutide

(ALX-0600), a dipeptidyl peptidase IV resistant glucagon-like

peptide 2 analogue, improves intestinal function in short bowel

syndrome patients. Gut. 2005;54(9):1224–1231.

34. Wallis K, Walters JR, Forbes A. Review article: glucagon-like

peptide 2–current applications and future directions. AlimentPharmacol Ther. 2007;25(4):365–372.

35. Kjellev S, Lundsgaard D, Poulsen SS, Markholst H. Reconstitu-

tion of Scid mice with CD4?. Int Immunopharmacol. 2006;6(8):

1341–1354.

36. Hare KJ, Hartmann B, Kissow H, Holst JJ, Poulsen SS. The

intestinotrophic peptide, GLP-2, counteracts intestinal atrophy in

mice induced by the epidermal growth factor receptor inhibitor,

gefitinib. Clin Cancer Res. 2007;13(17):5170–5175.

37. Stringer AM, Gibson RJ, Bowen JM, Logan RM, Yeoh AS,

Keefe DM. Chemotherapy-induced mucositis: the role of gas-

trointestinal microflora and mucins in the luminal environment.

J Support Oncol. 2007;5(6):259–267.

38. Jeppesen PB, Hartmann B, Thulesen J, et al. Glucagon-like

peptide 2 improves nutrient absorption and nutritional status in

short-bowel patients with no colon. Gastroenterology. 2001;

120(4):806–815.

39. Wojdemann M, Wettergren A, Hartmann B, Holst JJ. Glucagon-

like peptide-2 inhibits centrally induced antral motility in pigs.

Scand J Gastroenterol. 1998;33(8):828–832.

40. Wojdemann M, Wettergren A, Hartmann B, Hilsted L, Holst JJ.

Inhibition of sham feeding-stimulated human gastric acid secre-

tion by glucagon-like peptide-2. J Clin Endocrinol Metab. 1999;

84(7):2513–2517.

41. Nagell CF (2004) Glucagon-like peptide-2 inhibits antral

emptying in man, but is not as potent as glucagon-like

peptide-1.

42. Boushey RP, Yusta B, Drucker DJ. Glucagon-like peptide 2

decreases mortality and reduces the severity of indomethacin-

induced murine enteritis. Am J Physiol. 1999;277(5 Pt 1):E937–

E947.

43. Boushey RP, Yusta B, Drucker DJ. Glucagon-like peptide (GLP)-

2 reduces chemotherapy-associated mortality and enhances cell

survival in cells expressing a transfected GLP-2 receptor. CancerRes. 2001;61(2):687–693.

Dig Dis Sci (2010) 55:2785–2796 2795

123

44. Tavakkolizadeh A, Shen R, Abraham P, et al. Glucagon-like

peptide 2: a new treatment for chemotherapy-induced enteritis.

J Surg Res. 2000;91(1):77–82.

45. Thulesen J, Hartmann B, Hare KJ, et al. Glucagon-like peptide 2

(GLP-2) accelerates the growth of colonic neoplasms in mice.

Gut. 2004;53(8):1145–1150.

46. Koehler JA, Harper W, Barnard M, Yusta B, Drucker DJ. Glu-

cagon-like peptide-2 does not modify the growth or survival of

murine or human intestinal tumor cells. Cancer Res. 2008;

68(19):7897–7904.

2796 Dig Dis Sci (2010) 55:2785–2796

123