liposomes, part a

Preface The origins of liposome research can be traced to the contributions of Alec Bangham and colleagues in the mid 1960s. The description of lecithin disper- sions as containing ‘‘spherulites composed of concentric lamellae’’ (A. D. Bangham and R. W. Horne, J. Mol. Biol. 8, 660, 1964) was followed by the observation that ‘‘the diffusion of univalent cations and anions out of spontan- eously formed liquid crystals of lecithin is remarkably similar to the diffusion of such ions across biological membranes (A. D. Bangham, M. M. Standish and J. C. Watkins, J. Mol. Biol. 13, 238, 1965). Following early studies on the biophysical characterization of multilamellar and unilamellar liposomes, inves- tigators began to utilize liposomes as a well-defined model to understand the structure and function of biological membranes. It was also recognized by pioneers, including Gregory Gregoriadis and Demetrios Papahadjopoulos, that liposomes could be used as drug delivery vehicles. It is gratifying that their efforts and the work of those inspired by them have led to the development of liposomal formulations of doxorubicin, daunorubicin, and amphotericin B, now utilized in the clinic. Other medical applications of liposomes include their use as vaccine adjuvants and gene delivery vehicles, which are being explored in the laboratory as well as in clinical trials. The field has progressed enormously since 1965. This volume describes methods of liposome preparation, and the physico- chemical characterization of liposomes. I hope that these chapters will facilitate the work of graduate students, post-doctoral fellows, and established scientists entering liposome research. Subsequent volumes in this series will cover additional subdisciplines in liposomology. The areas represented in this volume are by no means exhaustive. I have tried to identify the experts in each area of liposome research, particularly those who have contributed to the field over some time. It is unfortunate that I was unable to convince some prominent investigators to contribute to the volume. Some invited contributors were not able to prepare their chapters, despite generous extensions of time. In some cases I may have inadvertently overlooked some experts in a particular area, and to these individuals I extend my apologies. Their primary contributions to the field will, nevertheless, not go unnoticed, in the citations in these volumes and in the hearts and minds of the many investigators in liposome research. ix In the last five years, the liposome field has lost some of its major members. Demetrios Papahadjopoulos (one of Alec Bangham’s proteges and one of my mentors) was a significant mover of the field and an inspiration to many young scientists. He organized the first conference on liposomes in 1977 in New York. He was also a co-founder of a company to attempt to commercialize liposomes for medical purposes. Danilo Lasic brought in his sophisticated biophysics background to help understand liposome behavior, wrote and co-edited nu- merous volumes on various aspects of liposomes, and helped their widespread appreciation with short reviews. David O’Brien was a pioneer in the field of photoactivatable liposomes, most likely inspired by his earlier work on rhod- opsin. He was to have contributed a chapter to the last volume of ‘‘Liposomes’’ in this series. For all their contributions to the field, this volume is dedicated to the memories of Drs. Papahadjopoulos, Lasic and O’Brien. I would like to express my gratitude to all the colleagues who graciously contributed to these volumes. I would like to thank Shirley Light of Academic Press for her encouragement for this project, and Noelle Gracy of Elsevier Science for her help at the later stages of the project. I am especially thankful to my wife Diana Flasher for her understanding, support and love during the endless editing process, and my children Avery and Maxine for their unique curiosity, creativity, cheer, and love. Nejat Du ¨ zgu ¨ nes Mill Valley x preface METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA FOUNDING EDITORS Sidney P. Colowick and Nathan O. Kaplan Contributors to Volume 367 Article numbers are in parentheses and following the names of contributors. Affiliations listed are current. Patrick Ahl (80), Bio Delivery Sciences International, Inc., UMDNJ-New Jersey Medical School, 185 South Orange Avenue, ADMC4, Newark, New Jersey 07103 Juha-Matti Alakoskela (129), Institute of Biomedicine, P.O. Box 63, Biomedcum Haartmaninkatu 8, University of Helsinki, Helsinki, FIN 00014, Finland Miglena Angelova (15), Institute of Bio- medicine, P.O. Box 63, Biomedicum Haartmaninkatu 8, University of Helsinki, Helsinki, FIN 00014, Finland Klaus Arnold (253), Institute for Medical Physics and Biophysics, Faculty of Medicine, University of Leipzig, D-04103 Leipzig, Germany Jesus Arroyo (213), Facultad Farmacia y Bioquı ´ mica, Universidad de Buenos Aires,Junin 956 2P, Buenos Aires 11113, Argentina Andrew Bacon (70), School of Pharmacy, Lipoxen Technologies Ltd., University of London, 29-39 Brunswick Square, London WC1N 1AX, England Luis A. Bagatolli (233), MEMPHYS- Center for Biomembrane Physics, Department of Biochemistry and Molecu- lar Biology, Campusvej 55, DK-5230 Odense M, Denmark Yechezkel Barenholz (270), Laboratory of Membrane and Liposome Research, Hebrew Univeristy-Hadassah Medical School, Jerusalem 91120, Israel Delia L. Bernik (213), Facultad Farmacia y Bioquı ´ mica, Universidad de Buenos Aires, Junin 956 2P, Buenos Aires 11113, Argentina Wilson Capparo ´ s-Wanderley (70), School of Pharmacy, Lipoxen Technolo- gies Ltd., University of London, 29-39 Brunswick Square, London WC1N 1AX, England Laurie Chow (3), Inex Pharmaceutical Copre, Glenlyon Business Park1, 100- 8900 Glenlyon Parkway, Burnaby, British Columbia, Canada V5J 5J8 Joel A. Cohen (148), Department of Physiology, University of the Pacific School of Dentistry, 2155 Webster Street, San Francisco, California 94115 Rivka Cohen (270), Laboratory of Membrane and Liposome Research, Hebrew Univeristy-Hadassah Medical School, Jerusalem 91120, Israel E. Anibal Disalvo (213), Facultad Farm- acia y Bioquı ´ mica, Universidad de Buenos Aires, Junin 956 2P, Buenos Aires 11113, Argentina Nejat Du ¨ zgu ¨ nes (23), Department of Microbiology, University of the Pacific School of Dentistry, 2155 Webster Street, San Francisco, California 94115 Simcha Even-Chen (270), Laboratory of Membrane and Liposome Research, Hebrew Univeristy-Hadassah Medical School, Jerusalem 91120, Israel Gregory Gregoriadias (70), School of Pharmacy, Lipoxen Technologies Ltd., University of London, 29-39 Bruns- wick Square, London WC1N 1AX, England Sadao Hirota (177), Tokyo Denki Univer- sity, 6-6-18 Higashikaigan-Minami, Chigasaki-Shi 253-0054, Japan vii Juha M. Holopainen (15), Institute of Biomedicine, P.O. Box 63, Biomedicum Haartmaninkatu 8, University of Helsinki, Helsinki, FIN 00014, Finland Reuma Honen (270), Laboratory of Mem- brane and Liposome Research, Hebrew Univeristy-Hadassah Medical School, Jerusalem 91120, Israel Michael Hope (3), Inex Pharmaceutical Copre, Glenlyon Business Park1, 100- 8900 Glenlyon Parkway, Burnaby, British Columbia, Canada V5J 5J8 Jana Jass (199), The Lawson Health Re- search Institute, 268 Grosvenor Street, London, Ontario, Canada N6A 4U2 Andrea Kas ˇ na ´ (111), Veterinary Research Institute, Department of Im- munology, Hudcova 70, 62132 Brno, Czech Republic Paavo K. J. Kinnunen (15, 129), Institute of Biomedicine, P.O. Box 63, Biomedicum Haartmaninkatu 8, Univer- sity of Helsinki, Helsinki, FIN 00014, Finland Peter Laggner (129), Institute of Bio- physics and X-Ray Structure Research, Austrian Academy of Sciences, Schmiedl- strasse 6,A-8042 Graz, Austria Brenda McCormack (70), School of Phar- macy, Lipoxen Technologies Ltd., University of London, 29-39 Brunswick Square, London WC1N 1AX, England Barbara Mui (3), Inex Pharmaceutical Copre, Glenlyon Business Park1, 100-8900 Glenlyon Parkway, Burnaby, British Columbia, Canada V5J 5J8 Jir ˇ ı ´ Nec ˇ a (111), Veterinary Research Insti- tute, Department of Immunology, Hudcova 70, 62132 Brno, Czech Republic Shinpei Ohki (253), Department of Physi- ology and Biophysics, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214 Walter R. Perkins (80), Transave, Inc., 11 Deerpark Drive, Suite 117, Monmouth Junction, New Jersey 08552 Gertrud Puu (199), Swedish Defense Re- search Agency, NBC Defence, SE 90182 Umei, Sweden Ramon Barnadas i Rodrı ´ guez (28), Uni- tat de Biofisica, Facultat de Medicine, Universitat Auto ` noma de Barcelona, Catalonia, 08193 Cerdanolya del Valle ` s, Spain Rolf Schubert (46), Pharmazeutisches Institut, Lehrstuhl fu ¨ r Pharmazeutische Technologie, Albert-Ludwigs-Universita ¨ t- Freiburg, Hermann-herder Strasse 9, D-79104 Freiburg, Germany Hilary Shmeeda (270), Shaare Zedek Medical Center, Department of Experimental Oncology, POB 3235, Jerusalem 91031, Israel Torbjo ¨ rn Tja ¨ rnhage (199), Swedish De- fense Research Agency, NBC Defence, SE 90182 Umei, Sweden Jaroslav Tura ´ nek (111), Veterinary Re- search Institute, Department of Immun- ology, Hudcova 70, 62132 Brno, Czech Republic Carmela Weintraub (270), Laboratory of Membrane and Liposome Research,- Hebrew Univeristy-Hadassah Medical- School, Jerusalem 91120, Israel Ewoud C. A. Van Winden (99), Regulon Gene Pharmaceuticals A.E.B.E., Auxentiou Grigoriou 7, Alimos, 17455 Athens, Greece Manuel Sabe ´ siXamanı ´ (28), Unitat de Biofisica, Facultat de Medicine, Universitat Auto ` noma de Barcelona, Catalonia, 08193 Cerdanolya del Valle ` s, Spain Dana Za ´ luska ´ (111), Veterinary Research Institute, Department of Immunology, Hudcova 70, 62132 Brno, Czech Republic viii contributors to volume 367 [1] Extrusion Technique to Generate Liposomes of Defined Size By Barbara Mui,Laurie Chow and Michael J. Hope Introduction Liposome extrusion is a widely used process in which liposomes are forced under pressure through filters with defined pore sizes to generate a homogeneous population of smaller vesicles with a mean diameter that reflects that of the filter pore. 1 This technique has grown in popularity and has become the most common method of reducing multilamellar lipo- somes, usually called multilamellar vesicles (MLVs), to large unilamellar vesicles (LUVs) for model membrane and drug delivery research. The extrusion concept was initially introduced by Olson et al., 2 who de- scribed the sequential passage of a dilute liposome preparation through polycarbonate filters of decreasing pore size, using a hand-held syringe and filter holder attachment, in order to produce a homogeneous size dis- tribution. This procedure was further developed and made more practical by the construction of a robust, metal extrusion device that employed medium pressures (800 lb=in 2 ) to rapidly extrude MLV suspensions dir- ectly through polycarbonate filters with pore diameters in the range of 50 to 200 nm to generate LUVs. 1 At the time this process represented a major advance for those routinely preparing LUVs. Other size reduction methods, such as the use of ultrasound or microfluidization techniques, tend to generate significant populations of ‘‘limit size’’ vesicles that are sub- ject to lipid-packing constraints 3 and also suffer from lipid degradation, heavy metal contamination, and limited trapping efficiencies. Reversed phase evaporation (REV) methods were also common in the 1980s and usually involved the formation of aqueous–organic emulsions followed by solvent evaporation to produce liposome populations with large trapped volumes and improved trapping efficiencies. 4 However, these methods are restricted by lipid solubility in solvent or solvent mixtures; moreover, 1 M. J. Hope, M. B. Bally, G. Webb, and P. R. Cullis, Biochim. Biophys. Acta 812, 55 (1985). 2 F. Olson, C. A. Hunt, F. C. Szoka, W. J. Vail, and D. Papahadjopoulos, Biochim. Biophys. Acta 557, 9 (1979). 3 M. J. Hope, M. B. Bally, L. D. Mayer, A. S. Janoff, and P. R. Cullis, Chem. Phys. Lipids 40, 89 (1986). 4 F. Szoka, F. Olson, T. Heath, W. Vail, E. Mayhew, and D. Papahadjopoulos, Biochim. Biophys. Acta 601, 559 (1980). [1] extrusion technique 3 Copyright 2003, Elsevier Inc. All rights reserved. METHODS IN ENZYMOLOGY, VOL. 367 0076-6879/03 $35.00 removal of residual solvent can be tedious. Detergent dialysis tech- niques are also subject to similar practical difficulties associated with lipid solubility and complete removal of detergent. Consequently, the convenience and speed of extrusion became a major advantage over other techniques. Extrusion can be applied to a wide variety of lipid species and mixtures, it works directly from MLVs without the need for sequential size reduction, process times are on the order of minutes, and it is only marginally limited by lipid concentration compared with other methods. Manufacturing issues related to removal of organic solvents or de- tergents from final preparations are eliminated and the equipment available for extrusion scales well from bench volumes (0.1 to 10 mL) through pre- clinical (10 mL to 1 liter) to clinical (>1 liter) volumes employing relatively low-cost equipment, especially at the research and preclinical levels. Extrusion and Extrusion Devices MLVs form spontaneously when bilayer-forming lipid mixtures are hy- drated in excess water, but they exhibit a broad size distribution ranging from 0.5 to 10 m in diameter and the degree of lamellarity varies depending on the method of hydration and lipid composition. These factors restrict severely the practical application of MLVs for membrane and drug delivery research, as discussed in detail elsewhere. 3 In general, <10% of the total lipid present in a normal multilamellar liposome is present in the outer monolayer of the externally exposed bilayer compared with 50% in the outer monolayer of a large unilamellar system. 1 Consequently, the LUV better reflects the bilayer structure of a typical plasma or large organelle membrane. Other limitations of MLVs include their large diam- eter, size heterogeneity, multiple internal compartments, low trap volumes, and inconsistencies from preparation to preparation. Therefore, sizing MLV preparations by extrusion is an effective way to overcome some of these problems and to generate reproducible model membrane systems for basic research, applied research, and clinical applications. Only moderate pressures (typically 200–800 lb=in 2 ) are required to force liquid crystalline MLVs through polycarbonate filters with defined pore sizes. The majority of laboratories specializing in liposome research, particularly as applied to drug delivery, use a heavy-duty device com- mercially available from Northern Lipids (Vancouver, BC, Canada; www.northernlipids.com). The Lipex extruder is an easy-to-use, robust stainless steel unit, which can operate up to pressures of 800 lb=in 2 (Fig. 1). A quick-fit sample port assembly allows for rapid and convenient cycling of preparations through the filter holder. The sequential use of large to small pore filters 2 to reduce back pressure is not necessary for 4 methods of liposome preparation [1] Fig. 1. A research-scale extrusion device (Lipex extruder) manufactured by Northern Lipids (Vancouver, BC, Canada) has a 10-mL capacity and can be operated over a wide range of temperatures when used in combination with a circulating water bath. The quick-release sample port at the top of the unit allows for rapid cycling of sample through the filters. [1] extrusion technique 5 the majority of lipid samples, and large multilamellar systems can be ex- truded directly through filter pore sizes as small as 30 nm. The equipment is also fitted with a water-jacketed, sample-holding barrel that enables the extrusion of lipids with gel–liquid crystalline phase transitions above room temperature, an important feature as gel-state lipids will not extrude (see Effect of Lipid Composition on Extrusion, later). Extrusion can also be performed with a hand-held syringe fitted with a standard sterilization filter holder or purpose-built hand-held units, such as those supplied by Avanti Polar Lipids (Alabaster, AL; www.avanti- lipids.com) and Avestin (Ottawa, ON, Canada; www.avestin.com). These devices are suitable only for small-volume applications (typically <1 mL); one example consists of two Hamilton syringes connected by a filter holder, allowing for back-and-forth passage of the sample. 5 Using this technique, a dilute suspension of liposomes (composed of liquid crystalline lipid) can be passed through the filters to reduce vesicle size. This method, however, is limited by the back pressure that can be tolerated by the syringe and filter holder, as well as the pressure that can be applied manually. Gener- ally, phospholipid concentrations must be less than 30 mM in order to comfortably extrude liposomes manually. A variety of filters suitable for reducing the mean diameter of liposome preparations are available from scientific suppliers. The most commonly used are standard polycarbonate filters (with straight-through pores). Other filter materials can be used, but the polycarbonate type has proved to be reliable, inert, durable, and easy to apply to filter supports without damage. Pore density influences extrusion pressure. In our experience there is usually little variation between filters from the same manufacturer. However, on occasion users may notice changes in vesicle diameter pre- pared when using filters from different batches from the same supplier or when using filters in which the pores are created by different manufactur- ing processes. Tortuous path type filters do not have well-defined pore diameters like the straight-through type, and back pressure tends to be higher when using these filters for liposome extrusion. However, adequate size reduction can still be achieved. Mechanism of Extrusion and Vesicle Morphology As the concentric layers of a typical MLV squeeze into the filter pore under pressure during extrusion, a process of membrane rupture and resealing occurs. The practical consequence of this is that any solute trapped 5 R. C. MacDonald, R. I. MacDonald, B. P. Menco, K. Takeshita, N. K. Subbarao, and L. R. Hu, Biochim. Biophys. Acta 1061, 297 (1991). 6 methods of liposome preparation [1] inside an MLV or large liposome before size reduction will leak out during the extrusion cycle. Therefore, when specific solutes are to be encapsulated, extrusion is nearly always performed in the presence of medium containing the desired final solute concentration and external (unencapsulated) solute is removed only when sizing is complete. In a study on the mechanism of liposome size reduction by extrusion, Hunter and Frisken 6 demonstrated that the pressure needed to reduce the particle size of vesicles during pas- sage through a 100-nm pore correlated with the force needed to rupture the lipid membrane and not the force required simply to deform the bilayer. Interestingly, these authors also noted that as flow rate through the filter in- creased the mean vesicle size decreased. This is attributed to the thickness of the lubricating layer formed by fluid associated with the sides of the pore from which particles are excluded. As the velocity of the fluid increases the thickness of the lubricating layer also increases, effectively reducing the pore diameter experienced by vesicles traversing the membrane. 6,7 The rupture and resealing process can also give rise to oval or sausage- shaped vesicles, and Mui et al. 8 showed that this shape deformation is dic- tated largely by osmotic force. As vesicles are squeezed through the pores they elongate and lose internal volume through transient membrane rupture to accommodate the increase in surface area-to-volume ratio asso- ciated with the nonspherical morphology. On exiting the pore the mem- brane wants to adopt a spherical shape, thermodynamically the lowest energy state for the bilayer, but the required increase in trapped volume is opposed by osmotic force. Therefore, in the presence of impermeable or semipermeable solutes (e.g., common buffers and salts) oval or saus- age-shaped vesicles are produced, whereas vesicles made in pure water are spherical (Fig. 2A and B). Sausage-like and dimpled vesicle morphology is observed when extru- sion occurs even in solutions of relatively low osmolarity, such as 10 mM NaCl. It should be noted that these vesicle morphologies have been ob- served only when employing cryoelectron microscopy techniques, in which vesicles are visualized through thin films of ice in the absence of cryopro- tectants. Freeze–fracture methods do not reveal sausage-like morphology under the same conditions, which may be due to the high concentrations of membrane-permeable glycerol (25%, v=v), used as a cryoprotectant, affecting the osmotic gradient. Rounding up of vesicles is readily achieved by simply lowering the ionic strength of the external medium. 8 6 D. G. Hunter and B. J. Frisken, Biophys. J. 74, 2996 (1998). 7 G. Gompper and D. M. Kroll, Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics 52, 4198 (1995). 8 B. L. Mui, P. R. Cullis, E. A. Evans, and T. D. Madden, Biophys. J. 64, 443 (1993). [1] extrusion technique 7 [...]... centrifuged at 100,000g for 1 h, preferably in a swinging bucket rotor, to eliminate any remaining large [3] preparing small and large unilamellar liposomes 25 liposomes The supernatant is used as the small unilamellar liposome preparation Preparation of Large Unilamellar Liposomes by Reverse-Phase Evaporation The reverse-phase evaporation technique was developed by Szoka and Papahadjopoulos,5 and refined... generate liposomes of different size and characteristics Here we describe the preparation of two types of liposomes: (1) small unilamellar liposomes prepared by bath sonication, (2) large unilamellar liposomes prepared by reverse-phase evaporation Preparation of Small Unilamellar Liposomes Small unilamellar liposomes were developed by Papahadjopoulos and Miller2 and characterized thoroughly by Huang3 and... sonicated in a bath-type sonicator (Laboratory Supply) for 2–5 min, ensuring that the surface of the mixture breaks up into small droplets The ether and aqueous phases should not separate and should form a stable emulsion After opening the screw cap, preferably under a hood, the tube is sealed again with Teflon tape and placed inside the larger glass tube that fits onto 5 F C Szoka, Jr and D Papahadjopoulos,... methods have been developed to generate liposomes of different size and characteristics Here we describe the preparation of two types of liposomes: (1) small unilamellar liposomes prepared by bath sonication, (2) large unilamellar liposomes prepared by reverse-phase evaporation Preparation of Small Unilamellar Liposomes Small unilamellar liposomes were developed by Papahadjopoulos and Miller2 and characterized... Oberholzer, and P Walde, in ‘‘Giant Vesicles’’ (P L Luisi and P Walde, eds.), p 37 John Wiley & Sons, New York, 1999 [3] Preparation and Quantitation of Small Unilamellar Liposomes and Large Unilamellar Reverse-Phase Evaporation Liposomes ¨ ¨ By Nejat Duzgunes, Introduction Since the publication of the first article on the preparation and characterization of multilamellar liposomes,1 numerous methods have been... 32 A Fischer, P L Luisi, T Oberholzer, and P Walde, in ‘‘Giant Vesicles’’ (P L Luisi and P Walde, eds.), p 37 John Wiley & Sons, New York, 1999 [3] Preparation and Quantitation of Small Unilamellar Liposomes and Large Unilamellar Reverse-Phase Evaporation Liposomes ¨ ¨ By Nejat Duzgunes, Introduction Since the publication of the first article on the preparation and characterization of multilamellar liposomes,1 ... Papahadjopoulos, Proc Natl Acad Sci USA 75, 4194 (1978) F Szoka, F Olson, T Heath, W Vail, E Mayhew, and D Papahadjopoulos, Biochim Biophys Acta 601, 559 (1980) 7 ¨ ¨ N Duzgunes,, J Wilschut, R Fraley, and D Papahadjopoulos, Biochim Biophys Acta 642, 182 (1981) 6 26 methods of liposome preparation [3] the rotary evaporator About 1 ml of water is included in the outer tube, both to maintain thermal contact and to minimize... Biomembranes, Vancouver, BC, Canada) or syringe extruder (Avestin, Ottawa, ON, Canada; Avanti Polar Lipids, Alabaster, AL) to achieve a uniform size distribution.6,8 Two stacked membranes separated by a drain disk may be used with the high-pressure extrusion device, and the liposome suspension may be passed through the membranes five times The syringe extruders are more convenient for multiple extrusions, and... small enough not only to circulate without becoming trapped in tissue microvasculature but also to accumulate at tumor and inflammation sites by extravasation through endothelial cell pores and gaps associated with these areas.14,15 Furthermore, vesicles of this size have good drug-carrying capacity but are small enough to pass through sterilizing filters without damage 14 S K Hobbs, W L Mosky, F Yuan,... using glass Hamilton syringes Any fluorescent lipids to be incorporated in the liposome membrane are included in the chloroform mixture at this stage Teflon tape is ‘‘sealed’’ over the top of the tube, the tube is placed in a larger glass tube with a fitting appropriate ¨ for a rotary evaporator (Buchi, with attached vacuum gauge and purge tubing connected to an argon tank), and the lipid is evaporated to . Unitat de Biofisica, Facultat de Medicine, Universitat Auto ` noma de Barcelona, Catalonia, 08193 Cerdanolya del Valle ` s, Spain Dana Za ´ luska ´ (111), Veterinary Research Institute, Department. Northern Lipids (Vancouver, BC, Canada) has a 10-mL capacity and can be operated over a wide range of temperatures when used in combination with a circulating water bath. The quick-release sample port at the. Sweden Ramon Barnadas i Rodrı ´ guez (28), Uni- tat de Biofisica, Facultat de Medicine, Universitat Auto ` noma de Barcelona, Catalonia, 08193 Cerdanolya del Valle ` s, Spain Rolf Schubert (46), Pharmazeutisches Institut,