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Acute Effects of the Glucagon-Like Peptide 2 Analogue, Teduglutide, on Intestinal Adaptation in Short Bowel Syndrome

Thymann, Thomas*; Stoll, Barbara; Mecklenburg, Lars; Burrin, Douglas G.; Vegge, Andreas*; Qvist, Niels§; Eriksen, Thomas||; Jeppesen, Palle B.; Sangild, Per T.*

Journal of Pediatric Gastroenterology and Nutrition: June 2014 - Volume 58 - Issue 6 - p 694–702
doi: 10.1097/MPG.0000000000000295
Original Articles: Hepatology and Nutrition
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ABSTRACT Neonatal short bowel syndrome following massive gut resection is associated with malabsorption of nutrients. The intestinotrophic factor glucagon-like peptide 2 (GLP-2) improves gut function in adult patients with short bowel syndrome, but its effect in pediatric patients remains unknown. Our objective was to test the efficacy of the long-acting synthetic human GLP-2 analogue, teduglutide (ALX-0600), in a neonatal piglet jejunostomy model. Two-day-old pigs were subjected to resection of 50% of the small intestine (distal part), and the remnant intestine was exteriorized on the abdominal wall as a jejunostomy. All pigs were given total parenteral nutrition for 7 days and a single daily injection of the following doses of teduglutide: 0.01 (n = 6), 0.02 (n = 6), 0.1 (n = 5), or 0.2 mg · kg−1 · day−1 (n = 6), and compared with placebo (n = 9). Body weight increment was similar for all 4 teduglutide groups but higher than placebo (P < 0.05). There was a dose-dependent increase in weight per length of the remnant intestine (P < 0.01) and fractional protein synthesis rate in the intestine was increased in the 0.2 mg · kg−1 · day−1 group versus placebo (P < 0.001); however, functional and structural endpoints including activity of digestive enzymes, absorption of enteral nutrients, and immunohistochemistry (Ki67, villin, FABP2, ChgA, and GLP-2R) were not affected by the treatment. Teduglutide induces trophicity on the remnant intestine but has limited acute effects on functional endpoints. Significant effects of teduglutide on gut function may require a longer adaptation period and/or a more frequent administration of the peptide. In perspective, GLP-2 or its analogues may be relevant to improve intestinal adaptation in pediatric patients with short bowel syndrome.

*Department of Nutrition, Exercise, and Sports, University of Copenhagen, Frederiksberg C, Denmark

Childrens Nutrition Research Center, Baylor College of Medicine, Houston, TX

Takeda GmbH, Konstanz, Germany

§Department of Surgery, Gastroenterology, Pediatrics and Endocrinology, Odense University Hospital, Odense C

||Department of Small Animal Clinical Sciences, University of Copenhagen, Frederiksberg C

Department of Gastroenterology, Rigshospitalet, Copenhagen, Denmark.

Address correspondence and reprint requests to Thomas Thymann, Unit of Clinical and Experimental Nutrition, Department of Nutrition, Exercise and Sports, University of Copenhagen, 30 Rolighedsvej, DK-1958 Frederiksberg C, Denmark (e-mail: ttn@life.ku.dk).

Received 29 December, 2013

Accepted 29 December, 2013

This study was funded by Takeda GmbH and the UNIK grant from University of Copenhagen.

T.T. and P.B.J. have received honoraria from Takeda GmbH for consultancy service. L.M. is employed by Takeda GmbH. The other authors report no conflicts of interest.

Short bowel syndrome (SBS) is common in neonates and children after excessive resection of the small bowel. Indications for resection include a variety of etiologies such as atresia, volvulus, and necrotizing enterocolitis (1). Following surgery, the concentration of the meal-stimulated intestinotrophic peptide, glucagon-like peptide 2 (GLP-2), in peripheral blood is decreased and this may contribute to the reduced digestive and absorptive function of the remnant intestine and the need for parenteral nutrition (PN) (2). Consequently, exogenous GLP-2 may stimulate intestinal growth, adaptation, and function in children with SBS who are dependent on PN for maintenance of fluid and nutrient balances; however, such a treatment is difficult as the native GLP-2 peptide has an extremely short half-life and therefore may require repeated daily injections (3). The long-acting synthetic human GLP-2 analogue, teduglutide (ALX-0600, [gly 2] – hGLP-2; Takeda GmbH, Konstanz, Germany), has an amino acid substitution at the N-terminal end that makes it resistant to degradation by dipeptidylpeptidase 4 (4). In adult patients with SBS, teduglutide increases nutrient balance and has recently been approved for clinical use (5). It, however, remains unknown whether neonatal patients with SBS display similar sensitivity to teduglutide treatment as adult patients with SBS.

Different rodent and pig models of SBS are available (6–12), but only few studies have tested the effects of GLP-2; in 1 study on juvenile SBS pigs, GLP-2 induced adverse effects (10). In this and other animal model studies, the remnant intestine was anastomosed to the colon or ileum following intestinal resection (6–12). Although this model is technically easier and improves fluid and nutrient balances, the placement of a jejunostomy, without a functioning colon, more accurately reflects the condition for most human patients just after resection. A functioning colon may allow increases in endogenous GLP-2 secretion because enteroendocrine L-cells are most abundant in the ileum and colon (10). This may, however, not always be the case, as shown in infants with SBS with a functioning colon (2). We have recently developed a piglet jejunostomy model and documented marked structural and functional gut responses to daily intravenous (IV) infusions of the native human GLP-2 peptide (13). On the basis of these observations, we hypothesized that teduglutide, administered once daily by subcutaneous injection, dose-dependently would stimulate remnant intestinal growth and function in pigs subjected to intestinal resection and establishment of a jejunostomy. The jejunostomy makes it possible to assess the possible effects of teduglutide on nutrient balances, together with effects on a series of structural and functional markers in the remnant intestine. The study was designed to investigate the adaptation of the remnant intestine following daily injections with teduglutide during the first week after resection of 50% of the small intestine.

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METHODS

Surgical Procedures

Two-day-old suckling female pigs (Landrace X Large White X Duroc) were separated from their mothers and equipped with a permanent jugular catheter (silastic tubing, ID 0.2 mm), which was passed subcutaneously and exteriorized on the dorsal part of the neck. An orogastric feeding tube was placed and exteriorized through the cheek. During these surgical procedures universal anesthesia and analgesia were induced and maintained with a combination of zolazepam/tiletamine (Zoletil 50; Virbac, Kolding, Denmark), xylacin (Narcoxyl 20 mg/mL; MSD Animal Health, Ballerup, Denmark), ketamine (Ketaminol 100 mg/mL; MSD Animal Health), and butorphanol (Torbugesic 10 mg/mL; ScanVet, Fredensborg, Denmark). Local infiltration analgesia in the skin was ensured with lidocaine 2%.

After recovery from anesthesia, the pigs were housed individually in heated cages with perforated floor, and fed through the orogastric feeding tube with a milk replacer at 15 mL · kg−1 · 3 h−1 for 6 to 9 hours (the macronutrient composition of milk replacer used in the study is outlined in Table 1). Then all pigs were switched to total parenteral nutrition (TPN) (Kabiven; Fresenius Kabi, Bad Homburg, Germany), which was infused into the jugular vein catheter at 4 mL · kg−1 · h−1 (∼70 kcal · kg−1 BW · day−1) for 3 to 6 h to ensure that the gastrointestinal tract was empty before abdominal surgery. Analgesia was then induced with an injection of butorphanol, and 15 minutes later, universal anesthesia was induced and maintained with isoflurane inhalation. Local infiltration analgesia was applied to the abdominal wall before incision. Following skin preparation and draping for aseptical surgery, a 5-cm celiotomy incision was made. The cecum and the plica iliocecalis were located, the ilium was transected close to the iliocecal junction, and ostium iliocecalis was ligated and closed using Monocryl 4.0 (Ethicon, Blue Ash, OH). Advancing in an oral direction, the distal small intestine was then separated from the mesentery with an electrocautery scalpel, and when an estimated 50% of the small intestinal length was excised (using the equation: total intestinal length (cm) = 300 × body weight [kg]0.65), the distal end of the remnant intestine was exteriorized through an incision in the dorsolateral part of the abdomen. An everted stoma was created on the skin surface, using 2–3 sutures (Monocryl 4.0). Finally, a silastic tube was inserted approximately 5 cm into the intestine through the stoma and fixed to the skin with suture and elastic tape with the distal part of the silastic tube left hanging between the hind legs to allow drainage. A tissue sample for later histology and enzyme analyses was collected from the proximal part of the resected intestine, corresponding to the middle part of the intact intestine (designated “mid-distal”).

TABLE 1

TABLE 1

A prophylactic treatment with antibiotics was initiated after jugular catherization using 0.125 mL · kg−1 · day−1 of gentamicin (Hexamycin; Sandoz, Odense, Denmark) and 0.175 mL · kg−1 · 12 h−1 of pentrexyl (Pentrexyl, Bristol-Myers Squibb, Bromma, Sweden) for 3 days. Postsurgical analgesia was ensured with daily IV infusions of 0.1 mL · kg−1 · day−1 of meloxicam (Metacam, Boehringer Ingelheim, Copenhagen, Denmark) for 3 days. All procedures were done in accordance with a protocol (2009/561-1731 C2) approved by the Danish Experimental Animal Inspectorate.

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Nutrition, Fluid Homeostasis, and Teduglutide Treatment

Immediately after gut resection, all pigs were given TPN at 4 mL · kg−1 · h−1 increasing to 6 mL · kg−1 · h−1 after 12 hours, further increasing to 8 mL · kg−1 · h−1 at 24 hours and onward. Body fluid homeostasis was maintained by continuous coinfusion of isotonic saline at 2 mL · kg−1 · h−1. Saline infusion was started 12 hours after gut resection and continued throughout the experiment, with the exception of a 24-hour enteral nutrition period wherein it was increased to 4 mL · kg−1 · h−1 (see detailed description later).

Following intestinal resection, the pigs were allocated into the following groups receiving placebo (n = 9) or increasing doses of GLP-2 (teduglutide, Takeda GmbH): 0.01 mg · kg−1 · day−1 (n = 6), 0.02 mg · kg−1 · day−1 (n = 6), 0.1 mg · kg−1 · day−1 (n = 5), or 0.2 mg · kg−1 · day−1 (n = 6). The pigs were allocated to these groups ensuring equal mean body weight among the groups. Dilutions of teduglutide and placebo were prepared daily, and samples were analyzed to verify accuracy and precision, relative to the target concentrations. Starting 1 to 3 hours after recovery from gut resection, the pigs were injected subcutaneously in the lateral part of the neck with either placebo or teduglutide. This was followed by subcutaneous injections every morning for 7 days.

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Blood Samples and Pharmacokinetics

To document endogenous levels of GLP-2, a baseline blood sample was taken after gut resection and before the first injection of teduglutide. The samples were collected in 2-mL syringes, immediately transferred to ethylenediaminetetraacetic acid–coated tubes, and centrifuged within 30 minutes (4°C, 1270g, 10 minutes), and plasma was isolated and stored at −80°C. To document the pharmacokinetic profile of teduglutide, a set of blood samples was collected following injection on day 5 after gut resection. Specific blood sampling time points were 0, 0.5, 1, 2, 8, and 24 h after injection. Plasma was isolated and analyzed using a validated liquid-chromatography-mass spectrometry method with a limit of detection of 1 μg/L. Noncompartmental pharmacokinetic analysis of the data was done using version 5.2 of the WinNonlin program (Pharsight, St Louis, MO). Maximum concentrations in plasma (Cmax) and time to maximum concentrations (tmax) were determined from the concentration-time profiles. The area under the curve (AUC) was calculated using a linear trapezoidal method up to the last sample point above the lower limit of quantification, and extrapolation to infinity was performed assuming a monoexponential decrease in the terminal phase of the PK profile. Finally, half-life (t½) was calculated from the terminal elimination coefficient.

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Nutrient Balance

Starting immediately after the last blood sample in the pharmacokinetic study, stoma bags (Wound Pouches, Eakin County Down, Northern Ireland) were glued to the skin around the stoma and a stocking was prepared from tube gaze to protect the stoma bag. A milk formula was fed enterally at 10 mL · kg−1 · 2 h −1 (∼130 kcal · kg−1 BW · day−1) for 24 hours through the orogastric tube, and intestinal output was collected quantitatively into the stoma bag and stored at −20°C until further analysis. The constituents of the milk formula were Seravit, 12 g/L and Calogen, 140 g/L (Nutricia Advanced Medical Nutrition, Wiltshire, UK); Polycose, 20 g/L (Abbott Nutrition, Lake Forest, IL); Variolac, 20 g/L; and Lacprodan alpha 10, 65 g/L (Arla Foods Ingredients, Viby, Denmark). At the end of the balance period the total volume of intestinal output from each pig was recorded and the stoma output was analyzed using bomb calorimetry (energy) and the Englyst method (carbohydrate), Kjeldahl method (protein), and Vander Kamer titration (fat), as previously described (14). Absolute intestinal absorption of fluid, energy, protein, carbohydrate, and fat was calculated as input from milk formula minus stoma output. The relative intestinal absorption was calculated as [(input − stoma output)/input] × 100 (Table 2).

TABLE 2

TABLE 2

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Intestinal Permeability, Protein Synthesis, and Tissue Collection

Exactly 3 hours before euthanasia, each pig was given an oral bolus (15 mL/kg) of a 2% mannitol, 2% lactulose solution (Sigma, St Louis, MO). A urine sample was collected at the time of euthanasia and stored at −80°C until further analyses of mannitol and lactulose concentration using spectrophotometric techniques.

To study tissue protein synthesis, the piglets from the placebo groups and the 0.2 mg · kg−1 · day−1 teduglutide group were given an IV injection of L-phenylalanine (1.5 mmol/kg) containing 0.15 mmol/kg of L-[ring-13C6]phenylalanine (Cambridge Isotopes, Woburn, MA). Exactly 30 minutes after isotope injection, the pigs were killed and tissue samples from the stomach, small intestine, and colon were collected and snap-frozen in liquid nitrogen and stored at −80°C until analysis for tracer enrichment, protein, and DNA concentration, as previously described (15). Accordingly, fractional protein synthesis rate (FSR), representing the amount of protein synthesized per day as a percentage of the total protein pool, was calculated from the amount of free L-[ring-13C6]phenylalanine relative to protein bound L-[ring-13C6]phenylalanine. Absolute synthesis rate (ASR), representing the total amount of synthesized protein in each organ (stomach, small intestine, colon) per day, was calculated as the fractional synthesis rate multiplied by the total protein content in each organ and divided by body weight.

Following the intestinal permeability and tissue protein synthesis protocols, the pigs were anaesthetized and subsequently euthanized with an intracardiac injection of sodium pentobarbitone. Organs were weighed and tissue was collected from longissimus dorsi, intestine, mesenteric lymph node, kidney, and liver. The length of the remnant intestine was measured, and tissue samples were collected from the proximal and distal parts (corresponding to proximal and middle of the intact intestine). From each organ 1 set of samples was snap-frozen in liquid nitrogen and stored at −80°C for later analysis. Another set was fixed in a cryoprotective embedding medium (Tissue-Tek OCT, Ted Pella, Redding, CA) and snap-frozen in liquid nitrogen, and a third set was fixed in a 4% paraformaldehyde solution. Finally, 10-cm sections were collected from each end of the remnant intestine (designated “proximal” and “mid-proximal”). They were weighed and slit longitudinally to expose the mucosa. The mucosa was scraped off with a plastic slide and weighed separately to calculate the proportion of mucosa.

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Villus Height, Crypt Depth, and Immunohistochemistry

Paraformaldehyde-fixed samples were dehydrated, infiltrated with paraffin, sliced, and stained with hematoxylin and eosin. Villus height and crypt depth were measured in samples from the proximal jejunum from all groups. Measurement was conducted using the analySIS docu software system (Olympus Soft Imaging Solutions GmbH, Münster, Germany). Briefly, 2 tissue samples per animal were photographed using a ×1.25 objective. The lengths of villi and crypts (6–25 villi/crypts per sample) were measured, and an average length per animal was calculated.

Immunohistochemistry was conducted using paraffin sections of proximal jejunum from selected animals from the placebo and the 0.2 mg · kg−1 · day−1 group. The following antibodies were used Ki-67 (DCS, KI68IC0I), villin (DCS, V1616R06), fatty acid binding protein 2 (FABP2) (Abcam, ab7805), Chromogranin A (Neomarker, RB-MS-324-PO), and GLP-2 receptor (GLP-2R) (LSBio, lS-A1312). The Ki-67 antigen was chosen as a cellular marker of proliferation as it is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but absent in resting cells (G0). Villin was investigated as a marker of mucosal architecture as it is associated with the actin core bundle of the brush border. FABP2 was investigated as an intestinal carrier protein for fatty acids and other lipophilic substances. Chromogranin A was chosen as it is a member of the granin family of neuroendocrine secretory proteins located in secretory vesicles of neurons and neuroendocrine cells, including enteroendocrine cells. Finally, the immunohistochemical location of the GLP-2R was investigated as it is the target for teduglutide.

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Digestive Enzyme Analysis

Activities of lactase (EC 3.2.1.23–62), sucrase (EC 3.2.1.48–10), maltase (EC 3.2.1.20), dipeptidyl-peptidase 4 (EC 3.4.14.5), aminopeptidase N (EC 3.4.11.2), and aminopeptidase A (EC 3.4.11.7) in tissue homogenates were determined spectrophotometrically using lactose, sucrose, maltose, glycyl-L-prolin-4-nitroanilide, L-alanine-4-nitroanilide, and α-L-glutamic acid 4-nitroanilide as substrates (16). Enzyme activities were expressed per gram of intestine and a hydrolytic rate of 1 μmol substrate released per minute at 37°C represented 1 U of enzyme activity.

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Statistical Analyses

Data collected over time were analyzed as repeated measures using a Gaussian model of spatial correlation in the MIXED procedure of SAS (version 9.1; SAS Institute, Cary, NC). All other statistical analyses of continuous data were based on regression analysis in the SAS software program. “Intestinal region” was considered as a fixed variable, and “pig” and “series” as random variables. Probability levels <0.05 were considered significant. Data not belonging to a normal distribution were analyzed using the Kruskal-Wallis and Dunn multiple comparison test.

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RESULTS

Clinical Observations

The initial surgical procedures (jugular catherization and bowel resection) were well tolerated by all pigs. During the initial 12 to 24 hours postresection, there was little stoma output, probably because of postsurgical paralysis of the intestine. The inserted jejunostomy tubes generally drained well but had to be repositioned in a few pigs after being displaced from rubbing against the cage wall. Some leakage from the stoma could not be avoided, and this induced skin irritation and inflammation for some pigs. To reduce skin irritation, zinc oxide ointment was applied to the skin around the stoma.

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Body and Organ Weights

Body weight was measured on days 1, 4, 6, and 9, and weight increments were calculated relative to weight on day 1. The 4 teduglutide groups showed similar body weights at the end of the experiment. The pooled value for the 4 teduglutide groups was significantly higher than that for the placebo group (Fig. 1). This effect was most pronounced on days 6 and 9.

FIGURE 1

FIGURE 1

The length of the intestine was accurately predicted from the established equation as indicated by the similar lengths of the resected and the remnant intestine among the placebo and teduglutide groups (Fig. 2). Hence, there was no indication of an effect of teduglutide on intestinal length after resection.

FIGURE 2

FIGURE 2

In some cases, dilation of the remnant intestine as a result of substomal stricture was observed. The dilations resulted from accumulation of digestive fluid and seemed to increase gut weight in the distal region. Data from pigs with strictures were excluded, that is, 2 from the placebo group, 2 from the 0.01 mg · kg−1 · day−1 group, and 1 from the 0.2 mg · kg−1 · day−1 group. There was no apparent association between teduglutide treatment and the development of these strictures.

On the basis of the regression analyses, teduglutide tended to increase (P = 0.12, Table 2, Fig. 3, upper panel) the absolute weight of the remnant intestine in a dose-dependent manner; however, values in each dose group were associated with a relatively large variation making all pairwise comparisons nonsignificant. Conversely, the weight of the isolated 10-cm section collected from the proximal remnant intestine showed a clear dose-dependent increase in weight per length (P = 0.01, Table 2, Fig. 3 middle panel). Supporting this observation, protein concentration, FSR, and ASR were all markedly higher (∼100% higher) in dose group 0.2 mg · kg−1 · day−1 versus placebo (Fig. 3, bar graphs).

FIGURE 3

FIGURE 3

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Digestive Enzymes and Nutrient Balances

The increase in weight per length and protein synthesis rate of the remnant intestine induced by teduglutide was not associated with any changes in the specific activities of 6 digestive enzymes (lactase, maltase, sucrase, aminopeptidase A, aminopeptidase N, and dipeptidylpeptidase 4), neither in the proximal part of the remnant intestine (Table 2) nor in the distal part (data not shown). These data indicate a proportional increase in enzyme activity as the intestinal weight per length increased, that is, maintaining specific activity expressed on a per gram basis. Gut permeability, as assessed by the urinary lactulose to mannitol ratio, tended to decrease with increasing dose of teduglutide (P = 0.15, Table 2), which may indicate marginally better gut integrity relative to placebo.

All animals were given a milk formula at 10 mL · kg−1 · 2 h−1 for 24 hours, corresponding to 130 kcal/kg during the nutrient balance study. During these 24 hours, the rate of IV infusion of isotonic saline was increased to account for an anticipated loss of sodium through the stoma. This was confirmed as there was a large stomal loss of sodium for all groups, most pronounced in the 0.02 mg · kg−1 · day−1 group, relative to the other groups (Table 2, P < 0.05). The sodium recovered from the stoma was generally ∼10 times higher than the oral intake. Neither the absolute nor the relative absorption of fluid, fat, carbohydrate, protein, potassium, sodium, calcium, and magnesium showed any significant correlation to increasing teduglutide doses. Consistent with the observed higher loss of sodium in the 0.02 mg · kg−1 · day−1 group, there was a relatively high loss of fluid in this group, compared with all other groups, and not related to the balances for macronutrients or other ions (data for potassium, calcium, and magnesium not shown).

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Pharmacokinetics

The teduglutide concentrations measured in the solutions for injection were within 10% of their predicted target concentrations. Teduglutide plasma concentrations consistently peaked at tmax = 0.5 hour for all 4 dose groups (Fig. 4, upper panel). Plasma levels of teduglutide were only detectable at 0.5, 1, and 2 hours after injection, and half-life during the elimination phase was 0.4 to 0.6 hour. Although there was clear linearity between Cmax, AUCinf, and teduglutide dose (Fig. 4, middle panel and lower panel), it was not possible to calculate AUCinf for the 0.01 mg/kg group because we found a detectable level in only 1 pig. Hence, this point in the graph represents only 1 observation (no standard deviation).

FIGURE 4

FIGURE 4

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Villus Height, Crypt Depth, and Immunohistochemistry

In the proximal jejunum, mean villus height differed slightly between groups (Fig. 5) but with no statistical difference among groups. Likewise in the mid-proximal jejunum, villus height and crypt depth both showed a trend toward increased mean values in the 0.2 mg · kg−1 · day−1 group, compared with placebo, but again without statistical significance (data not shown).

FIGURE 5

FIGURE 5

The immunohistochemical detection of various markers in the epithelium showed no apparent difference among the 5 treatment groups. Strong intranuclear reactivity for the Ki-67 protein was detected in the crypt epithelium from all animals (Fig. 6, panels A and B). Moderate expression was also observed in scattered cells located in the lamina propria of the mucosa and within the tunica muscularis. Villin, a tissue-specific actin-binding protein that associates with the actin core bundle of the brush border, labels all enterocytes of the intestinal mucosa, regardless of their stage of maturation. In this study, villin immunohistochemistry showed a pancytoplasmic reactivity in all animals (Fig. 6, panels C and D). FABP2 expression was detected within the cytoplasm of all villus enterocytes (Fig. 6, panels E and F). Chromogranin A was located in secretory vesicles of neurons and neuroendocrine cells, including enteroendocrine cells, and in single cells within the villus epithelium and within the crypt epithelium (Fig. 6, panels G and H). Immunoreactivity against the GLP-2 receptor was detected in single cells within the crypt and the villus epithelium (representing neuroendocrine cells), and in single cells within the mucosal lamina propria (representing myofibroblasts), again with no distinctive difference among treatment groups (Fig. 6, panels I and J).

FIGURE 6

FIGURE 6

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DISCUSSION

There is a need to identify treatments that improve intestinal adaptation following small bowel resection in infants that are dependent on adequate nutrient uptake for normal growth and development. Using a piglet jejunostomy model, the present results show that the GLP-2 analogue, teduglutide, exerts acute trophic effects on the remnant small intestine and increases body growth in the days after resection. Although strictures were encountered, there seemed to be no correlation to teduglutide treatment, as they were equally present in the placebo group. In this study, most of the stimulating effects on the growth of the proximal small intestine were achieved already with the lowest dose of teduglutide used (0.01 mg · kg−1 · day−1). The modest trophic effect was associated with a marked increase in protein synthesis and a decrease in intestinal DNA concentration, indicating that gut adaptation in the teduglutide-treated pigs was mediated by intestinal hypertrophy, rather than hyperplasia. The enhanced intestinal growth may result from an increased amount of intra- or extracellular structural proteins in the mucosa, leading to a lower number of cells within a given mucosal volume. This may also lead to a dilution of some functional proteins and explain that no effects were observed on nutrient uptake on day 5 or brush border enzyme activities and intestinal permeability by the end of the protocol. Regardless, the modest intestinotropic effect resulted in a significantly higher body growth rate in teduglutide-treated pigs compared with placebo controls.

In previous studies, teduglutide was shown to possess hyperplastic effects by stimulating enterocyte mitosis in the crypt area and reducing apoptosis at villus tips (17–20). Given the effects of teduglutide on both hyperplasticity and hypertrophicity, the risk of possible dysplastic effects of teduglutide has recently been studied in human adult patients with SBS (21). In this study, patients received 0.05 to 0.1 mg · kg−1 · day−1 for 6 months before intestinal biopsies were collected. Neither the placebo group nor the teduglutide groups showed indications of dysplasia within this treatment period. Although possible intestinal cell dysplasia and development of malignant cells should remain a concern, data from this human study did not indicate safety concerns regarding teduglutide treatment.

In contrast to our earlier studies in preterm and term TPN-fed pigs, with and without SBS (13,22,23), the GLP-2-induced hypertrophic effects were not in this study paralleled by teduglutide-induced improvements in intestinal functional indices. Accordingly, immunohistochemical stainings did not indicate major tissue changes in teduglutide groups versus placebo. Collectively these observations indicate that under the given experimental circumstances growth of the mucosal cell populations may have preceded any (later) effects on mucosal functions. Sucrase and maltase activities are generally extremely sensitive to GLP-2 stimulation in newborn pigs (24), and associate with increased nutrient absorption in preterm SBS pigs (13), but such effects were absent in the present study. We speculate that the lacking effects are because of both the relatively short period of treatment and the limited exposure to elevated bioactive GLP-2 levels, compared with these earlier studies.

In a recent study, provision of enteral nutrition potentiated the effect of GLP-2 treatment in a rodent SBS model (25). This observation may offer a potential for patients with SBS to increase enteral food intake during GLP-2 treatment and to become less dependent on PN support. There may, however, be substantial variation between patients with SBS as some have small bowel stomas and others have a preserved and functioning colon. Especially patients with SBS with intestinal failure, as defined by massive loss of gut mass, have reduced absorption of enteral nutrients and are dependent on PN support. Relative to patients with SBS with a jejunostomy, adult patients with a functional colon show better retention of sodium and fluid and have improved energy balance from absorption of short-chain fatty acids in the colon. On the one hand, this helps these patients to maintain body fluid homeostasis, but, on the other hand, a functioning colon in patients with SBS with an extremely short small intestine can induce excessive fermentation of undigested nutrients in the colon and production of D-lactic acid (26). Potential downstream pathological effects of D-lactic acid metabolic acidosis include intestinal paralysis and poor digestive function. Moreover, microorganisms originating from the colon may populate the distal small intestine causing small bowel bacterial overgrowth, which is associated with inflammation and reduced nutrient digestion. The benefits of a preserved colon, therefore, depend on a careful management of enteral nutrition and the function of the remnant small intestine.

In the present study we resected approximately 50% of the distal small intestine. Although this ensured extremely low secretion of endogenous GLP-2, the remnant small intestine may still have been sufficiently long to digest and absorb the enteral test diet equally well for the placebo and the 4 teduglutide groups. A more extensive intestinal resection may have provided better evidence of functional teduglutide effects. In contrast, this clinical situation requires extremely close monitoring of body fluid homeostasis, and effective means to correct and maintain it with saline infusions, which adds to the technical challenges in this preclinical jejunostomy animal model.

From the pharmacokinetic study results, we observed that teduglutide plasma levels peaked already within half an hour and the half-life was approximately 30 minutes. An extremely short exposure time may explain that the effects of teduglutide in this study were mainly seen on structural intestinal endpoints, with limited effects on intestinal function in contrast to the earlier studies in term pigs using continuous infusion of the native peptide (27–29). The total exposure time, as indicated by AUC, increased linearly with increasing doses, but still, further effects on intestinal growth and function were limited. As teduglutide in the circulation is resistant toward degradation by dipeptidylpeptidase 4, the observed half-life is mainly accounted for by renal glomerular filtration as the route of excretion (30). Maintaining GLP-2 levels high for an extended period may be required to achieve maximal structural and functional intestinal response to GLP-2.

We conclude that within the given dose range, daily injections of teduglutide to newborn SBS pigs induced a significant and acute trophic response in the remnant small intestine that translated into improved body weight gain within a few days of treatment. This intestinal trophicity was mainly accounted for by higher FSR, whereas functional endpoints, including specific digestive enzyme activity and nutrient digestion, remained unchanged. Although continuous infusion of GLP-2 into patients may be most effective in stimulating gut growth and function, such treatments are not clinically feasible. More studies are required to better define the optimal dose regimen and exposure time for various groups of patients that could benefit from the intestinotrophic effects of GLP-2 and degradation-resistant analogues, such as teduglutide.

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Keywords:

adaptation; infants; intestinal failure; short bowel syndrome

© 2014 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology,