WO2015104404A1

WO2015104404A1 - Method for the treatment or prevention of gastrointestinal disease caused by chorioamnionitis

Info

Publication number: WO2015104404A1
Application number: PCT/EP2015/050373
Authority: WO
Inventors: Tim Guillaume Anna Marie WOLFS; Boris Willy Werner KRAMER
Original assignee: Universiteit Maastricht; Academisch Ziekenhuis Maastricht
Priority date: 2014-01-09
Filing date: 2015-01-09
Publication date: 2015-07-16

Abstract

The invention is in the field of medical treatments. In particular it provides means and methods for the treatment of gastrointestinal disease, in particular in a fetal or newborn animal. The invention provides means and methods for treating, ameliorating or preventing adverse intestinal outcome as a consequence of chorioamnionitis, wherein the composition is administered to a fetal or a newborn animal. More in particular, the invention provides a method for the treatment of necrotizing enterocolitis of the fetal gut caused by chorioamnionitis wherein a suitable dose of IL-2 is administered to a fetal or a newborn animal.

Description

METHOD FOR THE TREATMENT OR PREVENTION OF GASTROINTESTINAL DISEASE CAUSED BY CHORIOAMNIONITIS.

Field of the invention

The invention is in the field of medical treatments. In particular it provides means and methods for the treatment of neonatal disease. The invention provides means and methods for treating, ameliorating or preventing of adverse intestinal outcomes. Such disease is multifactorial and among the most frequently and important risk factors is chonoamnionitis in the pregnant female. In particular, the invention relates to the prevention and treatment of necrotizing enterocolitis of the newborn gut.

Background of the invention

Preterm delivery is the primary cause of neonatal morbidity and mortality (1 ). The most frequent cause of preterm delivery is chonoamnionitis, a bacterial infection of the amniotic fluid, placenta and fetal membranes, often initiated by

Ureaplasma species (2,3). Since the fetus swallows the amniotic fluid, infected amniotic fluid results in premature gut exposure to bacteria or their components. Chonoamnionitis and prematurity are both associated with compromised postnatal outcomes such as poor nutritional uptake, failure to thrive and subsequent postnatal growth restriction (4-7).

Moreover, it was shown in a recent meta-analysis that chonoamnionitis is a risk factor for Necrotizing Enterocolitis (8), a serious gastrointestinal disorder frequently affecting preterm neonates (5).

The mechanisms underlying perturbation of gut development in premature babies suffering from chonoamnionitis remain largely unresolved. To study the effects of chonoamnionitis on antenatal organ development, a fetal sheep model of chonoamnionitis was developed, since the developmental biology of the ovine gut resembles closely the human situation (9-1 1 ). Using this translational model, it was recently shown that antenatal exposure to Ureaplasma parvum resulted in a pro- inflammatory response of the fetal intestine, which was characterized by influx of neutrophils and effector T-cells (12). This inflammatory reaction was preceded by depletion of regulatory T- cells which disturbed the effector/regulatory T-cell balance (10). The inflammatory process in the fetal gut was associated with a compromised gut barrier, impaired proliferation, differentiation and maturation of enterocytes, ultimately resulting in severe villus atrophy (12). Notwithstanding the above progress in understanding of mechanisms of disease origin and development, the exact immunological mechanisms which are involved in the adverse intestinal outcomes for the fetus or the newborn caused by chorioamnionitis in the pregnant female are not fully understood yet.

At present, pharmacological interventions aimed at preventing or curing or dampen adverse intestinal outcomes as a consequence of chorioamnionitis are not available. In particular there is a need in the art for means and methods to treat, prevent or dampen necrotizing enterocolitis. Infants that develop postnatal pathologies are treated after onset of these diseases. The current treatment is symptomatic and consists of supportive therapy in blood pressure and ventilation.

Summary of the invention

It has now surprisingly been found that adverse intestinal outcome as a consequence of chorioamnionitis may be treated by administering a suitable dose of IL-2 to the fetus or the newborn. The invention therefore relates to a method for the treatment, amelioration or prevention of adverse intestinal outcome as a consequence of chorioamnionitis wherein a suitable dose of IL-2 is administered to a fetal or newborn animal. Detailed description of the invention

We have now found that administration of IL-2 to a fetal animal protects the fetus or the newborn (and its gut in particular) against adverse outcomes of inflammatory responses in the course of chorioamnionitis, such as necrotizing enterocolitis.

The term "newborn" as used herein refers to a period after birth, preferably a period of a few day, such as 1 , 2, 3, 4, 5, 6, or 7 days. The period may also be a few weeks, such as 1 , 2, 3, or 4 weeks. Preferably, the period is not longer than 30 days.

In the context of the present invention, the term "administration to a fetal animal" or equivalents thereof, is to be understood as referring to the administration of a compound directly to a fetus, excluding the indirect administration to the fetus by administering the compound to the pregnant female. Direct administration of a compound to a fetus may be achieved by means known in the art, such as venous, arterial or gastric catheters. It is advantageous to administer the compound to the fetus or the newborn systemically, not into the gastrointestinal tract for instance, such as disclosed in WO2012/033459. It is also important to notice that the treatment as disclosed herein, is sufficient to obtain the effects as described herein, i.e. without the addition of other compounds, such as enzymes, for instance enzymes with pancreatic activity as disclosed in WO2012/033459. Hence, in a preferred embodiment, the invention relates to a composition comprising IL-2 for use in the treatment, amelioration or prevention of adverse intestinal outcome as a consequence of chorioamnionitis, wherein the composition is administered to a fetal or a newborn animal and wherein the composition does not comprise an enzyme with pancreatic activity.

Chorioamnionitis is herein defined as a mostly bacterial infection of the amniotic fluid, placenta and fetal membranes. Ureaplasma is commonly associated with chorioamnionitis.

The term adverse outcomes of inflammatory responses in the course of chorioamnionitis or adverse intestinal outcome as a consequence of chorioamnionitis as used herein, is to be understood as comprising all adverse outcomes involving inflammatory responses as a consequence of chorioamnionitis. The adverse outcome may be selected from the group consisting of necrotizing enterocolitis, malabsorption of food nutrients across the gastrointestinal tract, perforation of the gut, inflammation of the fetal gut, feeding intolerance, ileus and peristaltic dysfunction.

Without wanting to be bound by theory, we postulate that our invention targets the processes that predispose to adverse outcomes prenatally, i.e. direct from their onset which means in utero. The intervention with IL-2 restores an impaired

Teffector/T regulatory cell imbalance and prevents against a compromised gut barrier, both important risk factors for adverse postnatal intestinal outcomes.

Hence, in a more specific embodiment, the invention relates to a method of treatment directed at preventing an adverse intestinal outcome associated with a compromised gut barrier and with disturbed effector T-cell/T-reg balance such as NEC, feeding intolerance, sepsis and spontaneous intestinal perforation.

Clinical chorioamnionitis which is characterized by maternal fever, leukocytosis, tachycardia, uterine tenderness, and preterm rupture of membranes, is less common than subclinical/histologic chorioamnionitis, which is asymptomatic and defined by inflammation of the chorion, amnion, and placenta. Chorioamnionitis is often associated with a fetal inflammatory response. The fetal inflammatory response syndrome (FIRS) is defined by increased systemic inflammatory cytokine concentrations, funisitis, and fetal vasculitis.

In an experimental chorioamnionitis model, we have exposed pregnant sheep to intra-amniotic injections of endotoxin with or without prior intravenous IL-2 infusion of the fetus, before caesarean delivery at 133d gestational age (GA) (term 150d GA).

We found that inflammation of the fetal or newborn gut was diminished or even completely prevented when fetuses or newborns of pregnant sheep with chonoamnionitis were treated with a compound comprising IL-2 in a dose comparable to the usual human dose. These observations were based on a number of markers indicative for inflammation of the fetal gut as described in the Examples section herein.

Hence, the invention relates to a composition comprising IL-2 for use in diminishing or preventing inflammation of the fetal or newborn gut wherein the composition is systemically administered to a fetal or newborn animal born from a mother with chonoamnionitis.

In addition, we observed that the gut structure was significantly impaired in lambs suffering from chonoamnionitis as compared to control animals, which predisposes to intestinal pathologies including necrotizing enterocolitis. This gut barrier loss or mucosal injury was prevented by IL-2 treatment.

We also found that an amount of IL-2 of approximately 80.000 lU/kg fetal body weight was suitable for inducing FoxP3+ T-reg cell expansion without toxic or adverse effects such as the vascular leak syndrome. It was concluded that the data showed that intravenous IL-2 administration, in a similar concentration as used in human (16) induces preferential regulatory T-cell expansion, thereby ameliorating or preventing or treating adverse intestinal outcome as a consequence of chonoamnionitis, such as necrotizing enterocolitis.

Furthermore, we found that combined IL-2 and IL-1 RA treatment acted synergistically in the prevention of gut inflammation and thus prevented associated adverse outcomes of the gut, even better than each of these two components on their own.

Legend to the figures

Figure 1 : Graphs showing that antenatal exposure to Ureaplasma parvum resulted in a pro-inflammatory response of the fetal intestine characterized by influx of a number of infiltrating inflammatory cells. panel A: MPO+ cells, panel B: CD3+ cells, panel C: CD4+ cells, panel D: FoxP3+ cells.

Figure 2: Graph showing the gastrointestinal transit of dextran as measured by rhodamine-B-labeled dextran in several intestinal segments in lambs suffering from chorioamnionitis (squares) and controls (filled circles).

Examples

Example 1 : Experimental model.

The sheep model for chorioamnionitis has been described before. In brief, at 1 18 or 123d of gestational age, fetuses have been exposed by median laparotomy under general anesthesia. Fetal venous, arterial and gastric catheters were introduced in the fetus and one catheter was placed in the amnion, followed by closure of the uterus.

A venous line was placed in a hind leg of the ewe for maternal blood sampling and antibiotics administration. Four days after recovery of instrumentation, 6,25 x 10e5 IU of human IL-2 (Proleukin, Emeryville, CA) were administered by continuous infusion during four consecutive days (n=32) (or saline for controls n=32).

At 126 or 131 d, Ureaplasma (or media for controls) was injected in the amnion through the amniotic catheter. Animals were sacrificed directly at 2 or 7d after Ureaplasma administration.

Example 2: Isolation and characterization of fetal FoxP3+ T-reg cells

Heparinized fetal arterial blood was drawn daily to study the dynamics of circulating CD4+/CD25high/FoxP3+ regulatory T-cells following IL-2, rhILI ra and

Ureaplasma exposure by flow cytometry. Ovine regulatory T- cells were identified using sheep specific antibodies detecting CD4, CD25 and intracellular FoxP3. CD4+/CD25high regulatory T-cells were be isolated from blood and mesenteric lymph nodes by fluorescence-activated cell sorting (FACS). The proliferation capacity and cytokine profile was studied by culturing isolated regulatory T-cells in an established mitogen-induced proliferation assay. The immuno-suppressive capacity of isolated circulating and intestinal regulatory T-cells was assessed in an established co-culture suppression assay. The dynamics of cytokine levels in fetal plasma and in supernatant of the in vitro assays were determined with established ovine specific ELISAs including SAA, TNFa, IL-6 and IL-10.

Example 3: Intestinal inflammation.

After delivery and sacrifice, ileal inflammatory responses were analyzed immunohistochemically for myeloid peroxidase (MPO), CD3, CD4 and FoxP3. Established real-time PCR was used to determine ileal mRNA expression levels for cytokines (TNFa, IL-4, IL-10, IL-17 and interferon-gamma) and Toll-like receptor-1 , 2 and 6 which can detect and sense Ureaplasma components (18).

Example 4: Gut wall integrity, motility and development:

To assess intestinal permeability, inverted isolated ileal segments were filled with physiological buffer, ligated at both ends and incubated in physiological buffer containing HRP. After incubation, luminal content was measured

spectrophotometrically (19). Gut barrier integrity was further studied by analyzing the distribution and regulation of the tight junction proteins (Zonula occludens-1 and Claudin- 3) which play a crucial role in paracellular barrier sealing. In addition, the amount of circulating intestinal-Fatty Acid Binding Protein (l-FABP) was analyzed by ELISA as biomarker for intestinal mucosal damage (20).

Gastrointestinal transit was measured by evaluating the gastrointestinal distribution of rhodamine-B-labeled dextran. Rhodamine was

administered 24h before sacrifice via a gastric tube. Following autopsy, the digestive tract was divided in 20 equal parts and segments were opened and mixed vigorously in

PBS to obtain the rhodamine-containing gut content. Total recovered rhodamine content was quantified spectrophotometrically.

Markers that were further tested are: Intestinal proliferation (p-Histon- P3 and KI67), differentiation (Kruppel-like factor-5), apoptosis (caspase-3),

morphological changes and damage (Haematoxylin Eosin staining) and distribution of

Enterocytes, Goblet and Paneth cells (PAS/Alcian blue).

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Claims

1 . Composition comprising IL-2 for use in the treatment, amelioration or prevention of adverse intestinal outcome as a consequence of chorioamnionitis, wherein the composition is administered to a fetal or a newborn animal.

2. Composition for use according to claim 1 wherein the adverse intestinal outcome as a consequence of chorioamnionitis is selected from the group consisting of necrotizing enterocolitis, malabsorption of food nutrients across the gastrointestinal tract, perforation of the gut, feeding intolerance, ileus and peristaltic dysfunction.

3. Composition for use according to claim 2, wherein the adverse intestinal outcome as a consequence of chorioamnionitis is necrotizing enterocolitis of the fetal or newborn gut.

4. Composition for use according to any one of claims 1 - 3 wherein the fetal animal is inside the uterus of a pregnant female animal diagnosed with chorioamnionitis.

5. Composition for use according to any one of claims 1 - 3 wherein the newborn animal is born from a pregnant female animal diagnosed with chorioamnionitis.

6. Composition for use according to any one of claims 1 - 5 wherein the animal is a human.

7. Composition for use according to any one of claims 1 - 6 wherein the composition is administered intravenously.

8. Method for the treatment, amelioration or prevention of adverse intestinal outcome as a consequence of chorioamnionitis of the fetal gut caused by chorioamnionitis wherein a suitable dose of IL-2 is administered to a fetal or newborn animal.

9. Method according to claim 8 wherein the adverse intestinal outcome as a

consequence of chorioamnionitis is selected from the group consisting of necrotizing enterocolitis, malabsorption of food nutrients across the gastrointestinal tract, perforation of the gut, feeding intolerance, ileus and peristaltic dysfunction.

10. Method according to claim 8 wherein the adverse intestinal outcome as a consequence of chorioamnionitis is necrotizing enterocolitis.

1 1 . Method according to any one of claims 8 - 10 wherein the fetal animal is carried by a pregnant female animal diagnosed with chorioamnionitis.

12. Method according to any one of claims 8 - 10 wherein the newborn animal is born from a female animal diagnosed with chorioamnionitis.

13. Method according to any one of claims 8 - 12 wherein the animal is a human.

14. Method according to any one of claims 7 - 1 1 wherein the IL-2 is administered intravenously.

15. Use of a composition comprising IL-2 in the manufacture of a medicament for the treatment, amelioration or prevention of adverse intestinal outcome as a consequence of chorioamnionitis, in particular necrotizing enterocolitis in a fetal or newborn animal.