The Long-Acting Glucagon-Like Peptide-2 Analog Apraglutide Enhances Intestinal Protection and Survival After Chemotherapy and Allogeneic Transplantation in Mice

Introduction

Acute graft-versus-host disease (aGvHD) is a frequent complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) that may be life-threatening [1–4]. Every year, approximately 26,000 patients receive allo-HSCT, a standard of care and a potentially curative treatment for many hematological cancers [5–8], across the US, Europe, and Japan [1,9]. The proportion of patients who develop aGvHD after allo-HSCT despite prophylaxis with immunosuppressive therapy [10] varies from 20% to 80% [11,12].

Gastrointestinal (GI) tract involvement in aGvHD is associated with significant morbidity and mortality [13]. The vital intestinal L-cells are directly impacted by tissue damage triggered by conditioning chemotherapy or aGvHD; these cells secrete glucagon-like peptide-2 (GLP-2), an essential endocrine hormone that maintains GI integrity and nutrient/fluid absorption and repairs and regenerates cells that sustain healthy intestinal epithelium [14].

Endogenous GLP-2 is a 33-amino acid peptide derived from post-translational processing of proglucagon in intestinal L-cells that is released into the circulation following nutrient ingestion [15–18]. GLP-2 acts locally to stimulate intestinal nutrient transport and intestinal blood flow, while enhancing gut barrier function. GLP-2 also promotes organ-specific stimulation of intestinal growth through increased proliferation and decreased apoptosis of mucosal epithelial cells [19–23]. A decline in endogenous GLP-2 secretion disrupts the repair and regeneration of intestinal stem cells, Paneth cells, and goblet cells [14,24].

This profound deletion of intestinal stem cells and Paneth cells associated with aGvHD leads to disrupted GI homeostasis and a higher incidence of steroid-refractory aGvHD in patients who have less than one L-cell per crypt [14,24]. Additionally, low Paneth cell count at the onset of aGvHD in the GI tract (GI aGvHD) is an important risk factor for increased non-relapse mortality [25,26].

Consequently, GLP-2-based therapy is of interest as a protective approach in pathologic conditions where the integrity of the intestinal mucosa or its absorptive function are compromised by disease or cytotoxic therapy, to protect from the secondary life-threatening consequences of these regimens [25,26].

High-dose chemotherapy, in particular, and radiotherapy, given alone or as part of bone marrow transplantation (BMT) conditioning, act on rapidly dividing cells of the gut and induce profound damage of the GI epithelium [27] This results in a breakdown of functional epithelial cells, which, in turn, contributes to symptoms such as vomiting, abdominal pain, and diarrhea [13,28]. Critically, loss of the GI-protective barrier permits the translocation of intestinal bacteria into the systemic circulation [27]. Coupled with drug- or radiotherapy-induced neutropenia, this frequently results in life-threatening sepsis and, occasionally, death [29]. In addition, the use of antibiotics as prophylaxis or therapy for sepsis results in the development of multidrug-resistant bacteria and changes in the microbiome, which consequently alters the functioning of the immune system [27,30].

Preclinical and clinical data have shown that GLP-2 analogs improve intestinal absorption and clinical signs of GI aGvHD [31,32]. Apraglutide (Ironwood Pharmaceuticals, Basel, Switzerland) is a synthetic GLP2 analog with a unique pharmacologic profile, rationally designed to be more potent, selective, and longer acting than human GLP-2 (hGLP-2) and other GLP-2 analogs, resulting in enhanced intestinotrophic effect [33]. Apraglutide also binds to rat, monkey and pig GLP-2 receptors with a potency similar to, or within approximately twice, that of native hGLP-2 and with 90% maximal efficacy [33]. The selectivity of apraglutide for the GLP-2 receptor has been further confirmed using a panel of 80 receptors, ion channels, and transporters. Apraglutide was specifically designed to be resistant to cleavage by dipeptidyl peptidase-4 (DPP4) and to have increased protein binding, which significantly prolonged the in vivo half-life of the peptide (72 hours in rats) vs native hGLP-2 (7 minutes) [33].

In this study, we investigated how pretreatment with apraglutide preserved intestinal function in murine models of chemotherapy (cytarabine or melphalan) and of irradiation followed by allogeneic transplantation. The GI-protective effect of apraglutide against chemotherapy-induced GI damage was assessed by multiple modalities, including citrulline measurements – a plasma biomarker of enterocyte mass that is reduced after intestinal mucosal damage [34–36] – in 2 different animal models for cytarabine and high-dose melphalan – 2 chemotherapeutic agents that are routinely used in the treatment of various hematological cancers (eg leukemia, multiple myeloma, and lymphomas) [37–39].

Material and Methods

ANIMALS AND HOUSING:

Studies were conducted at the Department of Medical Oncology and Hematology of the Princess Margaret Hospital (University Health Network, Toronto, Canada; impact of apraglutide on cytarabine-induced intestinal damage) and at ITR Laboratories (ITR Laboratories Canada Inc, Montreal, Québec, Canada; impact of apraglutide on melphalan-induced intestinal damage and effect on allogeneic transplantation). Study plans and in-life procedures were approved by the local Animal Care Committees and studies were conducted according to the Canadian Council on Animal Care’s Guide to the Care and Use of Experimental Animals and the US National Institutes of Health’s Guide for the Care and Use of Laboratory Animals [40,41]. Female and male BALB/cAnNCRL mice (Charles River Canada Inc, St-Constant, Québec, Canada, and The Jackson Laboratory, Maine, USA) were used in the studies of chemotherapy-induced gastrointestinal (GI) damage.

The study investigating the effect of apraglutide in a model of GI damage resulting from allogeneic transplantation after total body irradiation was conducted at the Maisonneuve-Rosemont Hospital (Montreal, Québec, Canada). BALB/cJ (n=20 for Experiment 1 [8.5 Gy] and n=25 for Experiment 2 [7.0 Gy]) and C57BL/6J male mice (n=10 for each experiment) aged 7–8 weeks (The Jackson Laboratory, Maine, USA) were maintained in a specific pathogen-free barrier facility. All experimental procedures were approved by the local ethics committee, overseen by the Canadian Council for Animal Protection.

HOUSING CONDITIONS:

Mice were held at no more than 4 mice per cage in ventilated cages with appropriate bedding material (Envigo [Harlan] Teklad Sani-Chips 7090) as well as food (Envigo [Harlan] Teklad 2018) and water ad libitum. Sterile water was purified and filtered at 0.2 μm, with pH adjusted to 2.5. Housing rooms had controlled environmental conditions of temperature (23.5±2°C) and humidity (60±12%), with a 12-hour light/dark cycle and with ad libitum access to standard certified commercial rodent chow and water, in HEPA-filtered air (8–12 air exchanges per hour with no recirculation). During the acclimation period, animals were assigned to their respective dose groups by block randomization based on body weights.

ANIMAL MONITORING:

Mortality checks were performed at least twice a day during all study phases. Mice were weighed daily from day +1 to day +14 during the treatment period, between 10am and 11am. Weight control protocols followed established guidelines for diet control in laboratory animals from the Canadian Council on Animal Care’s Guide to the Care and Use of Experimental Animals and the US National Institutes of Health’s Guide for the Care and Use of Laboratory Animals [40,41]. Following ethical guidelines, mice with weight loss ≥25% or reaching other limit points were sacrificed. Cage side clinical signs were monitored daily, except on days of detailed clinical examination. Specific attention was paid to diarrhea, signs of dehydration, and cachexia (wasting syndrome). Animals whose health status was judged to warrant additional evaluation were examined by a clinical veterinarian or by a technician working under the supervision of a clinical veterinarian.

EUTHANASIA:

Euthanasia of animals at the facility in Toronto was performed by gas anesthesia over-dosage (isoflurane) followed by exsanguination. Euthanasia of animals at the Montreal facility was performed by cervical dislocation.

TREATMENTS:

Treatments, dosing regimens, and group sizes are shown in Table 1. Apraglutide was provided by the manufacturer as a lyophilized powder and was reconstituted with vehicle (sterile water containing 4.5% D-Mannitol, 20mM glycine, 20mM L-histidine; pH 8.6–8.8).

Cytarabine and melphalan were both purchased from Sigma-Aldrich (Missouri, USA). Cytarabine was dissolved in sterile 0.9% NaCl solution with the pH adjusted to 7.35, while melphalan was prepared in acidified alcohol.

ETHICAL APPROVAL:

In-life procedures were approved by the Canadian Council for Animal Care Committee of the University Health Network, Toronto, Canada, under Animal Utilization Protocol #: 859.14, and of the Canadian Council for Animal Care Committee of the CIUSSS of l’Est-de-l’Ile-de-Montréal, Montréal, Canada, under protocols numbers 2017-MA-012 and 2017-MA-013.

EFFECT OF APRAGLUTIDE ON CHEMOTHERAPY-INDUCED GI DAMAGE: Sixty-two BALB/c male mice (21–26 g) aged 12–18 weeks were assigned to 1 of 4 treatment groups of 6–18 animals each, to compare the efficacy of apraglutide in reducing intestinal damage and protecting from bacterial infection of intestinal mucositis induced by cytarabine. Apraglutide 3.3 mg/kg [42] was administered subcutaneously before and during chemotherapy (from day −4 to day +12; either once daily or every 48–72 hours) or after initiation of treatment with cytarabine (from day 0 to day +12). Cytarabine (30 mg/kg) was administered to all groups twice a day for 5 consecutive days (days 0 to day +4). To evaluate recovery from neutropenia, 3 additional animals were added in selected groups (Table 1, Experiment 1).

The intestine-protective effect of apraglutide during preconditioning was also assessed in a different animal model, using 34 BALB/c male mice (10 animals per group); immunoablation was achieved by intraperitoneal administration of a single high dose of melphalan (17.5 mg/kg) on day 0, alone or in combination with apraglutide, and was compared with vehicle controls. The pharmacologic effect of subcutaneously administered apraglutide 3.3 mg/kg was assessed, with dosing every 48 hours starting at day −8 and continuing until day +6 (Table 1).

In a separate dose-response study, 132 BALB/c female mice (17–24 g) aged 8–11 weeks were assigned to 1 of 6 treatment groups of 16 animals each (plus 6 extra animals per group for serum citrulline measurements), to assess the pharmacologic effect of increasing doses of apraglutide (0.11, 0.3, and 1.1 mg/kg) administered subcutaneously. Cytarabine was administered intraperitoneally at 30 mg/kg twice daily for 5 days (day 0 to day +4). Apraglutide was administered daily (in all but 1 group, in which it was administered every 48–72 hours) for a total of 9 days, 4 days before and 5 days during the administration of chemotherapy (day −4 to day +4). The effect of apraglutide was compared with that of unmodified hGLP-2, which was administered twice daily from day −4 to day +4 at a nominal dose of 160 nmol/kg and combined with cytarabine (Table 1, Experiment 2).

MEASUREMENTS:

Parameters monitored during the in-life portion of these studies included mortality, clinical signs, and body weight. Mortality checks were performed once or twice daily for the duration of the studies. Clinical signs were recorded during daily cage side observations, and detailed clinical examination (DCE) was performed before initiation of treatment, during treatment days, and prior to autopsy. For animals in recovery groups, DCE was performed once weekly and before termination. The DCE focused on the presence of diarrhea, signs of dehydration, and cachexia wasting syndrome. Body weight was recorded on selected days during treatment and during the recovery period (when applicable), as well as at autopsy for all animals. Mice with weight loss greater than 25% or reaching other limit points were euthanized. All other mice were euthanized as previously scheduled. To perform bacterial phenotyping, fecal samples from selected animals were collected over approximately 24 hours pretreatment and on the day before scheduled euthanasia. Animals were euthanized the day after the last scheduled dose of apraglutide. At the end of the study, blood samples were collected (0.3 mL in K₃EDTA) to measure the levels of circulating citrulline, which is known as a biomarker proportional to the intestinal mass. The analytical method for determining citrulline levels in mouse plasma was validated at York Bioanalytical Solutions (York, UK). The method employed protein precipitation with tandem mass spectrometry using turbo ionspray, in positive ion, multiple reaction monitoring mode. The lower limit of quantification of the method was 0.500 μg/mL; the sample aliquot volume was 25 μL.

Other blood samples (0.75 mL in EDTA) were dedicated to hematologic analysis. The impact of apraglutide on recovery from cytarabine-induced neutropenia was also assessed, by comparing the evolution of hematologic parameters for white blood cells following a 4-week recovery period for designated groups.

The composition of the fecal bacterial microbiota was investigated using high-throughput sequencing of variable regions of the 16S rDNA bacteria gene (taxonomic metasequencing analysis). Representations of the relative proportions of taxons in each sample were shown according to Phylum, Family, and Genus.

External examination, including identification of all clinically recorded lesions, and detailed internal examination were performed during autopsy. Intestinal segments were evaluated for signs of hemorrhage or inflammation, weighed, and their length measured; they were then fixed in formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin, to evaluate intestinal mucosa integrity.

EFFECT OF APRAGLUTIDE ON IRRADIATION- OR TRANSPLANTATION-INDUCED GI DAMAGE: Using a mouse model previously described by Thompson et al [43], the effect of apraglutide on GI protection following irradiation and allogeneic transplantation was assessed. Twenty (Experiment 1 [8.5 Gy]) and 25 (Experiment 2 [7.0 Gy]) BALB/cJ mice were weighed and randomized into 3 groups based on matching weights, so that each group contained a similar distribution of mouse weights. Mice received subcutaneous injections of apraglutide 3 mg/kg or vehicle from day −9 to day +7 post-allogenic transplantation. At day −1, all BALB/cJ mice from 2 out of the 3 groups were irradiated (7.0 or 8.5 Gy, LINAC X-ray source). At day 0, bone marrow and spleen T cells from C57BL/6J mice were collected, prepared, and transplanted into BALB/cJ mice (intravenous injection of 10 million bone marrow cells extracted from femurs and 2.5 million spleen cells; due to dehydration, intravenous injection was not possible in the mice receiving the cells by subcutaneous injection). To quantify the proportion of T cells in the spleen, 1×106 spleen cells were stained with anti-TCRβ PE antibody (control: 1×106 unstained spleen cells) and quantified by flow cytometry. The percentage of TCR+ cells was quantified with FlowJo Software (Becton, Dickinson and Company, USA). On the day of euthanasia, chimerism in BALB/c mice was measured by flow cytometry, by staining blood or spleen cells with antibodies to H2b (C57BL/6). The samples were assessed by flow cytometry at the Maisonneuve-Rosemont Hospital Research Center (Québec, Canada). The percentage of H2b+ cells was quantified with FlowJo Software. All irradiated mice showed presence of chimerism. The mice from the third group were neither irradiated nor transplanted, and they received subcutaneous injections of vehicle every second day starting from day −9 to day +7, matched with the irradiated groups (Table 1).

Intestinal symptoms, such as diarrhea and/or presence of blood in the feces, were recorded. At autopsy, the intestine was examined macroscopically for the presence of inflammation, necrosis, blood, or other alteration. Intestinal segments were collected and prepared for histopathology analysis. Results were presented as percentages of animals with pathologic severity scores from 0 to 4, with 4 being the most severe.

STATISTICAL ANALYSIS:

Animal survival data were analyzed using Kaplan-Meier plots (GraphPad Prism), with a log-rank test for trend and a log-rank (Mantel-Cox) test used to compare treatment groups. Mean and standard error of the mean (SEM) values were calculated for animal body weight throughout the studies. The body weight change on day 7 was calculated as:

Data for day 7 body weight change were statistically analyzed by one-way analysis of variance (ANOVA) with a Newman-Keuls multiple comparison post hoc test. Mean and SEM values for serum citrulline concentration were calculated for each of the 6 treatment groups and statistically analyzed, using one-way ANOVA with a Newman-Keuls multiple comparison post hoc test. Mean and SEM values for blood polymorphonuclear (PMN) cell count were calculated for each of the 6 treatment groups.

Results

Effect of Apraglutide on Chemotherapy-Induced GI Damage

IMPACT ON SURVIVAL: In the group treated with cytarabine only, some animals began to die 1 day after the last dose of chemotherapy, and no mice in this group were alive at 3 days. In all other groups (except control group), all animals had died by day +8, either due to toxicity or due to being euthanized for weight loss or other severe symptoms; mean survival was 7 days from start of treatment (Figure 1A).

Physical condition and survival rates were significantly (P<0.0001) improved when cytarabine treatment was combined with apraglutide injections administered prior to or at the same time as chemotherapy. When apraglutide treatment was initiated before chemotherapy and continued after initiation of chemotherapy, all animals but 1 survived to scheduled termination. However, when apraglutide administration was initiated on the first day of chemotherapy, 3/10 animals died on day 2 (Figure 1A).

Overall survival was significantly and dose dependently improved in the apraglutide treatment groups compared with animals treated with vehicle plus cytarabine. While only 5/16 animals survived in the vehicle plus cytarabine group at the end of the study, there were no deaths in the 3.3 mg/kg group (P<0.01), 14/16 survivors in the 1.1 mg/kg group (P<0.01), and 11/16 survivors in the 0.33 mg/kg group (P<0.05); 9/16 animals survived in the hGLP-2 plus cytarabine group (Figure 1B). No median survival time could be calculated. Improvement in survival with unmodified hGLP-2 was not statistically significant (Figure 1B).

Melphalan-only treatment resulted in premature euthanasia of 5/10 animals between days 4 and 7 post-chemotherapy, because of the severely compromised physical condition of the animals (Figure 1C). Survival was improved when melphalan was combined with pretreatment or concomitant treatment with apraglutide (Figure 1C).

EFFECT ON BODY WEIGHT: Cytarabine treatment alone induced body weight loss that was associated with a gradual deterioration in the clinical condition of the mice, and with a significant loss of body weight starting on the day following the first treatment with cytarabine (Figure 2A); body weight reached nadir on day 7. Preventive treatment with apraglutide 1.1 mg/kg or 3.3 mg/kg attenuated weight loss relative to cytarabine/vehicle-treated animals. On day 7, the body weight loss of the animals treated with the therapeutic regimens of apraglutide (1.1 mg/kg and 3.3 mg/kg) was significantly lower as compared with cytarabine alone. No difference was observed for the 0.33 mg/kg apraglutide dose level or for the group treated with hGLP-2 (Figure 2A). Treatment with apraglutide initiated pre-chemotherapy and continued after was associated with reduced body weight loss. Apraglutide treatment initiated concomitantly with cytarabine had no significant effect on the decrease of body weight (Figure 2B). Similar results were obtained when cytarabine was combined with increasing doses of apraglutide administered before initiation of chemotherapy and not continued after (data not shown). Overall survival was improved in the treatment groups relative to animals treated with vehicle plus cytarabine; a significant, dose-dependent difference compared with animals treated with vehicle plus cytarabine, as determined by a log-rank (Mantel-Cox) test, was observed for all apraglutide-treated groups but not for the group treated with hGLP-2. The median survival time for animals treated with vehicle plus cytarabine was 8 days, but it was not possible to calculate a survival time for the other groups because of the low level of mortality.

The mean body weight loss, between day 1 and day 4, of animals treated with melphalan alone was accompanied by a marked deterioration of clinical condition (Figure 2C). At day 4, the mean body weight of animals treated with melphalan 17.5 mg/kg was significantly decreased compared with controls (−17.3%; 20.9±2.3 g vs 27.0±0.9 g for controls; P<0.001). From day 5, mean body weight and clinical condition of surviving animals gradually improved. When the animals received melphalan in combination with apraglutide (3.3 mg/kg), body weight loss was less severe than for animals in the melphalan-only group at day 4 (−12.0%; 23.0±2.0 g vs 27.0±0.9 g for the controls; P<0.001). Body weight loss was further attenuated when animals were treated with melphalan in combination with apraglutide 17.5 mg/kg, with a mean body weight loss at day 4 of 2.5% (24.6±1.1 g vs 27.0±0.9 g for the control; P<0.001). In addition, mean body weight in this group was higher compared with that of the melphalan-only group (24.6±1.1 g vs 20.9±2.3 g).

EFFECT ON CYTARABINE-INDUCED LEUKOPENIA: Figure 3A shows the effect of cytarabine with and without apraglutide treatment on PMN cell count. With or without apraglutide administered before and during chemotherapy, there was an 80% decrease in leukocyte count at the end of cytarabine treatment (Figure 3B). The leukocyte counts returned to control levels after 4 weeks, indicating no detrimental effect of apraglutide on recovery of myelopoiesis and lymphocyte count. Similar results were also obtained with animals treated with melphalan 17.5 mg/kg, alone or in combination with apraglutide (not shown).

EFFECT ON INTESTINAL MORPHOLOGY: Treatment with cytarabine induced pathological changes in intestinal morphology, including villous atrophy (duodenum, jejunum, and ileum), villous epithelium vacuolation (duodenum, jejunum, and ileum), regenerative cryptal hyperplasia (jejunum), cryptal necrosis/degeneration (duodenum, jejunum, and ileum), cryptal atrophy (jejunum and ileum), and mucosal atrophy/submucosal edema (cecum) (Figure 4A). In mice that received cytarabine in combination with apraglutide 3.3 mg/kg, there were no duodenal findings and a low incidence and severity of jejunal changes. Intraperitoneal administration of melphalan was associated with degenerative/inflammatory mucosal changes in the intestine (duodenum, jejunum, ileum, cecum, and/or colon). The degenerative and inflammatory mucosal changes were characterized by degeneration and necrosis of the crypt epithelium, mixed or mononuclear infiltrate in the lamina propria, decreased number of crypts, shortening of villi (villous atrophy), vacuolation of the villous epithelium, regenerative cryptal hyperplasia, and/or mucosal erosion (Figure 4A). Such pathological features were greatly reduced or absent in mice treated with melphalan in combination with apraglutide and in control mice not treated with melphalan (Figure 4B).

EFFECT ON INTESTINAL EPITHELIUM MASS: Serum citrulline was significantly decreased in all mice receiving either cytarabine or melphalan (not shown) compared with control mice. Improvements in citrulline concentrations were observed when cytarabine was administered in combination with apraglutide 0.33 mg/kg or 1.1 mg/kg, compared with cytarabine alone (Figure 5).

Chemotherapy with melphalan was also associated with a 50% decrease in serum citrulline compared with control (29.2±19.3 μM vs 58.5±16.8 μM, respectively). Plasma levels of citrulline were unaffected when melphalan was given concomitantly with apraglutide (administered before and during chemotherapy).

EFFECT ON THE INTESTINAL MICROBIOTA: In laboratory mice, the intestinal microbiota is largely composed of Firmicutes spp. (gram-positive, aerobic and anaerobic bacteria) and Bacteroidetes spp. (gram-negative, anaerobic bacteria), the latter consisting of several families of opportunistic pathogenic bacteria. As intestinal microbiota and fecal microbiota have been shown to be closely related in other GI conditions, the fecal microbiota can be used as an indicative measure of the intestinal microbiota [44]. Treatment with cytarabine alone induced a profound modification of the composition of the intestinal microbiota in all taxonomic levels, with a significant decrease in the Firmicitus population and an increase in the Bacteroidetes at the Phylum level (Figure 6). In contrast, the relative proportion of fecal bacterial populations tended to normalize when treatment with cytarabine was combined with treatment with apraglutide starting before chemotherapy and continuing during chemotherapy. Preventive treatment with apraglutide also resulted in the physical preservation of the intestinal structure and in preservation of the global homeostatic environment of the intestinal microbiota.

EFFECT ON SURVIVAL FOLLOWING ALLO-HSCT: BALB/cJ mice that received no irradiation and no BMT had a survival of 100% at day 14 (Figure 7). All BALB/cJ mice that received pre-transplant total body irradiation (TBI) at a dose of 7 Gy and cells from C57BL/6J mice (TBI-BMT), but that did not receive apraglutide, died by day 9. These survival results are similar to those reported by Thompson et al [43]. TBI-BMT mice receiving apraglutide 3.3 mg/kg showed prolonged survival compared with mice not receiving apraglutide. Median survival was similar between the 2 groups: 9 days for the TBI-BMT plus apraglutide group (40% of apraglutide-treated animals survived after day 9) vs 8.5 days for the TBI-BMT group. Animals having long-term survival after day 9 also showed a gain in weight (data not shown).

Overall, the GI pathological score was more favorable in TBI-BMT animals treated with apraglutide 3.3 mg/kg (group A; 4/10 animals with score of 0) vs vehicle (group B; 1/10 animals with score of 0) (Figure 8).

At 8.5 Gy TBI, histological examination of the small intestine (jejunum only) showed that TBI treatment followed by allo-HSCT induced significant morphological modification of the cellular components characteristic of aGvHD (Figure 9). In TBI/transplanted animals treated with apraglutide 3.3 mg/kg, the overall mean severity score of mucosal degenerative/inflammatory changes (shortening of villi [villous atrophy], mononuclear [lymphohistiocytic]/neutrophilic cell infiltrate in the lamina propria/intra-cryptal epithelium, decreased number of crypts, and cryptal abscess) was decreased in the intestine compared with the TBI/transplanted animals treated with vehicle only. Additionally, the mean score of regenerative crypt hyperplasia was slightly increased compared with animals treated with vehicle only. The data presented in Figure 10 represent the percentages of animals corresponding to each histological grade (minimal [+1], mild [+2], moderate [+3], marked [+4], and severe [+5]) for each of the histological parameters (single cell necrosis, necrosis of all crypts, shortening of villi, cryptal hyperplasia, cell infiltrate, cryptal abscess, etc.).

EFFECT OF APRAGLUTIDE ON COLON LENGTH FOLLOWING IRRADIATION AND ALLOGENEIC TRANSPLANTATION:

Colon length measured at euthanasia is depicted for each mouse in Figure 11. The mean length of the colon of TBI-BMT mice (7.0 Gy) treated with apraglutide was similar to the length of animals that had not been irradiated and transplanted (8.6±0.35 cm vs 9.6±0.33, respectively). In contrast, the length of the colon of TBI-BMT mice treated only with vehicle was significantly shorter (7.19±0.10 cm) as compared with the 2 other groups.

Discussion

In allo-HSCT recipients, the GI tract is a tissue system damaged by conditioning regimens and aGvHD. These dual mechanisms of GI damage markedly contribute to the observed morbidity and mortality among patients with aGvHD after allo-HSCT [13,45]. The vital intestinal L-cells, which secrete GLP-2, an essential endocrine hormone that maintains GI integrity and nutrient/fluid absorption as well as that repairs and regenerates cells that sustain healthy intestinal epithelium, are directly impacted [31].

In the series of experiments presented here, the data show that the GLP-2 analog apraglutide, when administered in combination with chemotherapy, significantly improves the survival of chemotherapy-exposed mice, promotes recovery from chemotherapy-induced weight loss, exerts protective effects on epithelial structure by reducing chemotherapy-induced degenerative intestinal changes, maintains plasma citrulline levels, which is indicative of mucosal mass preservation, and preserves intestinal bacterial homeostasis during chemotherapy. In the context of chemotherapy alone, the administration of apraglutide preserved the intestinal epithelium from damage induced by standard clinical approaches used in the treatment of hematological cancers and significantly improved the physical condition of animals.

Apraglutide showed a significant protective effect against total body irradiation and allogeneic transplant-induced mortality, potentially through preserving intestinal integrity, as evidenced by reduced villi atrophy and decreased colon shortening.

Previous studies have examined the beneficial effects of GLP-2 on survival following radiation burn injuries in mice [46]. In the study presented here, adding apraglutide treatment to cytarabine or melphalan as well as to irradiation followed by allogeneic transplantation improved survival rates. As far as we are aware, this is the first time that improved survival with GLP-2 peptides has been reported for the chemotherapy agents cytarabine and melphalan, which are commonly used in the patient population of interest; however, improved survival has been reported with GLP-2 in mice following chemotherapy with irinotecan, a topoisomerase inhibitor [47].

Apraglutide treatment reduced body weight loss experienced with cytarabine or melphalan chemotherapy but did not impact weight loss following irradiation and allogeneic transplantation. The reduction of chemotherapy-induced weight loss did not appear to relate to food consumption; therefore, it is likely that this effect is due to preserved intestinal function in the way of nutrient absorption, and lack of chemotherapy-induced diarrhea, following apraglutide treatment. This is supported by evidence that endogenous GLP-1 and GLP-2 are key to gut healing after intestinal injury in mice [48], as demonstrated by preservation and/or restoration of citrulline levels, a biomarker indicative of the functional and mass integrity of the intestinal mucosa. It is unclear why apraglutide did not impact irradiation and allogeneic transplant-induced weight loss, as functional improvements in the intestine, in terms of increased colon length, were observed in this model.

In other studies, the authors have suggested that the protective effects of GLP-1 and GLP-2 on radiation-induced oxidative injury of the intestine are due to the inhibition of neutrophil infiltration and subsequent inflammatory responses [49]. The results presented here support an alternate explanation: apraglutide prevents direct drug- or irradiation-induced damage of epithelial cells and therefore preserves the intestinal mucosa, which maintains the normal microbiota structure and limits bacterial invasion below the epithelium [50] – the latter limits neutrophil response, infiltration, and further inflammation within the GI tract. The lack of protection seen with irradiation and allo-HSCT may be due to the damage to other parts of the GI tract, such as mouth, esophagus, and stomach and bladder, which are not protected by GLP-2, allowing for bacterial translocation and death due to sepsis.

Administration of cytarabine suppresses myelopoiesis and decreases blood counts, ie it elicits severe neutropenia. As noted, apraglutide did not affect the depletion of PMN cells when administered before and in conjunction with cytarabine. These results suggest that the improvements in animal survival and body weight do not result from improved neutrophil granulocyte count but, rather, from maintenance of GI integrity and a lack of neutrophil-induced inflammation and bacterial invasion that result in the death of animals.

Around 80% of individuals undergoing cancer treatment experience chemotherapy-induced GI toxicity (CIGT), which is thought to reflect the interaction of changes in the gut microbiome [51]. Apraglutide attenuates the negative impact of cytarabine on the intestinal microbiota, suggesting that it might have a beneficial impact on CIGT.

In addition to GI-associated infections, damage of intestinal tissue during preconditioning for allogeneic transplantation is reported as contributing to the ‘cytokine storm’ that is associated with the pathophysiologic mechanism leading to aGvHD. In fact, increasing evidence from experimental and clinical BMT settings suggests that damage to the GI tract plays a major pathophysiologic role in the development and amplification of aGvHD [52]. Studies have demonstrated that during aGvHD, the intestinal stem cell compartment is particularly injured, affecting several cell types, including the Paneth cells, which are responsible for the secretion of antimicrobial molecules (such as REG3A). Decrease in these cells levels correlates with the severity of the disease [52]. Therefore, a reduction in GI tract damage would be predicted to prevent systemic lipopolysaccharide translocation and thus reduce inflammatory cytokine production, culminating in decreased GI tract damage and mortality from GvHD [51,52].

Conclusions

In conclusion, apraglutide protects intestinal function and improves survival following chemotherapy with cytarabine or melphalan as well as after irradiation plus allogeneic transplant. Given that GI toxicity is a common problem with cancer treatments, the potential benefits of apraglutide in this area warrant further investigation.