Pharmacogenetics

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Our research aims at understanding the basis for differences between individuals in liver function, human drug metabolism, drug toxicity and drug response thereby facilitating a more effective personalized drug treatment at the clinics.

The emphasis is on genetic polymorphism of the genes encoding drug transporters, drug metabolizing enzymes and drug targets.

Furthermore, our research aims at understanding the epigenome and miRNA dependent regulation of hepatic genes, hepatocyte de-differentiation, re-differentiation and to develop novel in vitro systems for studying liver function and for novel treatment of liver diseases.

Section members

Pedro Gil Senior researcher, Research group leader
Delilah Hendriks PhD student
Magnus Ingelman-Sundberg Professor, Head of Section
Inger Johansson Senior lecturer
Marin Jukic Postdoc
Mikael Kozyra PhD Student
Volker Lauschke Assistant Professor, Research group leader
Souren Mkrtchian Senior researcher
Åsa Nordling Biomedical scientist
Sabine Vorrink Postdoc

Projects

1. Mimicking liver function and liver disease in vitro for drug development

Main responsible: Magnus Ingelman-Sundberg and Volker M Lauschke

1.1 Summary

We use 220 different livers and 3D hepatic spheroid systems obtained from human liver donations or cryopreserved hepatocyte cultures in combination with different types of non-parenchymal cells to:

  • Evaluate the usefulness of these spheroid systems for studying properties and functions of diseased liver models and their potential for use during drug development including validation of existing drugs and development of new drugs.
  • Evaluate in detail mechanisms of formations and treatment of steatosis, NAFLD and NASH in the spheroids systems including screening for novel drug therapies using the spheroids as validation system.
  • Evaluate chronic drug toxicity in diseased liver systems as well as novel mechanisms for drug induced enzyme induction

This information will be of importance for i) increased possibilities to avoid the development of hepatotoxic drugs, ii) understanding the bases for interindividual differences in drug metabolism and drug hepatotoxicity, iii) understanding different liver diseases and iv) possibly show proof of principle for an in vitro system for validation of drug targets aimed for treatment of liver diseases.

1.2 Experimental platform

In our lab, we have established and further developed novel spheroid based 3D systems for studies of liver function and properties in vitro (Gunness et al., 2013; Bell CC et al., 2016). Starting with hepatocytes obtained from the cell transplantation unit at Huddinge University Hospital (in collaboration with Dr Ewa Ellis) or from commercial sources, we form spheroids from primary human hepatocyte fractions (PHH spheroids) using the Corning ultra low attachment plates and defined media. In the spheroids, the cells have relevant cell-cell interactions, a physiological cell-extracellular matrix (ECM) interface, improved cell polarity and a functional bile canaliculi network (see Figure 1).

Indeed, we showed that relevant enzyme expression can be preserved for at least 5 weeks in culture and that periportal and perivenous hepatocytes retain their phenotype during this period (Bell et al., 2016). The hepatocytes in 3D spheroids exhibit molecular phenotypes on transcriptomic, proteomic and metabolomic level similar to the livers from which they originate (Figure 1). We can transfect the spheroids with virus mimicking viral induced hepatitis or supplement the media with bile acids, allowing the faithful identification of drugs with cholestatic liabilities (Hendriks et al., 2016). 

Figure 1: 3D spheroids from PHH closely resemble the in vivo liver hepatocytes at the proteome level and remain functionally stable for at least 5 weeks in culture. a, Heatmap visualizing whole proteome analysis of primary human liver samples (n=5) after 24h and 7d in 2D monolayer culture and spheroids after aggregation (7d 3D). Only differentially expressed proteins (n=574, F-test p<0.05) are shown. In vivo liver samples and spheroids cluster closely together while the proteomes of samples cultured in 2D are distinctly different (b). c. Importantly, the inter-individual variability observed in vivo is preserved in 3D culture, with each of the 3D samples clustering with the respective liver piece from the same donor. d. Chronic drug toxicity in spheroid systems; e. The spheroids retain morphology as evident from H&E staining and from EM microscopy (e, f) and express bile acid transporters in bile canaliculi (g) and express periportal and perivenous proteins stably. From Bell et al., Sci Rep, 2016.

Moreover, we can induce steatosis by drugs or by altering the media composition and we can grow the spheroids in the presence of non-parenchymal cells and induce inflammation and hepatitis.

1.3 Pathological liver systems

Using these diseased liver systems we want to elucidate the drug hepatotoxicity in diseased liver as compared to healthy liver, study factors causing steatosis, and fibrosis in the liver and new regimens to inhibit such development.

The transition from steatosis to NASH, fibrosis and HCC is characterized by an intricate interplay of a multitude of nutritional and genetic factors, pathways and cell types. We propose to validate the utility of the 3D spheroid culture system in which PHH can be co-cultured with Kupffer and stellate cells in 96-well format (Bell et al., 2016) as an in vitro disease model for human NAFLD compatible with high-throughput analyses

We will develop culture conditions that closely mimic the nutritional and hormonal status of hepatocytes in vivo with regards to e.g. glucose and glucagon levels, fatty acids and insulin. In this system, we will extensively characterize 3D cultured hepatocytes with regards to expression levels and activities of gene products involved in liver homeostasis and causing insulin resistance and metabolic diseases. We have previously shown that our human 3D culture system can recapitulate lipid accumulation, when exposed to elevated levels of fatty acids, fructose and insulin, as is the case in NAFLD in vivo (Figure 2).

Figure 2: A. Induction of steatosis using 2 x plasma concentration of FFA and induction of steatosis in the spheroids under metabolic syndrome conditions (Low insulin, 10 mM glucose, 10 mM fructose). B. Induction of steatosis using 0-2 x plasma concentration of FFA in three different spheroids.

We are developing systems mimicking fibrosis and regimens to treat this condition.

2. Liver regeneration and clinical implications

Main responsible: Volker M Lauschke

2.1 Background

Liver failure is a life-threatening condition in which the liver loses the ability to perform its physiological metabolic functions. For most cases, orthotopic liver transplantation (OLT) is the only therapeutic option. Because of the lack of liver donors, hepatocyte cell transplantations emerged as a powerful alternative approach that has been successfully employed in treatment of a variety of genetic liver disorders as well as acute and chronic liver failure. However, the clinical application of hepatocyte transplantations still faces important limitations:  Firstly, cells for transplantations are obtained primarily from livers that are non-transplantable due to advanced donor age or hepatic steatosis. Thus, the quality of the cells that are available for transplantation poses a major obstacle for current hepatocyte transplantation strategies. Secondly, transplantation of allogenic cells requires the use of immunosuppressive treatment with detrimental effects on patient morbidity.

2.2 Aims

  • Understanding the mechanisms of liver de- and redifferentiation:
    Assess the molecular basis that conveys hepatocyte plasticity to change differentiation states in 2D and spheroid cultures. Specifically, we will investigate critical coding genes, miRNAs and other ncRNAs in the processes of liver de- and re-differentiation and hepatic gene expression as well as the major signal transduction systems involved in these processes
  • Development of novel autologous treatment paradigms for liver failure:
    Develop and optimize protocols for proliferation and redifferentiation to open up new possibilities to generate the necessary quantities of functional hepatocytes for autologous transplantations.

2.3 Project description

We will generate functional hepatocytes in vitro that can be utilized for therapeutic transplantation purposes, thus alleviating the paucity of transplantable liver cells. To achieve this objective, we will tap the proliferative potential of primary human hepatocytes (PHH) by transiently dedifferentiating them into a fetal-like progenitor state, proliferating them via growth factors and cytokines and, after the cells sufficiently multiplied, to redifferentiate them back to functional mature hepatocytes using a 3D spheroid culture system (Figure 3).

Figure 3. Graphical Summary. PHH are isolated from patients with end-stage liver disease. Subsequently, they are dedifferentiated and proliferation is induced.  If applicable, gene correction using targeted endonucleases is performed, and then the cells are redifferentiated into fully functional mature hepatocytes using an integrated 3D culture system that we recently developed. These cells are potentially useful for therapeutic applications, for in vitro studies of disease mechanisms and for drug target validation and mechanistic evaluations.

The liver is an organ with an astonishing regenerative capacity. Upon partial hepatectomy, an operation in which up to three-quarters of the liver is surgically removed, the residual lobes enlarge and recover the original weight of the liver within 1-2 weeks.

We recently found that dedifferentiation is controlled by complex rearrangements of non-coding RNAs, particularly miRNAs, which precedes the loss of hepatic markers (Lauschke et al., Hepatology, 2016). We also found that strikingly, during spheroid aggregation stages, hepatocytes first dedifferentiate, followed by rapid redifferentiation, providing an ideal ex vivo experimental paradigm to study the full spectrum of differentiation state changes that occur in vivo during liver regeneration. 

Besides extending our mechanistic understanding, this finding opened possibilities for the development of therapeutic approaches as a substitute for orthotopic liver transplantations. To this end, we work on the establishment of protocols in which PHH, directly isolated from patients, proliferate and, after cells sufficiently multiplied, are induced to redifferentiate into functional hepatocytes using our 3D spheroid culture system. Using genomic editing tools, we aspire to correct genetic alterations in hepatocytes from patients with heritable liver disorders, such as hyperoxalosis or alpha1-antitrypsin deficiency.

Subsequently, we aim to expand recombinant cells and use the spheroid system to redifferentiate them into functional hepatocytes that in the future could serve as sources for autologous hepatocyte transplantations.

3. CYP2W1, a novel target for colon cancer therapy

Main responsible: Magnus Ingelman-Sundberg and Inger Johansson

We have found and characterized novel specific forms of P450 in cancer cells and in extrahepatic tissues. One interesting enzyme is CYP2W1, which is expressed in fetal colon but not in adult tissue, except for colon tumors. We have found in collaboration with David Edler found that the CYP2W1 expression can serve as a prognostic marker for malignancy.

In collaboration with Laurence Pattersson in Bradford, we have also been able to identify chemicals that can be bioactivated by CYP2W1 in the cancer cells to effectively kill the cells at 300 nM concentration. We are making xenograft models for treatment of colon cancers in vivo using these substances and found very promising results with respect to killing of colon cancer tumors in vivo with these agents. We will in due time develop chemical structures that can possibly be used in vivo in human for treatment of colon cancer.

The CYP2W1 gene is evolutionary conserved; it is expressed in embryonic gastrointestinal tract, and remained for a short period in the neonatal tissues, followed by epigenetic silencing throughout the adult life. The Cyp2w1 null mouse model is established, we are currently studying its endogenous function in embryogenesis, particularly in the gut development.

Furthermore we are in the start of a project aiming at develop novel prodrugs for CYP2W1 to be used in clinical trials

4. Role of CYP2C19 in brain phenotypes

Main responsible: Marin Jukic

CYP2C19 enzyme metabolizes endogenous and synthetic psychoactive compounds including steroid hormones, cannabinoids, SSRIs, tricyclic antidepressants, and benzodiazepines. Two major variant alleles are known, CYP2C19*2 is defective and CYP2C19*17, which is identified by our group, is causing increased enzyme expression. This project aims to reveal the role of CYP2C19 enzyme in prenatal brain development, as well as its role in adulthood in affective disorders and metabolism of psychopharmaceuticals.

4.1 Involvement of endogenous CYP2C19 in depressive phenotypes and hippocampal homeostasis

We detected CYP2C19 expression in fetal human brain and in collaboration with Nancy Pedersen, we previously showed that the presence of two defective (CYP2C19*2) alleles causes a decrease in depressive symptoms.We found that transgenic mice containing human CYP2C19 gene also show expression of this gene in developing brain, as well as decreased hippocampal volume, anxious phenotype, and increased hippocampal activation after acute stress.

In humans, we found that the absence of CYP2C19 is associated with a bilateral hippocampal volume increase in two independent healthy cohorts and a lower prevalence of major depressive disorder and depression severity in African-Americans (N=3848) (Jukic et al., 2016). Transgenic 2C19 mice showed high stress sensitivity, impaired hippocampal Bdnf homeostasis in stress, and more despair-like behavior in the forced swim test (FST).

So indeed several of the phenotypes originally described to be caused by overexpression of CYP2C19 in mice were also found to be influenced by CYP2C19 in humans which indicates that elevated CYP2C19 expression is associated with depressive symptoms, reduced hippocampal volume and impairment of hippocampal serotonin and BDNF homeostasis.

4.2 Establishing of CYP2C19 genotype-driven dosing regimen of escitalopram

Well-replicated in vitro and clinical studies indicate that escitalopram (ESC) is the most selective and most efficient among selective serotonin reuptake inhibitors (SSRIs). SSRIs are the cornerstone of the modern antidepressant pharmacotherapy; however, a large proportion of depressed patients do not respond adequately to the treatment with these drugs.

According to a large number of independent studies, the initial biotransformation step of ESC is catalysed by CYP2C19, with a minute contribution of CYP3A4. The variation in the disposition of ESC due to CYP2C19 polymorphism may significantly contribute to the inter-individual variability in antidepressant response among the patients. In this project, we aim to determine the exact contribution of CYP2C19 polymorphism to ESC plasma levels and clearance, as well as to establish the improved CYP2C19 genotype-driven dosing regimen, which ensures an appropriate perpetual ESC plasma concentration for every patient.

4.3 Involvement of CYP2C19 in developing brain in physiology and degradation of dopaminergic neurons

Movement disorders, including Parkinson’s disease, significantly contribute to the morbidity caused by diseases worldwide; however their etiology and pathophysiology are still largely unclear. Animals overexpressing the human CYP2C19 gene (2C19TG) exhibit an increased dopamine (DA) concentration in the brain and hyperkinesia in the young age and degeneration of dopaminergic neurons in the old age. Conversely, healthy human subjects of genotype connected with elevated CYP2C19 expression show lower grey matter scores in substantia nigra (SN), indicative of DA neuronal degradation in this brain region.  The aims of this project are to:

  1. Track the state and function of DA neurons in SN of 2C19TG mice to understand the succession of their early hyperactivity and late (potential) degradation in a systematic manner
  2. Elucidate the alteration in biochemical cascades behind the reduction in dopaminergic neurons in 2C19TG mutants, and
  3. Validate the clinical significance of these findings by using neuroimaging techniques on healthy individuals.

Overall, the investigation through which CYP2C19 affects dopaminergic neuronal physiology and degradation is aimed to lead to the establishment of genetic and neuroimaging biomarkers, as well as to provide novel drug-targets for the treatment of dopamine-mediated motoric deficits.

5. Evaluation of the importance of rare genetic variants on hepatic metabolism and drug response

Main responsible: Magnus Ingelman-Sundberg and Volker M Lauschke

Genetic variants primarily encoding drug and metabolite transporters, phase I and phase II drug metabolizing enzymes and nuclear receptors can influence drug response by modulating drug absorption, distribution, metabolism and excretion (ADME).

Importantly, while in the past decades an ever-growing arsenal of genetic variants with demonstrated impacts on human drug response has been identified in these pharmacogenes, a substantial fraction of the heritable variability in drug response remains unexplained. Rare genetic variants that only occur in very few individuals and are hence missed in genome-wide association studies have been proposed to contribute to this missing heritability.

We integrate data from recent population-wide Next-Generation Sequencing (NGS) projects to quantify the extent of genetic variability in pharmacogenes on a population level and, using an arsenal of in silico techniques, quantify the impact on hepatic metabolism and pharmacokinetics and -dynamics. Overall, such rare allelic variants are estimated to cause 40-50 % of the interindividual variability in drug response seen in the clinics.

This project is linked to our participation in the H2020 project Ubiquitous pharmacogenomics in which we have the primary responsibility to analyse the genetic background of outlier patients with abnormal drug response.

Financial support

Selected publications

 Massive rearrangements of cellular MicroRNA signatures are key drivers of hepatocyte dedifferentiation.
Lauschke V, Vorrink S, Moro S, Rezayee F, Nordling , Hendriks D, et al
Hepatology 2016 Nov;64(5):1743-1756

The Importance of Patient-Specific Factors for Hepatic Drug Response and Toxicity.
Lauschke V, Ingelman-Sundberg M
Int J Mol Sci 2016 Oct;17(10):

Novel 3D Culture Systems for Studies of Human Liver Function and Assessments of the Hepatotoxicity of Drugs and Drug Candidates.
Lauschke V, Hendriks D, Bell C, Andersson T, Ingelman-Sundberg M
Chem. Res. Toxicol. 2016 Dec;29(12):1936-1955

Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression.
Ivanov M, Kals M, Lauschke V, Barragan I, Ewels P, Käller M, et al
Nucleic Acids Res. 2016 Aug;44(14):6756-69

Elevated CYP2C19 expression is associated with depressive symptoms and hippocampal homeostasis impairment.
Jukić M, Opel N, Ström J, Carrillo-Roa T, Miksys S, Novalen M, et al
Mol. Psychiatry 2016 Nov;():

Hepatic 3D spheroid models for the detection and study of compounds with cholestatic liability.
Hendriks D, Fredriksson Puigvert L, Messner S, Mortiz W, Ingelman-Sundberg M
Sci Rep 2016 Oct;6():35434

The CYP2W1 enzyme: regulation, properties and activation of prodrugs.
Guo J, Johansson I, Mkrtchian S, Ingelman-Sundberg M
Drug Metab. Rev. 2016 Aug;48(3):369-78

Rare genetic variants in cellular transporters, metabolic enzymes, and nuclear receptors can be important determinants of interindividual differences in drug response.
Kozyra M, Ingelman-Sundberg M, Lauschke V
Genet. Med. 2017 Jan;19(1):20-29

A multicenter assessment of single-cell models aligned to standard measures of cell health for prediction of acute hepatotoxicity.
Sison-Young R, Lauschke V, Johann E, Alexandre E, Antherieu S, Aerts H, et al
Arch. Toxicol. 2016 Jun;():

Requirements for comprehensive pharmacogenetic genotyping platforms.
Lauschke V, Ingelman-Sundberg M
Pharmacogenomics 2016 Jun;17(8):917-24

Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease.
Bell C, Hendriks D, Moro S, Ellis E, Walsh J, Renblom A, et al
Sci Rep 2016 May;6():25187

Genetic variation in the human cytochrome P450 supergene family.
Fujikura K, Ingelman-Sundberg M, Lauschke V
Pharmacogenet. Genomics 2015 Dec;25(12):584-94

Membrane topology and search for potential redox partners of colon cancer-specific cytochrome P450 2W1.
Guo J, Thiess S, Johansson I, Mkrtchian S, Ingelman-Sundberg M
FEBS Lett. 2016 Feb;590(3):330-9

Precision Medicine and Rare Genetic Variants.
Lauschke V, Ingelman-Sundberg M
Trends Pharmacol. Sci. 2016 Feb;37(2):85-6

Cytostatic Effect of Repeated Exposure to Simvastatin: A Mechanism for Chronic Myotoxicity Revealed by the Use of Mesodermal Progenitors Derived from Human Pluripotent Stem Cells.
Peric D, Barragan I, Giraud-Triboult K, Egesipe A, Meyniel-Schicklin L, Cousin C, et al
Stem Cells 2015 Oct;33(10):2936-48

Pharmacogenomic information in drug labels: European Medicines Agency perspective.
Ehmann F, Caneva L, Prasad K, Paulmichl M, Maliepaard M, Llerena A, et al
Pharmacogenomics J. 2015 Jun;15(3):201-10

Expression and Function of mARC: Roles in Lipogenesis and Metabolic Activation of Ximelagatran.
Neve E, Köfeler H, Hendriks D, Nordling , Gogvadze V, Mkrtchian S, et al
PLoS ONE 2015 ;10(9):e0138487

Developmental regulation and induction of cytochrome P450 2W1, an enzyme expressed in colon tumors.
Choong E, Guo J, Persson A, Virding S, Johansson I, Mkrtchian S, et al
PLoS ONE 2015 ;10(4):e0122820

Interview with Magnus Ingelman-Sundberg.
Ingelman-Sundberg M
Trends Pharmacol. Sci. 2015 Feb;36(2):65-7

Whole-exome sequencing reveals defective CYP3A4 variants predictive of paclitaxel dose-limiting neuropathy.
Apellániz-Ruiz M, Lee M, Sánchez-Barroso L, Gutiérrez-Gutiérrez G, Calvo I, García-Estévez L, et al
Clin. Cancer Res. 2015 Jan;21(2):322-8

Epigenetic mechanisms of importance for drug treatment.
Ivanov M, Barragan I, Ingelman-Sundberg M
Trends Pharmacol. Sci. 2014 Aug;35(8):384-96

Ontogeny, distribution and potential roles of 5-hydroxymethylcytosine in human liver function.
Ivanov M, Kals M, Kacevska M, Barragan I, Kasuga K, Rane A, et al
Genome Biol. 2013 Aug;14(8):R83

Decreased hippocampal volume and increased anxiety in a transgenic mouse model expressing the human CYP2C19 gene.
Persson A, Sim S, Virding S, Onishchenko N, Schulte G, Ingelman-Sundberg M
Mol. Psychiatry 2014 Jun;19(6):733-41

Colon cancer-specific cytochrome P450 2W1 converts duocarmycin analogues into potent tumor cytotoxins.
Travica S, Pors K, Loadman PM, Shnyder SD, Johansson I, Alandas MN, Sheldrake HM, Mkrtchian S, Patterson LH, Ingelman-Sundberg M.
Clin Cancer Res. 2013 Jun 1;19(11):2952-61

3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies.
Gunness P, Mueller D, Shevchenko V, Heinzle E, Ingelman-Sundberg M, Noor F
Toxicol. Sci. 2013 May;133(1):67-78

In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes.
Ivanov M, Kals M, Kacevska M, Metspalu A, Ingelman-Sundberg M, Milani L
Nucleic Acids Res. 2013 Apr;41(6):e72

Scaling of embryonic patterning based on phase-gradient encoding.
Lauschke V, Tsiairis C, François P, Aulehla A
Nature 2013 Jan;493(7430):101-5

Contact us

Professor

Magnus Ingelman-Sundberg

Phone: +46-(0)8-524 877 35
Organizational unit: Ingelman-Sundberg Magnus group - Pharmacogenetics
E-mail: Magnus.Ingelman-Sundberg@ki.se

Pharmacology