| 1. |
- Abrantes, João A.
(författare)
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Pharmacometric Approaches to Improve Dose Individualization Methods in Hemophilia A
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hemophilia A is a bleeding disorder caused by the lack of functional coagulation factor VIII (FVIII). The overall aim of this thesis was to improve dose individualization of FVIII replacement therapy in hemophilia A using pharmacometric approaches.A population pharmacokinetic (PK) model of FVIII activity following the administration of moroctocog alfa was developed based on data from a large heterogeneous cohort of moderate to severe hemophilia A patients. Body weight, age, neutralizing anti-FVIII inhibitors, race, and analytical assay were found to be significant predictors of FVIII activity PK. In addition, large inter-individual variability (IIV) and inter-occasion variability (IOV) was identified highlighting the need for dose individualization.High magnitudes of IOV are known to impair model-based therapeutic drug monitoring. Using a population PK model of FVIII activity, several approaches to handle IOV in Bayesian forecasting of individual PK parameters were assessed across a wide range of features. Considering IOV in Bayesian forecasting, but ignoring IOV in dose calculation, led to the most precise individualized doses, in particular, when sparse data was used.The dose-exposure-response relationship of FVIII replacement therapy remains unclear. A parametric repeated time-to-categorical event (RTTCE) model was developed to characterize the relationship between the dose of octocog alfa, plasma FVIII activity, bleeding frequency and severity, and covariates, using data from clinical trials. The bleeding hazard was found to decrease throughout time and to be affected by plasma FVIII activity and number of previous bleeds. Unexplained IIV in the bleeding hazard was found to be large.Bayesian forecasting based on the RTTCE model was used to predict the future occurrence of bleeds, and to contrast the predicted outcome using individual i) PK, ii) bleeding, and iii) PK, bleeding and covariate information, from data collected in clinical trials. The results support that individual bleed information can inform the optimization of prophylactic dosing regimens in severe hemophilia A patients.In summary, the pharmacometric approaches presented provide a valuable quantitative framework to improve dose individualization in hemophilia A. Furthermore, enhanced dosing has the potential to reduce bleeding frequency and to lower the high costs associated to treatment.
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| 2. |
- Ahnfelt, Emelie
(författare)
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In vitro evaluation of formulations used in the treatment of hepatocellular carcinoma
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hepatocellular carcinoma (HCC) causes ~ 600,000 deaths annually, making it the second most deadly cancer form. HCC is classified into five stages and for the intermediate HCC treatment, the two most commonly used drug delivery systems (DDSs) are lipiodol-based emulsions and drug-eluting beads. The aims of this thesis were to develop in vitro methods suitable for studying these DDSs. It is important to investigate the release mechanisms and release rates with relevant in vitro methods, as this can improve the understanding of the in vivo performance. Miniaturized in vitro methods with sample reservoirs separated from the release medium by a diffusion barrier were developed and shown to be suitable for studying drug release from particle DDSs (Paper I). In Paper II these methods were further developed and used to study the release of doxorubicin (DOX) from the clinically used drug-eluting beads. DOX release rates were affected by the method set-up and the characteristics of the release medium. The choice of method and volume of release medium could improve the in vivo-likeness of the in vitro release profiles. Applied theoretical models suggested a film-controlled type of DOX release mechanism from the beads when self-aggregation, DOX-bead interaction, and DOX deprotonation were taken into account.A micropipette-assisted microscopy method was used to further improve the understanding of the release mechanism of amphiphilic molecules from the beads (Paper III). A detailed analysis suggested an internal depletion-layer model dependent on molecular self-aggregation for the release. It was further suggested that a simple ion-exchange mechanism is unrealistic in physiological conditions.The important pharmaceutical factors for the emulsion-based formulations were investigated in Paper IV. DOX solubility, lipid phase distribution, and emulsion stability increased when the contrast agent iohexol was added. Also, an increase in release half-life (h) was observed from emulsions with iohexol.The in vitro methods and theoretical models presented in this thesis can be used during development and optimization of future DDSs.
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| 3. |
- Almokhtar, Mokhtar, 1977-
(författare)
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Expression and regulation of steroid metabolizing enzymes in cells of the nervous and skeletal systems : Special focus on vitamin D metabolism
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Little is known about the mechanisms of vitamin D actions in the brain and bone. In this study, the metabolism of vitamin D and its regulation in various cell cultures of the nervous and skeletal systems were examined.Human osteosarcoma Saos-2 cells, human primary osteoblasts (hOB) and murine motor neuron-like NSC-34 cells were found to express mRNA for all enzymes required in vitamin D3 metabolism as well as the vitamin D receptor (VDR) that mediates vitamin D actions. Also, production of 24,25-dihydroxyvitamin D3 was found in these cells. Studies on vitamin D metabolism in NSC-34 cells and in primary neuron-enriched cells from rat cerebral cortex indicate formation of a previously unknown major metabolite formed from 25-hydroxyvitamin D3. Evaluation of the NSC-34 cells suggests that this cell line could be a novel model for studies of neuronal vitamin D metabolism and its regulation by endogenous and exogenous compounds.Treatment with glucocorticoids down regulated mRNA expression for the CYP24A1 gene in Saos-2 and hOB cells. Additionally, the glucocorticoid prednisolone showed suppression of CYP24A1-mediated metabolism and CYP24A1 promoter activity in Saos-2 cells. In NSC-34 cells, CYP24A1 mRNA levels were up-regulated by prednisolone, 1α,25-dihydroxyvitamin D3 and its synthetic analogues, EB1089 and tacalcitol. Formation of an endogenous glucocorticoid, 11-deoxycortisol, was observed in Saos-2 cells. Effects of glucocorticoids on the vitamin D system in bone cells may contribute to the adverse side effects in long-term treatment with glucocorticoids. Also, there may be a correlation between the administration of corticosteroids and adverse effects in the CNS.Expression and effects of vitamin D on steroidogenic enzymes were studied in primary neuron-enriched rat cortex cells, primary rat astrocytes and human neuroblastoma SH-SY5Y cells. These different cell cultures all expressed CYP17A1, whereas only astrocytes expressed 3β-hydroxysteroid dehydrogenase (3β-HSD). 1α,25-Dihydroxyvitamin D3 suppressed mRNA levels and enzyme activity of CYP17A1 in SH-SY5Y cells and astrocytes. 1α,25-Dihydroxyvitamin D3 suppressed enzyme activity and mRNA levels of 3β-HSD in astrocytes. The results suggest that vitamin D-mediated regulation of CYP17A1 and 3β-HSD may play a role in the nervous system.The results presented here contribute to our understanding of vitamin D metabolism and effects of glucocorticoids in the brain and bone.
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| 4. |
- Alogheli, Hiba
(författare)
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Computational Studies of Macrocycles and Molecular Modeling of Hepatitis C Virus NS3 Protease Inhibitors
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Computational tools are utilized in the drug discovery process to discover, design, and optimize new therapeutics. One important approach is structure-based drug design which relies on knowledge about the 3D structure of the biological target. The first part of this work focuses on applying structure-based drug design for binding mode prediction of HCV NS3 protease inhibitors. The NS3 protease is a challenging target from a computational perspective as it contains an extended binding site. Binding mode predictions were performed for various classes of new acyclic and macrocyclic HCV NS3 protease inhibitors and was used in the design of new inhibitors. None of the synthetized inhibitors have been co-crystallized yet, which has made the evaluation of the suggested binding mode predictions challenging.Macrocycles are an interesting compound class in drug discovery due to their unique structural architecture, which can enable access to new chemical space. Macrocycles can successfully modulate difficult therapeutic targets, as exemplified in the development of protease inhibitors. Furthermore they can improve drug-like properties, such as cell permeability and bioavailability. The second part of this thesis focuses on macrocycles from a computational point of view. A data set of 47 clinically relevant macrocycles was compiled and used in these studies. First, two different docking protocols rigid docking of pre-generated conformers and flexible docking in Glide were evaluated and compared. The results showed that flexible docking in Glide was sufficient for docking of macrocycles with respect to accuracy and speed.The aim of the second study was to evaluate and compare the performance of the more general conformational analysis tools, MCMM and MTLMOD, with the recently developed macrocycle-specialized conformational sampling tools, Prime-MCS and MMBS. In most cases, the general conformational analysis tools (with enhanced parameter settings) performed equally well as compared to the macrocycle-specialized conformational sampling techniques. However, MMBS was superior at locating the global energy minimum conformation.Finally, calculation of the conformational energy penalty of protein-bound macrocycles was performed. The macrocycle data set was complemented with linear analogues that are similar either with respect to physicochemical properties or 2D fingerprints. The conformational energy penalties of these linear analogues were calculated and compared to the conformational energy penalties of the macrocycles. The complete data set of macrocycles and non-macrocycles in this study differ from previously published work addressing conformational energy penalties, since it covers a more extended area of chemical space. Furthermore, there was a weak correlation between the calculated conformational energy penalties and the flexibility of the structures.
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| 5. |
- Alskär, Linda C., 1985-
(författare)
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Improved Molecular Understanding of Lipid-Based Formulations : for Enabling Oral Delivery of Poorly Water-Soluble Drugs
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The majority of emerging drug candidates are not suited for conventional oral dosage forms, as they do not dissolve in the aqueous environment of the gastrointestinal (GI) tract. Consequently, a large number of enabling formulation strategies have emerged. One such strategy is to deliver the drug pre-dissolved in a lipid-based formulation (LBF), thereby bypassing the rate-limiting dissolution step. To date, only about 4% of the marketed oral drugs are delivered as LBFs. The limited use of this strategy is a result of the incomplete understanding of drug solubility in lipid vehicles, the reduced chemical stability of pre-dissolved drug, and the complex interplay between drug and formulation undergoing intestinal lipid processing. Hence, this thesis targeted an improved molecular understanding of lipid-based drug delivery to make an informed formulation development. In the first part of the thesis, drug solubility in LBF excipients and composed formulations was assessed. Through experimental studies of nearly forty compounds in nine excipients drug physicochemical properties related to solubility in these excipients were identified. The obtained data was used to develop in silico tools for prediction of drug solubility in excipients and formulations. The second part of the thesis focused on LBF performance in vitro and in vivo. Factors associated with the type of solid form that is precipitating during digestions was revealed, which provides an initial framework for understanding drug precipitation behaviour under physiological conditions. It was also shown that clinically relevant doses of LBF significantly increases intestinal drug solubilization as a result of GI lipid processing and bile secretion. Moreover, simultaneous assessment of digestion and absorption in vitro provided the same rank order of absorbed drug as the in vivo studies. Coadministration of LBF and drug was shown to be a promising alternative to pre-dissolved drug in the LBF. In summary, this thesis has improved the molecular understanding of factors that govern drug solubility in lipid vehicles and solid form of precipitated drug under digestive conditions. It was also proved that clinically relevant doses of LBFs significantly increase the intestinal drug solubilization, and proof-of-concept was shown for coadministration of LBF with solid drug as an alternative to drug-loaded LBF.
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| 6. |
- Alskär, Oskar
(författare)
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Mechanism-Based Modelling of Clinical and Preclinical Studies of Glucose Homeostasis
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Glucose is an important nutrient and energy source in the body. However, too high concentration in the blood is harmful and may lead to several complications developing over time. It was estimated that 5 million people in the world died from complications related to diabetes during 2015. Several hormones and physiological factors are involved in the regulation of glucose homeostasis. To evaluate different aspects of glucose homeostasis and the effect of interventions, such as pharmacological treatment, glucose tolerance tests can be performed. In a glucose tolerance test glucose is administered either orally or intravenously, blood is sampled frequently and analyzed for different biomarkers. Mechanism-based pharmacometric models is a valuable tool in drug development, which can be applied to increase the knowledge about complex systems such as glucose homeostasis, quantify the effects of drugs, generate more information from clinical trials and contribute to more efficient study design. In this thesis, a new comprehensive mechanism-based pharmacometric model was developed. The model is capable of describing the most important aspects of glucose homeostasis during glucose tolerance test in healthy individuals and patients with type 2 diabetes, over a wide range of oral and intravenous glucose doses. Moreover, it can simultaneously describe regulation of gastric emptying and glucose absorption, regulation of the incretin hormones GLP-1 and GIP, hepatic extraction of insulin and the incretin effect, regulation of glucagon synthesis and regulation of endogenous glucose production. In addition, an interspecies scaling approach was developed by scaling a previously developed clinical glucose insulin model to describe intravenous glucose tolerance tests performed in mice, rats, dogs, pigs and monkeys. In conclusion, the developed mechanism-based models in this thesis increases the knowledge about short term regulation of glucose homeostasis and can be used to investigate combination treatments, drugs with multiple effects, and translation of drug effects between species, leading to improved drug development of new antidiabetic compounds.
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| 7. |
- Alvarsson, Jonathan, 1981-
(författare)
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Ligand-based Methods for Data Management and Modelling
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Drug discovery is a complicated and expensive process in the billion dollar range. One way of making the drug development process more efficient is better information handling, modelling and visualisation. The majority of todays drugs are small molecules, which interact with drug targets to cause an effect. Since the 1980s large amounts of compounds have been systematically tested by robots in so called high-throughput screening. Ligand-based drug discovery is based on modelling drug molecules. In the field known as Quantitative Structure–Activity Relationship (QSAR) molecules are described by molecular descriptors which are used for building mathematical models. Based on these models molecular properties can be predicted and using the molecular descriptors molecules can be compared for, e.g., similarity. Bioclipse is a workbench for the life sciences which provides ligand-based tools through a point and click interface. The aims of this thesis were to research, and develop new or improved ligand-based methods and open source software, and to work towards making these tools available for users through the Bioclipse workbench. To this end, a series of molecular signature studies was done and various Bioclipse plugins were developed.An introduction to the field is provided in the thesis summary which is followed by five research papers. Paper I describes the Bioclipse 2 software and the Bioclipse scripting language. In Paper II the laboratory information system Brunn for supporting work with dose-response studies on microtiter plates is described. In Paper III the creation of a molecular fingerprint based on the molecular signature descriptor is presented and the new fingerprints are evaluated for target prediction and found to perform on par with industrial standard commercial molecular fingerprints. In Paper IV the effect of different parameter choices when using the signature fingerprint together with support vector machines (SVM) using the radial basis function (RBF) kernel is explored and reasonable default values are found. In Paper V the performance of SVM based QSAR using large datasets with the molecular signature descriptor is studied, and a QSAR model based on 1.2 million substances is created and made available from the Bioclipse workbench.
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| 8. |
- Bazov, Igor
(författare)
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Epigenetic Dysregulations in the Brain of Human Alcoholics : Analysis of Opioid Genes
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Neuropeptides are special in their expression profiles restricted to neuronal subpopulations and low tissue mRNA levels. Genetic, epigenetic and transcriptional mechanisms that define spatiotemporal expression of the neuropeptide genes have utmost importance for the formation and functions of neural circuits in normal and pathological human brain. This thesis focuses on regulation of transcription of the opioid/nociceptin genes, the largest neuropeptide family, and on identification of adaptive changes in these mechanisms associated with alcoholism as model human pathology. Two epigenetic mechanisms, the common for most cells in the dorsolateral prefrontal cortex (dlPFC) and the neuron-subpopulation specific that may orchestrate prodynorphin (PDYN) transcription in the human dlPFC have been uncovered. The first, repressive mechanism may operate through control of DNA methylation/demethylation in a short, nucleosome size promoter CpG island (CGI). The second mechanism may involve USF2, the sequence–specific methylation–sensitive transcription factor which interaction with its target element in the CpG island results in USF2 and PDYN co-expression in the same neurons. The short PDYN promoter CGI may function as a chromatin element that integrates cellular and environmental signals through changes in methylation and transcription factor binding. Alterations in USF2–dependent PDYN transcription are affected by the promoter SNP (rs1997794: T>C) under transition to pathological state, i.e. in the alcoholic brain. This and two other PDYN SNPs that are most significantly associated with alcoholism represent CpG-SNPs, which are differentially methylated in the human dlPFC. The T, low risk allele of the promoter SNP forms a noncanonical AP-1–binding element. JUND and FOSB proteins, which may form homo- or heterodimers have been identified as dominant constituents of AP-1 complex. The C, non-risk variant of the PDYN 3′ UTR SNP (rs2235749 SNP: C>T) demonstrated significantly higher methylation in alcoholics compared to controls. PDYN mRNA and dynorphin levels significantly and positively correlated with methylation of the PDYN 3′ UTR CpG-SNP suggesting its involvement in PDYN regulation. A DNA–binding factor with differential binding affinity for the T allele and methylated and unmethylated C alleles of the PDYN 3′ UTR SNP (the T allele specific binding factor, Ta-BF) has been discovered, which may function as a regulator of PDYN transcription. These findings emphasize the complexity of PDYN regulation that determines its expression in specific neuronal subpopulations and suggest previously unknown integration of epigenetic, transcriptional and genetic mechanisms that orchestrate alcohol–induced molecular adaptations in the human brain. Given the important role of PDYN in addictive behavior, the findings provide a new insight into fundamental molecular mechanisms of human brain disorder. In addition to PDYN in the dlPFC, the PNOC gene in the hippocampus and OPRL1 gene in central amygdala that were downregulated in alcoholics may contribute to impairment of cognitive control over alcohol seeking and taking behaviour.
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| 9. |
- Belfrage, Anna Karin, 1977-
(författare)
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Design and Synthesis of Hepatitis C Virus NS3 Protease Inhibitors : Targeting Different Genotypes and Drug-Resistant Variants
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Since the first approved hepatitis C virus (HCV) NS3 protease inhibitors in 2011, numerous direct acting antivirals (DAAs) have reached late stages of clinical trials. Today, several combination therapies, based on different DAAs, with or without the need of pegylated interferon-α injection, are available for chronic HCV infections. The chemical foundation of the approved and late-stage HCV NS3 protease inhibitors is markedly similar. This could partly explain the cross-resistance that have emerged under the pressure of NS3 protease inhibitors. The first-generation NS3 protease inhibitors were developed to efficiently inhibit genotype 1 of the virus and were less potent against other genotypes.The main focus in this thesis was to design and synthesize a new class of 2(1H)-pyrazinone based HCV NS3 protease inhibitors, structurally dissimilar to the inhibitors evaluated in clinical trials or approved, potentially with a unique resistance profile and with a broad genotypic coverage. Successive modifications were performed around the pyrazinone core structure to clarify the structure-activity relationship; a P3 urea capping group was found valuable for inhibitory potency, as were elongated R6 residues possibly directed towards the S2 pocket. Dissimilar to previously developed inhibitors, the P1’ aryl acyl sulfonamide was not essential for inhibition as shown by equally good inhibitory potency for P1’ truncated inhibitors. In vitro pharmacokinetic (PK) evaluations disclosed a marked influence from the R6 moiety on the overall drug-properties and biochemical evaluation of the inhibitors against drug resistant enzyme variants showed retained inhibitory potency as compared to the wild-type enzyme. Initial evaluation against genotype 3a displayed micro-molar potencies. Lead optimization, with respect to improved PK properties, were also performed on an advanced class of HCV NS3 protease inhibitors, containing a P2 quinazoline substituent in combination with a macro-cyclic proline urea scaffold with nano-molar cell based activities.Moreover, an efficient Pd-catalyzed C-N urea arylation protocol, enabling high yielding introductions of advanced urea substituents to the C3 position of the pyrazinone, and a Pd-catalyzed carbonylation procedure, to obtain acyl sulfinamides, were developed. These methods can be generally applicable in the synthesis of bioactive compounds containing peptidomimetic scaffolds and carboxylic acid bioisosteres.
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| 10. |
- Bender, Brendan, 1967-
(författare)
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Pharmacometric Models for Antibody Drug Conjugates and Taxanes in HER2+ and HER2- Breast Cancer
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In oncology, there is a need to optimize drug treatment for efficient eradication of tumors, minimization of adverse effects (AEs), and prolonging patient survival. Pharmacometric models can be developed to streamline information between drug development phases, describe and quantify response to treatment, and determine dose regimens that balance toxicity and efficacy. In this thesis, data from trastuzumab emtansine (T-DM1) and taxane drug treatment were used to develop pharmacometric models of pharmacokinetics (PK), AEs, anti-tumor response, and survival, supporting drug development.T-DM1 is an antibody-drug conjugate (ADC) for treatment of human epidermal growth factor receptor 2 (HER2)–positive breast cancer. ADCs are a relatively new class of oncologic agents, and contain multiple drug-to-antibody ratio (DAR) moieties in their dose product. The complex distribution of T-DM1 was elucidated through PK models developed using in vitro and in vivo rat and cynomolgus monkey DAR data. Mechanism–based PK/pharmacodynamic (PKPD) models were also developed for T-DM1 that described the AEs thrombocytopenia (TCP) and hepatotoxicity in patients receiving T-DM1. Variable patterns of platelet and transaminase (ALT and AST) response were quantified, including an effect of Asian ethnicity that was related to higher incidences of TCP. Model simulations, comparing dose intensities (DI) and Grade 3/4 incidences between the approved T-DM1 dose (3.6 mg/kg every three weeks) and weekly regimens, determined that 2.4 mg/kg weekly provided the highest DI.Docetaxel and paclitaxel are taxane treatment options for HER2–negative breast cancer. Tumor response data from these treatments were used to develop a mechanism–based model of tumor quiescence and drug–resistance. Subsequently, a parametric survival analysis found that tumor baseline and the model–predicted time to tumor growth (TTG) were predictors of overall survival (OS). This tumor and OS modeling approach can be applied to other anticancer treatments with similar patterns of drug–resistance.Overall, the pharmacometric models developed within this thesis present new modeling approaches and provide understanding on ADC PK and PKPD (TCP and hepatotoxicity), as well as drug–resistance tumor response. These models can inform simulation strategies and clinical study design, and be applied towards dose finding for anticancer drugs in development, especially ADCs.
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