http://repub.eur.nl/res/aut/14002/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/32949/ 2012-06-12T00:00:00Z A large proportion of prescribed drugs to children are administered orally. Age-related change in factors affecting oral absorption can have consequences for drug dosing. Areas covered: For each process affecting oral drug absorption, a systematic search has been performed using Medline to identify relevant articles (from inception till February 2012) in humans. This review presents the findings on age-related changes of the following processes affecting oral drug absorption: gastric pH, gastrointestinal motility, bile salts, pancreatic function, intestinal pH, intestinal drug-metabolizing enzymes and transporter proteins. Expert opinion: Clinicians should bear in mind the ontogeny of oral drug absorption processes when prescribing oral drugs to children. The authors’ review shows large information gaps on almost all drug absorption processes. It is important that more knowledge is acquired on intestinal transit time, intestinal pH and the ontogeny of intestinal drug-metabolizing enzymes and drug transporter proteins. Furthermore, the ultimate goal in this field should be to predict more precisely the oral disposition of drugs in children across the entire pediatric age range. http://repub.eur.nl/res/pub/24731/ 2009-07-01T00:00:00Z Objectives: Previous studies have shown that the intestine uses a major part of the dietary threonine intake for the synthesis of the structural component of the protective intestinal mucus layer, the secretory mucin Muc2. In this context, the high intestinal demand for dietary threonine probably results from its incorporation into secretory mucins rich in threonine residues. Therefore, we compared threonine utilization in the colon of Muc2 knockout (Muc2-/-) and wild-type (Muc2+/+) mice to investigate the intestinal dietary threonine metabolism in the absence of Muc2, which results in inflammation of the colon. Materials and Methods: Concentrations and isotopic enrichment of threonine were measured by gas chromatographyisotope ratio mass spectrometry in the serum, colon, and colonic content of mice given a bolus [U-13C]threonine enterally. Results: We retrieved 37.8% and 40.9% of dietary threonine in Muc2+/+and Muc2-/-mice, respectively, either as free or incorporated threonine. There were no major differences in the availability and concentration of free or incorporated threonine recovered in both serum and colon in both types of mice. However, the Muc2-/-mice did show overall significantly higher threonine oxidation rates compared with Muc2+/+mice. Conclusions: In the absence of Muc2, dietary threonine is mainly used for constitutive protein synthesis or becomes a substrate for metabolic oxidation. This indicates that inflammation also requires high threonine amounts. JPGN 49:99-107, 2009. http://repub.eur.nl/res/pub/10865/ 2008-01-09T00:00:00Z Part 1 focuses primarily on the pathophysiology of mucositis, in order to gain more insight different experimental mouse models were used. Chapter 2 describes mucositis induced by high dose doxorubicin (DOX)- treatment. DOX is a frequently used cytostatic drug in childhood cancer, often causing severe mucositis. DOX-induced mucositis closely resembles the characteristics of previously studied methotrexate (MTX)- induced mucositis. Both drugs induce severe damage to the epithelial morphology, characterized by severe villus atrophy, changes in epithelial proliferation and loss of epithelial differentiation. We did not expect these similarities in morphological damage as DOX attacks epithelial cells much closer to the stem cell than MTX does. DOX was suspected to have a more severe influence on intestinal homeostasis in comparison to MTX. The resemblance suggests a general mechanism in intestinal damage and repair. The time-line however, in which both drugs induced their damage to the intestine was different. DOX-treatment leads to immediate hyper-proliferation (day 1 and 2) with subsequent inhibition of proliferati! on during severe morphological damage (day 3). MTX causes proliferation inhibition within one day, followed by a period of hyper- proliferation during severe intestinal damage. Furthermore, we studied changes in epithelial-mesenchymal cross talk during DOX-induced mucositis. The expression of the intestinal morphogene and TCF4, the main Wnt pathway transcription factor in the intestinal epithelium were followed by immunohistochemistry during the different stages of DOX-induced mucositis. BMP4- and TCF4 expression appeared to be linked, shown by the fact that BMP signaling seem to suppress Wnt signaling and visa versa during mucositis development and regeneration. This suggests a balance between epithelial proliferation and subsequent intestinal differentiation. Chapter 3 The objective of this study was to investigate the expression of the small intestinal transcription factors HNF-1a, Cdx2, GATA-4 in an experimental model of MTX-induced intestinal damage, and to correlate these alterations with histological damage, epithelial proliferation and differentiation. HNF-1a, Cdx2 and GATA-4 are critical transcription factors in epithelial differentiation, and in combination they act as promoting factors of the sucrase-isomaltase (SI) gene, an enterocyte-specific differentiation marker which is distinctly down regulated after MTX-treatment. Intestinal damage was most severe at day 3 and was associated with decreased expression of the transcriptional factors HNF-1a, Cdx2 and GATA-4, which correlated well with decreased expression of SI, and seemed inversely correlated with enhanced proliferation of epithelial crypt cells. During severe damage, the epithelium was preferentially concerned with proliferation rather than differentiation, most l! ikely in order to restore the small intestinal barrier function rather than maintaining its absorptive function. In Chapter 4 we show that there were no major differences found in intestinal pathology or protein expression during MTX-induced mucositis in Muc2+/+ mice in comparison to MTX-induced mucositis in Muc2-/- mice. Mucositis regeneration however, could not be assessed in the absence of Muc2 as almost all mice died spontaneously 1 day prior to sacrifice for evaluation. Surprisingly, however, the intestine of the Muc2 deficient mice evaluated just a few days after MTX-treatment showed already increased regeneration compared to the wild type mice. In addressing this question it became clear that the cytokine production by the mucosal immune system of Muc2 deficient mice was different compared to wild type littermates. Both the pro-inflammatory cytokine TNF-a as the anti-inflammatory cytokine Il-10 was increased in naïve Muc2 deficient mice, indicating that Muc2 deficiency leads to induction of an inflammatory response. This suggests that MTX induced damage in the Muc2-/- mice may ! be tempered by triggering the immune system to release IL-10, an anti- inflammatory cytokine, prior to MTX-treatment. Chapter 5 MTX is associated with severe damage of the intestinal epithelium. As a result, the mucosal immune cells become increasingly exposed to a vast amount of microbial stimuli. In this study we aimed at determining if and to what extent these cells are still functional during MTX treatment. Furthermore, we assessed whether activation of the mucosal immune system would play a role in the pathogenesis of mucositis. The fact that the adaptive immune system contributes to mucositis was established by showing that lamina propria lymphocytes that were derived from MTX-treated mice responded by an enhanced production of various cytokines to ex vivo polyclonal (anti-CD3e and anti-CD28 mAb)stimulation. Next, in vitro experiments revealed that macrophages, either a cell-line or cells isolated from the murine peritoneal cavity, were not affected by MTX in the capacity to produce TNF-α and IL-10 upon lipopolysaccharide (LPS) exposure. Moreover, in vivo experiments showed that peritoneal macrophages isolated from MTX treated mice produced more IL-10 and TNF-α upon LPS stimulation, compared to cells derived from control mice. These data indicate persistence of both innate and adaptive immune responses in this model. The clinical relevance of these findings was further established by the fact that LPS exposure prior to MTX treatment aggravated the course of mucositis. Furthermore, LPS responsive ! mice recovered more slowly compared to LPS unresponsive mice during MTX induced intestinal damage. Finally, we found an increase in weight loss and intestinal damage upon MTX treatment in IL-10 deficient mice in comparison to wild type (WT) controls, which suggests a protective role for IL-10 in mucositis. Part 2 focuses on intestinal metabolism during mucositis and mucositis prophylaxis in childhood cancer patients. In Chapter 6 we validate a new method for collecting breath samples that simplifies the collection of breath samples in young children in order to use this method in studies described in chapter 7. Stable isotope tracers are used in clinical studies to measure (intestinal) metabolism of various substrates. Nowadays, the oxidation of [13C] labeled substrates to 13CO2 and the measurement of the appearance of excess 13CO2 in expiratory air is a common method. The collection of respiratory CO2, occurs via trapping of CO2 in sodium hydroxide (trapping method) sometimes in conjunction with indirect calorimetry. The aim of the present study was to determine the accuracy of direct nasal-pharyngeal sampling method for the collection of breath samples in preterm infants compared with the currently used trapping method. Seven pre-term infants were studied while receiving full enteral feeding. A primed constant 3-h intragastric infusion of [13C] bicarbonate was given and breath samples ! were collected by means of direct nasal-pharyngeal sampling and by a sodium hydroxide trap simultaneously. Breath CO2 isotopic enrichments rose rapidly to reach a plateau by 120 min with < 5% variation of plateau in both methods. 13CO2 breath isotopic enrichments obtained by the direct nasal-pharyngeal sampling method correlated highly with the trapping method, showing that direct nasal-pharyngeal sampling for the collection of breath samples is as accurate as the trapping method. Chapter 7 The aim of this study was to evaluate systemic availability of dietary amino acids (leucine) during chemotherapy-induced mucositis. We studied eight childhood cancer patients (age 1.5 to 16 years) on two days, i.e. the day before chemotherapy and 3-5 days after. Chemotherapy-induced oral mucositis and diarrhea were scored on a WHO toxicity scale. Stable isotope tracers were used to measure first-pass splanchnic leucine uptake and whole-body leucine kinetics. Patients showed increased mucositis and/or diarrhea toxicity scores after chemotherapy. Systemic availability of enterally administered leucine was not significantly affected by chemotherapy. Interestingly however was that most of the children were already catabolic prior to start of a new cycle of chemotherapy. Therefore, all efforts should be directed at initiating enteral feeding even before start of chemotherapy in order to reduce catabolic state. Our data imply that this might be accomplished best by hydrolyzed formula. In Chapter 8 the efficacy and feasibility of a TGF-b2-enriched feeding for preventing oral and gastro-intestinal mucositis in childhood cancer patients were studied. The study was designed as a 2-period crossover, randomized, double-blinded, placebo controlled trial. Patients who had a high risk for developing mucositis and who would receive two comparable cycles of chemotherapy were eligible to the study. During one cycle of chemotherapy TGF-b2-enriched feeding was administered; during the other a ‘placebo’ (not enriched) feeding was used. WHO toxicity scales of diarrhea, oral mucositis, fever, anal lesions and nausea/vomiting were scored daily. In addition, the incidence of occurrence of blood cultures, antibiotic therapy and interventions or diagnostics related to mucositis were measured. The feasibility of the study was good: 83% of the patients completed two cycles and 86% of the study feeding was consumed. Administration of TGF-b2 was safe, as serum TGF-b2 did not ! increase and renal and liver function were not affected. The degree of toxicity, scored during the whole observation period and the number of days with WHO 3/4 toxicity did not significantly differ between cycles with TGF-b2 enriched and normal feeding. These studies do not provide evidence that TGF- decreases the incidence or degree of mucositis induced by combination therapy in childhood cancer-patients. In Part 3 all studies presented in this thesis are summarized, and new insights for future studies are discussed. http://repub.eur.nl/res/pub/35293/ 2007-08-01T00:00:00Z Chemotherapy will frequently induce intestinal damage (mucositis). Enteral nutrition is then often withheld for fear of impaired intestinal absorption as shown in animal models. There is no clinical evidence, however, that absorption is indeed compromised during chemotherapy-induced mucositis. The aim of this study was to evaluate systemic availability of dietary amino acids (leucine) during chemotherapy-induced mucositis. We studied eight childhood cancer patients (age 1.5-16 y) on 2 d, i.e. the day before chemotherapy and 3-5 d after. Chemotherapy-induced oral mucositis and diarrhea were scored on a World Health Organization toxicity scale. Stable isotope tracers were used to measure first-pass splanchnic leucine uptake and whole-body leucine kinetics. Patients showed increased mucositis and/or diarrhea toxicity scores (p < 0.0001) after chemotherapy. Systemic availability of enterally administered leucine was not significantly affected by chemotherapy (before 60%, after 90%, p = 0.46). Interestingly, five patients already showed a negative leucine balance before chemotherapy. In conclusion, most children receiving chemotherapy are already catabolic before start of a new cycle of chemotherapy. Amino acid transport as measured by leucine uptake in the intestine is not affected by chemotherapy-induced mucositis. http://repub.eur.nl/res/pub/35753/ 2007-08-01T00:00:00Z In the current study we aimed to gain insight into epithelial-mesenchymal cross-talk and progenitor compartment modulation during doxorubicin (DOX)-induced mucositis in mice. Intestinal segments were collected on various days after DOX treatment. DOX-induced damage at day 1-2 was characterized by increased epithelial proliferation and apoptosis and a decrease in the expression of epithelial differentiation markers. Concurrently, T-cell factor-4 (TCF4) levels increased and the epithelial differentiation enhancing factor, bone morphogenic protein-4 (BMP4), decreased. During severe damage (day 3), BMP4 levels were significantly increased, which inversely correlated with epithelial proliferation. At the same time, the expression of the epithelial differentiation markers was increasing again. At day 7, BMP4 levels were down-regulated, while the levels of the epithelial differentiation markers and TCF4 were normalized again. These data suggest that in response to DOX-induced damage, BMP4 and TCF4 are modulated in such a way that homeostasis of the progenitor compartment is partly preserved. http://repub.eur.nl/res/pub/37028/ 2007-05-01T00:00:00Z Background. Mucositis is one of the most frequent and severe side-effect of chemotherapy in childhood-cancer patients for which there is no prophylaxis available. The efficacy and feasibility of a TGF-β2-enriched feeding for preventing oral and gastro-intestinal-mucositis in childhood-cancer patients were studied. Procedure. The study was designed as a two-period cross-over, randomized, double-blinded, placebo, controlled trial. Patients who had a high risk for developing mucositis and who would receive two comparable cycles of chemotherapy were eligible for the study. During one cycle of chemotherapy, TGF-β2-enriched feeding was administered; during the other, a 'placebo' (not enriched) feeding was used. WHO toxicity scales of diarrhea, oral mucositis, fever, anal lesions and nausea/vomiting were scored daily. In addition, the incidence of occurrence of blood cultures, antibiotic therapy, and interventions or diagnostics related to mucositis were measured. Results. The feasibility of the study was good: 83% of the patients completed two cycles and 86% of the study-feeding was effectively consumed. Administration of TGF-β2was safe as serum TGF-β2did not increase, and renal and liver function were not affected during TGF-β2consumption compared to normal feeding. Differences in toxicity, scored during the whole observation period and the number of days with WHO 3/4 toxicity, were not significantly different between cycles with TGF-β2enriched and normal feeding. Conclusions. TGF-β2administration via feeding is well tolerated and safe. Although this study might have had limitations to show potential benefit of TGF-β2, it does not provide evidence that TGF-β2decreases the incidence or degree of mucositis induced by combination chemotherapy in childhood-cancer patients. http://repub.eur.nl/res/pub/35637/ 2007-01-01T00:00:00Z The mucin Muc2 or Mycin2 (Muc2), which is the main structural component of the protective mucus layer, has shown to be upregulated during chemotherapy-induced mucositis. As Muc2 has shown to have protective capacities, upregulation of Muc2 may be a counter reaction of the intestine protecting against mucositis. Therefore, increasing Muc2 protein levels could be a therapeutic target in mucositis prevention or reduction. Our aim was to determine the role of Muc2 in chemotherapy-induced mucositis. Mucositis was induced in Muc2 knockout (Muc2-/-) and wild type (Muc2+/+) mice by injecting methotrexate (MTX). Animals were weighed and sacrificed on Days 2-6 after MTX treatment and jejunal segments were analyzed. Before MTX treatment, the small intestine of Muc2+/+and Muc2-/-mice were similar with respect to epithelial morphology and proliferation. Moreover, sucrase-isomaltase and trefoil factor-3 protein expression levels were comparable between Muc2+/+and Muc2-/-mice. Up to Day 3 after MTX treatment, percentages of weight-loss did not differ. Thereafter, Muc2+/+mice showed a trend towards regaining weight, whereas Muc2-/-mice continued to lose weight. Surprisingly, MTX-induced intestinal damage of Muc2-/-and Muc2+/+mice was comparable. Prior to MTX-injection, tumor necrosis factor-α and interleukin-10 mRNAs were upregulated in Muc2-/-mice, probably due to continuous exposure of the intestine to luminal antigens. Muc2 deficiency does not lead to an increase in chemotherapy-induced mucositis. A possible explanation is the mechanism by which Muc2 deficiency may trigger the immune system to release interleukin-10, an anti-inflammatory cytokine before MTX-treatment. http://repub.eur.nl/res/pub/13256/ 2004-01-01T00:00:00Z In clinical studies, the oxidation of 13C-labeled substrates to 13CO2 and the measurement of the appearance of excess 13CO2 in expiratory air has progressed to an increasingly common method as it is noninvasive and lacks the radiation exposure associated with the use of 14C. The collection of respiratory CO2 currently used occurs via trapping of CO2 in sodium hydroxide (trapping method), sometimes in conjunction with indirect calorimetry. The aim of the present study was to determine the accuracy of our direct nasopharyngeal sampling method for the collection of breath samples in preterm infants compared with the currently used trapping method. We present a method that simplifies the collection of breath samples in preterm infants. Seven preterm infants with a gestational age of 26-29 wk were studied on different postnatal days (range, 8-52 d) while receiving full enteral feeding. A primed constant 3-h intragastric infusion of [13C]bicarbonate was given, and breath samples were collected by means of direct nasopharyngeal sampling and by a sodium hydroxide trap simultaneously. Breath CO2 isotopic enrichments rose rapidly to reach a plateau by 120 min with <5% variation of plateau in both methods. 13CO2 breath isotopic enrichments obtained by the direct nasopharyngeal sampling method correlated highly (r2 = 0.933; p < 0.0001) with the trapping method. The Bland-Altman analysis showed no significant variability between the two methods and demonstrated that the 95% confidence interval is within +/- 4.68 delta per thousand. These findings validate the simple method of direct nasopharyngeal sampling of expired air in neonates.