Strategies to Reduce Postsurgical Adhesion Formation

There are essentially three ways to reduce adhesion formation: [1] reduce tissue damage during a procedure; [2] use a therapeutic that will affect fibrin resorbtion, such as a fibrinolytic or anti-inflammatory agent; or [3] use a barrier that will keep tissue planes separated until the fibrin clot has been resorbed and normal wound healing has taken place. Adhesion formation can be influenced by surgical technique (54,62-64) and the use of surgical irrigants such as Ringer's Lactate (65), Sepracoat™ (32), and solutions of icodextrins (66). Fibrinolytics, such as tPA (67,68), urokinase (67), streptokinase (67,69-71) and anti-inflammatory agents, such as naproxen (72), tolmetin (67,73), diphenhydramine (74), and aspirin (75), are examples of compounds that have been shown in animal models to reduce adhesions. Anti-proliferatives, such as paclitaxel, have also been shown to significantly reduce post-surgical adhesions in animal models (76). We will limit our discussion to the development of adhesion reduction barriers for the peritoneal cavity based on chemically modified hyaluronate.

C. Preparation of Modified Hyaluronates for Adhesion Reduction

Adhesion reduction using a barrier requires that the barrier reside long enough at the target site to keep the damaged tissue separated for about 3 -7 days thereby allowing normal wound healing to occur (60). Unmodified hyaluronate will not remain at the target site long enough to keep tissue planes separated (26-28,77). Therefore, chemical modification of hyaluronate is required to extend the residence at the placement site long enough to affect adhesion formation.

A method to modify hyaluronate so that it can serve as a barrier is by interchain covalent cross-linking. There are four functional groups on hyaluronate that can react with cross-linking reagents. These groups are the hydroxyl, carboxyl, acetamido and the reducing end of the polymer (Fig. 1). The poor reactivity of the acetamido group in hyaluronate has resulted in a paucity of examples of derivatives prepared from the functional group. A novel hyaluronate

Figure 1 Functional groups on hyaluronate that can be chemically modified. Group 1, carboxyl; group 2, hydroxyl; group 3, acetamido; group 4, reducing end of polymer.

Acetamido Group

Figure 1 Functional groups on hyaluronate that can be chemically modified. Group 1, carboxyl; group 2, hydroxyl; group 3, acetamido; group 4, reducing end of polymer.

hydrogel has been prepared by first deacetylating hyaluronate with hydrazine then cross-linking the resulting amines with glutaraldehyde (78). The reducing end of hyaluronate does not lend itself to cross-linking hyaluronate because there is only one group per polymer. The most extensively published examples of hyaluronate cross-linking have been through the most prevalent functional group on the polymer, which are the hydroxyl or carboxyl groups.

Cross-linking through the hydroxyl groups, with bifunctional cross-linking reagents such as epichlorohydrin (79) and divinyl sulfone (80-83), has led to the development of some commercially available products for viscosupple-mentation (Synvisc®) (23,84-87) and soft tissue augmentation (Hylaform®) (88). These cross-links give functional groups that are hydrolytically quite stable under physiological conditions resulting in gels having residence times of months in the skin and years in the vitreous (80). Clearance of these derivatives from a placement site in vivo will depend almost entirely on the depolymerization of the hyaluronate chain.

Cross-linking via the hyaluronate carboxyl groups offers chemical diversity in that amide, acyl hydrazides or esters can be used thereby giving versatility in stability and degradation time. A very elegant method of cross-linking hyaluronate by an amide linkage has been done using either the Ugi or the Passerini reaction (Fig. 2). Reaction of hyaluronate, under Ugi conditions, with lysine, cyclohexylisocyanide, and formaldehyde yield an amide cross-linked hyaluronate (89).

Acyl hydrazide derivatives of hyaluronate form a derivative that possesses a nitrogen atom that is sufficiently nucleophilic at physiological pH to react with bifunctional cross-linking reagents (90,91) to give cross-linked hydrogels of hyaluronate. These molecules, despite their novelty and potential versatility, have not as of yet been developed into commercial products.

With regard to adhesions reduction one of the reasons for this lack of development might be that these materials by virtue of their high covalent cross-linking have residence time far in excess of the required 3 -7 days postulated to affect adhesion formation. A promising variant on the technology is the introduction of bio-labile bond, such as a disulfide, in the cross-linking reagent into the cross-linked acyl hydrazide (92).

There have been some interesting examples of cross-linked hyaluronate based materials that reduce adhesions in vivo. A novel example of such a gel is the ferric ion complex with the carboxyl groups of hyaluronate (93). This iron-hyaluronate hydrogel successfully reduced adhesions in women undergoing peritoneal surgery under laparotomy for preservation of fertility in separate studies both in the United States (94,95) and Europe (96). This product, however, has been withdrawn from sale to the global market (97).

Another interesting and novel hydrogel for adhesion reduction has been produced by affecting internal, self-reaction of hydroxyl groups with the carboxyl moieties in hyaluronan (Fig. 3). This hydrogel made from this auto-crosslinked-polysaccharide (ACP) reduced surgical adhesions in rats (98) and rabbits (99). A recent human clinical trial in women undergoing hysteroscopic adhesiolysis

Thc Metabolites
Figure 2 Putative reaction products for cross-linking of hyaluronate under the Ugi reaction conditions.

showed that patients that received the ACP gel at the time of surgery had significantly fewer adhesions at a 3 month follow-up (100).

The very first adhesion reduction device made from hyaluronate was approved in 1997 and is the Seprafilm® Bioresorbable Adhesion Barrier (34). This product is made from chemically derivatized sodium hyaluronate and sodium carboxymethyl cellulose (101). This device achieves the targeted residence time of 3-7 days, not by covalently cross-linking the polymer chains,

Edc Degridation
Figure 3 Putative structure of ACP hyaluronate.

but by chemically derivatizing the polymers so that there is a significant decrease in water solubility. This insolubilization is accomplished by derivatizing the carboxyl groups of both hyaluronate and carboxymethyl cellulose by the reaction of the water soluble carbodiimide, 1-ethyl-3-(30-dimethylaminopropyl)carbo-diimide hydrochloride (EDC) (Fig. 4). Under acidic conditions this reagent results in the modification of both hyaluronate and carboxymethyl cellulose to give an N-acylurea (102,103). The propylimido isomer has been shown, by isotopic labeling, to be the predominant of the two structural N-acylureas (104).

The partial modification of the carboxyl groups' results in a positive charge that can form a salt-bridge with residual unreacted carboxyl on other polymer chains. This charge-charge interaction decreases water solubility and in turn extends the residence times in vivo of devices made from this material.

Seprafilm®, prepared from this process, has been shown to be very effective in reducing the incidence of post-surgical adhesions both in abdominal (105) and pelvic surgical procedures (106). The abdominal study was particularly compelling in that more than half (51%) of Seprafilm® recipients were adhesion-free, versus only 6% of untreated patients. Recently, it has been shown in a large multicenter study that there was no significant difference between Seprafilm® treated and control groups for abscess formation, peritonitis, and foreign body reaction (107). This safety with respect to peritonitis is consistent with results seen in a severe animal model for intra-abdominal sepsis where Seprafilm® did not adversely affect the progression of acute peritonitis or abscess formation (108). Seprafilm®, by virtue of its well demonstrated safety and efficacy in preclinical and human clinical studies, remains the only product approved for general surgical indications in the abdomen to this date.

Seprafilm® has been shown to reduce the incidence of adhesions to non-degradable, permanent tissue augmentation implants like polypropylene hernia repair meshes (109). This has led to the use of the diimide-modified hyaluronate and carboxymethyl cellulose materials in the Sepramesh™ Bioresorbable Composite. Sepramesh™ is a polypropylene mesh that has had laminated to one side of the mesh the same material that is in Seprafilm® (110). The placement of Sepramesh™ in the abdominal wall, with the adhesion reducing-side facing

Cellulose And Water Adhesion
Figure 4 Scheme for the reaction of water soluble diimde, EDC with hyaluronate and carboxylmethyl cellulose under acidic reaction conditions (pH < 7). N-acylurea, B, is the predominant isomer under these reaction conditions.

the peritoneal cavity, has been shown to significantly reduce the incidence of adherence of the underlying viscera to the mesh (29,111). Reduction in the adherence of the viscera to a hernia repair mesh is thought to reduce the incidence of bowel fistula formation.

Additional derivatives have been introduced into the market to reduce sinus synechia (adhesions) following sinonasal surgery or trauma. Seprapak® Bioresorbable Nasal Packing is a foam that is made from the same material that is in Seprafilm® (101). This material functions to fill sinus cavities following surgery or trauma so that mucosal surfaces can remain separated during the healing process. A related product is Sepragel® Sinus which is prepared from divinyl sulfone cross-linked hylan (hylan B) (81). In a recently published study Sepragel® Sinus significantly improved the outcomes of patients that received this product following bilateral endoscopic ethmoidectomy (112). Lastly, an ethyl ester derivative of hyaluronate, Merogel®, is also commercially available. This material has been shown clinically to reduce the incidence of sinus adhesions following minimally invasive surgery to correct bilateral chronic sinusitis in patients (113).

III. Tissue Augmentation

A rapidly expanding area for the use of chemically modified hyaluronate has been in the use of these derivatives for soft tissue augmentation. The majority of applications for these types of products are in the aesthetics market to correct wrinkles and facial blemish such as acne scars. The hyaluronate-based products offer a significant advantage in that they can be used without a prerequisite skin test for sensitivity to bovine collagen products. Currently there are several hyaluronate-derived dermal fillers, such as Hylaform®, Restylane®, Perlane®, and Juvederm® that are sold in Europe. Hylaform is divinyl sulfone cross-linked hyaluronate-derived from chicken comb (hylan A) (Fig. 5) (80, 81).

Restylane®, Perlane® and Juvederm® are all prepared from bacterial-derived hyaluronate that is cross-linked with 1,4-butanediol diglycidyl ether under basic conditions (Fig. 6) (114). Both cross-linking reagents react with

Figure 5 Cross-linking of hyaluronate with divinyl sulfone under basic conditions.

Divinyl Sulfone

Figure 5 Cross-linking of hyaluronate with divinyl sulfone under basic conditions.

Carbodiimide Crosslink
Figure 6 Cross-linking of hyaluronate with 1,4-butanediol diglycidyl ether under basic conditions.

the hydroxyl groups in hyaluronate to give chemically stable ether linkages that significantly extend the residence time at the placement site for several months. These products will afford aesthetic correction for about 3-6 months in humans (115-117). These products have the potential to provide mechanical correction of other soft tissues (e.g., vocal folds) in the body (88).

The regulatory approval of both cross-linked and chemically derivatized hyaluronates provides a unique platform to expand the application of these devices. One such expanded use would be to combine these devices with drugs in an attempt to alter the biology around the implant site or to serve as a drug delivery platform.

IV. Conjugation of Therapeutic Agents to Hyaluronate

In this section, we will discuss several strategies for coupling biologically active agents to hyaluronate and discuss the putative mechanisms for drug release both in vitro and in vivo. Specifically we will focus on the development of hyaluronate conjugates to anti-inflammatory agents and anti-proliferatives.

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