Non-clinical safety of novel cross-linked chondroitin sulfate (SI-449) as an anti-adhesion barrier for abdominal and pelvic surgeries

Abstract

Postoperative adhesions result from abnormal tissue repair and most frequently occur in patients following abdominal and pelvic surgeries. Prevention of adhesion formation, including the use of anti-adhesion barriers during surgery, is considered the most effective clinical strategy for postoperative adhesions. However, no commercial product is commonly used worldwide because of its insufficient efficacy, complexity, and difficulty of use. We developed a new anti-adhesion barrier using novel biomaterial (SI-449), a cross-linked chondroitin sulfate, in an easy-to-apply powder form. Our previous study using animal models showed that the anti-adhesion efficacy of SI-449 was superior to that of an existing product. In this study, the safety of SI-449 was evaluated based on the results of systemic toxicity tests in rats after intraperitoneal application and a wound healing test in laparotomized rats to mimic its clinical use in abdominal and pelvic surgeries. In the systemic toxicity test, minimal and transient hematological changes and muscular regeneration in the abdominal wall and ileum were observed and were considered to be related to systemic toxicity or local irritation due to SI-449. However, these findings were within the safety margin and not associated with serious toxicity. Other changes were considered physiological reactions to SI-449 or its degradants. In the wound healing test, SI-449 was found to have no effect on the healing of the incisional abdominal wall. In conclusion, the non-clinical safety of SI-449 was confirmed, and this novel biomaterial is expected to be used as an anti-adhesion barrier in abdominal and pelvic surgeries.

Keywords

postoperative adhesion anti-adhesion barrier chondroitin sulfate cross-linking technology medical device

Introduction

Postoperative adhesions occur widely in soft tissues including the peritoneum, pericardium, uterus, dural sac, and tendons.^1–4 Adhesions result from abnormal tissue repair, leading to fibrous connections between the wound tissues and surrounding tissues, and are most commonly caused by inflammation after surgery or infection.^4,5 The pathophysiology of adhesions remains unclear; however, all types of adhesions have similarities, such as excessive fibrin deposition, fibroblast/myofibroblast proliferation, and collagen/collagen fiber deposition.^4,6 Regardless of the procedure or anatomical location, postoperative adhesions are frequently found, accounting for 50–95% of all surgeries.⁷ Abdominal or pelvic surgeries are the common causes of peritoneal adhesions, and the incidence of adhesions after these surgeries has been reported to be 67–97%, with 70–90% attributable to a previous surgery.^5,8 Postoperative adhesions cause considerable morbidity, leading to clinical complications such as intestinal obstruction, chronic pain, and female infertility, which significantly reduce quality of life and can be life-threatening.^4,5,9,10

Adhesiolysis is usually performed to release existing adhesions. However, adhesiolysis is complicated and time-consuming for surgeons, and poses an increased risk to patients.^2,11,12 Furthermore, owing to the high recurrence rate of adhesions (80%), inpatient care is necessary for patients, which increases medical costs.⁴ According to the literature, up to 60% of the surgeries conducted today are reoperations, and up to 20% of patients undergo inadvertent enterotomy during adhesiolysis.¹² Therefore, the prevention of adhesion formation is considered the most effective clinical strategy for postoperative adhesions.⁴ To prevent postoperative adhesions, resorbable anti-adhesion barriers are commonly used in abdominal and pelvic surgeries. Seprafilm^®, INTERCEED^®, TENALEAF^® (barrier film or sheet), and Adspray^® (barrier spray) have been approved in Japan.¹³ These act as physical barriers to separate the wound from surrounding tissues.³ However, no commercial product is commonly used worldwide because the incidence of adhesion remains at approximately 50%, even when using existing products.^14–18 Another issue with the existing products is the difficulty of their use. The film or sheet products are hard to handle during laparoscopic surgery, and the spray product requires multiple steps of preparation before use. Therefore, next-generation anti-adhesion barriers are expected to be clinically effective in preventing adhesions and are easy to use during laparoscopic surgery.^18–20

Given this background, we have developed a new resorbable anti-adhesion barrier using novel biomaterial (SI-449) for the United States and Japan, and recently submitted a marketing approval application as a medical device for Japanese health authority. SI-449 is a cross-linked chondroitin sulfate (CS) -C synthesized by the amidation of CS with a crosslinking reagent. CS is a largely heterogeneous glycosaminoglycan composed of alternate and variously sulfated disaccharides of glucuronic acid and N-acetyl-D-galactosamine linked by β (1 → 3) bonds. CS is used in various medical applications owing to its safety and unique biological properties. For example, anti-adhesion properties of CS were suggested in previous researches that CS inhibits cellular adhesion by its hydrophilicity and that CS prevents postoperative adhesion by reducing fibrin deposition following an intra-abdominal administration of CS to coat injured tissue surfaces.^21,22 Based on the properties of CS, SI-449 has been produced as a biomaterial to exert high tissue separation ability and physical obstruction of fibrin deposition.²³ After application to the wound tissue, SI-449 absorbs the surrounding water and swells to form a gel which prevents adhesions by covering the wound tissue and separating it from the surrounding tissues for a critical period for adhesion (3–5 days after surgery).^6,23–25 SI-449 is a powdered product that can be easily applied to uneven tissue surfaces during surgery. In a previous study, we demonstrated the efficacy of SI-449 as an anti-adhesion barrier in the gastrointestinal and gynecological fields in rat cecum-abraded and uterine horn adhesion models.²³ In this study, the safety of SI-449 was evaluated based on the results of systemic toxicity tests after intraperitoneal application and wound healing tests in laparotomized rats.

Materials and methods

Test article and animals

SI-449, a cross-linked CS sterilized by gamma ray radiation, manufactured by Seikagaku Corporation (Tokyo, Japan), was used as the test article in this study, which includes three systemic toxicity tests and a wound healing test. All tests were conducted in compliance with Good Laboratory Practice, and the experimental protocols were approved by the Institutional Animal Care and Use Committees of Seikagaku Corporation and testing facilities (BoZo Research Center Inc. [Tokyo, Japan] for systemic toxicity tests and CMIC Pharma Science Co., Ltd. [Yamanashi, Japan] for a wound healing test). Sprague–Dawley (Crl:CD) rats were purchased form Charles River Japan, Inc. (Kanagawa, Japan), and housed individually in plastic cages with wire lids and bedding materials in an environmentally controlled animal room with a temperature of 23°C ± 3°C, a relative humidity of 50% ± 20%, a ventilation rate of 10 to 15 times/hr, and a light: dark cycle of 12:12 h. Food pellets (CR-LPF, systemic toxicity tests; CRF-1, wound healing test) (Oriental Yeast Co., Ltd. [Tokyo, Japan]) and tap water were provided ad libitum throughout the experimental period. After quarantine and acclimatization, the animals allotted to the groups were anesthetized with isoflurane inhalation (concentration: 0.5–5%, flow rate: 0.5–3 L/min) and underwent surgery on Day 0. Pre- and post-operative management were performed with analgesic by intramuscular or subcutaneous injection of buprenorphine (Otsuka Pharmaceutical Co., Ltd. [Tokyo, Japan]; Nissin Pharmaceutical Industries Co., Ltd. [Yamagata, Japan]) at approximately 4 μg/kg.

Systemic toxicity tests

In this study, the systemic toxicity and local irritation of SI-449 were evaluated using data from three independent experiments (Table 1). Four-week-old male and female rats were quarantined and acclimatized for 14–16 days and then randomly allotted into groups based on body weight so that the mean body weight of each group was approximately equal (mean body weights at Day 0 were 201–239 g for males and 153–176 g for females in each group). On Day 0, under isoflurane inhalation anesthesia, the abdominal muscle was dissected approximately 0.5 cm. Subsequently, SI-449 was applied directly into the abdominal cavity using a syringe filled with SI-449 under the midline incision to mimic the clinical use of SI-449. The application of SI-449 was based on the “Guidance for Resorbable Adhesion Barrier Devices for Use in Abdominal and/or Pelvic Surgery Guidance for Industry”.²⁶ In clinical settings, SI-449 is applied directly to organs/tissues beneath the abdominal wall incision before closing the abdomen during laparotomy. Our previous non-clinical efficacy research of SI-449 demonstrated clear effectiveness at an application density of 4 mg/cm².²³ Although the required amount varies depending on the length of the incision, 2 g is generally considered sufficient to cover the target area of approximately 500 cm² in typical laparotomies. Even when the entire abdominal wall is incised, a maximum of 3 g is believed to adequately meet the requirement. Therefore, in systemic toxicity tests conducted on rats, the highest dose was set at 10 times the clinical dose assuming a human body weight of 50 kg. Specifically, for the acute and subacute systemic toxicity test, a dose equivalent to 10 times the maximum clinical usage of 60 mg/kg (3 g/50 kg) was set, i.e., 600 mg/kg. For subchronic and chronic systemic toxicity tests, a dose equivalent to 10 times the most commonly used dose of 40 mg/kg (2 g/50 kg) was set, i.e., 400 mg/kg. After the application of SI-449, the abdominal wall and skin incisions were sutured, and the animals were observed until necropsy. In the control group, the same procedure was performed with an unfilled syringe. According to the International Organization for Standardization (ISO) standard 10993-11,²⁷ necropsy time points were set at 3, 7, 30, 91, and 183 days after application to evaluate acute, subacute, subchronic, and chronic systemic toxicity. Because SI-449 is biodegradable and absorbable, from the perspective of evaluating the local effects after implantation, these necropsy time points corresponded to the following, in accordance with ISO 10993-6²⁸:

- Day 3: early to mid-time frame (early to ongoing degradation)

- Day 7: mid to late time frame (degradation to complete absorption)

- Days 30, 91, and 183: late time frame (absorption to complete excretion from the body)

Table 1.

Group composition of the systemic toxicity tests.

Group		No. of animals necropsied
Group		Day 3	Day 7	Day 30	Day 91	Day 183
Acute and subacute systemic toxicity test
Control		M: 5, F: 5	M: 5, F: 5	M: 5, F: 5	—	—
SI-449	60 mg/kg	M: 5, F: 5	M: 5, F: 5	M: 5, F: 5	—	—
SI-449	200 mg/kg	M: 5, F: 5	M: 5, F: 5	M: 5, F: 5	—	—
SI-449	600 mg/kg	M: 5, F: 5	M: 5, F: 5	M: 5, F: 5	—	—
Subchronic systemic toxicity test
Control		—	—	—	M: 10, F: 10	—
SI-449	40 mg/kg	—	—	—	M: 10, F: 10	—
SI-449	400 mg/kg	—	—	—	M: 10, F: 10	—
Chronic systemic toxicity test
Control		—	—	—	—	M: 10, F: 10
SI-449	40 mg/kg	—	—	—	—	M: 10, F: 10
SI-449	400 mg/kg	—	—	—	—	M: 10, F: 10

Abbreviation: -, no animal; F, female; M, male.

According to the ISO standard 10993-11²⁷, evaluations consist of clinical observations, measurements of body weight, food consumption, ophthalmology, urinalysis, hematology, blood chemistry, organ weights, gross pathology, and histopathology. The histopathological examination of the abdominal wall was performed by collecting samples from an area slightly distant from the incision site. The local irritation caused by SI-449 was evaluated by examining the intraperitoneal organs and the abdominal wall, which were directly in contact with SI-449. For histopathological analysis, organs/tissues were fixed with phosphate-buffered 10% formalin, embedded in paraffin, and sectioned at a thickness of 3–5 µm. The specimens were stained with hematoxylin and eosin, then examined microscopically.

Wound healing test

Based on previous reports,^29–31 the effect of SI-449 on wound healing in abdominal wall (abdominal muscles and peritoneum) incisions was evaluated using a rat peritoneal wound-healing model (Table 2). Male rats (8 weeks old) were quarantined and acclimatized for 8 days and then randomly allotted into groups based on body weight so that the mean body weight of each group was approximately equal (mean body weight at Day 0 was 359–402 g in each group). On Day 0, under isoflurane anesthesia, a midline incision of approximately 2.5 cm was made in the abdominal wall using a scalpel blade (No. 23; FUTABA Co., Ltd., Tokyo, Japan). Subsequently, SI-449 was applied once at a dose of 14 mg/site to the intra-abdominal organs directly below the incision. In the control group, only the surgical procedures were performed. The abdominal incision was sutured, and the animals were observed and weighed up to the necropsy time points (Days 7 and 14). The dose of SI-449 was based on a previous study²³ where SI-449 prevented adhesion at a dose of 4 mg/cm² (48 mg/12 cm²) in rat adhesion models; the laparotomy wound area was estimated to be 3.5 cm² considering the effects of a 2.5 cm-incision on its surrounding area.

Table 2.

Group composition of the wound healing test.

Group	No. of animals necropsied
	Day 7		Day 14
	Measurement of tensile strength	Histopathology	Measurement of tensile strength	Histopathology
Control	M: 10	M: 5	M: 10	M: 5
SI-449 (14 mg/site)	M: 10	M: 5	M: 10	M: 5

Abbreviation: M, male.

On Days 7 and 14, animals were necropsied, and the abdominal wall was collected (1.0 cm × 4.0 cm) centered on the middle incision. Immediately after the collection, tensile strength of the collected tissue was measured based on the previous reports^29,30 evaluating the ultimate tensile strength of the abdominal wall in rats after laparotomy. Both sides of the collected tissue were pulled in a direction perpendicular to the midline incision at a speed of 20 cm/min using a tensile tester (EZ-SX 100N, SHIMADZU Corporation, Kyoto, Japan), and the maximum tensile strength (unit: N) was recorded. For histopathology, the collected tissues were fixed in phosphate-buffered 10% formalin, and the fixed tissues were cut, embedded in paraffin, thin-sectioned (3–5 µm), and stained with hematoxylin-eosin and Masson’s trichrome. The specimens were examined microscopically to determine the regeneration of the incision wound, fibrosis, and collagen deposition. The tensile strength measurement and histopathology were performed under blinded conditions.

Statistical analyses

Numerical data were statistically analyzed for systemic toxicity tests. A multiple comparison was conducted between the control and each test article group at each evaluation time point. First, group mean value with standard deviation was calculated and homogeneity of variances was analyzed by Bartlett’s test (significance level: 0.01). Homogenous data were analyzed using Dunnett’s test, and heterogeneous data were analyzed using Steel’s test between the control group and each test article group (significance levels: 1% and 5%, two-tailed). For the wound healing test, numerical tensile strength data were statistically analyzed between the control and test article group at each evaluation time point. First, an F-test for equality of variances was conducted (significance level: 5%). When variances were homogeneous, Student’s t-test was performed; when heterogeneous, the Aspin-Welch test was performed (significance level: 5%, two-tailed). Analyses were performed using the integrated statistical package SAS Release 9.1.3 or 9.3 (SAS Institute Inc., NC, USA).

Results

Systemic toxicity tests

No changes related to SI-449 were found in clinical signs, body weight, food consumption, ophthalmology, urinalysis, blood chemistry, or organ weight. Statistically significant hematological changes were found in the 600 mg/kg group on Day 3 compared to the control group, as follows: increased white blood cell (WBC) and platelet (PLT) counts, prolonged prothrombin time (PT), and activated partial thromboplastin time (APTT) (Table 3).

Table 3.

Hematological changes at Day 3 in the acute and subacute toxicity test.

Group		SI-449
Findings	Control	60 mg/kg	200 mg/kg	600 mg/kg
WBC count (10E2/µL)	M: 79.7 ± 5.1 F: 44.6 ± 17.0	M: 90.5 ± 36.1 F: 35.4 ± 6.2	M: 68.3 ± 22.0 F: 46.8 ± 16.5	M: 128.5 ± 26.3* F: 120.1 ± 25.4**
PLT count (10E4/µL)	M: 110.8 ± 41.8 F: 160.6 ± 12.6	M: 133.9 ± 15.4 F: 147.4 ± 19.5	M: 138.3 ± 9.2 F: 144.3 ± 6.5	M: 150.3 ± 13.7* F: 142.0 ± 18.8
PT (second)	M: 11.8 ± 0.5 F: 11.6 ± 0.6	M: 11.5 ± 0.3 F: 11.2 ± 1.0	M: 11.6 ± 0.5 F: 11.7 ± 0.4	M: 13.5 ± 1.5* F: 14.4 ± 0.6**
APTT (second)	M: 13.3 ± 1.3 F: 12.0 ± 0.6	M: 13.7 ± 1.2 F: 11.5 ± 2.2	M: 14.8 ± 1.5 F: 12.5 ± 1.2	M: 27.1 ± 3.7 F: 33.8 ± 3.1

Values are indicated as mean ± standard deviation from 5 animals/sex/group.

Abbreviation: APTT, activated partial thromboplastin time; F, female; M, male; PLT, platelet; PT, prothrombin time; WBC, white blood cell.

*p ≤ .05; **p ≤ .01, comparison with the control group (Dunnett’s test, two tailed test).

Gross pathology revealed no organ abnormalities in the SI-449 group at any necropsy time point. Retention of ascites was observed in the 200 and 600 mg/kg groups on Day 3, and its incidence increased in accordance with the dose level (four males, 200 mg/kg; five males and two females, 600 mg/kg). Histopathological changes related to SI-449 were observed from Days 3 to 30 (Table 4). Representative histopathological changes observed in the abdominal wall, pancreas and liver are shown in Figure 1. Muscular regeneration in the abdominal wall (Figure 1(A)) and ileum was sporadically observed on Day 3 in the SI-449-treated groups. This change was not observed after Day 7. On Days 3, 7, and 30, foamy macrophage infiltration was observed in the peritoneum (serosa and capsule) of the abdominal organs or their surrounding tissues. In some animals, foamy macrophages infiltrated the muscle layer of the abdominal wall or ileum. Thickening of the capsule was observed sporadically in the pancreas on Day 3 (Figure 1(B)) and spleen on Day 7. In the 600 mg/kg group, foamy Kupffer cells were sporadically observed in the liver on Days 7 and 30 (Figure 1(C)). No SI-449-related changes were noted in any of the evaluations on Days 91 and 183. The no-observed-adverse-effect level for the systemic toxicity of SI-449 was determined to be 200 mg/kg in both sexes, as hematological changes were observed in the 600 mg/kg group.

Table 4.

Histopathological findings observed in the acute and subacute systemic toxcity test.

Findings	No. of animals with finding
	Group	Control		SI-449
	Group	Control		60 mg/kg		200 mg/kg		600 mg/kg
	Grade	+	++	+	++	+	++	+	++
Day 3
Regeneration, muscular:
Abdominal wall		—	—	F: 1	—	M: 1 F: 1	—	M: 4 F: 1	—
Intestine, ileum		—	—	—	—	—	—	M: 1	—
Foamy macrophage infiltration: around tissues, peritoneal, muscular layer
Abdominal wall, stomach, intestine, liver, pancreas, spleen, adrenal, testis, epididymis, ovary, oviduct, and urinary bladder		—	—	M: 5 F: 4	F: 1	M: 4 F: 5	M: 1	M: 1 F: 2	M: 4 F: 3
Thickening: pancreas: capsular		—	—	—	—	M: 4	—	M: 4 F: 1	—
Day 7
Foamy macrophage infiltration: around tissues, peritoneal, muscular layer
Abdominal wall, stomach, intestine, liver, pancreas spleen, testis, epididymis, and urinary bladder		—	—	M: 4 F: 3	M: 1	M: 1 F: 3	M: 4 F: 2	F: 3	M: 5 F: 2
Thickening: pancreas, spleen: capsular		—	—	—	—	M: 1	—	—	—
Foamy Kupffer cell: liver		—	—	—	—	—	—	M: 1 F: 2	—
Day 30
Foamy macrophage infiltration: around tissues, peritoneal^a
Abdominal wall, stomach, intestine, pancreas, spleen, and epididymis		—	—	M: 3 F: 1	—	M: 5 F: 4	—	M: 5 F: 4	—
Foamy Kupffer cell: liver		—	—	—	—	—	—	F: 1	—

The histopathological findings were graded by the following criteria: -, not remarkable; +, minimal; ++, mild; +++, moderate; ++++, severe.

Abbreviation: F, female; M, male.

^aFoamy macrophage infiltration to the peritoneum is observed only in the abdominal wall.

Wound healing test

No changes in clinical signs or body weight related to SI-449 were found in necropsied animals on Days 7 and 14. No significant differences in tensile strength were observed between the control and SI-449 groups on Days 7 and 14 (Figure 2). The histopathology of the abdominal wall at the incision site revealed inflammation, granulation tissue, fibrosis, and muscle tissue regeneration on Day 7 in the control and SI-449 groups (Table 5, Figure 3). On Day 14, fibrosis remained, but other findings decreased in both groups compared with those on Day 7, suggesting that the wound healing process progressed from Day 7 to Day 14. Foamy macrophage infiltration was observed on Days 7 and 14 only in the SI-449 group, which was thought to be a physiological reaction to SI-449. SI-449 had no effect on wound healing, as no difference in the frequency or degree of histopathological changes related to inflammation was observed between the control and SI-449 groups at any time point.

Table 5.

Histopathological findings of the incisional abdominal wall.

Findings	No. of animals with finding
	Group	Control			SI-449
	Grade	+	++	+++	+	++	+++
Day 7
Inflammation		5	—	—	5	—	—
Granulation tissue		—	4	1	1	4	—
Fibrosis		5	—	—	5	—	—
Regeneration, muscle		5	—	—	5	—	—
Foamy macrophage aggregation		—	—	—	2	3	—
Day 14
Inflammation		1	1	—	2	—	—
Granulation tissue		—	—	—	—	—	—
Fibrosis			4	1	1	4	—
Regeneration, muscle		—	—	—	1	—	—
Foamy macrophage aggregation		—	—	—	4	—	—

The histopathological findings were graded by the following criteria as shown below.

Inflammation:

-, no animal with finding; +, slight inflammation with a few lymphatic and plasma cells; ++, moderate inflammation with higher levels of lymphocytes, neutrophils, eosinophils and plasma cells; +++, severe inflammation with massive infiltration of inflammatory cells.

Fibrosis, granulation tissue, muscle regeneration, and macrophage aggregation:

-, no animal with finding; +, minimal changes; ++, moderate changes; +++, severe changes.

Discussion

Several anti-adhesion barriers are commercially available; however, there are no highly effective products for gastroenterological and gynecological surgery. With an increase in the number of laparoscopic surgeries, easily applicable products are required, due to the complexity and difficulty of their use. SI-449 is expected to be used as a new anti-adhesion barrier to resolve these issues, and our previous study demonstrated its efficacy in rat adhesion models. However, anti-adhesion barriers are classified as high-risk medical devices that require high levels of safety. In the U.S., INTERGEL^® Adhesion Prevention Solution, a 0.5% ferric hyaluronate gel, was withdrawn from the market in 2003 due to concerns of adverse events.³² Currently, in the U.S., the non-clinical safety of anti-adhesion barriers must meet the requirements of the U.S. Food and Drug Administration (FDA) guidances, in addition to those of ISO 10993 standards.^26,33,34 Therefore, we evaluated the non-clinical safety of SI-449 to confirm its potential use in abdominal and pelvic surgeries.

SI-449 is composed of CS and amino acids. When applied intraperitoneally, SI-449 absorbs the surrounding water and swells, exerting an anti-adhesion effect. Subsequently, its cross-linked structure and CS backbone are degraded through hydrolysis and other processes, leading to systemic absorption. CS, the main component widely used as medical devices and additive. In the U.S., CS is also used as a food additive and is a generally recognized as safe (GRAS) compound.³⁵ Furthermore, biocompatibility of CS oligosaccharides, enzymatic hydrolysates of CS, was demonstrated in a 90-days repeated oral toxicity study in rats.³⁶ Regarding cross-linked CS products similar to SI-449, in situ cross-linked injectable CS hydrogels showed good biocompatibility without foreign body reactions in various rat models, including cecum-abdominal wall and hepatic adhesion, as well as subcutaneous implantation tests in mice.³⁷ Other CS-based biomaterials, including cross-linked CS, have also been reported to be biocompatible, although some synthetic additives may induce toxicity or immunological reactions, especially upon prolonged application.³⁸ SI-449 contains no synthetic additives amd its degradants are considered to be composed of CS and amino acid residues generated by hydrolysis of ester bonds in cross-linking structure of SI-449. Both SI-449 and its degradants would disappear from the application site within a short period.

In the present study, the safety of SI-449, including its degradation products likely generated during clinical use, was evaluated by intraperitoneal administration in rats. No serious toxicity related to SI-449 was observed in the present study, supporting that SI-449 is a safe compound with high biocompatibility. Because a relationship with SI-449 application cannot be ruled out, some changes were considered to be caused by systemic toxicity or local irritation by SI-449 (Table 6). However, these are unlikely to cause clinical safety concerns for several reasons.

- Hematological changes (prolongation of PT and APTT, and increased WBC and PLT counts) were observed on Day 3 in the 600 mg/kg group, without any changes suggestive of inflammation or systemic dysfunction. It is known that antithrombin, one of the blood coagulation factors, is regulated by glycosaminoglycans, and CS has been reported to exhibit potential anticoagulant activity, although weaker than that of heparin.^39–41 Because SI-449 and its degradants are systemically exposed after intraperitoneal administration (data not shown), transient anticoagulant effects may have occurred due to the high dose administration. Similarly, increased WBC and PLT counts may be related to systemic exposure to SI-449 and its degradants, but the detailed mechanism remains unclear. Although changes in the WBC and PLT counts are generally associated with infection or inflammation, no other test results indicated the presence of infection or inflammatory responses. Because the hematological changes observed in the present study were minimal and transient, and there was a safety margin relative to the expected clinical dose, they were considered unlikely to cause clinical concerns. However, alterations in coagulation parameters are clinically relevant in the surgical setting, and considering the potential risk of bleeding, administration after adequate hemostasis might be recommended.

- Muscular regeneration, which indicates a recovery response to the local irritation of SI-449 administration, was observed in the abdominal wall and ileum (Day 3, 60 mg/kg or higher groups); however, because this change was transient and minimal, it is unlikely to cause any clinical concerns.

Table 6.

Summary of the noteworthy findings related to SI-449 in the systemic toxicity tests.

Dose	60,200 and 600 mg/kg			40 and 400 mg/kg
Necropsy time point	Day 3	Day 7	Day 30	Days 91, 183
Hematology	Systemic toxicity
Hematology	Increased WBC and PLT counts, prolongation of PT and APTT (600 mg/kg)	—	—	—
Gross pathology	Physiological reaction
Gross pathology	Retention of ascites (200 and 600 mg/kg)	—	—	—
Histopathology	Local irritation
	Muscular regeneration^a (60 to 600 mg/kg)	—	—	—
	Physiological reaction	Physiological reaction	Physiological reaction
	Foamy macrophage infiltration^b (60 to 600 mg/kg)	Foamy macrophage infiltration^b (60 to 600 mg/kg)	Foamy macrophage infiltration^b (60 to 600 mg/kg)	—
	Thickening of the capsule^c (200 and 600 mg/kg)	Thickening of the capsule^c (200 mg/kg)	Foamy Kupffer cells (600 mg/kg)
		Foamy Kupffer cells (600 mg/kg)

Abbreviation: -, no noteworthy findings; APTT, activated partial thromboplastin time; PLT, platelet; PT, prothrombin time; WBC, white blood cell.

^aMuscular regeneration was observed in the abdominal wall and ileum.

^bFoamy macrophage infiltration was observed in the peritoneal of abdominal organs or their surrounding tissues.

^cThickening of the capsule was observed sporadically in the pancreas at Day 3 and in the pancreas and spleen at Day 7.

The other changes shown below were thought to be physiological reactions caused by SI-449 or its degradants, but not toxic changes. These physiological reactions refer to non-pathological responses and include processes such as the degradation of SI-449 and its processing by phagocytic cells, without adverse tissue reactions such as inflammation, granuloma formation, or fibrosis.

- Transient retention of ascites (Day 3, 200 mg/kg or higher groups) could be due to a mixture of residual SI-449 or its degradants and body fluid. This is supported by a separate rat pharmacokinetic study, in which residual SI-449 and its degradants were detected in peritoneal lavage fluid on Day 3 after administration of 600 mg/kg SI-449 (data not shown). Importantly, although the characteristics of the ascitic fluid were not analyzed, no findings suggestive of malignant ascites were observed, based on clinical signs, necropsy, and histopathological examination (e.g., changes in peritoneal space osmolality, portal hypertension, plasma protein levels, peritonitis, or organ dysfunction).⁴²

- Foamy macrophage infiltration in the peritoneum (Days 3 to 30, 60 mg/kg or higher groups) was considered to indicate phagocytosis of SI-449 and its degradants, and was considered a physiological reaction to SI-449.²³

- Thickening of the capsule in the pancreas and spleen (Day 3, 200 mg/kg or higher groups; Day 7, 200 mg/kg group) could be associated with foreign-substance processing of SI-449. This change was not associated with tissue necrosis or inflammation; in some cases, foamy macrophage infiltration was observed, which decreased and disappeared over time.

- Foamy Kupffer cells (Days 7 and 30, 600 mg/kg group) could be due to foreign-substance processing of SI-449 after resorption from the abdominal cavity. The pharmacokinetic study showed SI-449 degradants were detected in the liver until Day 30 (data not shown).

Changes considered to be related to SI-449 application were found up to Day 30 in the systemic toxicity tests; however, they were unlikely to cause adverse clinical events. In addition, no results raised safety concerns in other biocompatibility tests (Supplemental material 1). Therefore, SI-449 was considered biocompatible and meets the requirements of the ISO 10993 standards. Furthermore, SI-449 did not have an effect on wound healing, and our results showed that SI-449 did not enhance infection in rats following cecal content inoculation (cecal slurry model of sepsis^43,44) (Supplemental material 2). The FDA recommends non-clinical evaluation of its impact on wound healing and infection.²⁶ These results suggest that SI-449 meets the safety requirements of the FDA guidance as an anti-adhesion barrier for abdominal and pelvic surgeries.

This study has several limitations, and future directions are provided below. Although no severe adverse effects of SI-449 on the abdominal wall were noted in the histopathological evaluation or ultimate failure strength measurement, to more clearly assess the relationships of the histopathological changes and mechanical properties, it would be better to measure tissue stiffness by elastic (Young’s) modulus,^45–48 which correlates with pathological conditions such as fibrosis and inflammation.^45,46 In addition, species differences should be acknowledged. For example, species differences in anatomical features and rate of peritoneal healing^49,50 may limit the direct extrapolation of our results in the rat model to humans. Furthermore, species differences in peritoneal macrophage populations (resident tissue and infiltrating macrophages) should be considered.^51–54 Because macrophages play crucial roles in inflammation, tissue remodeling, wound healing, and phagocytic clearance of pathogens,^4,51 differences in macrophage populations may influence the processes of peritoneal wound healing, macrophage phagocytic efficiency toward SI-449, and the nature of peritoneal fluid, and consequently affecting the degradation kinetics and immune responses to SI-449. Given the aforementioned limitations, the local and systemic effects of SI-449 observed in the present study cannot be fully extrapolated to humans, and future studies are expected to address these points. However, since a sufficient safety margin was confirmed in the present study, the risk of safety concerns upon human use is considered low.

Conclusion

In conclusion, the non-clinical safety of SI-449 was confirmed in the present study; therefore, this novel biomaterial is expected to be used as an anti-adhesion barrier in abdominal and pelvic surgeries. A pivotal clinical trial in Japan for SI-449 as a postoperative anti-adhesion barrier in the gastroenterological field (Trial ID, jRCT1080225247) (https://jrct.mhlw.go.jp/en-latest-detail/jRCT1080225247) showed favorable results, and we are preparing to provide evidence for the efficacy and safety of SI-449 in the future.

Supplemental material

Supplemental material - Non-clinical safety of novel cross-linked chondroitin sulfate (SI-449) as an anti-adhesion barrier for abdominal and pelvic surgeries

Supplemental material for Non-clinical safety of novel cross-linked chondroitin sulfate (SI-449) as an anti-adhesion barrier for abdominal and pelvic surgeries by Ayako Shiraki, Shino Ito, Dai Muramatsu, Tomoki Sasaki, Akira Otsuka, Tomomi Miyahara, Katsuya Takahashi, Keiji Yoshioka, Miki Suehiro in Journal of Biomaterials Applications

Supplemental material

Supplemental material - Non-clinical safety of novel cross-linked chondroitin sulfate (SI-449) as an anti-adhesion barrier for abdominal and pelvic surgeries

Footnotes

Acknowledgments

We are grateful to Dr. Aisuke Nii for the histological interpretation of tissue sections.

ORCID iDs

Ayako Shiraki

Shino Ito

Dai Muramatsu

Tomomi Miyahara

Keiji Yoshioka

Ethical considerations

Ethical approval for all animal studies were obtained from the Animal Experiment Ethics Committee of Seikagaku Corporation and testing facilities (BoZo Research Center Inc. and CMIC Pharma Science Co., Ltd.).

Author contributions

All authors have contributed to study conception, design, acquisition and interpretation of data. A.S., S.I and D.M. contributed to drafting and revision of the article with support from M.S and K.Y. All the authors have reviewed and approved the final version of the manuscript. All authors have also reviewed and approved the data presented in the manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by Seikagaku Corporation.

Declaration of conflicting interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The test material (SI-449) was produced by Seikagaku Corporation. All authors are employees of Seikagaku Corporation working in the Research & Development Division.

Data Availability Statement

The dataset supporting the conclusions of this study is stored at Seikagaku Corpation. Further inquiries regarding these data can be submitted to Ayako Shiraki (ayako.shiraki@seikagaku.co.jp).

Supplemental material

Supplemental material for this article is available online.

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