Core Project 3: In vitro modeling

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Core Project 3: Mechanisms and in vitro prediction of non-allergic idiosyncratic toxicity

Introduction:
Idiosyncratic toxicity of drugs is rare and is not related to their pharmacological action. This type of toxicity can therefore so far not be predicted and is not detected by the usual screening methods during preclinical and clinical drug development. Although being rare, the consequences of this type of toxicity can be very severe, both for the patients affected and for the pharmaceutical industry. Recent examples include benzbromarone, an uricosuric agent associated with hepatic toxicity, troglitazone, an insulin-sensitizer associated with hepatotoxicity, nefazadon, an antidepressent associated with hepatotoxicity, cerivastatin, a serum cholesterol-lowering drug associated with skeletal muscle toxicity and rofecoxib, an analgesic and anti-inflammatory drug associated with cardiovascular toxicity. All of these drugs were associated with severe adverse effects in patients, eventually resulting in a fatal outcome. The pharmaceutical companies producing these drugs had therefore to withdraw them from the market, with serious financial consequences for the companies with reputation-damaging effects for the whole pharmaceutical industry. The ability to predict such adverse effects would therefore represent a large step towards safer medicines.
Clinically, two types of idiosyncratic toxicity can be distinguished: the allergic and the non-allergic (or metabolic) type. Concerning the non-allergic type of idiosyncratic toxicity, certain risk factors are assumed to render affected persons more susceptible to certain drugs. Such risk factors may for instance be polymorphisms of drug-metabolizing enzymes, leading to the formation and accumulation of more toxic metabolites, mitochondrial dysfunctions, leading to apoptosis and/or necrosis in the presence of mitochondrial toxins, and “cellular stress” such as inflammation, also increasing the toxicity of certain drugs.
In order to be able to screen for this type of toxicity, the concept regarding the risk factors has to be validated using suitable systems and methods, and assays have to be produced which contain these risk factors and can be used for screening. Such assays would be most suitable if they were based on cell culture models, since cells can chemically and genetically be manipulated, can be grown in large quantities and can be assessed easily. Since hepatotoxicity, skeletal and cardiac muscle toxicity as well as neurotoxicity appear to be the most important types of idiosyncratic toxicity, we will focus on the corresponding cell types.

Questions:
1. Can the concept of underlying risk factors rendering cells, animals or individuals more susceptible to drug toxicity be confirmed experimentally?
2. Can human cell lines be derived and characterized; and can these cell lines be manipulated for the introduction of such risk factors?
3. Can the methods produced be used for high throughput screening?

Enabling technologies
There are several cytotoxicity assays on the market which we use routinely. These assays are mostly based on the leakiness of the plasma membrane with the possibility to determine enzymes or other substances spilling out of the cells. Assays for apoptosis and necrosis are also established, both in cell cultures and in animals. We can also assess the function of mitochondria in isolated organelles and in cell cultures. New technologies, which we consider to be important for our purposes, are cell imaging systems (with the possibility to assess the accumulation of specific marker substances in cell organelles) and the possibility to determine oxygen consumption and glycolysis simultaneously in cell cultures as a sensitive marker of mitochondrial function. The molecular biology techniques used are all established.

Objectives:
1. To demonstrate that dysfunction of skeletal muscle mitochondria is a risk factor for statin-associated muscle toxicity in mice
2. To demonstrate that statins are more toxic for fibroblasts from patients with mitochondrial diseases than for control fibroblasts
3. To demonstrate that induction of CYP3A renders amiodarone more toxic towards hepatocytes than under basal conditions using the same cell line
4. To demonstrate that “cellular stress” can be a risk factor for drug cytotoxicity
5. To develop and characterize cell lines for different organs suitable for screening for drug toxicity

Plan:
1. In vivo studies in mice with a specific mitochondrial defect
Simvastatin will be administered via an osmotic pump implanted under the skin in a rising dosage, starting from 0.1 mg/kg/day in control mice, until toxic effects on skeletal muscle are achieved in >50% of the mice (not more than 10 mg/kg/day). The experiment will then be repeated in mice with impaired b-oxidation/with a mitochondrial defect (mice with carnitine deficiency due to a mutation in the carnitine carrier OCTN2 and long-chain acyl-CoA dehydrogenase knock-out mice) until >50% of the mice show skeletal muscle toxicity. The corresponding TD50 values will be calculated and be compared between control mice and mice with mitochondrial dysfunction. Toxicity will be assessed by the determination of the activities of aspartate aminotransferase and creatine kinase in plasma and the myoglobin and creatinine concentrations in urine and plasma. Mice will be treated for up to 2 months and measurements will be performed every 2 weeks.
2. In vitro studies using cells with a specific mitochondrial defect
We will use fibroblasts from patients with CPT II deficiency and long-chain acyl-CoA dehydrogenase deficiency and compare the results with control fibroblasts. We have also prepared C2C12 cells with a stable knock-down of carnitine:acylcarnitine translocase. These cells will be incubated with rising concentrations of specific statins, and viability and specific mitochondrial functions (oxygen uptake) and glycolysis will be assessed. If statins show an increased toxicity on myocytes and fibroblasts with mitochondrial impairment than in control cells, we have another indication for the correctness of our hypothesis. Furthermore, we have shown that it is possible to create in vitro models for investigating risk factors for metabolic (mitochondrial) idiosyncratic toxicity.
3. CYP3A activity and toxicity of amiodarone and other drugs
We have created HepG2 cells with stable overexpression of CYP3A. In addition, we have shown previously that that the cytoxicity of the amiodarone metabolites N-mono- and N-di-desethyl-amiodarone is increased compared to the parent drug. We therefore predict that cytotoxicity of amiodarone is increased in cells with a high CYP3A activity compared to normal HepG2 cells, which have only a small activity. Since we know that amiodarone is a mitochondrial toxin, not only cytotoxicity, but also the mitochondrial membrane potential, oxygen consumption and glycolysis will be assessed. If our hypothesis is correct, the molecular mechanisms underlying this finding are going to be studied using synthesized N-mono- and N-di-desethyl-amiodarone. Similar studies will then be performed using other tertiary amines, e.g. tricyclic antidepressants.
4. “Cellular stress” as a potential risk factor for drug toxicity
We will focus on the impact of activation of the glucocorticoid receptor, NF-kB and/or Nrf-2 on cytotoxicity in the presence of model drugs such as amiodarone, valproate, antidepressants or neuroleptics. Furthermore, the role of changes in the cellular redox potential on cytotoxicity associated with the above mentioned drugs will be investigated. A close collaboration of the groups in Basel and Lausanne is a prerequisite for this project.
5. Preparation and validation of cell models for mitochondrial toxicity
Since we have all necessary permissions to obtain and work human embryonal stem cells (hESC) and a number of research groups in our network are experienced in working with hESC, this cell type represents one of our starting points. Regarding the strong network of research groups working in this field, we can formulate ambitious aims. The final aim is certainly to obtain culturable human cells with major characteristics of neurocytes, hepatocytes or myocytes. To reach this aim, both a long-term commitment for this project and a close collaboration of the groups involved (in Basel, Lausanne and Geneva) are necessary. Our access to hESC lines, our experience with transdifferentiation of ovarian stem cells into other cell types, such as osteoblasts, chondrocytes and neurons, and our experience with three-dimensional culture systems provide a sound basis for the establishment of transdifferentiation of hESC into hepatocytes and possibly myocytes and for the elaboration of long-term culture systems of such cells. Such cells will then be manipulated by the risk factors characterized in the above mentioned studies and used as models for toxicity screening. In collaboration with specialized institutions, those systems can be further developed for high-throughput drug toxicity testing systems. In the mean time, existing cell lines will be used for this purpose, such as the HepG2 and C2C12 cells described above. These cell lines will be characterized in detail and manipulated as described above.

Milestones:
To validate clearly that certain risk factors are associated with idiosyncratic toxicity. Establishment of animal and cellular models to screen for certain types of metabolic idiosyncratic toxicity.

Endpoints:
Better tools for screening for metabolic idiosyncratic toxicity, improved drug safety, better understanding of idiosyncratic toxicity.

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