Refine
Has Fulltext
- yes (5)
Is part of the Bibliography
- yes (5)
Document Type
- Doctoral Thesis (5) (remove)
Language
- English (5) (remove)
Keywords
- biomarker (5) (remove)
Conjugation of reactive intermediates of drugs with proteins or DNA may result in toxic effects such as hepatotoxicity, agranulocytosis, allergies, tumors, etc. From 1975 to 1999, 2.9% of drugs were withdrawn from the market due to such severe adverse drug reactions. Thus, formation of chemically reactive intermediates is a widely discussed problem in drug development processes. Early detection of potentially toxic compounds is required for drug discovery and drug development. Conjugation of such electrophilic compounds with glutathione (GSH) is one of the most important detoxifying reactions in vivo. Processing of these GSH-conjugates ultimately leads to the formation of renally cleared mercapturic acids, which may also be oxidized to sulfoxides. Thus, mercapturic acids may be generated and detected in vitro and non-invasively in vivo in urine to assess the reactivity of a compound in early stages of drug development processes. Therefore, the aim of this work was to develop and evaluate a HPLC-MS/MS screening method for simple and rapid detection and characterization of known and unknown mercapturic acids and application of the method to several different matrices. Based on the common constant neutral loss (CNL) of 129 Da of all mercapturic acids tested (in negative ion mode), a CNL survey scan was performed using a linear ion trap instrument and was combined with two enhanced product ion (EPI) scans with different collision energies to characterize the detected signals. The CNL resulted from the cleavage between the sulfur and the carbon atom in the N-acetyl-L-cysteine moiety. After optimization of the experimental parameters, the detection limits of the reference substances in rat urine ranged from 0.3 to 15.5 pmol on column (i.e. 20 ng/ml to 800 ng/ml). For in vitro evaluation of the method, the model compounds acetaminophen, diclofenac, bifonazole, clozapine, troglitazone, carbamazepine, and bisphenol A were screened for formation of reactive intermediates and, hence, detection of the corresponding mercapturic acids. To determine possible species- and tissue-specific toxicities, the model compounds were incubated with stimulated neutrophils and with liver microsomes from rats and humans. Species-specific differences were observed in incubations of acetaminophen and diclofenac with rat and human hepatic microsomes. Tissue-specific differences in biotransformation of the model compounds in incubations with human neutrophils and human liver microsomes were observed for diclofenac, carbamazepine, clozapine, and bifonazole. The developed HPLC-MS/MS method was also evaluated in vivo by analysis of rat and human urine. Drug-related mercapturic acids were detected in urine of rats orally treated with acetaminophen (20 mg/kg and 640 mg/kg b.w.) or diclofenac (10 mg/kg and 20 mg/kg b.w.). Human urine samples were analyzed before and after oral administration of a clinically used dose of 500 mg and 50 mg of acetaminophen. Besides detection of the mercapturic acid of N-acetylbenzoquinoneimine (AAP-MA), a second mercapturic acid with m/z 327 occurred dose-dependently in rat and human urine samples after administration of acetaminophen. Further investigations on identification of this metabolite using authentic compounds and comparing their MS/MS mass spectra demonstrated oxidation of AAP-MA to stereoisomeric sulfoxides in vivo. For diclofenac, a novel mercapturic acid with m/z 441 was detected in rat urine samples that was identical to a metabolite obtained in incubations with human neutrophils before. The in vivo formation of this diclofenac metabolite is described here for the first time. In addition, three endogenously formed mercapturic acids were detected and identified. In conclusion, the results of the in vitro and in vivo evaluation demonstrate the advantages of the rapid and generic HPLC-MS/MS screening method for the detection of mercapturic acids, that can be obtained with a minimum of sample preparation and a high throughput in diverse matrices.
Lewy bodies and Lewy neurites are neuropathological hallmarks of Parkinson’s disease (PD). These depositions in the brain mostly consist of aggregated α-synuclein (α-syn) phosphorylated at Ser129. A number of studies reported detection of phosphorylated α-syn (p-α-syn) in the dermal nerve fibers in Parkinson’s disease. The objective of this study was to investigate whether pathological α-syn accumulations detected in the skin represent aggregated protein. A number of methods aimed at detecting α-syn oligomers and aggregates were first tested and optimized on the brain samples in PD and normal control. These methods included proximity ligation assay (PLA), PET-blot, immunohistochemical (IHC) stains with α-syn aggregate (5G4) or oligomer specific (ASyO5) antibodies and a stain against native α-syn (syn211) after proteinase K (PK) digestion. Subsequently, the most specific methods (stains with 5G4, ASyO5 and syn211 after PK digestion) were studied in two separate patient and control cohorts. Anti-p-α-syn stain was performed in parallel.
Single sections from at least 2 biopsy sites from 44 patients and 22 controls (cohort 1) as well as serial sections of 4 biopsy sites from 27 patients and 5 controls (cohort 2) were systematically studied for presence of aggregated and oligomeric α-syn. In total, 5G4 positive deposits were found in 24% (cohort 1) and 37% (cohort 2), ASyO5 positive lesions in 17,7% (cohort 1) and 33% (cohort 2), syn211 positive lesions after PK digestion in 38,7% (cohort 1) and 48% (cohort 2) of cases. There was a major overlap among positivity for a particular staining on the patient level and in most cases, the same nerve fiber was found to be positive for all 4 markers in neighboring sections. Among the skin biopsies which contained p-α-syn accumulation, 59% were also PK resistant, 41% were 5G4 positive and 45% were ASyO5 positive. The samples belonging to normal controls did not show any positive signal in either of the newly established stainings or in the anti-p-α-syn staining.
Using 3 distinct IHC methods, α-syn oligomers and aggregates were detectable in the majority of p-α-syn positive skin biopsies. This finding supports the hypothesis that α-syn aggregation occurs in the peripheral (i.e. dermal) nerves and can be specifically detected using skin biopsy.
Oncolytic virotherapy represents a promising approach to revolutionize cancer therapy. Several preclinical and clinical trials display the safety of oncolytic viruses as wells as their efficiency against solid tumors. The development of complementary diagnosis and monitoring concepts as well as the optimization of anti-tumor activity are key points of current virotherapy research. Within the framework of this thesis, the diagnostic and therapeutic prospects of beta-glucuronidase expressed by the oncolytic vaccinia virus strain GLV-1h68 were evaluated. In this regard, a beta-glucuronidase-based, therapy-accompanying biomarker test was established which is currently under clinical validation. By using fluorescent substrates, the activity of virally expressed beta-glucuronidase could be detected and quantified. Thereby conclusions about the replication kinetics of oncolytic viruses in animal models and virus-induced cancer cell lysis could be drawn. These findings finally led to the elaboration and establishment of a versatile biomarker assay which allows statements regarding the replication of oncolytic viruses in mice based on serum samples. Besides the analysis of retrospective conditions, this test is able to serve as therapy-accompanying monitoring tool for virotherapy approaches with beta-glucuronidase-expressing viruses. The newly developed assay also served as complement to routinely used plaque assays as well as reference for virally expressed anti-angiogenic antibodies in additional preclinical studies. Further validation of this biomarker test is currently taking place in the context of clinical trials with GL-ONC1 (clinical grade GLV-1h68) and has already shown promising preliminary results. It was furthermore demonstrated that fluorogenic substrates in combination with beta-glucuronidase expressed by oncolytic viruses facilitated the optical detection of solid tumors in preclinical models. In addition to diagnostic purposes, virus-encoded enzymes could also be combined with prodrugs resulting in an improved therapeutic outcome of oncolytic virotherapy. In further studies, the visualization of virus-induced immune reactions as well as the establishment of innovative concepts to improve the therapeutic outcome of oncolytic virotherapy could be accomplished. In conclusion, the results of this thesis provide crucial findings about the influence of virally expressed beta-glucuronidase on various diagnostic concepts in the context of oncolytic virotherapy. In addition, innovative monitoring and therapeutic strategies could be established. Our preclinical findings have important clinical influence, particularly by the development of a therapy-associated biomarker assay which is currently used in different clinical trials.
Mineralocorticoid-receptor antagonism and its metabolic consequences in haemodialysis patients
(2022)
Patients on haemodialysis are highly susceptible to different forms of heart failure. To date, the benefit of Mineralocorticoid-receptor antagonist (MRA) administration in haemodialysis patients remains subject to discussion. Biomarkers play an important role in therapy guidance and pose a promising tool to detect pathological processes of heart failure in an earlier stage. The randomised-controlled Mineralocorticoid-Receptor Antagonists in End-Stage Renal Disease (MiREnDa) trial was set up to investigate the effect of 50 mg of spironolactone once daily on left ventricular mass index in haemodialysis patients and several secondary endpoints. This dissertation reports findings from the MiREnDa trial on (a) the efficacy of spironolactone to influence serum levels of biomarkers of heart failure, fibrosis and inflammation and electrolytes and (b) the ability of N-terminal pro-B-type natriuretic peptide (NT-proBNP), Galectin-3 and soluble source of tumorigenicity 2 (sST2) to reflect left ventricular hypertrophy and diastolic dysfunction assessed by imaging characteristics. Treatment of spironolactone over a 40-week period did not alter serum levels of biomarkers of heart failure, fibrosis and inflammation including NT-proBNP, Galectin-3 and sST2. A small but significant effect on serum sodium but not potassium was observed. NT-proBNP was significantly different in the presence or absence of left ventricular hypertrophy (LVH) (normal vs. LVH (median [IQR]): 2,120 [810; 5,040] vs. 6,340 [2,410; 15,360] pg/ml, p<0.01) or moderate and severe diastolic dysfunction (DD) (normal diastolic function and DD grade I vs. DD grade II and DD grade III: 2,300 [850; 6,050] vs. 12,260 [3,340; 34,830] pg/ml, p=0.02). NT-proBNP further showed a significant correlation at baseline with LVMi (Spearman’s rho=0.41, p<0.001), LAVi (Spearman’s rho=0.55, p<0.001) and septal E/e’ (Spearman’s rho=0.45, p<0.001). No correlation was observed between Galectin-3 and the investigated functional and morphological parameters. sST2 was mildly correlated to LVMi at baseline (Spearman’s rho=0.21, p=0.05) and NT-proBNP at baseline (Spearman’s rho=0.37, p<0.001). In conclusion, spironolactone did not affect the investigated parameters but NT-proBNP proved to be significantly correlated to cardiac imaging measurements.
The high failure rate of new drug candidates in preclinical or clinical studies due to hepatotoxicity represents a considerable problem in the drug development. Hence, there is an urgent need to develop new approaches for early and reliable prediction of drug-induced hepatotoxicity that enables a better identification of drug candidates with high potential for toxicity at early stages of drug development. Therefore, the aim of this work was to improve the prediction of drug-induced liver injury in preclinical studies through evaluation of more reliable and sensitive biomarkers of hepatotoxicity and a better understanding of the underlying mechanistic basis for drug-induced toxicity. First, the ability of a set of potential markers (NGAL, thiostatin, clusterin, PON1) to detect early signs of liver injury was assessed in rats treated with drug candidates that were dropped from further development, in part due to toxic adverse effects in the liver. In summary, PON1 and clusterin were not consistently altered in response to liver injury and thus provide no additive information to the traditional liver enzymes in detecting drug-induced hepatotoxicity. In contrast, thiostatin and NGAL were increased in serum and urine of treated animals in a time- and dose-dependent manner. These changes correlated well with mRNA expression in the target organ and generally reflected the onset and degree of drug-induced liver injury. Receiver-operating characteristics analyses supported serum thiostatin, but not NGAL, as a better indicator of drug-induced hepatobiliary injury than conventional clinical chemistry parameters, such as ALP, ALT and AST. Although thiostatin, an acute phase protein expressed in a range of tissues, may not be specific for liver injury, our results indicate that thiostatin may serve as a sensitive, minimally-invasive diagnostic marker of inflammation and tissue damage in preclinical safety assessment. In the second part of this work, combined application of genomics profiling technology and RNAi to inhibit the pharmacological target of a drug candidate BAY16, a glucagon receptor (GCGR) antagonist, was used to determine if interference with the pharmacological target plays a role in the toxic response to BAY16, and to narrow down those molecular changes that are associated with toxicity, and not the pharmacological action of BAY16. In contrast to Bay 16, which was found to be cytotoxic at concentrations of 75 µM, silencing of the glucagon receptor did not affect cell viability in primary rat hepatocytes. Thus, it can be concluded that hepatotoxicity of Bay 16 was not related to the drugs inhibitory effect on the glucagon receptor in vitro and in vivo. These findings were supported by the fact that most of BAY16-induced changes in gene expression occurred independently of the pharmacological modulation of GCGR. These off-target effects include altered xenobiotic metabolism, oxidative stress, increased fatty acid synthesis, and alterations in cholesterol and bile acid metabolic processes. Although it was not possible to draw a final conclusion about the mechanism of BAY16 hepatotoxicity, changes in these molecular mechanisms appear contribute to progression of hepatic injury. With regard to drug safety assessment in preclinical studies, the utilization of siRNA technology in vitro represents a new approach to improve mechanistic understanding of the nature of drug’s toxicity, being either chemically mediated or due to primary or secondary pharmacological mode of action.