Review on the effect of chemotherapy on the intestinal barrier: Epithelial permeability, mucus and bacterial translocation.

Chemotherapy kills fast-growing cells including gut stem cells. This affects all components of the physical and functional intestinal barrier, i.e., the mucus layer, epithelium, and immune system. This results in an altered intestinal permeability of toxic compounds (e.g., endotoxins) as well as luminal bacterial translocation into the mucosa and central circulation. However, there is uncertainty regarding the relative contributions of the different barrier components for the development of chemotherapy-induced gut toxicity. This review present an overview of the intestinal mucosal barrier determined with various types of molecular probes and methods, and how they are affected by chemotherapy based on reported rodent and human data. We conclude that there is overwhelming evidence that chemotherapy increases bacterial translocation, and that it affects the mucosal barrier by rendering the mucosa more permeable to large permeability probes. Chemotherapy also seems to impede the intestinal mucus barrier, even though this has been less clearly evaluated from a functional standpoint but certainly plays a role in bacteria translocation. Combined, it is however difficult to outline a clear temporal or succession between the different gastrointestinal events and barrier functions, especially as chemotherapy-induced neutropenia is also involved in intestinal immunological homeostasis and bacterial translocation. A thorough characterization of this would need to include a time dependent development of neutropenia, intestinal permeability, and bacterial translocation, ideally after a range of chemotherapeutics and dosing regimens.

Anakinra and dexamethasone treatment of idarubicin-induced mucositis and diarrhoea in rats.

Chemotherapy-induced mucositis, characterized by diarrhoea and villous atrophy, is a severe side effect contributing to reduced quality of life and premature death in cancer patients treated with cytostatics. Despite its high incidence, there is no effective supportive therapy available. The main objective of this study was to determine if the anti-inflammatory drugs anakinra and/or dexamethasone-which have different mechanisms-of-action-might be used to effectively treat idarubicin-induced mucositis in rats. Mucositis was induced through a single injection with 2 mg/kg idarubicin (with saline as control), followed by daily treatments of anakinra (100 mg/kg/day), dexamethasone (10 mg/kg/day) or both for 3 days. After 72 h, jejunal tissue was collected for morphological, apoptotic and proliferative analyses, and colonic faecal water content and body weight change were determined. The diarrhoea that was induced by idarubicin (from 63.5% to 78.6% water content in faeces) was completely reversed by anakinra alone, and the jejunal villus height reduction by 36% was prevented by a combination of anakinra and dexamethasone. Dexamethasone reduced apoptosis in the jejunal crypts, both alone and in combination with anakinra. These positive effects encouraged further investigations into the use of anakinra and dexamethasone as supportive therapies for chemotherapy-induced intestinal mucositis and diarrhoea.

Study protocol for locoregional precision treatment of hepatocellular carcinoma with transarterial chemoembolisation (TACTida), a clinical study: idarubicin dose selection, tissue response and survival.

INTRODUCTION: Hepatocellular carcinoma (HCC) is a common cause of cancer-related death, often detected in the intermediate stage. The standard of care for intermediate-stage HCC is transarterial chemoembolisation (TACE), where idarubicin (IDA) is a promising drug. Despite the fact that TACE has been used for several decades, treatment success is unpredictable. This clinical trial has been designed believing that further improvement might be achieved by increasing the understanding of interactions between local pharmacology, tumour targeting, HCC pathophysiology, metabolomics and molecular mechanisms of drug resistance. METHODS AND ANALYSIS: The study population of this single-centre clinical trial consists of adults with intermediate-stage HCC. Each tumour site will receive TACE with two different IDA doses, 10 and 15 mg, on separate occasions. Before and after each patient's first TACE blood samples, tissue and liquid biopsies, and positron emission tomography (PET)/MRI will be performed. Blood samples will be used for pharmacokinetics (PK) and liver function evaluation. Tissue biopsies will be used for histopathology analyses, and culturing of primary organoids of tumour and non-tumour tissue to measure cell viability, drug response, multiomics and gene expression. Multiomics analyses will also be performed on liquid biopsies. PET/MRI will be used to evaluate tumour viability and liver metabolism. The two doses of IDA will be compared regarding PK, antitumour effects and safety. Imaging, molecular biology and multiomics data will be used to identify HCC phenotypes and their relation to drug uptake and metabolism, treatment response and survival. ETHICS AND DISSEMINATION: Participants give informed consent. Personal data are deidentified. A patient will be withdrawn from the study if considered medically necessary, or if it is the wish of the patient. The study has been approved by the Swedish Ethical Review Authority (Dnr. 2021-01928) and by the Medical Product Agency, Uppsala, Sweden. TRIAL REGISTRATION NUMBER: EudraCT number: 2021-001257-31.

Evaluation and validation of chemotherapy-specific diarrhoea and histopathology in rats.

Chemotherapy-induced mucositis is characterized by diarrhoea and villous atrophy. However, it is not well-understood why diarrhoea arises, why it only occurs with some chemotherapeutics and how it is related to villus atrophy. The objectives in this study were to determine (i) the relationship between chemotherapy-induced diarrhoea and villus atrophy and to (ii) establish and validate a rat diarrhoea model with clinically relevant endpoints. Male Wistar Han IGS rats were treated with saline, doxorubicin, idarubicin, methotrexate, 5-fluorouracil, irinotecan or 5-fluorouracil+irinotecan. After 72 h, jejunal tissue was taken for morphological, apoptotic and proliferative analyses, and faecal water content and change in body weight were determined. All treatments except methotrexate caused a similar reduction (≈42%) in villus height, but none of them altered mucosal crypt cell proliferation or apoptosis. Doxorubicin, idarubicin, irinotecan and 5-fluorouracil+irinotecan caused body weight reduction, but only irinotecan and idarubicin caused diarrhoea. No direct correlation between diarrhoea and villus height or body weight loss was observed. Therefore, studies of the mechanisms for chemotherapy-induced diarrhoea should focus on functional factors. Finally, the irinotecan and idarubicin diarrhoea models established in this study will be useful in developing supportive treatments of this common and serious adverse effect in patients undergoing chemotherapy.

Effects of α2-adrenoceptor stimulation on luminal alkalinisation and net fluid flux in rat duodenum.

The sympathetic nervous system is highly involved in the regulation of gastrointestinal functions such as luminal alkalinisation and fluid absorption. However, the exact mechanisms are not clear. This study aimed to delineate how α2-adrenergic receptor stimulation reduces duodenal luminal alkalinisation and induces net fluid absorption. This was tested by perfusing the duodenum of anesthetized rats with isotonic solutions devoid of Cl- and/or Na+, in the absence and presence of the α2-adrenoceptor agonist clonidine. The clonidine was also studied in rats treated with dimethylamiloride (a Na+/H+ exchange inhibitor), vasoactive intestinal peptide, and the nicotinic receptor antagonist hexamethonium. Clonidine reduced luminal alkalinisation and induced net fluid absorption. The Cl--free solution decreased luminal alkalinisation and abolished net fluid absorption, but did not prevent clonidine from doing so. Both the Na+-free solution and luminal dimethylamiloride increased luminal alkalinisation and abolished net fluid absorption, effects counteracted by clonidine. The NaCl-free solution (D-mannitol) did not affect luminal alkalinisation, but reduced net fluid absorption. Clonidine reduced luminal alkalinisation and induced net fluid absorption in rats perfused luminally with mannitol. However, clonidine did not affect the vasoactive intestinal peptide-induced increase in luminal alkalinisation or fluid secretion. Pre-treatment with hexamethonium abolished the effects of clonidine on luminal alkalinisation and net fluid flux. In summary, our in vivo experiments showed that clonidine-induced reduction in luminal alkalinisation and induction of net fluid absorption was unrelated to luminal Na+ and Cl-, or to apical Na+/H+ or Cl-/HCO3- exchangers. Instead, clonidine seems to exert its effects via suppression of nicotinic receptor-activated acetylcholine secretomotor neurons.

Chemotherapeutics Combined with Luminal Irritants: Effects on Small-Intestinal Mannitol Permeability and Villus Length in Rats.

Chemotherapy causes intestinal mucositis, which includes villous atrophy and altered mucosal barrier function. However, there is an uncertainty regarding how the reduced small-intestinal surface area affects the mucosal permeability of the small marker probe mannitol (MW 188), and how the mucosa responds to luminal irritants after chemotherapy. The aims in this study were to determine (i) the relationship between chemotherapy-induced villus atrophy and the intestinal permeability of mannitol and (ii) how the mucosa regulate this permeability in response to luminal ethanol and sodium dodecyl sulfate (SDS). This was investigated by treating rats with a single intraperitoneal dose of doxorubicin, irinotecan, or 5-fluorouracil. After 72 h, jejunum was single-pass perfused and mannitol permeability determined at baseline and after 15 min luminal exposure to 15% ethanol or 5 mg/mL SDS. Tissue samples for morphological analyses were sampled from the perfused segment. All three chemotherapeutics caused a similar 30% reduction in villus length. Mannitol permeability increased with irinotecan (1.3-fold) and 5-fluorouracil (2.5-fold) and was reduced with doxorubicin (0.5-fold), suggesting that it is not epithelial surface area alone that regulates intestinal permeability to mannitol. There was no additional increase in mannitol permeability induced by luminal ethanol or SDS in the chemotherapy-treated rats compared to controls, which may be related to the relatively high basal permeability of mannitol compared to other common low-permeability probes. We therefore suggest that future studies should focus on elucidating the complex interplay between chemotherapy in combination with luminal irritants on the intestinal permeability of other probes.

Melatonin-Activated Receptor Signaling Pathways Mediate Protective Effects on Surfactant-Induced Increase in Jejunal Mucosal Permeability in Rats.

A well-functional intestinal mucosal barrier can be compromised as a result of various diseases, chemotherapy, radiation, and chemical exposures including surfactants. Currently, there are no approved drugs targeting a dysfunctional intestinal barrier, which emphasizes a significant medical need. One candidate drug reported to regulate intestinal mucosal permeability is melatonin. However, it is still unclear if its effect is primarily receptor mediated or antioxidative, and if it is associated with enteric neural pathways. The aim of this rat intestinal perfusion study was to investigate the mechanisms of melatonin and nicotinic acetylcholine receptors on the increase in intestinal mucosal clearance of 51Cr-labeled ethylenediaminetetraacetate induced by 15 min luminal exposure to the anionic surfactant, sodium dodecyl sulfate. Our results show that melatonin abolished the surfactant-induced increase in intestinal permeability and that this effect was inhibited by luzindole, a melatonin receptor antagonist. In addition, mecamylamine, an antagonist of nicotinic acetylcholine receptors, reduced the surfactant-induced increase in mucosal permeability, using a signaling pathway not influenced by melatonin receptor activation. In conclusion, our results support melatonin as a potentially potent candidate for the oral treatment of a compromised intestinal mucosal barrier, and that its protective effect is primarily receptor-mediated.

Chemotherapeutics-Induced Intestinal Mucositis: Pathophysiology and Potential Treatment Strategies.

The gastrointestinal tract is particularly vulnerable to off-target effects of antineoplastic drugs because intestinal epithelial cells proliferate rapidly and have a complex immunological interaction with gut microbiota. As a result, up to 40-100% of all cancer patients dosed with chemotherapeutics experience gut toxicity, called chemotherapeutics-induced intestinal mucositis (CIM). The condition is associated with histological changes and inflammation in the mucosa arising from stem-cell apoptosis and disturbed cellular renewal and maturation processes. In turn, this results in various pathologies, including ulceration, pain, nausea, diarrhea, and bacterial translocation sepsis. In addition to reducing patient quality-of-life, CIM often leads to dose-reduction and subsequent decrease of anticancer effect. Despite decades of experimental and clinical investigations CIM remains an unsolved clinical issue, and there is a strong consensus that effective strategies are needed for preventing and treating CIM. Recent progress in the understanding of the molecular and functional pathology of CIM had provided many new potential targets and opportunities for treatment. This review presents an overview of the functions and physiology of the healthy intestinal barrier followed by a summary of the pathophysiological mechanisms involved in the development of CIM. Finally, we highlight some pharmacological and microbial interventions that have shown potential. Conclusively, one must accept that to date no single treatment has substantially transformed the clinical management of CIM. We therefore believe that the best chance for success is to use combination treatments. An optimal combination treatment will likely include prophylactics (e.g., antibiotics/probiotics) and drugs that impact the acute phase (e.g., anti-oxidants, apoptosis inhibitors, and anti-inflammatory agents) as well as the recovery phase (e.g., stimulation of proliferation and adaptation).

Antibody-Drug Conjugates and Targeted Treatment Strategies for Hepatocellular Carcinoma: A Drug-Delivery Perspective.

Increased understanding of cancer biology, pharmacology and drug delivery has provided a new framework for drug discovery and product development that relies on the unique expression of specific macromolecules (i.e., antigens) on the surface of tumour cells. This has enabled the development of anti-cancer treatments that combine the selectivity of antibodies with the efficacy of highly potent chemotherapeutic small molecules, called antibody-drug conjugates (ADCs). ADCs are composed of a cytotoxic drug covalently linked to an antibody which then selectively binds to a highly expressed antigen on a cancer cell; the conjugate is then internalized by the cell where it releases the potent cytotoxic drug and efficiently kills the tumour cell. There are, however, many challenges in the development of ADCs, mainly around optimizing the therapeutic/safety benefits. These challenges are discussed in this review; they include issues with the plasma stability and half-life of the ADC, its transport from blood into and distribution throughout the tumour compartment, cancer cell antigen expression and the ADC binding affinity to the target antigen, the cell internalization process, cleaving of the cytotoxic drug from the ADC, and the cytotoxic effect of the drug on the target cells. Finally, we present a summary of some of the experimental ADC strategies used in the treatment of hepatocellular carcinoma, from the recent literature.