Chemotherapy induced intestinal mucositis; from bench to bed

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

Review: Ontogeny of oral drug absorption processes in children

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

Standardized and Individualized Parenteral Nutrition Mixtures in a Pediatric Home Parenteral Nutrition Population

OBJECTIVES: Studies evaluating efficacy or safety of standardized parenteral nutrition (PN) versus individualized PN are lacking. We aimed to assess effects on growth and safety of standardized PN compared with individualized PN in our Home PN group. METHODS: Descriptive cohort study in Dutch children on Home PN, in which standardized PN was compared with individualized PN. Both groups received similar micronutrient-supplementation. Primary outcome was growth over 2 years, secondary outcomes were electrolyte disturbances and biochemical abnormalities. Additionally, patients were matched for age to control for potential confounding characteristics. RESULTS: Fifty patients (50% girls, median age 6.5 years) were included, 16 (32%) received standardized PN mixtures. Age (11 vs 5 years), gestational age (39.2 vs 36.2 weeks) and PN duration (97 vs 39 months) were significantly higher in the group receiving standardized PN (P: ≤0.001; 0.027; 0.013 respectively). The standardized PN group showed an increase in weight-for-age (WFA), compared with a decrease in the individualized PN group (+0.38 SD vs -0.55 SD, P: 0.003). Electrolyte disturbances and biochemical abnormalities did not differ. After matching for age, resulting in comparable groups, no significant differences were demonstrated in WFA, height-for-age, or weight-for-height SD change. CONCLUSIONS: In children with chronic IF, over 2,5 years of age, standardized PN mixtures show a comparable effect on weight, height, and weight for height when compared with individualized PN mixtures. Also, standardized PN mixtures (with added micronutrients) seem noninferior to individualized PN mixtures in terms of electrolyte disturbances and basic biochemical abnormalities. Larger studies are needed to confirm these conclusions. TRIAL REGISTRATION: Academical Medical Center medical ethics committee number W18_079 #18.103

Proteomics of human liver membrane transporters: a focus on fetuses and newborn infants

Background: Hepatic membrane transporters are involved in the transport of many endogenous and exogenous compounds, including drugs. We aimed to study the relation of age with absolute transporter protein expression in a cohort of 62 mainly fetus and newborn samples. Methods: Protein expressions of BCRP, BSEP, GLUT1, MCT1, MDR1, MRP1, MRP2, MRP3, NTCP, OCT1, OATP1B1, OATP1B3, OATP2B1 and ATP1A1 were quantified with LC-MS/MS in isolated crude membrane fractions of snap-frozen post-mortem fetal and pediatric, and surgical adult liver samples. mRNA expression was quantified using RNA sequencing, and genetic variants with TaqMan assays. We explored relationships between protein expression and age (gestational age [GA], postnatal age [PNA], and postmenstrual age); between protein and mRNA expression; and between protein expression and genotype. Results: We analyzed 36 fetal (median GA 23.4 weeks [range 15.3–41.3]), 12 premature newborn (GA 30.2 weeks [24.9–36.7], PNA 1.0 weeks [0.14–11.4]), 10 term newborn (GA 40.0 weeks [39.7–41.3], PNA 3.9 weeks [0.3–18.1]), 4 pediatric (PNA 4.1 years [1.1–7.4]) and 8 adult liver samples. A relationship with age was found for BCRP, BSEP, GLUT1, MDR1, MRP1, MRP2, MRP3, NTCP, OATP1B1 and OCT1, with the strongest relationship for postmenstrual age. For most transporters mRNA and protein expression were not correlated. No genotype-protein expression relationship was detected. Discussion and conclusion: Various developmental patterns of protein expression of hepatic transporters emerged in fetuses and newborns up to four months of age. Postmenstrual age was the most robust factor predicting transporter expression in this cohort. Our data fill an important gap in current pediatric transporter ontogeny knowledge

Ontogeny of human hepatic and intestinal transporter gene expression during childhood: Age matters

Many drugs prescribed to children are drug transporter substrates. Drug transporters are membrane-bound proteins that mediate the cellular uptake or efflux of drugs and are important to drug absorption and elimination. Very limited data are available on the effect of age on transporter expression. Our study assessed age-related gene expression of hepatic and intestinal drug transporters. Multidrug resistance protein 2 (MRP2), organic anion transporting polypeptide 1B1 (OATP1B1), and OATP1B3 expression was determined in postmortem liver samples (fetal n = 6, neonatal n = 19, infant n = 7, child n = 2, adult n = 11) and multidrug resistance 1 (MDR1) expression in 61 pediatric liver samples. Intestinal expression of MDR1, MRP2, and OATP2B1 was determined in surgical small bowel samples (neonates n = 15, infants n = 3, adults n = 14). Using real-time reverse-transcription polymerase chain reaction, we measured fetal and pediatric gene expression relative to 18S rRNA (liver) and villin (intestines), and we compared it with adults using the 22 -Delta;Delta;Ct method. Hepatic expression of MRP2, OATP1B1, and OATP1B3 in all pediatric age groups was significantly lower than in adults. Hepatic MDR1 mRNA expression in fetuses, neonates, and infants was significantly lower than in adults. Neonatal intestinal expressions of MDR1 and MRP2 were comparable to those in adults. Intestinal OATP2B1 expression in neonates was significantly higher than in adults. We provide new data that show organ- and transporter-dependent differences in hepatic and intestinal drug transporter expression in an age-dependent fashion. This suggests that substrate drug absorption mediated by these trans