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  Pharmaceutical Patents  


Title:  Biomaterials for preventing post-surgical adhesions comprised of hyaluronic acid derivatives
United States Patent: 
March 17, 2009

 Pressato; Daniele (Montegrotto Terme, IT), Pavesio; Alessandra (Padua, IT), Callegaro; Lanfranco (Thiene, IT)
  Fidia Advanced Biopolymers, S.r.l (Abano Terme, IT)
Appl. No.:
 March 29, 2004


Outsourcing Guide


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 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.


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 view.

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 dyspnea.

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 hemostasis.

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.


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.


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 HA).

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. 2478468).

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 collective term.

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 potassium.

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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