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Vol. 23, No. 3, 2010
Issue release date: March 2010
Free Access
Skin Pharmacol Physiol 2010;23:139–151
(DOI:10.1159/000270386)

Identification of Novel in vitro Test Systems for the Determination of Glucocorticoid Receptor Ligand-Induced Skin Atrophy

Schoepe S.a, b · Schäcke H.a · Bernd A.c · Zöller N.c · Asadullah K.b, d
aCommon Mechanism Research Berlin and bTarget Discovery, Global Drug Discovery, Bayer Schering Pharma AG, Berlin, and cDepartment of Dermatology and Venerology, J.W. Goethe University, Frankfurt/Main, and dGerman Skin Research Center, Berlin, Germany
email Corresponding Author

Abstract

Topical glucocorticoids (GCs) demonstrate good anti-inflammatory effects but are limited by their side effect potential, with skin atrophy being the most prominent one. Thus, determining the atrophogenic potential of novel compounds is important. The aim of this study was to establish an in vitro skin atrophy model. A screening cascade was applied and GCs with a known atrophogenic potential were used as tool compounds. Five rodent and human cutaneous cell types/cell lines and 2 human skin equivalents were tested. Known and suspected atrophy markers related to collagen metabolism and epidermal thickness were measured. Altogether, a combination of 7 different cellular assays with up to 16 markers each were investigated. A reproducible, more than 2-fold, regulation of the candidate markers by dexamethasone or clobetasol was found for: (a) matrix metalloproteinase (MMP) 1, 2, 3 and 9 expression in human keratinocytes, (b) COL1A1 and COL3A1 expression in 3T3 fibroblasts, and (c) epidermal thickness, collagen and MMP synthesis in the full-thickness skin model (FTSM). These 3 models were further investigated with a panel of 4–5 GCs, demonstrating dose dependency and correlation with the atrophogenic potential of the tool compounds, qualifying them as potentially suitable. Finally, the predictability of these models for the in vivo situation was analyzed, testing a novel selective GC receptor agonist (SEGRA) in comparison to clobetasol. The results from the in vitro models suggested less atrophogenic effects for the SEGRA compound, which indeed was confirmed in the hr/hr rat skin atrophy model. In conclusion, a combination of 3 in vitro models based on 3T3 cells, human keratinocytes and FTSM with several readouts is recommended to determine atrophogenicity of GC receptor ligands. Further experiments are necessary to eventually reduce this panel and to demonstrate the true predictability for the clinic.


 goto top of outline Key Words

  • In vitro test systems
  • Glucocorticoid receptor ligand
  • Skin atrophy

 goto top of outline Abstract

Topical glucocorticoids (GCs) demonstrate good anti-inflammatory effects but are limited by their side effect potential, with skin atrophy being the most prominent one. Thus, determining the atrophogenic potential of novel compounds is important. The aim of this study was to establish an in vitro skin atrophy model. A screening cascade was applied and GCs with a known atrophogenic potential were used as tool compounds. Five rodent and human cutaneous cell types/cell lines and 2 human skin equivalents were tested. Known and suspected atrophy markers related to collagen metabolism and epidermal thickness were measured. Altogether, a combination of 7 different cellular assays with up to 16 markers each were investigated. A reproducible, more than 2-fold, regulation of the candidate markers by dexamethasone or clobetasol was found for: (a) matrix metalloproteinase (MMP) 1, 2, 3 and 9 expression in human keratinocytes, (b) COL1A1 and COL3A1 expression in 3T3 fibroblasts, and (c) epidermal thickness, collagen and MMP synthesis in the full-thickness skin model (FTSM). These 3 models were further investigated with a panel of 4–5 GCs, demonstrating dose dependency and correlation with the atrophogenic potential of the tool compounds, qualifying them as potentially suitable. Finally, the predictability of these models for the in vivo situation was analyzed, testing a novel selective GC receptor agonist (SEGRA) in comparison to clobetasol. The results from the in vitro models suggested less atrophogenic effects for the SEGRA compound, which indeed was confirmed in the hr/hr rat skin atrophy model. In conclusion, a combination of 3 in vitro models based on 3T3 cells, human keratinocytes and FTSM with several readouts is recommended to determine atrophogenicity of GC receptor ligands. Further experiments are necessary to eventually reduce this panel and to demonstrate the true predictability for the clinic.

Copyright © 2009 S. Karger AG, Basel


 goto top of outline References
  1. Sterry W, Asadullah K: Topical glucocorticoid therapy in dermatology. Ernst Schering Res Found Workshop 2002;40:39–54.
  2. Niedner R: Glukokortikosteroide in der Dermatologie: Kontrollierter Einsatz erforderlich. Dtsch Ärztebl 1996;93:A2868–A2872.
  3. Luger T, Loske KD, Elsner P, Kapp A, Kerscher M, Korting HC, Krutmann J, Nickner R, Röcken M, Ruzicka T, Schwarz T: Topische Dermatotherapie mit Glukokortikoiden – Therapeutischer Index. J Dtsch Dermatol Ges 2004;2:629–634.
  4. Korting HC, Unholzer A, Schafer-Korting M, Tausch I, Gassmueller J, Nietsch KH: Different skin thinning potential of equipotent medium-strength glucocorticoids. Skin Pharmacol Appl Skin Physiol 2002;15:85–91.
  5. Kimura T, Doi K: Dorsal skin reactions of hairless dogs to topical treatment with corticosteroids. Toxicol Pathol 1999;27:528–535.
  6. Booth BA, Tan EM, Oikarinen A, Uitto J: Steroid-induced dermal atrophy: effects of glucocorticosteroids on collagen metabolism in human skin fibroblast cultures. Int J Dermatol 1982;21:333–337.
  7. Mills CM, Marks R: Side effects of topical glucocorticoids. Curr Probl Dermatol 1993;21:122–131.
  8. Kolbe L, Kligman AM, Schreiner V, Stoudemayer T: Corticosteroid-induced atrophy and barrier impairment measured by non-invasive methods in human skin. Skin Res Technol 2001;7:73–77.
  9. Kao JS, Fluhr JW, Man MQ, Fowler AJ, Hachem JP, Crumrine D, Ahn SK, Brown BE, Elias PM, Feingold KR: Short-term glucocorticoid treatment compromises both permeability barrier homeostasis and stratum corneum integrity: inhibition of epidermal lipid synthesis accounts for functional abnormalities. J Invest Dermatol 2003;120:456–464.
  10. Sheu HM, Yu HS, Sheen TC: Histochemical and ultrastructural studies on cutaneous side effects due to topical corticosteroids. 1. Changes in epidermis. Derm Sinica 1989;7:143–153.
  11. Saarni H, Hopsu-Havu VK: The decrease of hyaluronate synthesis by anti-inflammatory steroids in vitro. Br J Dermatol 1978;98:445–449.
  12. Lehmann P, Zheng P, Lavker RM, Kligman AM: Corticosteroid atrophy in human skin. A study by light, scanning, and transmission electron microscopy. J Invest Dermatol 1983;81:169–176.
  13. Cutroneo KR, Rokowski R, Counts DF: Glucocorticoids and collagen synthesis: comparison of in vivo and cell culture studies. Coll Relat Res 1981;1:557–568.
  14. Nuutinen P, Autio P, Hurskainen T, Oikarinen A: Glucocorticoid action on skin collagen: overview on clinical significance and consequences. J Eur Acad Dermatol Venereol 2001;15:361–362.
  15. Schoepe S, Schäcke H, May E, Asadullah K: Glucocorticoid therapy-induced skin atrophy. Exp Dermatol 2006;15:406–420.
  16. Schäcke H, Docke WD, Asadullah K: Mechanisms involved in the side effects of glucocorticoids. Pharmacol Ther 2002;96:23–43.
  17. Mirshahpanah P, Döcke WD, Merbold U, Asadullah K, Röse L, Schäcke H, Zollner TM: Superior nuclear receptor selectivity and therapeutic index of methylprednisolone aceponate versus mometasone furoate. Exp Dermatol 2007;16:753–761.
  18. Schäcke H, Schottelius A, Döcke WD, Strehlke P, Jaroch S, Schmees N, Rehwinkel H, Hennekes H, Asadullah K: Dissociation of transactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic effects from side effects. Proc Natl Acad Sci USA 2004;101:227–232.
  19. Wach F, Bosserhoff A, Kurzidym U, Nowok K, Landthaler M, Hein R: Effects of mometasone furoate on human keratinocytes and fibroblasts in vitro. Skin Pharmacol Appl Skin Physiol 1998;11:43–51.
  20. Ponec M, De Haas C, Bachra BN, Polano MK: Effects of glucocorticosteroids on cultured human skin fibroblasts. 3. Transient inhibition of cell proliferation in the early growth stages and reduced susceptibility in later growth stages. Arch Dermatol Res 1979;265:219–227.
  21. Schäcke H, Berger M, Rehwinkel H, Asadullah K: Selective glucocorticoid receptor agonists (SEGRAs): novel ligands with an improved therapeutic index. Mol Cell Endocrinol 2007;275:109–117.
  22. Kurzen H, Henrich C, Booken D, Poenitz N, Gratchev A, Klemke CD, Engstner M, Goerdt S, Maas-Szabowski N: Functional characterization of the epidermal cholinergic system in vitro. J Invest Dermatol 2006;126:2458–2472.
  23. Mewes KR, Raus M, Bernd A, Zöller NN, Sattler A, Graf R: Elastin expression in a newly developed full-thickness skin equivalent. Skin Pharmacol Physiol 2007;20:85–95.
  24. Schottelius AJ, Zugel U, Docke WD, Zollner TM, Rose L, Mengel A, Buchmann B, Becker A, Grutz G, Naundorf S, Friedrich A, Gaestel M, Asadullah K: The role of mitogen-activated protein kinase-activated protein kinase 2 in the p38/TNF-alpha pathway of systemic and cutaneous inflammation. J Invest Dermatol 2009, E-pub ahead of print.
  25. Li YY, Bao M, Meurer J, Skuballa W, Bauman JG, Doecke WD, Zollner TM: The identification of a small molecule inhibitor that specifically reduces T cell-mediated adaptive but not LPS-mediated innate immunity by T cell membrane-monocyte contact bioassay. Immunol Lett 2008;117:114–118.
  26. Zöller NN, Kippenberger S, Thaci D, Mewes K, Spiegel M, Sattler A, Schultz M, Bereiter-Hahn J, Kaufmann R, Bernd A: Evaluation of beneficial and adverse effects of glucocorticoids on a newly developed full-thickness skin model. Toxicol In Vitro 2008;22:747–759.
  27. Zerbe GO: On Fieller’s theorem and the general linear model. The Am Stat 1978;32:103–105.

    External Resources

  28. Dixon WJ: Processing data for outliers. Biometrics 1953;9:74–89.

    External Resources

  29. Wiedersberg S, Leopold CS, Guy RH: Bioavailability and bioequivalence of topical glucocorticoids. Eur J Pharm Biopharm 2008;68:453–466.
  30. Commandeur S, de Gruijl FR, Willemze R, Tensen CP, El Ghalbzouri A: An in vitro three-dimensional model of primary human cutaneous squamous cell carcinoma. Exp Dermatol 2009;18:849–856.

 goto top of outline Author Contacts

Prof. Dr. med. Khusru Asadullah
Target Discovery, Bayer Schering Pharma AG
DE–13342 Berlin (Germany)
Tel. +49 30 468 14853, Fax +49 30 468 192 374
E-Mail khusru.asadullah@bayerhealthcare.com


 goto top of outline Article Information

Received: September 25, 2009
Accepted: November 2, 2009
Published online: December 23, 2009
Number of Print Pages : 13
Number of Figures : 4, Number of Tables : 6, Number of References : 30


 goto top of outline Publication Details

Skin Pharmacology and Physiology (Journal of Pharmacological and Biophysical Research)

Vol. 23, No. 3, Year 2010 (Cover Date: March 2010)

Journal Editor: Lademann J. (Berlin)
ISSN: 1660-5527 (Print), eISSN: 1660-5535 (Online)

For additional information: http://www.karger.com/SPP


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

Topical glucocorticoids (GCs) demonstrate good anti-inflammatory effects but are limited by their side effect potential, with skin atrophy being the most prominent one. Thus, determining the atrophogenic potential of novel compounds is important. The aim of this study was to establish an in vitro skin atrophy model. A screening cascade was applied and GCs with a known atrophogenic potential were used as tool compounds. Five rodent and human cutaneous cell types/cell lines and 2 human skin equivalents were tested. Known and suspected atrophy markers related to collagen metabolism and epidermal thickness were measured. Altogether, a combination of 7 different cellular assays with up to 16 markers each were investigated. A reproducible, more than 2-fold, regulation of the candidate markers by dexamethasone or clobetasol was found for: (a) matrix metalloproteinase (MMP) 1, 2, 3 and 9 expression in human keratinocytes, (b) COL1A1 and COL3A1 expression in 3T3 fibroblasts, and (c) epidermal thickness, collagen and MMP synthesis in the full-thickness skin model (FTSM). These 3 models were further investigated with a panel of 4–5 GCs, demonstrating dose dependency and correlation with the atrophogenic potential of the tool compounds, qualifying them as potentially suitable. Finally, the predictability of these models for the in vivo situation was analyzed, testing a novel selective GC receptor agonist (SEGRA) in comparison to clobetasol. The results from the in vitro models suggested less atrophogenic effects for the SEGRA compound, which indeed was confirmed in the hr/hr rat skin atrophy model. In conclusion, a combination of 3 in vitro models based on 3T3 cells, human keratinocytes and FTSM with several readouts is recommended to determine atrophogenicity of GC receptor ligands. Further experiments are necessary to eventually reduce this panel and to demonstrate the true predictability for the clinic.



 goto top of outline Author Contacts

Prof. Dr. med. Khusru Asadullah
Target Discovery, Bayer Schering Pharma AG
DE–13342 Berlin (Germany)
Tel. +49 30 468 14853, Fax +49 30 468 192 374
E-Mail khusru.asadullah@bayerhealthcare.com


 goto top of outline Article Information

Received: September 25, 2009
Accepted: November 2, 2009
Published online: December 23, 2009
Number of Print Pages : 13
Number of Figures : 4, Number of Tables : 6, Number of References : 30


 goto top of outline Publication Details

Skin Pharmacology and Physiology (Journal of Pharmacological and Biophysical Research)

Vol. 23, No. 3, Year 2010 (Cover Date: March 2010)

Journal Editor: Lademann J. (Berlin)
ISSN: 1660-5527 (Print), eISSN: 1660-5535 (Online)

For additional information: http://www.karger.com/SPP


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

  1. Sterry W, Asadullah K: Topical glucocorticoid therapy in dermatology. Ernst Schering Res Found Workshop 2002;40:39–54.
  2. Niedner R: Glukokortikosteroide in der Dermatologie: Kontrollierter Einsatz erforderlich. Dtsch Ärztebl 1996;93:A2868–A2872.
  3. Luger T, Loske KD, Elsner P, Kapp A, Kerscher M, Korting HC, Krutmann J, Nickner R, Röcken M, Ruzicka T, Schwarz T: Topische Dermatotherapie mit Glukokortikoiden – Therapeutischer Index. J Dtsch Dermatol Ges 2004;2:629–634.
  4. Korting HC, Unholzer A, Schafer-Korting M, Tausch I, Gassmueller J, Nietsch KH: Different skin thinning potential of equipotent medium-strength glucocorticoids. Skin Pharmacol Appl Skin Physiol 2002;15:85–91.
  5. Kimura T, Doi K: Dorsal skin reactions of hairless dogs to topical treatment with corticosteroids. Toxicol Pathol 1999;27:528–535.
  6. Booth BA, Tan EM, Oikarinen A, Uitto J: Steroid-induced dermal atrophy: effects of glucocorticosteroids on collagen metabolism in human skin fibroblast cultures. Int J Dermatol 1982;21:333–337.
  7. Mills CM, Marks R: Side effects of topical glucocorticoids. Curr Probl Dermatol 1993;21:122–131.
  8. Kolbe L, Kligman AM, Schreiner V, Stoudemayer T: Corticosteroid-induced atrophy and barrier impairment measured by non-invasive methods in human skin. Skin Res Technol 2001;7:73–77.
  9. Kao JS, Fluhr JW, Man MQ, Fowler AJ, Hachem JP, Crumrine D, Ahn SK, Brown BE, Elias PM, Feingold KR: Short-term glucocorticoid treatment compromises both permeability barrier homeostasis and stratum corneum integrity: inhibition of epidermal lipid synthesis accounts for functional abnormalities. J Invest Dermatol 2003;120:456–464.
  10. Sheu HM, Yu HS, Sheen TC: Histochemical and ultrastructural studies on cutaneous side effects due to topical corticosteroids. 1. Changes in epidermis. Derm Sinica 1989;7:143–153.
  11. Saarni H, Hopsu-Havu VK: The decrease of hyaluronate synthesis by anti-inflammatory steroids in vitro. Br J Dermatol 1978;98:445–449.
  12. Lehmann P, Zheng P, Lavker RM, Kligman AM: Corticosteroid atrophy in human skin. A study by light, scanning, and transmission electron microscopy. J Invest Dermatol 1983;81:169–176.
  13. Cutroneo KR, Rokowski R, Counts DF: Glucocorticoids and collagen synthesis: comparison of in vivo and cell culture studies. Coll Relat Res 1981;1:557–568.
  14. Nuutinen P, Autio P, Hurskainen T, Oikarinen A: Glucocorticoid action on skin collagen: overview on clinical significance and consequences. J Eur Acad Dermatol Venereol 2001;15:361–362.
  15. Schoepe S, Schäcke H, May E, Asadullah K: Glucocorticoid therapy-induced skin atrophy. Exp Dermatol 2006;15:406–420.
  16. Schäcke H, Docke WD, Asadullah K: Mechanisms involved in the side effects of glucocorticoids. Pharmacol Ther 2002;96:23–43.
  17. Mirshahpanah P, Döcke WD, Merbold U, Asadullah K, Röse L, Schäcke H, Zollner TM: Superior nuclear receptor selectivity and therapeutic index of methylprednisolone aceponate versus mometasone furoate. Exp Dermatol 2007;16:753–761.
  18. Schäcke H, Schottelius A, Döcke WD, Strehlke P, Jaroch S, Schmees N, Rehwinkel H, Hennekes H, Asadullah K: Dissociation of transactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic effects from side effects. Proc Natl Acad Sci USA 2004;101:227–232.
  19. Wach F, Bosserhoff A, Kurzidym U, Nowok K, Landthaler M, Hein R: Effects of mometasone furoate on human keratinocytes and fibroblasts in vitro. Skin Pharmacol Appl Skin Physiol 1998;11:43–51.
  20. Ponec M, De Haas C, Bachra BN, Polano MK: Effects of glucocorticosteroids on cultured human skin fibroblasts. 3. Transient inhibition of cell proliferation in the early growth stages and reduced susceptibility in later growth stages. Arch Dermatol Res 1979;265:219–227.
  21. Schäcke H, Berger M, Rehwinkel H, Asadullah K: Selective glucocorticoid receptor agonists (SEGRAs): novel ligands with an improved therapeutic index. Mol Cell Endocrinol 2007;275:109–117.
  22. Kurzen H, Henrich C, Booken D, Poenitz N, Gratchev A, Klemke CD, Engstner M, Goerdt S, Maas-Szabowski N: Functional characterization of the epidermal cholinergic system in vitro. J Invest Dermatol 2006;126:2458–2472.
  23. Mewes KR, Raus M, Bernd A, Zöller NN, Sattler A, Graf R: Elastin expression in a newly developed full-thickness skin equivalent. Skin Pharmacol Physiol 2007;20:85–95.
  24. Schottelius AJ, Zugel U, Docke WD, Zollner TM, Rose L, Mengel A, Buchmann B, Becker A, Grutz G, Naundorf S, Friedrich A, Gaestel M, Asadullah K: The role of mitogen-activated protein kinase-activated protein kinase 2 in the p38/TNF-alpha pathway of systemic and cutaneous inflammation. J Invest Dermatol 2009, E-pub ahead of print.
  25. Li YY, Bao M, Meurer J, Skuballa W, Bauman JG, Doecke WD, Zollner TM: The identification of a small molecule inhibitor that specifically reduces T cell-mediated adaptive but not LPS-mediated innate immunity by T cell membrane-monocyte contact bioassay. Immunol Lett 2008;117:114–118.
  26. Zöller NN, Kippenberger S, Thaci D, Mewes K, Spiegel M, Sattler A, Schultz M, Bereiter-Hahn J, Kaufmann R, Bernd A: Evaluation of beneficial and adverse effects of glucocorticoids on a newly developed full-thickness skin model. Toxicol In Vitro 2008;22:747–759.
  27. Zerbe GO: On Fieller’s theorem and the general linear model. The Am Stat 1978;32:103–105.

    External Resources

  28. Dixon WJ: Processing data for outliers. Biometrics 1953;9:74–89.

    External Resources

  29. Wiedersberg S, Leopold CS, Guy RH: Bioavailability and bioequivalence of topical glucocorticoids. Eur J Pharm Biopharm 2008;68:453–466.
  30. Commandeur S, de Gruijl FR, Willemze R, Tensen CP, El Ghalbzouri A: An in vitro three-dimensional model of primary human cutaneous squamous cell carcinoma. Exp Dermatol 2009;18:849–856.