QT
`34
`861578
`
`1993
`
`I
`
`SURPLUS -1
`LIBRARY OF CONGRESS
`DUPLICATE
`
`Biotechnological
`Po lytners
`
`MEDICAL
`PHARMACEUTICAL
`and INDUSTRIAL
`APPLICATIONS
`A CONFERENCE IN PRINT
`
`Edited by
`Charles G. Gebelein, Ph.D.
`
`ALL 2068
`PROLLENIUM V. ALLERGAN
`IPR2019-01505 et al.
`
`

`

`Biotechnological Polymers
`a TECHNOMICpublication
`
`Puhlished ill the W('.\Ierti Hemisphere hy
`Technomic Publishing Company, Inc.
`851 New Holland Avenue, Box 3535
`Lancaster, Pennsylvania 17604 U.S.A.
`
`Distrihuted in the Rest of the »t"ld hy
`Technomic Publishing AG
`Missionsstrasse 44
`CH-4055 Basel, Switzerland
`
`Copyright © 1993 by Technomic Publishing Company, Inc.
`All rights reserved
`
`No part of this publication may be reproduced, stored in a
`retrieval system, or transmitted, in any form or by any means,
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`without the prior written permission of the publisher.
`
`Printed in the United States of America
`
`1098765432
`
`Main entry under title:
`Biotechnological Polymers: Medical, Pharmaceutical and Industrial Applications(cid:173)
`A Conference in Print
`
`A Technomic Publishing Company book
`Bibliography: p.
`
`Library of Congress Catalog Card No. 93-60079
`ISBN No. 1-56676-034-8
`
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`

`

`;
`
`J
`J
`
`TABLE OF CONTENTS
`
`,
`PART I: BUSINESS AND REGULATORy.cONSIDERATIONS
`Biotech Polymers: What They Are and What They Do ..... .
`Dr. Charles G. Gebelein, Lionfire, Inc.
`Biopolymers: Key Companies, Technologies, and Products ....... .
`Roger E. Shamel, President, Consulting Resources Corporation
`Regulatory Considerations for Biotechnology Applications ..
`M. R. Thomas, The Dow Chemical Company
`
`. .3
`
`. .17
`
`. ........... 22
`
`. ........... 34
`
`PART II: MEDICAL AND PHARMACEUTICAL APPLICATIONS
`Merging Frontiers of Biotechnology in Pharmaceutical
`and Polymer Applications .................................................... 29
`Malay Ghosh, Schering Plough Research Institute
`Biological Activities of Hydroxyl-Containing Natural Products
`Emphasizing Tin-Containing Products ......................... .
`Charles E. Carraher, Jr. and Cynthia Butler, Departments of Chemistry
`and Biological Sciences, Florida Atlantic University
`Applications of Hyaluronan and Its Derivatives ................................... 41
`Endre A. Balazs, Edward Leshchiner, Nancy E. Larsen and Philip Band,
`Biomatrix, Inc.
`Collagen Biotechnology and Its Medical Applications ......... .
`Shu-Tung Li, Ph.D., Vice President of R&D/Operations, ReGen Corporation
`Medical Applications of Bioelastic Materials ...................................... 82
`D. W. Urry, Director and A. Nico1, Laboratory of Molecular Biophysics,
`The University of Alabama at Birmingham
`D. C. Gowda and B. A. Cox, Bioelastics Research, Ltd.
`Lynne D. Hoban, Adam McKee and Taffy Williams, U.S. Department of the Navy,
`Naval Medical Research
`D. B. Olsen, Department of Biomedical Engineering, University of Utah
`Melanin, the Natural Biopolymer for Ultraviolet Protection ......................... 104
`Sergio Nacht, Ph.D., Senior Vice President, R&D, Advanced Polymer Systems, Inc.
`Polymer Coatings in Biomedical Applications ................................... 123
`Melvin H. Keyes, Ph.D., Vice President, David E. Albert, Mary B. Douglas,
`Don N. Gray, Marcia A. Hintz, Timothy J. Wolf and Christopher S. Youngen,
`Anatrace, Inc.
`BiotechlJological Diagnostic Polymers and Coatings .............................. 139
`Arthur M. Usmani, Boehringer Mannheim
`
`. .66
`
`iii
`
`

`

`. ................... . 175
`
`. .................... . 191
`
`PART III: BIODEGRADABILITY AND APPLICATIONS
`Plastics in the Environment-The Gathering Storm.
`Dr. W James Hammar, Staff Scientist, 3M Company
`Requirements to Define Biodegradable Polymers.
`Graham Swift, Rohm and Haas Company
`Polymers from Biotechnology: Bacterial Polyesters
`and y-Poly(glutamic acid) . ......... .
`Richard A. Gross, Gregory A. Birrer, Anne-Marie Cromwick
`and Stephen A. Giannos, Department of Chemistry, University of
`Massachusetts at Lowell
`Stephen P. McCarthy, Department of Plastics Engineering,
`University of Massachusetts at Lowell
`Biodegradable Polymers for Packaging. . . . . . . . . . . . . .. . ........... .
`Stephen P McCarthy, Department of Plastics Engineering,
`University of Massachusetts at Lowell
`
`. . 200
`
`....... . 214
`
`iv
`
`

`

`APPLICATIONS OF HYALURONAN
`AND ITS DERIVATIVES
`
`Endre A. Balazs, Edward Leshchiner, Nancy E. Larsen and Philip Band
`Biomatrix, Inc., Ridgefield, New Jersey
`
`INTRODUCTION
`\,
`During the past two decades hyaluronan (HA, hyaluronic acid) and 'it;&e~lvatives have become
`important therapeutic agents in medicine. This development was triggered oy the recognition in the
`late 1960's that the highly purified form of hyaluronan (the non-inflammatory fraction of Na(cid:173)
`hyaluronan - NIFNaHA) prepared from animal tissues, is extremely biocompatible when applied to
`such sensitive tissue compartments as the vitreus of the eye and the synovial space in joints { 1, 2}.
`A few years later this discovery led to the medical application of NIF-NaHA in ophthalmic surgery
`,
`and for the treatment of arthritis in humans and in horses (3, 4}.
`
`In the mid 1980's, the field of crosslinked hyaluronan derivatives began to develop. Two
`crosslinked forms of hyaluronan, hylan fluid and hylan gel, were invented and their biological
`activity and medical usefulness were widely explored (5}. They proved to be just as
`biocompatible as the native hyaluronan, but had enhanced rheological properties and longer
`residence time in the tissues than hyaluronan. The therapeutic application of the hylans in a broad
`spectrum of medical specialties is now in progress. These include: viscosurgery, arthritis therapy,
`adhesion management, topical administration, drug delivery and soft tissue augmentation (e.g.
`dermal, urological and reconstructive applications). This important development stimulated broad
`interest in new forms of crosslinked or otherwise modified forms of hyaluronan.
`
`This paper will review the chemistry of the various hylans and other crosslinked hyaluronans,
`focusing on their current usage and proposed medical applications.
`
`HYALURONAN
`
`This ubiquitous polysaccharide molecule is located in the intercellular space and fills the space
`between the collagen and elastin fibers, cell membranes and basal laminae. It is produced by many
`cell types in the cell membrane and is, therefore, never stored inside the cell. After synthesis it is
`It is considered as a space filling, structure
`extruded directly into the extracellular space.
`stabilizing, cell coating and cell protective polysaccharide. Its primary biological role is to stabilize
`the intercellular structure of fibrous and membranous proteins. It forms a structurally integrated
`system with the fibrous proteins of the intercellular space, creating the elastoviscous, protective,
`lubricating and stabilizing matrix in which cells are embedded. Hyaluronan solutions are extremely
`elastoviscous and pseudoplastic. Their exceptionally high rheological properties are present even
`in highly hydrated polymer systems (water content more than 99%). This combination of high
`elastoviscosity and low solids content pem1its unhindered diffusion of metabolites to and from the
`cells embedded in or separated by the viscoelastic hyaluronan molecular network.
`
`The unusual rheological properties of hyaluronan are based on a relatively simple structure. It is a
`linear, unbranched (not crosslinked) polyanionic molecular chain consisting of repeating
`glucuronic acid-N-acetyl glucosamine dimers with a large mass (4-5 million). In aqueous solution,
`the hyaluronan molecule behaves as a highly hydrated random coil with very large molecular
`volume, forcing the molecules to become entangled and interpenetrate at relatively low
`
`41
`
`

`

`concentrations «0.01 %), and thereby giving rise to hyaluronan's unusual rheological properties
`(6} .
`
`Hyaluronan solutions of low concentration and lower molecule mass than occurs naturally were
`found to influence various cell activities in in vitro studies (7}. These effects could not be
`demonstrated in in vivo systems. The highly elastoviscous hyaluronan solutions also have an
`effect on cells, especially on the movement of the cell membrane and the cytoskeleton of the cell
`[8}. The effect of viscoelastic hyaluronan sol utions on cells and the intercellular matrix forms the
`basis of their current medical applications.
`
`The need for enhanced viscoelastic properties and greater solidity than provided by native
`hyaluronan solutions triggered the interest in producing its crosslinked derivatives. The
`crosslinking derivation must produce not only rheologically useful, but also biologically
`compatible polymeric systems. We shall review here only those derivatives which have been
`shown to be useful as therapeutic agents.
`
`HY ALURONAN DERIVATIVES
`
`The hyaluronan polysaccharide chain contains three types of functional groups which can be used
`for derivitization, namely, hydroxy-, carboxy-, and acetamido groups. The reducing end groups
`need not be considered due to the extremely high molecular weight of the polymer). There is an
`enormous number of derivatives which may be obtained through chemical reactions of these
`functionalities with various reagents. The practical significance of any hyaluronan derivative is
`determined, mainly, by its utility in the medical field.
`
`The earliest synthetized derivative of hyaluronan was its sulfate ester (9}. A heterogeneous
`sulfation process for suspension of hyaluronan in pyridine by a mixture of pyridine and
`chlorosulfonic acid was developed. A highly substituted product was obtained by running the
`sulfation reaction repeatedly. The substance was characterized as having strong antienzymatic
`activity, i.e. strong antihyaluronidase and anticoagulant activities, and to be inhibitory of fibroblast
`growth. Single phase solution method to obtain hyaluronan sulfate esters was described in {9}
`which probably represents the first patented application of hyaluronan technology. Despite the
`beneficial properties of hyaluronan sulfates they never have been developed into commercial
`products, probably due to the availability of other sulfated glycosaminoglycans, especially heparin
`and chondroitin sulfate.
`
`A substantial number of crosslinked hyaluronan derivatives have been developed. According to
`the nomenclature suggested by Balazs et al. (5}, cross-linked derivatives of hyaluronan modified
`only through hydroxy groups (carboxyls and acetamido groups remain unreacted) are named
`hylans. The first cross-linked hyaluronan derivative of the hylan type was obtained using the
`bifunctional substance - 1, 2, 3, 4-diepoxybutane (12}. The diepoxide reacted with hyaluronan in
`the presence of alkali and sodium borohydride forming a gel which could swell in water or saline
`several hundred times. Obviously, the cross-linking was provided by reaction of hydroxy groups
`with epoxy groups of diepoxybutane.
`
`A large group of hyaluronans crosslinked via their hydroxy group reactivity was developed by
`Balazs and Leshchiner (13} Cross-linking agents of various functionality were used in the
`reactions including formaldehyde, dimethylol urea, dimethylolethylene urea, polyisocyanate, vinyl
`sulfone. Water insoluble products were obtained in the shape of a powder, a film, or a coating on
`various substrates. The products obtained with vinyl sulfone as cross-linking agent were
`developed further and the family of hylan gels with properties varying in broad limits were
`obtained {14}. The following competitive reactions determine the properties of vinyl sulfone
`crosslinked hyaluronan:
`
`42
`
`

`

`'" CH = CH2
`-t"'1-CH20CH2CH2S02CH = CH2
`I-CH20H + 02S "
`CH = CH2
`I-CH20CH2CH2S02CH = CH2 + I.CH20H ..... I.CH20CH2CH2S02CH2CH20CH2-1
`I·CH20CH2CH2S02CH = CH2 + H2o.1·CH20CH2CH2S02CH2CH20H
`/CH = CH2
`02S
`'CH = CH2
`
`CH2CH2
`",CH2CH20H
`+H20..., 02S,
`-.. 02S....
`"0
`CH = CH2
`"CH2CH2'"
`
`(I)
`
`(II)
`
`(III)
`
`(IV)
`
`To simplify the above equations, only primary hydroxy groups of hyaluronan are shown to take part
`in the reaction. As one can see, the consecutive reactions (I) and (II) lead to formation of bis-(ethyl)
`sulfone cross-links, whereas the reactions (I) and (III) give 2-hydroxy ethyl-sulfonyl-ethyl pendant
`groups. The reaction (IV) proceeds without polymer and results in formation of 1, 4-thioxane
`dioxide from vinyl sulfone and water. Such important properties of this hylan gel as swelling in
`various media and rheological properties are determined, mainly, by the ratio of cross-links and
`pendant groups. Modified hylan gels were also developed by including other polymers containing
`chemical groups reactive with vinyl sulfone {IS} or low molecular weight substances {16}. In
`general, the hylan gels obtained using this vinyl sulfone process possess an exceptional
`biocompatibility and other useful properties which make them excellent products for biomedical
`applications.
`
`A unique soluble hylan polymer (hylan fluid) was developed by Balazs et al {l7, 18} by cross(cid:173)
`linking hyaluronan chains to specific proteins molecules with formaldehyde during recovery of the
`polymer from animal tissues, such as combs of domestic fowl. Some of the important rheological
`properties of hylan fluid were described elsewhere {S}. This polymer remains soluble in aqueous
`media due to the fact that the small amount of formaldehyde combined with the polymer (it may vary
`in broad limits depending on the conditions of the process but usually is around 0.001 - 0.005%) is
`not enough for formation of an infinite network. The cross-linking is evident from the higher
`molecular weight of the hyaluronan polymers obtained in the range of 6-24 million. Solutions made
`from this polymer (hylan fluids), exhibit very high elastoviscous properties. {5}
`
`Soluble cross-linked hyaluronan preparations were obtained by Sakurai et al {19} with the use of
`polyfunctional epoxy compounds in alkaline media and in the presence of an organic solvent.
`Depending upon reaction conditions, especially the ratio of a polyepoxy compound to hyaluronan a
`
`43
`
`

`

`cross-linked insoluble polymer may be obtained as well. The solubility of the resulting polymer is
`determined mainly by cross-linking index (according to the authors' terminology) which is expressed
`as number of cross-links per 1000 disaccharide units. The products with a cross-linking index of
`about 5-10 are soluble and products with higher cross-linking index, e.g. 40, are insoluble.
`
`Similar cross-linked insoluble gels of hyaluronan produced by reaction with polyfunctional epoxy
`compounds, e.g. 1, 4 - butanediol diglycidil ether, in alkaline media were suggested for use as a
`vitreous substitute and for retinal detachment surgery {20}.
`
`The great reactivity of hydroxy groups of hyaluronan toward various reagents was recently used for
`obtaining new derivatives of hyaluronan, -phosphate esters {21}. These derivatives are obtained by
`esterification of hyaluronan with phosphoric acid derivatives such as, e.g. phosporyl chloride
`POCi3, in organic or aqueous solutions and in the presence of acid acceptors. The gel fonnation
`occurs very fast. The consistency of the gel depends greatly on the reaction conditions. A soft gel
`with the polymer concentration about I - 1.5% and the phosphorus content about 0.1 % is described.
`This gel is said to be autoclavable and it degrades spontaneously in aqueous media at physiological
`conditions.
`
`All hyaluronan derivatives described above are based on reactions in which hyaluronan plays the role
`of a polymeric polyhydroxy component. The other group of hyaluronan derivatives consists of
`substances obtained through reactions in which hyaluronan plays the role of polycarboxylic acid.
`These derivatives include, mainly, esters and amides and some intermediate products as well. A
`large group of esters ofhyaluronan and various alcohols is described in several patents {22-24}. A
`convenient method for obtaining hyaluronan esters is described and involves the reaction of
`tetrabutylammonium salt of hyaluronan in dimethyl sulfoxide with any substance possessing a
`halogen attached to a carbon. Tetraalkylammonium salts are known to be phase transfer catalysts.
`The use of tetrabutylammonium salt provides the solubility of hyaluronan in organic solvents and
`catalyzes its reaction with halogen compounds. The reaction goes according to the following
`scheme:
`
`+-
`-+
`HA-COO N (C4H9)4 + RX ~HA-COOR + (C4H9)4 NX
`
`where HA is the designation for hyaluronan polymer chain and X is a halogen, preferably, iodine or
`bromine. Partial or fully substituted esters of hyaluronan with a variety of alcohols are described.
`The properties of the esters such as solubility in aqueous or organic media, kinetics of hydrolysis in
`water, etc., are determined mainly by the nature of the alcohol component of the ester and the degree
`of substitution of the ester. By using a polyfunctional alkylating agent a cross-linked ester of
`hyaluronan can be obtained. Various substances characterized by pharmacological activity may be
`attached by this method to the hyaluronan macromolecules. One of such substances described in the
`patents is hydrocortizone which is attached to hyaluronan by reaction of its 21-bromo derivative with
`tetrabutylammonium hyaluronate. Various medical uses of these hyaluronan esters are suggested,
`including drug delivery, skin care, etc.
`
`Another method of obtaining hyaluronan esters in which the polymer plays a role of the acid
`component is described by Malson {25}. It is stated that the reaction of epoxy compounds with
`hyaluronan in the presence of an acid catalyst proceeds with formation of ester groups rather than
`ether groups which form from the reaction of hyaluronan with epoxy compounds in alkaline media.
`By using polyfunctional epoxy compounds and alternating acidic and alkaline conditions it is
`possible to obtain cross-linked polymers containing ether and ester groups simultaneously. Cross-
`
`44
`
`

`

`linked gels of hyaluronan obtained by reactions of the polymer with a polyfunctional epoxy
`compound such as I, 4-butanediol diglycidyl ether are suggested to be used for prevention of
`adhesion formation in surgery {26}.
`
`The other types of hyaluronan derivatives reported in the literature and have found some practical
`applications are obtained through a process which involves activation of carboxyl groups with a
`water-soluble carbodiirnide followed by the reaction of the intermediate substance with a nucleophile.
`When an amino group containing substance is used as a nucleophile the reaction results in formation
`of an amide boud. The uses of water-soluble carbodiimides for the synthesis of peptides in aqueous
`solution {27} and for modification of carboxyl groups in proteins {28} were reported. The general
`mechanism of the reaction was postulated by Khorana {29} as follows:
`a. A Carboxyl-containing substance reacts with a water-soluble carbodiimide with formation of an
`I
`I
`O-acylisourea:
`tV.; R
`It
`Co
`1\
`N'-R U
`
`II +
`,..,
`
`....
`
`b. The O-acylisourea may rearrange to give N-acylurea:
`
`1\Jf{ ....- R.
`()
`/ / ,
`,. R-c ... o-e
`f\ T
`NH
`IJ
`'I(-
`
`o
`I
`/R.
`II
`R -c.. ..... O-N
`r
`c.-:ZO
`t
`"'H
`n
`.... R
`
`1/ ()
`
`or
`c. may react with a nucleophile such as glycine methyl ester to form an amide derivative of carboxyl
`f1 t
`acid:
`l.l
`{V T1 ", l'\.
`1\-c. ~O-c.
`"+
`tl
`N44 --R
`~D
`R-c. - Nt{ - c...U.z. ~ool:+l~
`amide
`The application of this reaction to various glycosaminoglycans including hyaluronan was described
`by Danishefsky and Siskovic {30}. The yield for the reaction of hyaluronan with glycine methyl
`ester was about 39%. Water-soluble carbodiimides were used also for attachment to other amino
`acids as biocompatible spacers for drugs and to variolls glycosaminoglycans including hyaluronan
`which serve as carriers {31}.
`
`The use of carbodiimides for modifying hyaluronan was recently described in a patent publication
`{32}. The new feature in this work is the use of bis-carbodiimide which leads to formation of cross(cid:173)
`linked derivatives. A slightly modified method for obtaining amide derivatives of hyaluronan and
`various esters of amino acids is described in a recent patent {33}. These derivatives are described as
`water-insoluble polymers which form hydrogels that hydrolyze at a rate depending on the nature and
`the amount of the substituting groups. Polyfunctional reagents are not used in the described method
`and the resulting products are not cross-linked;
`the insolubility is provided by introduction of
`
`45
`
`

`

`hydrophobic alkyl ester groups. The products are recommended for preventing adhesions after
`surgery, for drug delivery and for other medical uses. A similar method is described in another
`patent publication {34} which uses other polyanionic polysaccharides together with hyaluronan.
`
`VISCOSURGERY
`
`The first medical use of NIF-NaHA {II} was in the field of ophthalmic surgery. The new surgical
`procedure developed during the 1970's {35, 36, 37} was aimed at protecting the tissues during
`surgery and providing the surgeon with a safe tool to make space and to move tissues. This surgical
`procedure was called viscosurgery {37} and was introduced into medical practice in the early 1980's
`with the first commercial product of NIF-NaHA, under the trademark Healon® (Kabi Pharrnacia,
`Uppsala, Sweden).
`
`The efficacy of viscosurgical devices is based on their elastoviscous properties. They have an
`important function after surgery as well. When left in the surgical site they become viscosurgical
`implants and provide a separation between tissue surfaces and a barrier against post-surgical
`exudation. The molecular network of hyaluronan forms a barrier to blood, fibrin and invading cells,
`thereby preventing adhesion and scar tissue formation and helping to restore normal mechanical
`function after surgery.
`
`The most important property of viscosurgicaI devices, besides their rheological properties, is their
`biocompatibility. NIF-NaHA is not recognized by the body as foreign and does not cause an
`immunogenic response. Since hyaluronan is present in those tissues where it is used as a
`viscosurgical device in the eye (anterior chamber and vitreous), its presence during and after surgery
`does not elicit tissue reaction.
`
`NIF-NaHA as a viscosurgical device is primarily used in cataract surgery. It minimizes the surgical
`trauma during the removal of the cataractous lens and the introduction of its replacement, the
`intraocular plastic lens. It is also used for replacement of the vitreous after vitreoretinal surgery,
`mostly in connection with the surgical treatment of retinal detachment. The elastoviscosity of
`hyaluronan solution has not proved to be sufficient in most cases to retain the retina in its reattached
`position. Therefore, the newly developed crosslinked hylan gel has been used in several clinical
`trials as a vitreous substitute. These studies demonstrate that hylan gel can successfully achieve a
`long-lasting reattachment of the retina.
`
`Healon was the first viscosurgical device marketed worldwide. It is still the most widely used of
`such devices because it has the greatest elastoviscosity compared with many imitations later
`developed for the same use. Since hylan fluid is several times more elastoviscous than hyaluronan
`solutions of comparable polymer concentration, its use as a more effective viscosurgical tool in
`ophthalmology is being explored.
`
`TREATMENT OF ARTHRITIS WITH VISCOSUPPLEMENTATION
`
`Viscosupplementation was introduced to medicine as a new therapeutic modality in the early 1970's
`(for review see 2). This therapeutic concept means that the elastoviscous properties of a tissue
`compartment are supplemented or augmented in order to influence the pathological process and
`restore normal physiological function. In various fomlS of arthritis, especially in osteoarthritis, the
`elastoviscosity of the intercellular matrix of the soft joint tissues (synovial fluid and tissues, and
`capsule) is decreased. This decrease is caused by two pathological events. Due to the inflammatory
`process in the joint, excess water diffuses from the blood vessels into the tissues. Thus a joint
`effusion develops and dilutes the normal matrix, decreasing its elastoviscosity. Alternatively or
`
`46
`
`

`

`accompanying this process the molecular mass of the hyaluronan in the joint fluid and tissues
`decreases for yet unknown reasons, causing a dramatic drop in the elastoviscous properties of the
`matrix of these tissues. Both mechanisms lead to a significant drop in the rheological properties of
`the synovial fluid and the intercellular matrix. This is a pathological event which is characteristic of
`nearly all arthritic conditions: traumatic arthritis, metabolic arthritis, osteoarthritis or rheumatoid
`arthritis. When this pathological chain of events was recognized (38}, the hypothesis was proposed
`that by restoring the homeostasis of elastoviscosity of the synovial Quid and matrix of the joint tissue
`one can expect improvement of the pathological conditions of the joint. Since the most important
`symptom of arthritis is pain, one would expect relief of pain after the rheological homeostasis is
`restored in the joint. This hypothesis was tested first in 1970-1971 in race horses with traumatic
`arthritis treated with NIF-NaHA (Healon® Biotrics, Inc., Arlington, Massachusetts). The
`elastoviscous 1 % solution of about 2 million molecular mass hyaluronan was injected once or
`repeatedly into the affected joint {39, 40}.
`
`With this viscosupplementation the low elastoviscosity of the joint fluid was immediately elevated
`and maintained at that level for a few days. The pain decreased, the joint mobility increased and
`often the rheological properties were restored to nOnllal level even after the injected hyaluronan was
`gone from the joint {41 }. These studies were then extended to the treatment of human osteoarthritis
`(42,43). The results obtained with two or three intra-articular injections one week ap.art confirmed
`the same analgesic effect in the human knee joint. Double-blind, controlled studies indicated an
`effect lasting several months in about fifty percent of the patients treated {44}.
`
`Based on these findings, in the late 1980's, two hyaluronan preparations were marketed for
`viscosupplementation of osteoarthritic joints: one in Japan (Arzt®, Seikagaku and Kaken) and one in
`Italy (Hyalgan®, Fidia). Both of these preparations consist of a 1 % solution of relatively low
`molecular mass (-700,000) hyaluronan; therefore, the elastoviscosity of these products is relatively
`low. Nevertheless, investigators from Japan {45}, Italy {46} and Great Britain {47} reported
`successful relief of pain with treatment consisting of 6-12 weekly injections.
`
`Studies in equine arthritis clearly demonstrated that the greater the elastoviscosity of the solution used
`for viscosupplementation, the fewer injections needed, with the result that greater and more lasting
`pain relief could be observed {48}.
`
`Since the crosslinked derivative of hyaluronan, hylan, is more elastoviscous, and has a longer inter(cid:173)
`articular residence time, a combination of hylan fluid and hylan gel (hylan gel-fluid 20, Synvisc®,
`Biomatrix, Inc., USA) was developed and tested in equine and human arthritis. The data showed
`that three injections, one week apart, is an efficacious treatment of osteoarthritic pain in the knee
`joint. A nearly complete (>80% improvement) pain relief is achieved in 60-70% of the treated
`patients for a four to six month period. Longer lasting effects were also observed. Most importantly
`no systemic side effects were observed, and local transient discomfort was noted in fewer than 1 %
`of the patients treated {49, 50, 51, 52}.
`
`The mode of action of viscosupplementation has been the subject of many studies. In vitro studies
`showed that hyaluronan and hylan have a protective effect on cartilage cells against mechanical and
`chemical damage. This protective effect depends on the elastoviscosity of the ~olution. Gr~ater
`rheological properties provide more protection. The protection was tested agamst. such noxIOUS
`factors as tissue degrading enzymes and other agents (interleukin -1) released by whIte blood cells,
`and oxygen-derived free radicals {53}. Since these enzymes and free radicals are considered as
`possible cause in osteoarthritis {54}, the implication is that such protective mechanisms may playa
`role. Direct, in vivo studies have not yet supported the existence of this mechanism.
`
`Another explanation for the analgesic effect of viscosupplementation could be through its direct effect
`on pain receptors of the joint capsule. Pain in the joint is triggered by sensory afferent nerve fibers
`
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`called nociceptors. Excitation of hociceptors or afferents of the joint is due to the transmission of
`mechanical forces to the nerve terminals that are normally activated only by noxious (abnormal,
`overextended) movement, producing a train of nerve impulses that constitutes a signal of pain for the
`central nervous system. These signals were produced and measured in normal animal joints which
`were overextended (noxious movement) and in arthritic animals joints which were moved within the
`normal range. In the joints of these animals, hylan gel-fluid 20 (Synvisc®) was injected, and within
`one hour a significant reduction of ongoing or stimulated nerve activity was observed. With
`nonelastovisous solutions of hylan or hyaluronan this effect was not observed {55}. These in vivo
`model studies on arthritic pain strongly suggest that the pain-reducing effect of visco supplementation
`is the result of the buffering and screening out of mechanical and chemical stimulatory factors from
`nociceptors. This effect seems directly related to the elastoviscous properties of the material used for
`viscosupplementation. Japanese authors using animal models also reported analgesic effects of
`hyaluronan solutions. They could demonstrate this effect only if the solution contained high
`molecular weight (>720,000) hyaluronan molecules {56, 57}.
`
`Probably the most important factor in the therapeutic process of viscosupplementation is the increase
`of joint mobility. The analgesic effect triggers a cascade of events, starting with increased joint
`mobility and resulting in the restoration of the normal flow of hyaluronan in the joint. It flows froin
`the synovial cells where it is synthetized, to the cartilage surface and through the soft tissues of the
`joint to the site of outflow, the lymph vessels. It is well known that an immobilized normal joint
`undergoes pathological changes and the hyaluronan metabolism becomes seriously impaired {58}.
`With movement of the joint the homeostasis of the fluid circulation (blood, lymph, synovial fluid) is
`restored. If the degenerative process in the joint did not progress too far, or if there is no excessive
`inflammation in the joint, viscosupplementation can restore the homeostasis of the joint.
`
`ADHESION MANAGEMENT
`
`Recently the use of HA and its derivatives in adhesion management has achieved widespread
`recognition, most notably by commercial ventures, as a potentially unique and effective approach
`to the prevention or reduction in formation of unwanted collagenous connective tissue following
`tissue injury. Over twenty years ago, Balazs et al. {59} reported on the effect ofHA on adhesion
`formation between tendon and tendon sheaths and between the conjunctiva and sclera. Viscoelastic
`solutions of HA and water soluble dry sheets of HA were found to reduce the in