Enzymatic degradation and drug release behavior of dense collagen implants [Elektronische Ressource] / vorgelegt von Iris Metzmacher

Iris Metzmacher

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• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Enzymatic Degradation and
Drug Release Behavior of Dense
Collagen Implants




Dissertation

zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München





vorgelegt von
Iris Metzmacher
aus München

München 2005

published by Verlag Dr. Hut, Munich, ISBN: 3-89963-253-2

ERKLÄRUNG:
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der
Promotionsordnung vom 29. Januar 1998 von Herrn Prof. Dr. Wolfgang Frieß
betreut.


EHRENWÖRTLICHE VERSICHERUNG
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe angefertigt.


München, den 28. September 2005


..............................................................................
(Iris Metzmacher)





Dissertation eingereicht am: 30. September 2005
1. Gutachter: Prof. Dr. Wolfgang Frieß
2. Gutachter: Prof. Dr. Karsten Mäder
Tag der mündlichen Prüfung: 25. Oktober 2005
published by Verlag Dr. Hut, Munich, ISBN: 3-89963-253-2
Acknowledgments

This thesis was written at the Department of Pharmacy, Pharmaceutical Technology
and Biopharmaceutics at the Ludwig-Maximilians-University in Munich. The co-
operation of several other parties contributed to the development of this work.
I would like to express my gratitude to my supervisor Prof. Dr. Wolfgang Frieß who
gave me the opportunity to join his working group and greatly encouraged my interest
in the field of biomaterials. I very much enjoyed the scientific discussions (which often
developed into conversations about many other topics), his ongoing interest in my work
and his scientific and personal advice. Thanks for providing such an outstanding
working climate.
I would also like to extend my appreciation to Prof. Dr. Gerhard Winter who has created
a very pleasant working atmosphere at the institute and who originally engaged my
interest in pharmaceutical technology during my study of pharmacy in Munich.
Over the years I was accompanied by Dr. Florin Radu and PD Dr. Markus Bause from
the Institute of Applied Mathematics at the Friedrich-Alexander University of Erlangen-
Nuremberg (working group of Prof. Dr. Peter Knabner). Based on our cooperation in
describing the drug release from dense collagen devices, a close working partnership
developed. Thanks for giving me a small insight into the wide (and sometimes very
confusing) field of mathematics and the chance to see problems through different eyes.
I really enjoyed this broadening of my scientific horizons.
I am indebted to Prof. Dr. Karsten Mäder who introduced me to the secrets of ESR and
who spent a lot of time making my investigations possible. In his working group at the
Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics at the
Martin-Luther-University in Halle/Saale, I would like to thank Kerstin Schwarz who
performed the in vitro ESR investigations and who provided every detail I needed to
deal with the data. I would also like to acknowledge Martin Bastrop who guided me
through my in vivo measurements. Thanks to the whole group for the warm welcome
and the very pleasant stay in Halle/Saale.
The in vivo investigations were performed in co-operation with the working group of
Prof. Dr. Dr. Stefan Schultze-Mosgau (Department of Oral and Maxillofacial
Surgery/Plastic Surgery at the Friedrich-Schiller-University, Jena). Dr. Falk Wehrhan is
published by Verlag Dr. Hut, Munich, ISBN: 3-89963-253-2
acknowledged for his support, even if the stress of the clinical work limited the time
available for discussions.
Dr. Martin Abel and Dr. Peter Ruth (both Lohmann & Rauscher GmbH & Co. KG,
Rengsdorf) are acknowledged for their scientific advice concerning the binding studies
on collagen wound dressings.
At the LMU Munich, I’m grateful to Prof. Dr. Joachim Rädler for the opportunity to use
the FCS at the Institute of Experimental Physics. Special thanks go to Dr. Laura Rusu
and Dr. Simon Keller who spent many hours with me at the spectrometer to perform
the measurements.
For the challenging investigation of the diffusion coefficient inside the implants, I would
like to thank Dr. André Pampel (Department of Physics and Earth Science, Group of
Physics of Dielectric Solids at the University Leipzig) for his great efforts.
I would like to thank all my colleagues who shared the time with me at our institute.
Imke Leitner who supported me whenever I needed help and all the other members of
the “Frießens” for their friendship and support. Special thanks go to my lab partners
Andreas Rutz and Dr. Daniel Schwartz who made the years in B.0.003 a pleasant
short-time stay and who always helped me when things weren’t working the way they
should. I am grateful for the assistance of Dr. Silke Mohl and Dr. Roland Schmidt who
answered every question patiently.
Guido, Mama, Papa and Gernot, thanks a lot for your love and support; I will always
remember the words “If you try hard enough, everything is possible!”.
There is not enough space to acknowledge all of the contributions to this work. Many
thanks go to all who contributed in one or another way to my work, but were not
explicitly listed here. This does not reduce my appreciation in any way.
Last but not least, I would like to thank the DFG for financially supporting this thesis
and Innocoll GmbH, Saal/Donau and Lohmann & Rauscher GmbH & Co. KG,
Rengsdorf for providing the collagen materials.
published by Verlag Dr. Hut, Munich, ISBN: 3-89963-253-2


















“As far as the laws of mathematics
refer to reality, they are not certain,
as far as they are certain, they do
not refer to reality.”

Albert Einstein (1879-1955)
published by Verlag Dr. Hut, Munich, ISBN: 3-89963-253-2

Table of Contents

1 INTRODUCTION 1
1.1 Classification of Polymers for Controlled Drug Release Devices 2
1.2 Classifications of Biodegradable Polymers 5
1.2.1 Origin of Polymer – Collagen: a Natural Polymer 5
1.2.1.1 Structure of Collagen 7
1.2.1.2 Cross-linking of Collagen 9
1.2.1.3 Collagen as a Biomaterial 12
1.2.2 Mechanism of Degradation 14
1.2.2.1 In vitro Degradation of Collagen 16
1.2.2.2 In vivo Degradation of Collagen 20
1.2.3 Mechanism of Drug Release 25
1.2.3.1 Erosion Controlled Drug Release 27
1.2.3.2 Drug Release from Collagen Devices 28
1.3 Introduction to Mathematical Models 30
1.3.1 Enzymatic Reaction 33
1.3.1.1 Adsorption 35
1.3.1.2 Degradation 37
1.3.2 Drug Release from Biodegradable Devices 42
1.3.2.1 Diffusion Controlled Drug Release 42
1.3.2.2 Swelling Controlled Drug Release 43
1.3.2.3 Erosion Controlled Drug Release 43
2 GOALS OF THIS THESIS 50
3 MATERIALS AND METHODS 52
3.1 Materials 52
3.1.1 Collagen 52
3.1.2 Wound Dressings 52
published by Verlag Dr. Hut, Munich, ISBN: 3-89963-253-2
3.1.3 Enzymes 52
3.1.3.1 Collagenase 52
3.1.3.2 Gelatinase A 53
3.1.3.3 Gelatinase B 53
3.1.4 Model Compounds 54
3.1.5 Reagents and Buffers 55
3.2 Methods 57
3.2.1 Matrix Preparation 57
3.2.1.1 Collagen Powder 57
3.2.1.2 Collagen Minirods 57
3.2.1.3 DHT Cross-linking 59
3.2.1.4 EDC 60
3.2.1.5 Lyophilization 60
3.2.2 Characterization of the Collagen Matrices 61
3.2.2.1 Macroscopic Studies 61
3.2.2.2 Scanning Electron Microscopy (SEM) 61
3.2.2.3 Determination of Density 61
3.2.2.4 Karl-Fischer Titration 61
3.2.2.5 Differential Scanning Calorimetry (DSC) 62
3.2.2.6 Swelling Studies 62
3.2.2.7 Pulsed Field Gradient Nuclear Magnetic Resonance Spectroscopy
(PFG-NMR) 62
3.2.3 Characterization of the Model Drugs 63
3.2.3.1 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-
PAGE) 63
3.2.3.2 Fluorescence Correlation Spectroscopy (FCS) 63
3.2.4 Characterization of the Enzymatic Reaction 64
3.2.4.1 Determination of Enzyme Activity 64
3.2.4.1.1 FALGPA Assay 64
3.2.4.1.2 EnzChek® Gelatinase / Collagenase Assay Kit 64
3.2.4.2 Binding studies 64
3.2.4.3 Sorption Studies 65
3.2.4.4 Determination of the Degradation Constants 65
3.2.5 In vitro Degradation Studies 66
3.2.6 In vitro Release Studies 66
published by Verlag Dr. Hut, Munich, ISBN: 3-89963-253-2
3.2.6.1 FITC Dextrans 66
3.2.6.2 BSA 66
3.2.7 In vivo Studies 67
3.2.7.1 Electron Spin Resonance Spectroscopy (ESR) 67
3.2.7.2 Histology 67
3.2.8 Mathematical Discretization 68
4 RESULTS AND DISCUSSION 69
4.1 Characterization of Collagen Matrices 69
4.1.1 Characterization of Dry Collagen Matrices 69
4.1.1.1 Matrix Density 70
4.1.1.2 Differential Scanning Calorimetry (DSC) 74
4.1.2 Transport of Water in Collagen Matrices 77
4.1.2.1 Swelling of Collagen Matrices 77
4.1.2.1.1 Swelling Without Addition of Collagenase 78
4.1.2.1.2 Swelling in the Presence of Collagenase 87
4.1.2.2 Dif