Klaus Seuwen

Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States

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Publications (59)341.02 Total impact

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    ABSTRACT: The pH-sensing receptor OGR1 (Ovarian Cancer G protein-coupled receptor 1, GPR68) is expressed in the gut. Inflammatory bowel disease (IBD) is typically associated with a decrease in local pH, which may lead to altered epithelial barrier function and subsequent gastrointestinal repair involving epithelial cell adhesion and migration. As the mechanisms underlying the response to pH changes are not well understood, we have investigated OGR1-mediated pH-dependent signaling pathways in intestinal epithelial cells. Caco-2 cells stably overexpressing OGR1 were created and validated as tools to study OGR1 signaling. Barrier function, migration and proliferation were measured using ECIS (Electric Cell-Substrate Impedance Sensing) technology. Localization of the tight-junction proteins ZO-1 and occludin, and the rearrangement of cytoskeletal actin were examined by confocal microscopy. Paracellular permeability, protein and gene expression analysis using DNA microarrays were performed on filter-grown Caco-2 monolayers. We report that an acidic pH shift from pH 7.8 to 6.6 improved barrier function and stimulated reorganization of filamentous actin (F-actin) with prominent basal stress fiber formation. Cell migration and proliferation during in vitro wound healing were inhibited. Gene expression analysis revealed significant up-regulation of genes related to cytoskeleton remodeling, cell adhesion, and growth factor signaling. We conclude that acidic extracellular pH can have a signaling function and impact the physiology of intestinal epithelial cells. Deconstructing OGR1-dependent signaling may aid our understanding of mucosal inflammation mechanisms. Copyright © 2014, American Journal of Physiology- Gastrointestinal and Liver Physiology.
    AJP Gastrointestinal and Liver Physiology 07/2015; DOI:10.1152/ajpgi.00408.2014 · 3.74 Impact Factor
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    ABSTRACT: Blood gas and tissue pH regulation depend on the ability of the brain to sense CO2 and/or H(+) and alter breathing appropriately, a homeostatic process called central respiratory chemosensitivity. We show that selective expression of the proton-activated receptor GPR4 in chemosensory neurons of the mouse retrotrapezoid nucleus (RTN) is required for CO2-stimulated breathing. Genetic deletion of GPR4 disrupted acidosis-dependent activation of RTN neurons, increased apnea frequency, and blunted ventilatory responses to CO2. Reintroduction of GPR4 into RTN neurons restored CO2-dependent RTN neuronal activation and rescued the ventilatory phenotype. Additional elimination of TASK-2 (K2P5), a pH-sensitive K(+) channel expressed in RTN neurons, essentially abolished the ventilatory response to CO2. The data identify GPR4 and TASK-2 as distinct, parallel, and essential central mediators of respiratory chemosensitivity. Copyright © 2015, American Association for the Advancement of Science.
    Science 06/2015; DOI:10.1126/science.aaa0922 · 31.48 Impact Factor
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    ABSTRACT: A novel family of proton-sensing G protein-coupled receptors, including OGR1, GPR4, and TDAG8, was identified to be important for physiological pH homeostasis and inflammation. Thus, we determined the function of proton-sensing OGR1 in the intestinal mucosa. OGR1 expression in colonic tissues was investigated in controls and patients with IBD. Expression of OGR1 upon cell activation was studied in the Mono Mac 6 (MM6) cell line and primary human and murine monocytes by real-time PCR. Ogr1 knockout mice were crossbred with Il-10 deficient mice and studied for more than 200 days. Microarray profiling was performed using Ogr1 and Ogr1 (WT) residential peritoneal macrophages. Patients with IBD expressed higher levels of OGR1 in the mucosa than non-IBD controls. Treatment of MM6 cells with TNF, led to significant upregulation of OGR1 expression, which could be reversed by the presence of NF-κB inhibitors. Kaplan-Meier survival analysis showed a significantly delayed onset and progression of rectal prolapse in female Ogr1/Il-10 mice. These mice displayed significantly less rectal prolapses. Upregulation of gene expression, mediated by OGR1, in response to extracellular acidification in mouse macrophages was enriched for inflammation and immune response, actin cytoskeleton, and cell-adhesion gene pathways. OGR1 expression is induced in cells of human macrophage lineage and primary human monocytes by TNF. NF-κB inhibition reverses the induction of OGR1 expression by TNF. OGR1 deficiency protects from spontaneous inflammation in the Il-10 knockout model. Our data indicate a pathophysiological role for pH-sensing receptor OGR1 during the pathogenesis of mucosal inflammation.
    Inflammatory Bowel Diseases 04/2015; 21(6). DOI:10.1097/MIB.0000000000000375 · 5.48 Impact Factor
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    ABSTRACT: Antagonism of the calcium-sensing receptor in the parathyroid gland leads to parathyroid hormone (PTH) release. Calcilytics are a new class of molecules designed to exploit this mechanism. In order to mimic the known bone-anabolic pharmacokinetic (PK) profile of s.c. administered PTH, such molecules must trigger sharp, transient and robust release of PTH. The results of two early clinical studies with the orally-active calcilytic AXT914, a quinazolin-2ne derivative are reported. These were GCP-compliant, single and multiple dose studies of PK/PD and tolerability in healthy volunteers and postmenopausal women. The first study, examined single ascending doses (4 to 120mg) and limited multiple doses (60 or 120mg q.d. for 12days) of AXT914. The second study was a randomized, double-blind, active- and placebo-controlled, 4-week repeat-dose parallel group study of healthy postmenopausal women (45 and 60mg AXT914, placebo, 20μg Forteo / teriparatide / PTH(1-34) fragment). AXT914 was well tolerated at all doses and reproducibly induced the desired PTH-release profiles. Yet, 4weeks of 45 or 60mg AXT914 did not result in the expected changes in circulating bone biomarkers seen with teriparatide. However total serum calcium levels increased above baseline in the 45 and 60mg AXT914 treatment groups (8.0 % and 10.7%, respectively), compared to that in the teriparatide and placebo groups (1.3% and 1.0%, respectively). Thus the trial was terminated after a planned interim analysis due to lack of effect on bone formation biomarkers and dose-limiting effects on serum calcium. In conclusion, AXT914 was well tolerated but the observed transient and reproducible PTH-release after repeat oral administration of AXT914 which showed an exposure profile close to that of s c. PTH, did not translate into a bone anabolic response and was associated with a persistent dose-related increase in serum calcium concentrations.
    Bone 04/2014; 64. DOI:10.1016/j.bone.2014.04.015 · 4.46 Impact Factor
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    ABSTRACT: Oxysterols have recently been identified as natural ligands for a G protein-coupled receptor called EBI2 (aka GPR183) 1, 2. EBI2 is highly expressed in immune cells 3 and its activation has been shown to be critical for the adaptive immune response and has been genetically linked to autoimmune diseases such as type I diabetes 4. Here we describe the isolation of a potent small molecule antagonist for the EBI2 receptor. First we identified a small molecule agonist 1 (NIBR51), which enabled identification of inhibitors of receptor activation. One antagonist called 2 (NIBR127) was used as a starting point for a medicinal chemistry campaign which yielded 4m (NIBR189). This compound was extensively characterized in binding and various functional signaling assays. Furthermore we have used 4m to block migration of a monocyte cell line called U937, suggesting a functional role of the oxysterol/EBI2 pathway in these immune cells.
    Journal of Medicinal Chemistry 03/2014; 57(8). DOI:10.1021/jm4019355 · 5.48 Impact Factor
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    ABSTRACT: Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.
    Nature Chemical Biology 03/2014; 10(5). DOI:10.1038/nchembio.1481 · 13.22 Impact Factor
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    ABSTRACT: Oxysterols such as 7 alpha, 25-dihydroxycholesterol (7α,25-OHC) are natural ligands for the Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183), a G protein-coupled receptor (GPCR) highly expressed in immune cells and required for adaptive immune responses. Activation of EBI2 by specific oxysterols leads to chemotaxis of B cells in lymphoid tissues. While the ligand gradient necessary for this critical process of the adaptive immune response is established by a stromal cells subset here we investigate the involvement of the oxysterol / EBI2 system in the innate immune response. First, we show that primary human macrophages express EBI2 and the enzymes needed for ligand production such as cholesterol 25-hydroxylase (CH25H), sterol 27-hydroxylase (CYP27A1), and oxysterol 7 α -hydroxylase (CYP7B1). Furthermore, challenge of monocyte-derived macrophages with lipopolysaccharides (LPS) triggers a strong up-regulation of CH25H and CYP7B1 in comparison to a transient increase in EBI2 expression. Stimulation of EBI2 expressed on macrophages leads to calcium mobilization and to directed cell migration. Supernatants of LPS-stimulated macrophages are able to stimulate EBI2 signaling indicating that an induction of CH25H, CYP27A1, and CYP7B1 results in an enhanced production and release of oxysterols into the cellular environment. This is a study characterizing the oxysterol / EBI2 pathway in primary monocyte-derived macrophages. Given the crucial functional role of macrophages in the innate immune response these results encourage further exploration of a possible link to systemic autoimmunity.
    Biochemical and Biophysical Research Communications 01/2014; 446(3). DOI:10.1016/j.bbrc.2014.01.069 · 2.28 Impact Factor
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    ABSTRACT: Antagonism of the calcium-sensing receptor in the parathyroid gland leads to parathyroid hormone (PTH) release. Calcilytics are a new class of molecules designed to exploit this mechanism. In order to mimic the known bone-anabolic pharmacokinetic (PK) profile of s.c. administered PTH, such molecules must trigger sharp, transient and robust release of PTH. The results of two early clinical studies with the orally-active calcilytic AXT914, a quinazolin-2ne derivative are reported. These were GCP-compliant, single and multiple dose studies of PK/PD and tolerability in healthy volunteers and postmenopausal women. The first study, examined single ascending doses (4 to 120 mg) and limited multiple doses (60 or 120 mg q.d. for 12 days) of AXT914. The second study was a randomized, double-blind, active- and placebo-controlled, 4-week repeat-dose parallel group study of healthy postmenopausal women (45 and 60 mg AXT914, placebo, 20 μg Forteo / teriparatide / PTH(1–34) fragment). AXT914 was well tolerated at all doses and reproducibly induced the desired PTH-release profiles. Yet, 4 weeks of 45 or 60 mg AXT914 did not result in the expected changes in circulating bone biomarkers seen with teriparatide. However total serum calcium levels increased above baseline in the 45 and 60 mg AXT914 treatment groups (8.0 % and 10.7%, respectively), compared to that in the teriparatide and placebo groups (1.3% and 1.0%, respectively). Thus the trial was terminated after a planned interim analysis due to lack of effect on bone formation biomarkers and dose-limiting effects on serum calcium. In conclusion, AXT914 was well tolerated but the observed transient and reproducible PTH-release after repeat oral administration of AXT914 which showed an exposure profile close to that of s c. PTH, did not translate into a bone anabolic response and was associated with a persistent dose-related increase in serum calcium concentrations.
    Bone 01/2014; · 4.46 Impact Factor
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    ABSTRACT: Background: The proton-activated G protein-coupled receptor GPR4 is expressed in many tissues including white adipose tissue. GPR4 is activated by extracellular protons in the physiological pH range (i.e. pH 7.7 - 6.8) and is coupled to the production of cAMP. Methods: We examined mice lacking GPR4 and examined glucose tolerance and insulin sensitivity in young and aged mice as well as in mice fed with a high fat diet. Expression profiles of pro- and anti-inflammatory cytokines in white adipose tissue, liver and skeletal muscle was assessed. Results: Here we show that mice lacking GPR4 have an improved intraperitoneal glucose tolerance test and increased insulin sensitivity. Insulin levels were comparable but leptin levels were increased in GPR4 KO mice. Gpr4(-/-) showed altered expression of PPARα, IL-6, IL-10, TNFα, and TGF-1β in skeletal muscle, white adipose tissue, and liver. High fat diet abolished the differences in glucose tolerance and insulin sensitivity between Gpr4(+/+) and Gpr4(-/-) mice. In contrast, in aged mice (12 months old), the positive effect of GPR4 deficiency on glucose tolerance and insulin sensitivity was maintained. Liver and adipose tissue showed no major differences in the mRNA expression of pro- and anti-inflammatory factors between aged mice of both genotypes. Conclusion: Thus, GPR4 deficiency improves glucose tolerance and insulin sensitivity. The effect may involve an altered balance between pro- and anti-inflammatory factors in insulin target tissues. © 2013 S. Karger AG, Basel.
    Cellular Physiology and Biochemistry 11/2013; 32(5):1403-1416. DOI:10.1159/000356578 · 3.55 Impact Factor
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    ABSTRACT: TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing has been shown to modulate energy metabolism, differentiation and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This report describes the use of a series of studies that show G protein-coupled receptor (GPCR)-mediated biological activity of TLQP-21 signalling in CHO-K1 cells. Unbiased genome wide sequencing of the transcriptome from responsive CHO-K1 cells identified a prioritized list of possible GPCRs bringing about this activity. Further experiments using a series of defined receptor antagonists as well as siRNAs led to the identification of the complement receptor C3AR1 as a target for TLQP-21 in rodents. We have not been able to demonstrate so far that this finding is translatable to the human receptor. Our results are in line with a large number of physiological observations in rodent models of food intake and metabolic control, where TLQP-21 shows activity. In addition, the sensitivity of TLQP-21 signalling to pertussis toxin is consistent with the known signalling pathway of the C3AR1 receptor. The binding of TLQP-21 to the C3AR1 not only has effects on signalling but it also modulates cellular functions as TLQP-21 was shown to have a role in directing migration of RAW264.7 mouse cells.
    Journal of Biological Chemistry 08/2013; 288(38). DOI:10.1074/jbc.M113.497214 · 4.57 Impact Factor
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    ABSTRACT: Lymphocyte trafficking is critically regulated by the Sphingosine 1-phosphate receptor-1 (S1P(1)), a G protein-coupled receptor that has been highlighted as a promising therapeutic target in autoimmunity. Fingolimod (FTY720, Gilenya) is a S1P(1) receptor agonist that has recently been approved for the treatment of multiple sclerosis (MS). Here, we report the discovery of NIBR-0213, a potent and selective S1P(1) antagonist that induces long-lasting reduction of peripheral blood lymphocyte counts after oral dosing. NIBR-0213 showed comparable therapeutic efficacy to fingolimod in experimental autoimmune encephalomyelitis (EAE), a model of human MS. These data provide convincing evidence that S1P(1) antagonists are effective in EAE. In addition, the profile of NIBR-0213 makes it an attractive candidate to further study the consequences of S1P(1) receptor antagonism and to differentiate the effects from those of S1P(1) agonists.
    Chemistry & Biology 09/2012; 19(9):1142-1151. DOI:10.1016/j.chembiol.2012.07.016 · 6.59 Impact Factor
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    ABSTRACT: The Ovarian cancer G protein-coupled Receptor 1 (OGR1; GPR68) is proton-sensitive in the pH range of 6.8 - 7.8. However, its physiological function is not defined to date. OGR1 signals via inositol trisphosphate and intracellular calcium, albeit downstream events are unclear. To elucidate OGR1 function further, we transfected HEK293 cells with active OGR1 receptor or a mutant lacking 5 histidine residues (H5Phe-OGR1). An acute switch of extracellular pH from 8 to 7.1 (10 nmol/l vs 90 nmol/l protons) stimulated NHE and H(+)-ATPase activity in OGR1-transfected cells, but not in H5Phe-OGR1-transfected cells. ZnCl(2) and CuCl(2) that both inhibit OGR1 reduced the stimulatory effect. The activity was blocked by chelerythrine, whereas the ERK1/2 inhibitor PD 098059 had no inhibitory effect. OGR1 activation increased intracellular calcium in transfected HEK293 cells. We next isolated proximal tubules from kidneys of wild-type and OGR1-deficient mice and measured the effect of extracellular pH on NHE activity in vitro. Deletion of OGR1 affected the pH-dependent proton extrusion, however, in the opposite direction as expected from cell culture experiments. Upregulated expression of the pH-sensitive kinase Pyk2 in OGR1 KO mouse proximal tubule cells may compensate for the loss of OGR1. Thus, we present the first evidence that OGR1 modulates the activity of two major plasma membrane proton transport systems. OGR1 may be involved in the regulation of plasma membrane transport proteins and intra- and/or extracellular pH.
    Cellular Physiology and Biochemistry 04/2012; 29(3-4):313-24. DOI:10.1159/000338486 · 3.55 Impact Factor
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    ABSTRACT: The G protein-coupled receptor GPR4 is activated by acidic pH and recent evidence indicates that it is expressed in endothelial cells. In agreement with these reports, we observe a high correlation of GPR4 mRNA expression with endothelial marker genes, and we confirm expression and acidic pH dependent function of GPR4 in primary human vascular endothelial cells. GPR4-deficient mice were generated; these are viable and fertile and show no gross abnormalities. However, these animals show a significantly reduced angiogenic response to VEGF (vascular endothelial growth factor), but not to bFGF (basic fibroblast growth factor), in a growth factor implant model. Accordingly, in two different orthotopic models, tumor growth is strongly reduced in mice lacking GPR4. Histological analysis of tumors indicates reduced tumor cell proliferation as well as altered vessel morphology, length and density. Moreover, GPR4 deficiency results in reduced VEGFR2 (VEGF Receptor 2) levels in endothelial cells, accounting, at least in part, for the observed phenotype. Our data suggest that endothelial cells sense local tissue acidosis via GPR4 and that this signal is required to generate a full angiogenic response to VEGF.
    Angiogenesis 11/2011; 14(4):533-44. DOI:10.1007/s10456-011-9238-9 · 4.41 Impact Factor
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    ABSTRACT: Parathyroid hormone (PTH), when injected daily as either the intact hormone PTH(1-84) or the active fragment PTH(1-34) (teriparatide), is an efficacious bone anabolic treatment option for osteoporosis patients. Injections lead to rapid and transient spikes in hormone exposure levels, a profile which is a prerequisite to effectively form bone. Oral antagonists of the calcium-sensing receptor (calcilytics) stimulate PTH secretion and represent thus an alternative approach to elevate hormone levels transiently. We report here on ATF936, a novel calcilytic, which triggered rapid, transient spikes in endogenous PTH levels when given orally in single doses of 10 and 30mg/kg to growing rats, and of 1mg/kg to dogs. Eight weeks daily oral application of 30mg/kg of ATF936 to aged female rats induced in the proximal tibia metaphysis increases in bone mineral density, cancellous bone volume and cortical and trabecular thickness as evaluated by computed tomography. In healthy humans, single oral doses of ATF936 produced peak PTH levels in plasma after a median time of 1h and levels returned to normal at 24-h post-dose. The average maximum PTH concentration increase from baseline was 1.9, 3.6, and 6.0-fold at doses of 40, 70, and 140mg. ATF936 was well tolerated. The sharp, transient increase in PTH levels produced by the oral calcilytic ATF936 was comparable to the PTH profile observed after subcutaneous administration of teriparatide. In conclusion, ATF936 might hold potential as an oral bone-forming osteoporosis therapy.
    Bone 08/2011; 49(2):233-41. DOI:10.1016/j.bone.2011.04.007 · 4.46 Impact Factor
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    ABSTRACT: Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2-oxysterol signalling pathway has an important role in the adaptive immune response.
    Nature 07/2011; 475(7357):524-7. DOI:10.1038/nature10280 · 42.35 Impact Factor
  • Gastroenterology 01/2011; 140(5). DOI:10.1016/S0016-5085(11)63474-4 · 13.93 Impact Factor
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    ABSTRACT: A series of novel benzimidazole derivatives has been designed via a scaffold morphing approach based on known calcilytics chemotypes. Subsequent lead optimisation led to the discovery of penta-substituted benzimidazoles that exhibit attractive in vitro and in vivo calcium-sensing receptor (CaSR) inhibitory profiles. In addition, synthesis and structure-activity relationship data are provided.
    Bioorganic & medicinal chemistry letters 09/2010; 20(17):5161-4. DOI:10.1016/j.bmcl.2010.07.016 · 2.33 Impact Factor
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    ABSTRACT: Parathyroid hormone (PTH) is an effective bone anabolic agent. However, only when administered by daily sc injections exposure of short duration is achieved, a prerequisite for an anabolic response. Instead of applying exogenous PTH, mobilization of endogenous stores of the hormone can be envisaged. The secretion of PTH stored in the parathyroid glands is mediated by a calcium sensing receptor (CaSR) a GPCR localized at the cell surface. Antagonists of CaSR (calcilytics) mimic a state of hypocalcaemia and stimulate PTH release to the bloodstream. Screening of the internal compound collection for inhibition of CaSR signaling function afforded 2a. In vitro potency could be improved >1000 fold by optimization of its chemical structure. The binding mode of our compounds was predicted based on molecular modeling and confirmed by testing with mutated receptors. While the compounds readily induced PTH release after iv application a special formulation was needed for oral activity. The required profile was achieved by using microemulsions. Excellent PK/PD correlation was found in rats and dogs. High levels of PTH were reached in plasma within minutes which reverted to baseline in about 1-2 h in both species.
    Journal of Medicinal Chemistry 02/2010; 53(5):2250-63. DOI:10.1021/jm901811v · 5.48 Impact Factor
  • Nature Chemical Biology 12/2009; 5(12). DOI:10.1038/nchembio1209-954a · 13.22 Impact Factor

Publication Stats

2k Citations
341.02 Total Impact Points

Institutions

  • 2009–2014
    • Novartis Institutes for BioMedical Research
      • Global Discovery Chemistry Group
      Cambridge, Massachusetts, United States
  • 1998–2014
    • Novartis
      Bâle, Basel-City, Switzerland
  • 2007
    • ETH Zurich
      • Institute for Operations Research
      Zürich, Zurich, Switzerland
  • 2003
    • Università degli Studi dell'Aquila
      • Department of Experimental Medicine
      Aquila, Abruzzo, Italy
  • 1993
    • LKC Switzerland Ltd.
      Basel-Landschaft, Switzerland