| 1. |
- Kullenberg, Fredrik
(författare)
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Anthracyclines : Toxicity and chemotherapy-induced mucositis
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Anthracyclines belong to a class of cytostatic compounds that are commonly used to treat various types of cancers, including lymphoma, breast cancer and primary liver cancer. The aim of this thesis was to study how two anthracyclines, doxorubicin and idarubicin, induced cytotoxicity in tumor cells, as well as their off-target effect on the gastrointestinal system. In Paper I, we exposed four different cancer cell lines to various concentrations of doxorubicin in two different formulations, and measured the cell viability and how much doxorubicin was taken up by the cells. We found that the cell lines differed in their uptake and sensitivity, and that the most resistant cell line had an intracellular exposure of doxorubicin that was about 100 times lower when compared to the more sensitive cell lines. To study the off-target gastrointestinal toxicity, we dosed rats with doxorubicin and studied how chemotherapy-induced mucositis developed during seven days in Paper II. The main effect parameter was intestinal villus atrophy, which was most severe after three days. This was preceded by an increased cell death and decreased proliferation in the crypts, which occurred within the first day after doxorubicin exposure. To study how different cytostatic drugs affected chemotherapy-induced mucositis, and to what extent they caused diarrhea, rats were dosed with one of six different chemotherapies, including doxorubicin and idarubicin, in Paper III. All selected chemotherapies caused similar villus atrophy three days after dosing, but only a third, including idarubicin, caused clear diarrhea. In paper IV the objective was to treat or prevent idarubicin-induced mucositis and diarrhea, by using the anti-inflammatory drugs anakinra and/or dexamethasone. Anakinra alone stopped the diarrhea, while the combination of anakinra and dexamethasone prevented villus atrophy. These positive effects encourage further investigations into the use of anakinra and dexamethasone as supportive therapies for chemotherapy-induced mucositis and diarrhea.The overall long-term impact of the different studies in this thesis is to increase the specific anti-tumoral effect of chemotherapies, while also alleviating the adverse effects. This would improve quality-of-life and treatment outcomes of cancer patients.
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| 2. |
- Peters, Karsten
(författare)
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Experimental Disruption of Intestinal Mucosal Homeostasis : Exploring the Protective Potential of Melatonin and Misoprostol
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The intestinal mucosa serves as a protective layer that separates the intestinal contents from the underlying tissues. It restricts harmful substances, pathogens, and undigested particles from entering the bloodstream. This mucosa also facilitates selective absorption of nutrients, electrolytes, and fluids, allowing essential substances to pass while maintaining a defense against potential threats. The integrity of the mucosa can be disrupted, such as in diseases or by off-target toxicities of chemotherapeutic drugs. A dysfunctional intestinal mucosa can result in inflammation, altered epithelial secretory and absorptive functions, as well as an increased mucosal permeability that may enable bacterial translocation. Chemotherapy-induced intestinal side effects may lead to dose reduction or even discontinuation of the treatment, but also decreasing the patient’s quality of life. The aim of this thesis was to explore the protective potential of melatonin and misoprostol on experimental disruption of small intestinal mucosal permeability and chemotherapy-induced mucositis. In Papers I and II an increase in intestinal mucosal permeability was induced by perfusing the jejunal segment with the surfactant sodium dodecyl sulfate (SDS) in rats. Melatonin and misoprostol were found to mitigate the induced increase in permeability. In Paper II it was shown that the melatonin receptor antagonist luzindole completely abolished the protective effect of melatonin on SDS-induced increase in mucosal permeability, showing that the effect of melatonin is receptor-mediated. In Papers III and IV off-target intestinal toxicity of the chemotherapeutic agents doxorubicin (DOX) and 5-fluorouracil (5-FU) were evaluated. In Paper III the progression of intestinal mucositis during seven consecutive days after a single injection of DOX was monitored. It was found that villus atrophy was most distinct after three days. In addition, within the first 24 hours after administration of DOX the most pronounced effect on a decrease in cell proliferation and an increase in crypt cell apoptosis was observed. In Paper IV it was found that daily administration of melatonin fully prevented villus atrophy and reduced the number of apoptotic crypts cells induced by a single injection of 5-FU. Administration of misoprostol increased colonic water contents but had no effect on 5-FU-induced villus atrophy or apoptosis. Furthermore, melatonin reduced 5-FU-induced cytotoxicity in murine intestinal organoids.In conclusion, the results suggest that melatonin might be a potential candidate for supportive therapy in diseases affecting the small intestinal mucosal barrier and in chemotherapy-induced mucositis.
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| 3. |
- Bergman, Ebba, 1977-
(författare)
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The Hepatobiliary Transport of Rosuvastatin In Vivo
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In vivo studies of hepatobiliary disposition are challenging. The hepatobiliary system is complex, as its physiological localization, complex cellular structure with numerous transporters and enzymes, and the interindividual variability in protein expression and biliary flow will all affect the in vivo disposition of a drug under investigation. The research included in this thesis has focused on the involvement of hepatic transport proteins in the hepatobiliary disposition of rosuvastatin. The impact that several transport inhibitors had on the pharmacokinetics of rosuvastatin was investigated in healthy volunteers and in pigs. The effects were considerable, following inhibition of sinusoidal transport proteins by cyclosporine and rifampicin. These inhibitors significantly reduced the hepatic extraction of rosuvastatin by 50 and 35%, respectively, and the plasma exposure increased by factors of 9.1 and 6.3, respectively. Drug-drug interactions (DDI) resulting in markedly higher plasma exposures are important from a drug safety perspective as increased extrahepatic exposure of statins is associated with an increased risk of severe side-effects, such as myopathy which in rare cases could develop into rhabdomyolysis. The DDI caused by cyclosporine and rifampicin can probably be attributed to inhibition of hepatic uptake transporters. In contrast, inhibition of canalicular transporters by imatinib did not significantly affect the pharmacokinetics of rosuvastatin, which suggests that the intracellular concentration of the inhibitor in the hepatocyte was insufficient to affect the transport of rosuvastatin, or that imatinib is not a sufficiently potent inhibitor in vivo. Furthermore, gemfibrozil administered as a single dose into the jejunum in healthy volunteers and pigs did not affect the plasma or biliary pharmacokinetics of rosuvastatin. The previously reported DDI in humans upon repeated dosing with gemfibrozil might be explained by the accumulation of metabolites able to affect the disposition of rosuvastatin. The investigations presented in this thesis conclude that transport proteins are of considerable importance for the hepatobiliary disposition of rosuvastatin in vivo. The Loc-I-Gut catheter can be applied for the investigation of biliary accumulation and to determine bile specific metabolites, however it has limitations when conducting quantitative measurements. In the porcine model, hepatic bile can be collected for up to six hours and enables the determination of the hepatic extraction in vivo.
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| 4. |
- Eriksson, Johanna, 1991-
(författare)
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Drug absorption in the lungs : studies in the isolated perfused rat lung model combined with physiologically based biopharmaceutics modelling
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pulmonary delivery of drugs is the preferred route of administration for treatment of local lung diseases like asthma and chronic obstructive pulmonary disease. Recently, there has also been increased interest in systemic delivery of drugs via the lungs to avoid problems with low and/or variable gastrointestinal absorption, and as a needle-free alternative for drugs that cannot be ingested. Both the pharmacological and the potentially adverse effects of inhaled drugs depend on the drug’s local and systemic concentrations, which in turn depend on the pulmonary absorption of the drug. Pulmonary drug absorption is governed by the dissolution, permeability, tissue retention, and non-absorptive clearance of the drug in the lungs. Predicting systemic and local exposure is necessary for developing an inhaled drug product, and these predictions can be based on data obtained from both in vitro and ex vivo methods, such as cell lines, solubility measurements, and the isolated perfused lung (IPL) model. Data obtained by these methods can then be used to inform physiologically based biopharmaceutics (PBB) models about drug-specific absorption parameters.The overall aim of this thesis was to increase the mechanistic understanding of pulmonary drug absorption, with a special focus on obtaining and analyzing ex vivo absorption parameters for different inhalation drugs and formulations, and evaluating the predictive power of these parameters in simulations of pulmonary drug absorption. In the first two papers of the thesis, drugs were formulated as solutions, suspensions, and dry powders, and pulmonary absorption of these were measured using the IPL model. The data from these experiments were then analyzed to obtain absorption parameters for each drug using a PBB model. Tissue retention was shown to be an important parameter for describing drug absorption in IPL, and particle wetting was shown to greatly affect the absorption of dry powders. Permeability in IPL correlated well with intrinsic permeability measured in cell monolayers, suggesting that passive transcellular transport is the main transport mechanism in the lungs. In the second two papers, the absorption parameters obtained from IPL data were used to simulate rat and human pulmonary drug absorption. The simulations predicted systemic exposure after inhalation well for both rat and human, suggesting that ex vivo parameters can be used to predict rat in vivo and human plasma concentrations. This thesis deepens our understanding of absorption parameters involved in pulmonary drug absorption, and suggests applications for these parameters in predictions of local and systemic exposure after inhalation.
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| 5. |
- Matsson, Elin, 1979-
(författare)
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In vivo Pharmacokinetics of Two New Thrombin Inhibitor Prodrugs : Emphasis on Intestinal and Hepatobiliary Disposition and the Influence of Interacting Drugs
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biliary excretion is an important elimination route for many drugs and metabolites. For such compounds, it is important to know the extent of excretion and drug exposure in the bile, e.g., for the risk assessment of drug interactions, liver toxicity and the effects of genetic variants. In this thesis, duodenal aspiration of bile was performed in healthy volunteers and complemented with experiments in an in vivo model in pigs to increase the understanding of the intestinal and hepatobiliary disposition of two direct thrombin inhibitors. The compounds investigated, ximelagatran and AZD0837, are both prodrugs that require bioactivation to exert their pharmacological effect. Upon co-administration with erythromycin and ketoconazole, respectively, altered plasma exposure to ximelagatran and AZD0837 and their respective metabolites has been observed. The main objective of this thesis was to characterize the biliary excretion of the compounds, and investigate whether this elimination route explains the observed drug-drug interactions. High plasma-to-bile AUC ratios were observed, in particular for ximelagatran, its active metabolite melagatran, and AR-H067637, the active metabolite of AZD0837. These high ratios indicate the involvement of active transporters in the biliary excretion of the compounds, which is important since transporters constitute possible sites for drug interactions. The effects of erythromycin and ketoconazole on the plasma exposure of the prodrugs and metabolites were confirmed in both the pig and the clinical studies. The changes seen in plasma for ximelagatran and its metabolites were partly explained by reduced biliary clearance. Inhibited CYP3A4 metabolism likely caused the elevated plasma levels of AZD0837, whereas reduced biliary clearance was seen for AR-H067637 suggesting an effect on its excretion into bile. In summary, the studies led to mechanistic insights in the hepatobiliary disposition of ximelagatran and AZD0837, and demonstrate the value of combined clinical and animal studies for the investigation of the biliary drug excretion.
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| 6. |
- Degerstedt, Oliver
(författare)
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Translational Tumor Drug Delivery : Doxorubicin formulation performance, intracellular uptake and molecular diffusion
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Globally, hepatocellular carcinoma (HCC) is the most common form of liver cancer and a leading cause of cancer death. One important risk factor is liver cirrhosis and the disease progression is characterized by deposition of extracellular matrix proteins that form a fibrous network, which increases liver stiffness and may limit the effectiveness of different treatment strategies. The overall aim of this thesis was to investigate the anticancer drug doxorubicin (DOX) and its clinically relevant drug delivery systems from an in vitro perspective. The focus was on developing and using qualitative and quantitative methods to better understand formulation performance, intracellular uptake and molecular diffusion. The experimental in vitro findings were then translated to clinical scenarios using physiologically based pharmacokinetic (PBPK) modelling.The performance of clinically employed emulsion formulations containing DOX and the tumor accumulating oil Lipiodol® were evaluated in terms of their stability (Paper I). The most stable emulsion (> 72 h) was achieved when using an aqueous phase containing the contrast agent iohexol and with an aqueous to lipid phase ratio of 1:4 to assure formation of a water-in-oil emulsion. This was followed by a cell-based study (Paper II) where nanoformulated DOX was compared to DOX in solution in terms of tumor cell toxicity, intracellular DOX uptake and intracellular formation of the main active metabolite doxorubicinol (DOXol). DOX in solution was more potent in all investigated cell lines, where the most sensitive cells (HepG2) displayed IC50 values that were approximately 100 times lower than the most resistant cell line (SNU449). This was explained by the rapid intracellular uptake in HepG2 cells which was also confirmed with a complimentary miniaturized chip technique in Paper IV. In papers III and IV the focus was on molecular diffusion across biomimetic hydrogels mimicking tissue properties of cirrhotic liver and early stage HCC. The diffusion of DOX was significantly reduced in biomimetic gels as compared with more commonly used agarose gels, however the presence of human liver tumor cells did not significantly influence diffusion. Simulations using a developed PBPK and spatio-temporal tissue concentration model suggested that a liver tumor resembling SNU449 cells would not reach therapeutic exposure levels in a clinical scenario while the diffusion of DOX required further reduction by the tumor extracellular matrix in order to generate tumor concentration-time curves consistent with in vivo observations.This thesis contributes to an increased understanding of using DOX and its drug delivery systems as a treatment option for HCC. The approach of translating in vitro experimental data to clinical scenarios using modelling will grow in relevance as methods become more complex and data more bio-relevant.
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| 7. |
- Dubbelboer, Ilse R
(författare)
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Biopharmaceutical investigations of doxorubicin formulations used in liver cancer treatment : Studies in healthy pigs and liver cancer patients, combined with pharmacokinetic and biopharmaceutical modelling
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- There are currently two types of drug formulation in clinical use in the locoregional treatment of intermediate hepatocellular carcinoma (HCC). In the emulsion LIPDOX, the cytostatic agent doxorubicin (DOX) is dissolved in the aqueous phase, which is emulsified with the oily contrast agent Lipiodol® (LIP). In the microparticular system DEBDOX, DOX is loaded into the drug-eluting entity DC Bead™.The overall aim of the thesis was to improve pharmaceutical understanding of the LIPDOX and DEBDOX formulations, in order to facilitate the future development of novel drug delivery systems. In vivo release of DOX from the formulations and the disposition of DOX and its active metabolite doxorubicinol (DOXol) were assessed in an advanced multisampling-site acute healthy pig model and in patients with HCC. The release of DOX and disposition of DOX and DOXol where further analysed using physiologically based pharmacokinetic (PBPK) and biopharmaceutical (PBBP) modelling. The combination of in vivo investigations and in silico modelling could provide unique insight into the mechanisms behind drug release and disposition.The in vivo release of DOX from LIPDOX is not extended and controlled, as it is from DEBDOX. With both formulations, DOX is released as a burst during the early phase of administration. The in vivo release of DOX from LIPDOX was faster than from DEBDOX in both pigs and patients. The release from DEBDOX was slow and possibly incomplete. The in vivo release of DOX from LIPDOX and DEBDOX could be described by using the PBBP model in combination with in vitro release profiles.The disposition of DOX and DOXol was modelled using a semi-PBPK model containing intracellular binding sites. The contrast agent Lipiodol® did not affect the hepatobiliary disposition of DOX in the pig model. The control substance used in this study, cyclosporine A, inhibited the biliary excretion of DOX and DOXol but did not alter metabolism in healthy pigs. The disposition of DOX is similar in healthy pigs and humans, which was shown by the ease of translation of the semi-PBPK pig model to the human PBBP model.
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| 8. |
- Lilienberg, Elsa, 1984-
(författare)
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Biopharmaceutical Evaluation of Intra-arterial Drug-Delivery Systems for Liver Cancer : Investigations in healthy pigs and liver cancer patients
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- There are currently two types of intra-arterial drug-delivery system (DDS) in clinical use in the palliative treatment of primary liver cancer. The chemotherapeutic drug doxorubicin (DOX) can be formulated into a drug-in-lipiodol emulsion (LIPDOX) or a microparticulate drug-eluting bead system (DEBDOX). To facilitate development of future DDSs, we need to understand the release and local distribution of drug from these DDSs into the complex, in vivo, pathological environment.The overall aim of this project was to assess and improve understanding of the in vivo release of DOX from LIPDOX and DEBDOX and its local disposition in the liver. These processes were investigated in detail in a multisampling-site, healthy pig model and in human patients with liver cancer. The mechanisms involved in DOX disposition were studied by examining potential interactions between DOX and lipiodol and/or cyclosporine A (CsA) in pigs. In this project, the main elimination pathway for DOX and its primary metabolite doxorubicinol (DOXol) was via bile; their extensive canalicular carrier-mediated transport (e.g. ATP-binding cassette transporters ABCB1, ABCC1, ABCC2 and ABCG2) was inhibited by CsA. CsA had no effect on the carbonyl and aldo-keto reductases responsible for the metabolism of DOX into DOXol. LIPDOX released DOX more rapidly and to a greater extent into the circulation than DEBDOX, which had only released 15% of the dose in patients after 24 hrs. The systemic exposure to DOX was lower for DEBDOX than for LIPDOX. Greater fractions of DOXol were formed in blood and bile with LIPDOX than with DEBDOX. This may have been because DOX was more widely distributed into regions with increased metabolic capacity or because of increased intracellular uptake when DOX was delivered in LIPDOX. The excipient lipiodol in the LIPDOX formulation did not interact with transporters, enzymes or membranes that would explain the increased cellular uptake of DOX.In conclusion, the release of DOX from DEBDOX is more controlled in vivo than that from LIPDOX, indicating that DEBDOX is a more robust pharmaceutical product. The formulations for future optimized DDSs should therefore be more similar to DEBDOX than to LIPDOX.
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| 9. |
- Lundahl, Anna, 1980-
(författare)
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In vivo Pharmacokinetic Interactions of Finasteride and Identification of Novel Metabolites
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The general aim of this thesis was to improve the understanding of the in vivo pharmacokinetics and, in particular, the metabolism of finasteride, a 5α-reductase inhibitor used in the treatment of enlarged prostate glands and male pattern baldness. CYP3A4 has been identified as the major enzyme involved in the sequential metabolism of finasteride to ω-OH finasteride (M1) and ω-COOH finasteride (M3). The consequences of induced and inhibited metabolism on the pharmacokinetics of finasteride and its metabolites were investigated in humans and pigs. Both studies included bile collection. The collected human and pig samples were used for the metabolite identification. As expected, induced metabolism led to reduced plasma exposure of finasteride and inhibited metabolism had the opposite effect. The interactions were investigated in detail and included examination of the biliary pharmacokinetics of finasteride and its metabolites. In pigs, the study included monitoring of the hepatic extraction over time, deconvolution and the development of a semi-physiological model for comparison of the effects on the gut wall and liver metabolism. For M3, the concentration ratios of bile to plasma and the renal clearance indicated that carrier-mediated processes are involved in the biliary and urinary excretion. This was not, however, the case for finasteride. The metabolite, M1, could not be quantified either in humans or pigs. Instead, two other OH metabolites, M1 isomers, were identified in humans. These metabolites were found to undergo glucuronide conjugation. In humans, one glucuronide was identified intact and in pigs, both glucuronides were identified intact in bile and in urine. In addition, a glucuronide of M3 was identified in human bile. In conclusion, advances have been made in the understanding of the pharmacokinetics of finasteride, in particular in relation to the metabolism. Hopefully, the findings of this comprehensive investigation can be applied to other drugs and novel chemical entities.
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| 10. |
- Thörn, Helena Anna, 1981-
(författare)
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First-pass Intestinal Metabolism of Drugs : Experiences from in vitro, in vivo and simulation studies
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The bioavailability of a drug can be described as the fraction of an orally administered dose that reaches the systemic circulation and is often limited by first-pass metabolism in the gut and the liver. It is important to have knowledge about these processes since the systemic blood drug concentration is tightly connected to the effect of the drug. The general aim of this project was to quantitatively examine the role of the intestine in relation to the liver in first-pass metabolism of orally administered drugs. The first-pass metabolism of verapamil and raloxifene was investigated in detail with in vivo, in vitro and simulation studies, using the pig as an experimental model. The intestine contributed to the same extent as the liver to first-pass metabolism of R/S-verapamil in vivo in pigs. The S-isomer of verapamil was found in lower plasma concentrations compared to the R-isomer after oral dosing. The in vitro metabolism of verapamil in pig and human liver showed interspecies similarity and indicated equal intrinsic clearance for R- and S-verapamil. Through physiologically based pharmacokinetic modeling the stereoselectivity was explained by a combination of several processes, including enantioselective plasma protein binding, blood-to-plasma partition, and gut and liver tissue distribution. For raloxifene the intestine was the dominating organ in first-pass glucuronidation in vivo in pigs. Furthermore, the raloxifene concentration entering the intestine or the dose administered in the gut did not influence the plasma PK of raloxifene and indicated that the intestinal metabolism was not saturable with clinical relevant doses. For both verapamil and raloxifene, a time-dependent hepatic metabolism was noted with major consequences to the pharmacokinetic of the drugs. This project has pointed out the importance of intestinal metabolism in the overall first-pass extraction of drugs and indicates that intestinal metabolism should be considered and evaluated early in drug development.
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