Biomaterials for preventing post-surgical adhesions comprised of
hyaluronic acid derivatives
United States Patent: 7,504,386
Issued: March 17, 2009
Daniele (Montegrotto Terme, IT), Pavesio; Alessandra (Padua, IT),
Callegaro; Lanfranco (Thiene, IT)
Assignee: Fidia Advanced
Biopolymers, S.r.l (Abano Terme, IT)
Appl. No.: 10/812,587
Filed: March 29, 2004
New biomaterials essentially constituted
by esterified derivatives of hyaluronic acid or by cross-linked
derivatives of hyaluronic acid for use in the surgical sector,
particularly for use in the prevention of post-surgical adhesions.
Description of the
OBJECT OF THE INVENTION
The present invention concerns new biomaterials essentially constituted by
esterified derivatives of hyaluronic acid or by cross-linked derivatives
of hyaluronic acid for use in the surgical sector, particularly for use in
the prevention of post-surgical adhesions.
FIELD OF THE INVENTION
Postoperative adhesion formation is a common complication in abdominal or
pelvic surgery which may lead to a substantial morbidity. Many factors may
influence the development of adhesions: mechanical trauma, chemical
agents, drying of serosa in combination with blood, ischemia, infection
and foreign material are all known to increase adhesion formation. Other
causes are intraabdominal inflammatory diseases and congenital
abnormalities. The pathophysiological mechanism still remain unclear, but
a central common pathway in which peritoneal fibrinolysis plays an
important role has been suggested.
The surgical trauma of the tissue causes the release of a serosanguinous
exudate which forms a fibrinous bridge that persists several days during
which cell growth occurs. If the exudate is not absorbed or lysed within
this period, it becomes ingrown with fibroblasts and subsequent collagen
deposition leads to the formation of a permanent scar connecting the two
adjacent surfaces, called an adhesion. Thus, adhesion formation seems to
be a result of an inflammatory response.
In this latter case, the research has mainly focused on the search for
bioabsorbable materials with a short time of in vivo persistence, to act
as barriers to adhesion formation until healing has occurred; in order to
obviate the problems caused by non-absorbable materials (infection,
calcification of the implants, scar formation, etc.).
One particularly promising polymer is Hyaluronic Acid (HA), a component of
extracellular matrix ubiquitously found within the human body. Hyaluronic
Acid solutions have been shown to reduce postoperative adhesion formation
after abdominal surgery (Urman, B. et al., Effect of Hyaluronic Acid on
Postoperative Intraperitoneal Adhesions Formation in the Rat Model, Fertil.
Steril. 1991; 56:563; Shushan A. et al., Hyaluronic Acid for Preventing
Experimental Postoperative-intraperitoneal Adhesions, J. Reprod. Med.
1994; 39:398) and orthopaedic operation (Hagberg, L, Gerdin, B., Sodium
Hyaluronate as an adjunctive in adhesion prevention after flexor tendon
surgery in rabbits, J. Hand. Surg. 1992; 17A:935).
Fidia Advanced Biopolymers has developed chemical derivatives of
hyaluronic acid, i.e., internal esters (ACP series) and esters with
non-active alcohols (HYAFF series) Rastrelli, A. et al., Hyaluronic Acid
Esters, A New Class of Semisynthetic Biopolymers: Chemical and Physico-chemical
Properties, Clinical Implant Materials, Advanced in Biomaterials, G.
Heinrike, V. Sollz and A J C Lee (Eds), Elsevier, Amsterdam 1990;
9:199-205, which display physico-chemical properties different from that
of HA (i.e. higher residence time and ability to be manufactured to
produce devices, but possessing tolerability and biocompatibility
properties typical of the original biological polymer). Moreover, these
derivatives are characterized from a chemical and toxicological point of
The aim of the present invention has been to develop batches of
derivatives of hyaluronic acid such as ACP gel and HYAFF in an attempt to
evaluate the effect in adhesion prevention.
The onset of adherences, or fibrous masses which form between adjacent
tissues affected by trauma or ischemia following surgery, is still one of
the most serious complications in numerous surgical procedures. A large
number of methods have been proposed to avoid this complication, but the
problem has remained mainly unsolved.
One proposed method has been the use of suspensions of dextran (diZerega
G. S., "Contemporary adhesion prevention" Fertility and Sterility, Vol.
61, No. 2, Feb. '94) injected into the peritoneal cavity after surgery.
The clinical results of the use of such dextran solutions have been
largely discordant. Moreover, the use of solutions of dextran has been
accompanied by frequent complications, including edema, abdominal pain and
The use of barriers in the form of defined structures (e.g. meshes,
membranes) (diZerega G. S., "Contemporary adhesion prevention" Fertility
and Sterility, Vol. 61, No. 2, Feb. '94) or viscous gels (Genzyme U.S.
Pat. No. 4,937,270-U.S. Pat. No. 5,017,229) placed between the injured
organs has also been proposed. However, these barriers have generally
proved ineffective because they provoke ischemic or inflammatory reactions
due to the presence of foreign bodies. The only materials currently
approved for clinical use are barriers based on oxidized regenerated
cellulose (INTERCEED.RTM.) and barriers based on expanded
polytetrafluorine ethylene (e-PTFE) (U.S. Pat. Nos. 4,478,665 and
4,482,516) or polyethylene or polypropylene.
In addition to the fact that clinical investigations into the efficacy of
such barriers have produced highly discordant results, it must also be
noted that both of the aforesaid materials are associated with major
contraindications. The use of barrier membranes of e-PTFE or polyethylene
or polypropylene involves the implantation of a synthetic material which
is foreign to the human body and not biodegradable, and which may require
a second surgical operation to remove or reposition the barrier membrane
because of undesirable inflammatory-type reactions.
In preclinical and clinical models, meshes based on oxidized regenerated
cellulose have proved to be efficacious in preventing the formation of
adherences, but only if their application is preceded by thorough
The use of viscous solutions of high-molecular-weight hyaluronic acid (HA)
has, therefore, been proposed as an aid in the prevention of adherence
(Grainger D. A. et al., "The use of hyaluronic acid polymers to reduce
postoperative adhesions", J. of Gynecol. Surg., Vol. 7, No. 2, 1991;
Hurman B. et al., "Effect of hyaluronic acid on postoperative
intraperitoneal adhesion formation in the rat model", Fertility and
Sterility, Vol. 56, No. 3, September 1991; Shushan A. et al., "Hyaluronic
acid for preventing experimental postoperative intraperitoneal adhesions:,
J. of Reproductive Med., Vol. 39, No. 5, May 1994; Mitchell J. D. et al.,
"Reduction in experimental pericardial adhesions using a hyaluronic acid
bioabsorbable membrane", Eur. J. Cardio-thorac. Surg., 8, 149-152, 1994).
Hyaluronic acid as such, however, is characterized by very rapid
absorption times which are incompatible with the residence time necessary
to prevent adhesion. Moreover, natural hyaluronic acid cannot be processed
and as such cannot be transformed into biomaterial form. In order to
prolong its degradation times and enable it to be processed into various
physical forms for use in different surgical sectors, esters of hyaluronic
acid and cross-linked derivatives of hyaluronic acid have been developed.
The preparation of esters of hyaluronic acid, wherein all or part of the
carboxy groups are esterified, the preparation of cross-linked derivatives
of hyaluronic acid, wherein part of the carboxy groups undergo
cross-linking and their uses in the pharmaceutical, cosmetic and surgical
sectors and in that of biodegradable plastic materials are described in
U.S. Pat. Nos. 4,851,521 and 4,965,353, EP 0 216 453 and EP 0 341 745.
SUMMARY OF THE INVENTION
The present invention provides biomaterials for use in the prevention of
post-surgical adhesions. The biomaterials are comprised of benzyl esters
of hyaluronic acid and/or internally cross-linked derivatives of
hyaluronic acid and may be in the form of gels, membranes, woven tissues
or meshes and nonwoven tissues.
DETAILED DESCRIPTION OF THE INVENTION
The present invention, therefore, describes the preparation of healthcare
and surgical articles based on a benzyl ester of hyaluronic acid or on
cross-linked derivatives of hyaluronic acid, used singly or in mixtures
with one another, characterized by high biocompatibility and transformable
into physical forms which make them suitable for various uses in surgery.
Surgical fields wherein hyaluronic acid derivatives and materials of the
present invention are useful include, but are not limited to, abdominal,
laparoscopic, laparotomic, intestinal, gynecological, abdominal pelvic,
peritoneal, urogential, orthopedic, spinal, such as dura mater,
tendon/nerve such as carpal tunnel, cardiovascular thoracic, oncologic,
plastic, esthetic, ENT, paranasal sinuses and transplantation. The
materials are also completely biodegradable and do not need to be removed
from the application site, thus avoiding a second surgical operation. When
prepared in the form of gels, the cross-linked derivatives present
materials with significantly greater viscosity than the unmodified polymer
and with variable degradation times. Moreover, both the benzyl ester-based
materials and the cross-linked derivative-based materials of the present
invention can be in the form of membranes, woven tissues or meshes and
nonwoven tissues (prepared according to procedures per se described in
U.S. Pat. Nos. 4,851,521; 4,956,353,; WO 93/11804; WO 93/11803; WO
94/17837 and EP 0 341 745) and are characterized by the following
technical specifications: the membranes vary in thickness between 10 .mu.m
and 1.5 mm, especially 20-50 .mu.m; the tissues or meshes vary in
thickness between 200 .mu.m and 1.5 mm; the nonwoven tissues are
essentially characterized by a basis weight which varies between 20
g/m.sup.2 and 500 g/m.sup.2 and by a thickness of between 0.2 mm and 5 mm,
especially <1 mm.
These materials can be used singly or in association with one another or
with other materials constituted by synthetic polymers (e.g. gels based on
cross-linked hyaluronic acid+polypropylene, or membranes essentially
constituted by esterified derivatives of HA+polypropylene or membranes
comprised of esterified derivatives of HA, coated with a gel of auto-crosslinked
Indeed, the present invention also concerns the use of composite materials
in the form of gels (for the cross-linked derivatives), membranes, woven
or nonwoven tissues, essentially constituted by the benzyl esters or
cross-linked derivatives of hyaluronic acid in association with
nonbiodegradable materials in the form of meshes or membranes or nonwoven
tissues such as e-PTFE, polyethylene, polypropylene, polyester (Dacron.RTM.).
The present invention, therefore concerns a new class of healthcare and
surgical articles to be used in the field of surgery for the prevention of
the formation of post-surgical adherence.
The materials of the present invention can also be used in association
with active agents useful in promoting healing, reducing inflammation,
preventing infection, etc. Such active agents include steroidal and
non-steroidal antiinflammatories, fibrinolytics, hemostatics,
antithrombotics, growth factors, antitumorals and antibiotics including
antibacterials, antivirals, antifungals. These active agents may be
applied separately and immediately prior to or simultaneously with the
hyaluronic derivation adhesion prevention material. Alternative, the
active agent may be impregnated into the adhesion prevention materials.
As noted above, the present invention is characterized by materials
comprised of derivatives of hyaluronic acid, especially benzyl ester
derivatives and internally cross-linked derivatives.
The term "hyaluronic acid" (also referred to as "HA" hereinafter) is used
in literature to designate an acidic polysaccharide with various molecular
weights constituted by resides of D-glucuronic acid and N-acetyl-D-glucosamine,
which naturally occur in cellular surfaces, in the basic extracellular
substances of the connective tissues of vertebrates, in the synovial fluid
of joints, in the vitreous humor of the eye, in the tissue of the human
umbilical cord and in cocks' combs.
Hyaluronic acid plays an important role in the biological organism,
firstly as a mechanical support of the cells of many tissues, such as the
skin, the tendons, the muscles and cartilage and it is therefore the main
component of the extracellular matrix. But hyaluronic acid also performs
other functions in the biological processes, such as the hydration of
tissues, lubrication, cellular migration, cell function and
differentiation. (See for example, A. Balazs et al., Cosmetics &
Toiletries, No. 5/84, pages 8-17). Hyaluronic acid may be extracted from
the above-mentioned natural tissues, such as cocks' combs, or also from
certain bacteria. Today, hyaluronic acid may also be prepared by
microbiological methods. The molecular weight of whole hyaluronic acid
obtained by extraction is in the region of 8-13 million. However, the
molecular chain of the polysaccharide can be degraded quite easily under
the influence of various physical and chemical factors, such as mechanical
influences or under the influence of radiation, hydrolyzing, oxidizing or
enzymatic agents. For this reason, often in the ordinary purification
procedures of original extracts, degraded fractions with a lower molecular
weight are obtained. (See Balazs et al., cited above). Hyaluronic acid,
its molecular fractions and the respective salts have been used as
medicaments and their use is also proposed in cosmetics (see for example,
the above-mentioned article by Balazs et al., and the French Patent No.
Although the term "hyaluronic acid" is commonly used in an improper sense,
meaning, as can be seen from above, a whole series of polysaccharides with
alternations of residues of D-glucuronic acid and N-acetyl-D-glucosamine
with varying molecular weights or even degraded fractions of the same, and
although the plural form "hyaluronic acids" may seem more appropriate, the
discussion herein shall continue to use the singular form to refer to
hyaluronic acid in its various forms including its molecular fractions,
and the abbreviation "HA" will also often be used to describe this
1. The Benzyl Ester Derivatives:
The first preferred material of the invention is based on the benzyl ester
of hyaluronic acid, particularly the 80-100% esters wherein 80% to 100% of
the HA carboxyl groups are esterified. Those benzyl esters wherein 80-99%
of the HA carboxyl groups are esterified with a benzyl group are referred
to as "partial esters", because only a portion of the carboxyl groups are
esterified and the remaining carboxyl groups are either free or salified
with an alkaline or alkaline earth metal, such as sodium, calcium or
Most preferred for the biomaterials of the invention are so-called "total"
benzyl esters wherein all of the HA carboxy groups are esterified. In
these total esters, all of the HA carboxy groups may be esterified with a
benzyl group (also referred to as HYAFF 11). A partial ester is one
wherein a portion (75 to 99%) but not all of the HA carboxy groups are
esterified with a benzyl group. "Mixed" esters are those where in the HA
carboxy groups are esterfied with more than one type of ester group. The
mixed esters may be total esters in that for example, a portion (75-99%)
may be esterfied with a benzyl group and all of the remaining carboxyl
groups esterfied with the lipid chain/alkyl residue from a C.sub.10-20
aliphatic alcohol. Of these aliphatic alcohols, palmitic alcohol
(C.sub.16-hexadecyl) and stearic alcohol (C.sub.18 octadecyl) are the most
preferred. The benzyl esters of hyaluronic acid according to the invention
may be prepared by methods known per se for the esterification of
carboxylic acids, for example by treatment of free hyaluronic acid with
the alcohol (benzyl and/or C.sub.10-C.sub.20 alcohol) in the presence of
catalyzing substances, such as strong inorganic acids or ionic exchangers
of the acid type, or with an etherifying agent capable of introducing the
desired alcoholic residue in the presence of inorganic or organic bases.
The benzyl hyaluronic esters may, however, be prepared to advantage
according to a particular method described in EP 0 216 453. This method
consists of treating a quaternary ammonium salt of hyaluronic acid with an
etherifying agent, preferably in an aprotic organic solvent.
For the preparation of the benzyl esters it is possible to use hyaluronic
acids of any origin, such as for example, the acids extracted from the
above mentioned natural starting materials, for example, from cocks'
combs. The preparation of such acids is described in literature:
preferably, purified hyaluronic acids are used. According to the
invention, especially used are hyaluronic acids comprising molecular
fractions of the integral acids obtained directly by extraction of the
organic materials with molecular weights varying within a wide range, for
example, from about 90%-80% (M=11.7 - 10.4 million) to 0.2% (M=30,000) of
the molecular weight of the integral acid having a molecular weight of 13
million, preferably between 5% and 0.2%. Such fractions may be obtained
with various procedures described in literature, such as by hydrolyzing,
oxidizing, enzymatic or physical procedures, such as mechanical or
radiational procedures. Primordial extracts are therefore often formed
during these same purification procedures (for example, see the article by
Balazs et al., quoted above in "Cosmetics & Toiletries"). The separation
and purification of the molecular fractions obtained are brought about by
known techniques, for example by molecular filtration.
One fraction of purified HA suitable for use according to the invention is
for example that known as "non-inflammatory-NIF-NaHA sodium hyaluronate
described by Balazs in the booklet "Healon"--A guide to its use in
Ophthalmic Surgery, D. Miller & R. Stegmann, eds. John Wiley & Sons, N.
Y., 81983: p 5.
Particularly important as starting materials for the benzyl ester are two
purified fractions obtainable from hyaluronic acid, for example the ones
extracted from cocks' combs, known as "Hyalastine" and "Hyalectin". The
fraction Hyalastine has an average molecular weight of about 50,000 to
100,000 while the fraction Hyalectin has an average molecular weight of
between about 500,000 and 730,000. A combined fraction of these two
fractions has also been isolated and characterized as having an average
molecular weight of about 250,000 to about 350,000. This combined fraction
may be obtained with a yield of 80% of total hyaluronic acid available in
the particular starting material, while the fraction Hyalectin may be
obtained with a yield of 30% and the fraction Hyalastine with a yield of
50% of the starting HA. The preparation of these fractions is described in
EP 0 138 572.
Claim 1 of 15 Claims
1. A method for preventing surgical
adhesions of tissue which comprises applying to tissue involved in surgery
a biomaterial comprised of at least one auto-crosslinked derivative of an
hyaluronic acid with an average molecular weight of 150,000 to 730,000
Daltons, wherein 4.5 to 5% of the carboxyl group of hyaluronic acid are
cross-linked to the hydroxyl group of the same or different hyaluronic
acid molecule, wherein said biomaterial comprising the cross-linked
derivative has a viscosity of at least 200 Pa*sec.sup.-1.
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