HIGH CONCENTRATED PROTEIN COMPOSITIONS FOR PREVENTING TISSUE ADHESION

Abstract

Disclosed herein is an anti-adhesion kit comprised of: (i) a fibrinogen solution component comprising: fibrinogen at a concentration of about 5 to 25 mg/ml; and free calcium ions at a concentration ranging from 0.1 M to 1 mM; and (ii) a thrombin component containing thrombin. Further disclosed is an anti-adhesion kit comprised of: (i) a fibrinogen solution component containing fibrinogen at a concentration of 8% to 25% of total protein by weight, and optionally free calcium ions at a concentration ranging from 0.1 M to 1 mM; wherein a total protein concentration ranges from about 80 to 120 mg/ml; and (ii) a thrombin component containing thrombin. Methods of using the kits e.g., to provide anti-adhesion curable compositions are also disclosed.

Claims

1. An anti-adhesion kit comprising: (i) a first container comprising a fibrinogen solution component comprising fibrinogen at a concentration of about 8% to about 25% of total protein by weight, and optionally free calcium ions at a concentration ranging from about 0.1 M to about 1 mM; wherein a total protein concentration ranges from about 80 to about 120 mg/ml; and (ii) a second container comprising a thrombin component comprising thrombin.

2. The anti-adhesion kit of claim 1, wherein said thrombin component comprises free calcium ions, optionally at a concentration of about 0.1 M to about 45 mM, or about 35 mM to about 45 mM.

3. The anti-adhesion kit of claim 1, wherein said thrombin component is devoid of chelating agent.

4. The anti-adhesion kit of claim 1, wherein said fibrinogen in said fibrinogen component is present at a concentration of more than about 7.7 to less than about 17 mg/ml.

5. The anti-adhesion kit of claim 1, wherein each of said fibrinogen component and said thrombin component is devoid of: potassium citrate, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetra-acetic acid (EGTA), and PBS.

6. The anti-adhesion kit of claim 1, wherein the fibrinogen solution component comprises albumin at a concentration of at least 20% of the total proteins by weight.

7. The anti-adhesion kit of claim 1, wherein the fibrinogen solution component comprises albumin at a concentration ranging from 60 to 110 mg/ml.

8. The anti-adhesion kit of claim 1, wherein the fibrinogen is derived from plasma concentrated cryo-precipitate, optionally, a viral inactivated plasma cryo-precipitate.

9. The anti-adhesion kit of claim 1, wherein the fibrinogen solution component further comprises factor XIII at a concentration of at least 1 IU/ml.

10. The anti-adhesion kit of claim 1, wherein the fibrinogen is present in said fibrinogen solution component at a concentration of below 14%, of total protein by weight.

11. The anti-adhesion kit of claim 1, wherein the thrombin component is in the form of a liquid solution, optionally at a concentration ranging from 100 to 300 IU/ml.

12. The anti-adhesion kit of claim 1, wherein said fibrinogen component does not comprises tranexamic acid or aprotinin.

13. The anti-adhesion kit of claim 1, for use in producing an anti-adhesive curable composition, optionally for use in preventing tissue adhesion following invasive procedures selected from post-operative procedures, or laparoscopic procedures, e.g., resolving intestinal obstruction, and abdominal or genecology surgery, optionally wherein the composition is used by spraying or dripping the composition onto a site to be treated.

14. The anti-adhesion kit of claim 1, for use in preventing tissue adhesion.

15. A mixture comprising fibrinogen, calcium, and thrombin, wherein the fibrinogen is present at a concentration of 3 to 15% of a total protein by weight, the total protein concentration ranges from about 40 to about 120 mg/ml, and the thrombin is present at a concentration of about 50-150 IU/ml.

16. A two-component composition for adhesion prevention comprising: component A comprising a fibrinogen solution comprising fibrinogen at a concentration of about 5 to 25 mg/ml; albumin at a concentration ranging from 60 to 110 mg/ml, and optionally comprising free calcium ions at a concentration ranging from 0.1 M to 1 mM; and component B comprising a thrombin component.

17. The two-component composition of claim 16, wherein said thrombin component comprises free calcium ions, optionally at a concentration ranging from 35 to 45 mM.

18. The two-component composition claim 16, wherein said thrombin component is devoid of chelating agent.

19. The two-component composition of claim 16, wherein said fibrinogen in said fibrinogen component is present at a concentration of 10 to 20 mg/ml.

20. The two-component composition of claim 16, wherein the thrombin component is in the form of a liquid solution, optionally at a concentration ranging from 100 to 300 IU/ml, optionally wherein said component A and said component B are present at a ratio ranging from 1.2:1 to 1:1.2, by volume.

Description

EXAMPLES

Example 1

Evaluation of Adhesions in a Rabbit Uterine Horn Model

[0386] This model has been used extensively for the determination of efficacy of putative anti-adhesion agents, and good correlations exist between data obtained in animals and that obtained clinically (Wiseman D M, Gottlick-Iarkowski L, Kamp L. Prevention of Bowel Adhesions Using Two Barriers of Oxidized Regenerated Cellulose (ORC). J Inv Surg 12:141-146 (1999); Wiseman, 1999).

[0387] Briefly, adhesion prevention was evaluated using the uterine horn model in rabbits as previously described (Wiseman D M, Gottlick L E, Diamond M P. Effect of thrombin-induced hemostasis on the efficacy of an absorbable adhesion barrier. J. Reprod. Med. 37:766-770 (1992); Wiseman 1992). Six female New Zealand White rabbits were used for each of the following groups: 1) No treatment; 2) Treatment with 32 mg/ml fibrinogen (derived from cryoprecipitate) and 512 IU/ml Thrombin; 3) Treatment with 12 mg/ml fibrinogen (derived from cryoprecipitate) and 260 IU/ml Thrombin; 4) Treatment with Seprafilm. Briefly, uterine horns were abraded by scraping with a scalpel blade 40 times on each side of each uterine horn and the experimental treatment was applied. After 15-16 days, adhesions were evaluated by estimating the extend of adhesions, the severity of adhesions and the degree of uterine convolution, a measure of anatomical distortion due to adhesions.

[0388] More details of exemplary procedures are provided below.

[0389] Materials and Methods

[0390] EVICEL-derived Formula #1 was an FDA approved commercially marketed product from which the biological components were taken and diluted by a factor of 2. The dilution was carried out by the addition of appropriate surrogate liquid to each of the biological components.

[0391] EVICEL-derived Formula #2 was an FDA approved commercially marketed product from which the biological components were taken and diluted by a factor of 4. The dilution was carried out by the addition of appropriate surrogate liquid to each of the biological components.

[0392] First Component of Test Material: Diluted BAC2

[0393] Denomination: BAC2, thawed and diluted by a factor of 2 (Formula #1 or by a factor of 4 (Formula #2) with BAC2 surrogate (see Table 1).

[0394] Supplier: Omrix Biopharmaceuticals Ltd.

[0395] Protein concentration: Target: 80-120 mg/ml. Actual: 85 mg/ml (Formula #1) and 83 mg/ml (Formula #2)

[0396] Clottable Fibrinogen: Target: 27-43 mg/ml (Formula #1) or 13-22 mg/ml (Formula #2). Actual: 32 mg/ml (Formula #1) and 12 mg/ml (Formula #2)

[0397] Quantity required: at least 12 vials2 ml per formula

[0398] Storage conditions: 18 C. until use.

[0399] Hazards: Handle material with gloves, as with all blood products.

[0400] Second Component of Test Material: Diluted Thrombin

[0401] Denomination: Thrombin 1000 IU/ml and diluted by a factor of 2 or by a factor of 4 with Thrombin surrogate (see Table 1).

[0402] Supplier: Omrix Biopharmaceuticals Ltd.

[0403] Protein concentration: Target: 5.5-6.5 mg/ml. Actual: 5.9 mg/ml (Formula #1) and 6.0 mg/ml (Formula #2)

[0404] Thrombin concentration: 400-600 IU/ml (Formula #1) or 200-300 IU/ml (Formula #2). Actual: 512 IU/ml (Formula #1) and 260 IU/ml (Formula #2)

[0405] Quantity required: at least 12 vials2ml per formula

[0406] Storage conditions: 18 C. until use.

[0407] Hazards: Handle material with gloves, as with all blood products.

[0408] BAC2 surrogate and Thrombin surrogate formulas are provided in Table 1 below.

TABLE-US-00001 TABLE 1 BAC2 surrogate and Thrombin surrogate formulas Material Reagent Concentration BAC2 Sodium Chloride 120 mM Surrogate Tri-Sodium Citrate dihydrate 10 mM Glycine 120 mM Arginine hydrochloride 9.5 mM Calcium chloride dihydrate 1 mM Human albumin 11% pH 7.0-7.2 Thrombin Sodium Acetate trihydrate 20 mM Surrogate D-Mannitol 0.1M Calcium chloride dihydrate 40 mM Human Albumin 0.6% Sodium Chloride ~90 mM pH 6.8-7.2

[0409] Following dilution, the samples were filtered through 0.2 m Polyethersulfone (PES) membrane filter, filled into 2 ml vials, capped with a manual crimper and stored at 18 C. until shipment to Synechion.

[0410] Reference Materials: No treatmentSurgery alone. Seprafilm Adhesion Barrier: Seprafilm Adhesion Barrier (Lot 7BYSEP041, exp 2020-05-31) was obtained from Sanofi-Aventis U.S. LLC, Bridgewater, N.J.

[0411] Applicator devices for EVICEL formulations: The EVICEL Application Device with the EVICEL Airless Spray Accessory was used to apply the EVICEL-derived formulations according to the Assembly guide and the manufacturer instructions.

[0412] Acclimation: Animals were examined by experienced animal care personnel shortly after arrival at the test facility. Animals were acclimated for a minimum of 5 days prior to initiation of the study and monitored by experienced animal care personnel daily. Animals supplied for this study were used only if they appeared grossly normal (i.e. showing a clean unruffled coat, bright clear eyes, no unusual exudate from body orifices, alert and active posture).

[0413] Housing environment and husbandry: Animals were individually housed in stainless steel cages, each labeled with an individual card indicating the study number and the individual animal number. The room environment was maintained at approximately 68 oF (20 oC) with 30-70% relative humidity and a light/dark cycle of 12 hours/12 hours.

[0414] Diet and Water: Purina Prolab Rabbit diet 15% protein (LabDiet, St. Louis, Mo.) and tap water was provided ad libitum to the animals for the duration of the study. Carrots and alfalfa cubes were provided to the rabbits.

[0415] Pre-Operative Preparation: Animals were weighed on the day of surgery. Anesthesia was induced and maintained by inhalation of isoflurane (5% and 3.5% concentration, respectively). Depilation of the surgical site was accomplished with an electric animal clipper. The area was vacuumed to remove hair clippings and debris, and then rinsed with isopropyl alcohol 70%. The bladder was expressed (i.e. manipulated externally to force urine out of it). The entire area was painted with an aqueous iodophor solution (iodine scrub) of 1% available iodine. The area was then be swabbed with 70% isopropyl alcohol solution.

[0416] Procedures were performed in a sterile manner in a room reserved for aseptic survival surgery. Sterile towels, drapes, and instruments were used. The anesthetized and surgically prepared rabbit was delivered to the operating table and restrained via the limbs in the desired recumbent position. A sterile incise drape was applied to the prepared area.

[0417] Animals were randomized in blocks of 4 to one of the study groups by lottery by a disinterested individual. Surgery was conducted on two study days with half the animals from each group being allocated to each study day.

[0418] Intra-Operative Procedure: The rabbit uterine horn simple abrasion model was conducted as described by Wiseman et al., 1992. The abdomen was entered via a midline laparotomy incision about 6 cm long. The bladder and uterus were delivered into the wound.

[0419] Only those animals with two uterine horns with a diameter of between 9 and 16 French, inclusive, were entered into the study. Using a number 10 scalpel blade, 5 cm lengths of uterine horn, approximately 1 cm from the uterine bifurcation, were scraped, 40 times per side. Hemostasis was controlled by tamponade with gauze. If necessary, small vessels are ligated.

[0420] After abrasion procedures were completed per animal, the group assignment was revealed to the surgeon. Test materials were applied to the uterine horn. Organs were repositioned anatomically. During the treatment, the animals were observed carefully to remove any animal with unexpected response to the anesthetic treatment.

[0421] Study Arms Application: Reference Control-Controls consisted of animals in which all surgical procedures have been performed, but without the application of test materials.

[0422] EVICEL derived Formula 1 and Formula 2 Treatments: at least 4 vials of 2 ml of each component per animal were thawed at room temperature before induction of anesthesia. Each product was handled according to the EVICEL manufacturer's instructions and delivered by spraying using the Omrix application Airless spray device. Enough product was applied so as to cover the traumatised surfaces with a thin film. The volume applied was recorded.

[0423] Seprafilm Standard Treatment Control: An approximately 23 piece of Seprafilm was draped over each uterine horn before replacing the uterus anatomically.

[0424] Post-Operative Procedure: Abdominal incisions were closed using a continuous Vicryl 4-0 suture. Fascia was closed loosely with 4-0 Vicryl and the skin closed with undyed 4-0 Vicryl (cutting needle) using a subcuticular suturing method.

[0425] Three doses of buprenorphine (Buprenex) (0.03 mg/kg, 0.4 ml0.3 mg/ml) were given by subcutaneous injection, one on the morning of surgery, one six to eight hours later and one the following morning. Further doses were given if deemed necessary by the veterinarian.

[0426] Clinical Signs of Animals: All animals were observed closely until vertical recumbency was resumed, when they were returned to their cages. Rabbits were placed under a heat lamp or on a heating blanket for this initial post-operative period.

[0427] Each rabbit was observed daily after surgery to determine its health status on the basis of general attitude and appearance, food consumption, weight loss, fecal and urinary excretion and presence of abnormal discharges. Animals were observed daily for incisional integrity, excessive bruising and infection, evaluation of pain and/or discomfort and were given analgesics additional to the standard doses described above. Animals were euthanatized if deemed necessary by the attending veterinarian and excluded from analysis.

[0428] Daily monitoring records were kept. Unusual findings were reported to the study director and/or the veterinarian and a decision made to treat an animal or, in the case of a moribund animal, to euthanize it. Moribund animals were euthanized and recorded.

[0429] Animal husbandry personnel were blinded as to the group assignment.

[0430] Evaluation of adhesions: 15-16 days following surgery, animals were euthanized by intravenous injection of 1.5 ml of Beuthanasia-D (sodium pentobarbital 390 mg/ml; phenytoin sodium 50 mg/ml) (Merck Animal Health, Madison, N.J.). Body weights of the animals were recorded. The abdomen was opened and the surgical site inspected. Adhesions were graded by a blinded observer.

[0431] The length of each uterine horn with adhesions was estimated. Results were expressed as incidence of adhesions (number of uterine horns with adhesions/total number) and extent of adhesions (% length of uterine horn with adhesions).

[0432] Tenacity (Severity) of Adhesions (Grade) were graded as 0 (absent), 1 (filmy adhesions), and 2 (tenacious, requiring sharp dissection).

[0433] The degree of uterine convolution, a measure of the anatomical distortion of the organ due to adhesions, was recorded according to the following scale: [0434] No convolution: straight lengths of adherent or nonadherent horns that are clearly seen [0435] Partly convoluted: horns have adhesions and 50%-75% of the horn length is entangled preventing discernment of the straight portions [0436] Completely convoluted: it is impossible to see the uterine anatomy because the horn is completely entangled.

[0437] Tissue samples were taken into formalin for possible future histology. No histology was performed. Representative photographs were taken during surgery and evaluation.

[0438] Data were entered onto a spreadsheet by hand and transcribed into a personal computer using Microsoft Excel for Windows, with double checking. For each animal, the average % extent of adhesions was calculated for the two horns. This average was used to calculate the mean extent (percent of the length of the uterus involved) of adhesions (SEM) for the group, displayed to one decimal place. Comparisons of all groups with the Control group were made using Dunnett's test (Dunnett C W. New tables for multiple comparisons with a control. Biometrics 20; 482-491 (1964); Dunnett, 1964). The incidence of adhesions was compared using Fisher's Exact Test, and the tenacity and degree of uterine convolution was compared using the .sup.2 test. For all tests, the level of statistical significance was taken as p<0.05.

[0439] Results

[0440] The results for evaluation of adhesion sites are presented in Table 2 below.

TABLE-US-00002 TABLE 2 Concentration in the fibrinogen component 2-fold dilution 4-fold dilution EVICEL (#1): EVICEL (#2): Negative Fib. 32 mg/ml Fib. 12 mg/ml Parameter Control Thr. 512 IU/ml Thr. 260 IU/ml Seprafilm % Adhesions* 77.6%.sup. 43.8%.sup. 21.9%.sup. 32.8%.sup. Severity** Absent 0% 8% 16.7%.sup. 8% Filmy 0% 42% 42% 25% Tenacious 100% 50% 42% 67% Convolution*** No 25% 50% 92% 58% Partially 50% 42% 8% 25% Completely 25% 8% 0% 16.7%.sup. Concentrations after mixing fibrinogen with Thrombin 1:1 2-fold dilution 4-fold dilution EVICEL (#1): Mixed components Mixed components EVICEL (#2): Negative Fib. 16 mg/ml Fib. 6 mg/ml Parameter Control Thr. 206 IU/ml Thr. 130 IU/ml Seprafilm % Adhesions* 77.6%.sup. 43.8%.sup. 21.9%.sup. 32.8%.sup. Severity** Absent 0% 8% 16.7%.sup. 8% Filmy 0% 42% 42% 25% Tenacious 100% 50% 42% 67% Convolution*** No 25% 50% 92% 58% Partially 50% 42% 8% 25% Completely 25% 8% 0% 16.7%.sup. 1) *% Adhesions = % length of uterine horn with adhesions 2) **The severity is presented as the % number of uterine with the following severity grades: absent/filmy adhesions/tenacious, requiring sharp dissection 3) ***The degree of uterine convolution, a measure of the anatomical distortion of the organ due to adhesions is presented as the % number of uterine with the following convolution grades: 4) No convolution: straight lengths of adherent or nonadherent horns that are clearly seen 5) Partly convoluted: horns have adhesions and 50%-75% of the horn length is entangled preventing discernment of the straight portions 6) Completely convoluted: it is impossible to see the uterine anatomy because the horn is completely entangled.

[0441] Extensive adhesions were observed in the Control group animals (784.4%, N=6). A statistically significant (Dunnett's t test) reduction in the % extent of adhesion formation was observed for animals treated with either EVICEL derived #1 (449.8%, N=6; p<0.05), EVICEL derived #2 (22 7.4%, N=6; p<=0.01) or Seprafilm (3311.6%, N=6; p<=0.01) as compared with the Control animals (no treatment).

[0442] The incidence of adhesions was 100% (12/12 horns) for the Control group, 92% (11/12 horns) for the EVICEL derived #1 Group, 83% (10/12 horns) for the EVICEL derived #2 group and 92% (11/12 horns) for the animals treated with Seprafilm. These differences were not statistically significant (Fisher's Exact Test).

[0443] Both EVICEL-derived formulations showed a statistically significant reduction in the tenacity of adhesions as compared with controls (p<0.05, .sup.2 test), whereas the reduction in the tenacity as compared to Seprafilm was insignificant (p=0.12).

[0444] Only EVICEL derived #2 group showed a statistically significant reduction in the degree of uterine convolution, a measure of anatomical distortion, as compared with the Control group (p=0.004, .sup.2 test). EVICEL derived #1 and Seprafilm showed a downshift in the degree of uterine convolution, but this was not statistically significant.

[0445] As can be seen from Tables 2A-2B, while the 32 mg/ml fibrinogen concentration provided an effect compared to the negative control, the lower concentration of fibrinogen of 12 mg/ml in the fibrinogen component (Table 2A), or concentration of fibrinogen of 6 mg/ml (Table 2B) after mixing with thrombin solution 1:1, provided a much greater effect, which was better than the commercially available product Seprafilm in every respect. The total % of adhesions was lower; the adhesions were less severe with more absent adhesions and fewer tenacious adhesions; and there was only one partly convoluted adhesion (and no completely convoluted adhesions), compared to 5 convoluted adhesions (2 complete) in the Seprafilm group.

Example 2

Effect of Fibronogen and Albumin in a Rabbit Uterine Horn Model

[0446] The purpose of this study was to characterize the efficacy of various formulations with different concentrations of fibrinogen and albumin in a rabbit uterine horn adhesion model.

Materials and Methods:

The Test System

[0447] Juvenile/young adult (13-15 weeks old at surgery) female New Zealand white rabbits (Oryctolagus cuniculus) were used in the study. Rabbits were obtained from Western Oregon Rabbit Co., PO Box 653, Philomath, Oreg. USA and were individually identified by unique, ear tags, applied by the supplier.

[0448] Animals were examined by experienced animal care personnel shortly after arrival at the test facility.

[0449] Animals were acclimated for a minimum of 5 days prior to initiation of the study and monitored by experienced animal care personnel daily. Animals supplied for this study were used only if they appeared grossly normal (i.e., showing a clean unruffled coat, bright clear eyes, no unusual exudate from body orifices, alert and active posture).

Environment and Husbandry:

[0450] Animals were individually housed in stainless steel cages, each labeled with an individual card indicating the study number and the individual animal number. The room environment was maintained at approximately 68 F. (approximately 20 C.) with 30-70% relative humidity and a light/dark cycle of 12 hours/12 hours.

[0451] Purina Prolab Rabbit diet 15% protein (LabDiet, St. Louis, Mo.) and tap water was provided ad libitum to the animals for the duration of the study and carrots and alfalfa cubes.

[0452] Pre-Operative Preparation: Adhesion was studied using surgery. Animals were weighed on the day of surgery. Anesthesia was induced and maintained by inhalation of isoflurane (5% and 3.5% concentration, respectively). Depilation of the surgical site was accomplished with an electric animal clipper. The area was vacuumed to remove hair clippings and debris, and then rinsed with isopropyl alcohol 70%. The bladder was expressed (i.e. manipulated externally to force urine out of it). The entire area was painted with an aqueous iodophor solution (iodine scrub) of 1% available iodine. The area was then swabbed with 70% isopropyl alcohol solution.

[0453] Procedures were performed in a sterile manner in a room reserved for aseptic survival surgery. Sterile towels, drapes, and instruments were used. The anesthetized and surgically prepared rabbit was delivered to the operating table and restrained via the limbs in the desired recumbent position. A sterile incise drape was applied to the prepared area. Since there is an approximate correlation between animal weight and uterine horn size, in order to avoid operating on animals with smaller or larger out-of-range horns, and since there were two consecutive surgery days in each of two consecutive weeks, animals were assigned to surgery in a sequential order based on their weight. The assignment of an animal to one of the study groups was made according to its sequential surgery assignment.

Justification of the Test System:

[0454] The rabbit uterine horn simple abrasion model is performed essentially as described in Wiseman 1992 (Effect of thrombin induced hemostasis on the efficacy of an absorbable adhesion barrier. The journal of reproductive medicine vol 37 No 9, September 1992), with and without the bleeding modification.

[0455] Six animals per group are to be entered into the study. Given the variances observed historically, these group sizes are sufficient to provide the study with sufficient power to detect statistical differences between treatment and control group.

[0456] This model has been used extensively for the determination of efficacy of putative anti-adhesion agents, and good correlations exist between data obtained in animals and that obtained clinically (Wiseman, 1999 Effect of different barriers of oxidized regenerated cellulose (ORC) on cecal and sidewall adhesions in the presence and absence of bleeding. Journal of Investigative Surgery 12:141-146).

[0457] The 42 sequentially assigned animals were randomized by lottery by a disinterested individual in blocks of 7 so that each of the study groups was represented in each block.

[0458] The rabbit uterine horn simple abrasion model was conducted as described by Wiseman et al., 1992.

[0459] Intra-Operative Procedure: The abdomen was entered via a midline laparotomy incision about 6 cm long. The bladder and uterus were delivered into the wound. Only those animals with two uterine horns with a diameter of between 9 and 16 French, inclusive, were entered into the study. Using a number 10 scalpel blade, 5 cm lengths of uterine horn, approximately 1 cm from the uterine bifurcation, were scraped, 40 times per side. Hemostasis was controlled by tamponade with gauze. If necessary small vessels were ligated. Organs were repositioned anatomically. During the treatment, the animals were observed carefully to remove any animal with unexpected response to the anesthetic treatment. Controls consisted of animals in which all surgical procedures have been performed, but without the application of test materials.

[0460] Tested formulas were used in vials of 5 ml of each component per animal were thawed at room temperature before induction of anesthesia. Each product was handled according to the manufacturer's instructions and delivered by spraying using the Omrix application airless spray device. Enough product was applied so as to cover the traumatised surfaces with a thin film. The volume applied was recorded.

[0461] Post-Operative Procedure: Abdominal incisions were closed using a continuous Vicryl 4-0 suture. Fascia was closed loosely with 4-0 Vicryl and the skin closed with undyed 4-0 Vicryl (cutting needle) using a subcuticular suturing method.

[0462] Three doses of buprenorphine (Buprenex) (0.03 mg/kg, 0.4 ml0.3 mg/ml) were given by subcutaneous injection, one on the morning of surgery, one six to eight hours later and one the following morning. Further doses were given if deemed necessary by the veterinarian.

[0463] Clinical Signs of Animals: All animals were observed closely until vertical recumbency was resumed, when they were returned to their cages. Rabbits were placed under a heat lamp or on a heating blanket for this initial post-operative period.

[0464] Each rabbit was observed daily after surgery to determine its health status on the basis of general attitude and appearance, food consumption, weight loss, fecal and urinary excretion and presence of abnormal discharges. Animals were observed daily for incisional integrity, excessive bruising and infection, evaluation of pain and/or discomfort and were given analgesics additional to the standard doses described above. Animals were euthanatized if deemed necessary by the attending veterinarian and excluded from analysis.

[0465] Daily monitoring records were kept. Unusual findings were reported to the study director and/or the veterinarian and a decision made to treat an animal or, in the case of a moribund animal, to euthanize it. Moribund animals were euthanized and recorded. Animal husbandry personnel were blinded as to the group assignment.

[0466] Adhesion and Tenacity Detection: Adhesion evaluation was done 11-16 days following surgery, animals were euthanized by intravenous injection of 1.5 ml of Beuthanasia-D (sodium pentobarbital 390 mg/ml; phenytoin sodium 50 mg/ml) (Merck Animal Health, Madison, N.J.). Body weights of the animals were recorded. The abdomen was opened, and the surgical site inspected. Adhesions were graded by a blinded observer.

Examinations Performed

[0467] Extent and Incidence of Adhesions: The length of each uterine horn with adhesions is estimated. Results are expressed as incidence of adhesions (number of uterine horns with adhesions/total number) and extent of adhesions (% length of uterine horn with adhesions).

[0468] Tenacity of Adhesions: Tenacity (Severity) of Adhesions (Grade) is graded as 0 (absent), 1 (filmy adhesions), and 2 (tenacious, requiring sharp dissection).

[0469] Degree of Uterine Convolution: The degree of uterine convolution, a measure of the anatomical distortion of the organ due to adhesions, is recorded according to the following scale:

[0470] No convolution: straight lengths of adherent or nonadherent horns that are clearly seen;

[0471] Partly convoluted: horns have adhesions and 50%-75% of the horn length is entangled preventing discernment of the straight portions;

[0472] Completely convoluted: it is impossible to see the uterine anatomy because the horn is completely entangled.

[0473] Data Analysis of Adhesions: For each animal, the average extent of adhesions is calculated for the two horns. This average is used to calculate the mean extent (percent of the length of the uterus involved) of adhesions (SEM) for the group, displayed to one decimal place. Comparisons of all groups with the Control group (no treatment) are made using Dunnett's test (Dunnett, 1964). The incidence of adhesions of each group is compared with controls using Fisher's Exact Test, and the tenacity and degree of uterine convolution is compared using the chi2 test. For all tests, the level of statistical significance is taken as p<0.05.

[0474] Exclusion from Analysis: upon examination of an animal at necropsy, but before inspection of the surgical site and evaluation of adhesions, a determination is made as to whether the animal should be excluded from the primary analysis. Animals are to be excluded from the primary analysis if there are signs of unusual occurrences that may affect the outcome. Such signs commonly include presence of infection within the abdominal cavity, or excessive weight loss (>10% of body weight). Any decision to exclude an animal is made without knowledge of the group assignment or of the presence or extent of adhesions.

TABLE-US-00003 TABLE 3 BAC2 and Thrombin Albumin-based buffer solution formulas* Material Reagent Concentration BAC2 buffer Sodium Chloride 120 mM (With Albumin) Tri-Sodium Citrate dihydrate 10 mM Glycine 120 mM Arginine hydrochloride 9.5 mM Calcium chloride dihydrate 1 mM Human albumin 11% pH 7.0-7.2 Thrombin buffer Sodium Acetate trihydrate 20 mM (With Albumin) D-Mannitol 0.1M Calcium chloride dihydrate 40 mM Human Albumin 0.6% Sodium Chloride ~90 mM pH 6.8-7.2 *(Thrombin and albumin surrogate)
Applicator devices for all formulations: The EVICEL Application Device with the EVICEL Airless Spray Accessory is used to apply the formulations according to the Assembly guide and the manufacturer instructions.

TABLE-US-00004 TABLE 4 with experimental details Thrombin component Total Total Fibrinogen % Albumin Fibrinogen Thrombin protein Fibrinogen albumin from total (Alb) mg/ml component (IU/ml) and (mg/ml) in (mg/ml) in (mg/ml) in protein in Total Albin Source of Source of Exp. Thr/BAC 2 Thr/BAC2 Thr/BAC2 Thr/BAC2 protein In BAC2 Thr Fibrinogen fibrinogen thrombin No. 1:1 1:1 1:1 1:1 (mg/ml) (mg/ml) (mg/ml) (mg/ml) and buffer buffer 1 45.5 11.5 42 25% 91 77 6 23 BAC2(1:2) 161 W/albumin W/albumin 2 47 4.5 50 9.5% 94 94 6 9 BAC2(1:6) 161 W/albumin W/albumin 3 18 11.5 3 67% 36 4 2 23 BAC2(1:2) 153 W/O albumin W/O albumin 4 10 5.5 1.5 55% 20 2 1 11 BAC2(1:6) 153 W/O albumin W/O albumin 5 55 7 58 13.7%.sup. 102 110 6 14 pure 161 fibrinogen W/albumin W/albumin 6 55 8.5 58 21.25% 80 110 6 17 pure 161 fibrinogen W/albumin W/albumin

TABLE-US-00005 TABLE 5 Results summary for the assessment of the effect of fibrin-based formulations on adhesion formation Representative Average Extent Adhesion Exp. (No.) (Adhesion)%.sup.1 p.sup.2 Free.sup.3 Grade.sup.4 P# Conv.sup.5 p# control 67 NA 0 0/1/11 3/5/4 1 32 0.039 6.3 1/5/10 12/4/0 0.012 2 33 0.047 0 0/5/7 10/2/0 0.012 5 33 0.07 7.1 1/6/7 0.078 8/4/2 6 59 0.67 7.1 1/4/9 9/1/4 0.063 .sup.1% of length of uterine horn with adhesions, mean of left and right horns .sup.2p value for Student's t- test against Control .sup.3% of uterine horns free of adhesions (number of uterine horns free of adhesions/total) .sup.4Number of horns with no adhesions/grade 1 adhesions/grade 2 adhesions .sup.5Number of horns with no convolution/partial convolution/full convolution * p < 0.05 Dunnett's t test vs. Control #p value 2 test, vs Control). Only values <0.1 shown.

TABLE-US-00006 TABLE 6 Summary of discrete data statistic for 6-8 samples used for the anti-adhesion experiments tested for each composition (fibrinogen component) #5 #6 (mg/ml) (mg/ml) #2 **FGPure FG Pure #1 (mg/ml) (mg/ml) Fibrinogen Fibrinogen *FGBAC2 FGBAC2 14 + total 17 + total 23 + total 9 + total protein protein Threshold set prot 91 + prot 94 + 102 + alb. 80 + alb. to above 35% Control alb 77 alb aprox 94 aprox. 110 approx. 110 Extent *** 70.0 45.0 35.0 75.0 42.5 Extent *** 90.0 40.0 41.5 15.0 100.0 Extent *** 60.0 33.5 60.0 11.0 7.5 Extent *** 10.5 65.0 8.0 11.0 45.0 Extent *** 92.5 10.0 35.0 5.0 90.0 Extent *** 77.5 16.0 20.0 80.0 25.0 Extent *** 4.0 33.0 100.0 Extent *** 41.5 Average 67 32 33 33 59 Extent *** individual 5 4 2 2 5 results above the threshold Overall No. 6 8 6 7 7 of results per group Proportion of 0.83 0.50 0.33 0.29 0.71 results above threshold *FGBACII = fibrinogen in BACH; **FGPure Fibrinogen = Fibrinogen in Pure fibrinogen; *** % adhesion

TABLE-US-00007 TABLE 7 Summary of discrete data statistic for 6-8 samples for the anti-adhesion experiments tested for each composition (fibrinogen and thrombin) #5 (mg/ml) #6 (mg/ml) FGPure FGPure #1 (mg/ml) #2 (mg/ml) fibrinogen fibrinogen FGBAC2 FGBAC2 7 + tot. 8.5 + tot. 11.5 + total 4.5 + total protein prot Threshold set to prot. 45.5 + prot. 47 + 54 + alb 43 + alb above 35% Control alb 42 alb approx. 50 aprox. 58 approx. 58 Extent* 70.0 45.0 35.0 75.0 42.5 Extent* 90.0 40.0 41.5 15.0 100.0 Extent* 60.0 33.5 60.0 11.0 7.5 Extent* 10.5 65.0 8.0 11.0 45.0 Extent* 92.5 10.0 35.0 5.0 90.0 Extent* 77.5 16.0 20.0 80.0 25.0 Extent* 4.0 33.0 100.0 Extent* 41.5 Average 67 32 33 33 59 Extent* individual 5 4 2 2 5 results above the threshold Overall No. of 6 8 6 7 7 results per group Proportion of 0.83 0.50 0.33 0.29 0.71 results above threshold *% adhesion

[0475] Table 3 shows the buffers used for dilution of fibrinogen and thrombin with albumin. Table 4 shows the concentrations of albumin (calculated), fibrinogen (measured), and total protein (measured) in the fibrinogen component and in the mixture of fibrinogen and thrombin in representative samples.

[0476] A threshold was set to 35% or below as indication to efficient antiadhesion extent (Tables 6 and 7). In samples 2 and 5 the majority of the tested samples were equal to or below this threshold. The results show that a fibrinogen component comprising total protein at a concentration of 94 and 102 mg/ml and a low concentration of fibrinogen of about 9 and 14 mg/ml samples 2 and 5 respectively; can be used in a kit for efficient prevention or to reduction of adhesion (Table 5 and Table 6).

[0477] It was found that a mixture of fibrinogen and thrombin comprising total protein at a concentration of 47 and 55 mg/ml and a concentration of fibrinogen of 4.5 and 7 mg/ml samples 2 and 5 respectively, can be used for efficient prevention or reduction of adhesion (Table 5 and Table 7).

[0478] It was found that a fibrinogen component comprising albumin at a concentration of 94 and 110 mg/ml and a low concentration of fibrinogen of 9 and 14 mg/ml, samples 2 and 5 respectively, can be used in a kit for efficient prevention or reduction of adhesion (Table 5 and Table 6).

[0479] It was found that a mixture of fibrinogen and thrombin comprising albumin at a concentration 50 and 58 mg/ml and a low concentration of fibrinogen of 4.5 and 7 mg/ml; samples 2 and 5 respectively, can be used for efficient prevention or reduction of adhesion (Table 5 and Table 7).

Example 3

Impact of the Formulations on the Water Retention Under Ambient Conditions

[0480] Further exemplary procedures were aimed at quantitating the % of water retention in the clots with the different formulations after 1-2 hours hydrogels formation and under ambient conditions.

[0481] Formulations 1-12 are as described in Table 8.

[0482] In formulations 9-12 TH04 is an in-process fraction in the thrombin manufacture in which thrombin source is devoid of albumin and calcium.

[0483] After one and up to two hours following hydrogel formation (formation of hydrogel is determined by a visual inspection) under ambient conditions the hydrogel was subjected to a mild centrifugation of 630 g, having a surface area of about 1 cm.sup.2 for 30 min inside a centricon tube and the amount of retained water in the hydrogel was assessed.

[0484] The results summarized in Table 8 show that the water retention capacity of the different clots is 37.4%-61.6%

TABLE-US-00008 TABLE 8 Impact of the formulations on the water retention under ambient conditions Fibrinogen component Thrombin component % water Formulation# Material Diluent Material Diluent retained 1 BAC2 (1:4) With Thrombin With With 56.11 2 BAC2 (1:8) albumin (1:4) albumin Calcium 37.40 3 BAC2 (1:4) W/O W/O With 58.05 4 BAC2 (1:8) albumin albumin Calcium 48.33 5 BAC2 (1:4) With With W/O calcium, 54.15 6 BAC2 (1:8) albumin albumin 5 mM EDTA 45.38 7 BAC2 (1:4) W/O W/O W/O calcium, 42.52 8 BAC2 (1:8) albumin albumin 5 mM EDTA 37.65 9 BAC2 (1:4) With TH04 W/O albumin W/O calcium, 61.59 10 BAC2 (1:8) albumin 100-300 5 mM EDTA 47.58 11 BAC2 (1:4) W/O IU/ml 59.99 12 BAC2 (1:8) albumin 52.18

Example 4

Impact of the Formulations on the Water Retention Under High Pressure

[0485] Further exemplary procedures were aimed at quantitating the % of water retention in the clots with the different formulations upon centrifugation (in an Eppendorf tube 0.7-1 cm.sup.2 at 31,514 g for 30 mins.), shortly after hydrogel formation (formation of hydrogel is determined by a visual inspection) under ambient conditions.

[0486] Formulations 1-12 are as described in Table 8.

[0487] The Results are summarized in Table 9 below.

TABLE-US-00009 TABLE 9 Impact of the formulations on the water retention under high pressure Albumin conc free % retained from % lost from in the total calcium initial weight initial weight Exp. Dilution of solution ions are after 1st after 1st No. fibrinogen mg/ml present centrifugation centrifugation 1 1:4 48.20 + 57.55 42.45 2 1:8 52.20 + 29.32 70.68 3 1:4 1.8 + 89.66 10.34 4 1:8 1.25 + 88.52 11.48 5 1:4 48.20 64.35 35.65 6 1:8 52.50 23.55 76.45 7 1:4 1.80 91.19 8.81 8 1:8 1.25 87.97 12.03 9 1:4 45.20 26.87 73.13 10 1:8 49.50 6.92 93.08 11 1:4 1.20 70.46 29.54 12 1:8 0.67 16.89 83.11

[0488] When albumin is present at high concentration (45-52 mg/ml) or when the total protein is high e.g. of about 50 mg/ml (or 35 to 60 mg/ml), at a fibrinogen dilution of 1:4 (in Exp 1, and 5) water retention is about 50%. When albumin is present at high concentration to (45-52 mg/ml) or when the total protein is about 50 mg/ml (or 35 to 60 mg/ml), and fibrinogen dilution is of 1:8 (Experiment 2, 6 and 10) water retention decreases to about 29% to about 7% of the initial weight. Complete depletion of calcium (thrombin used TH04 completely lacks calcium and EDTA was added as in Examples 9 and 10) water retention decreases. Water retention is minimal under conditions of fibrinogen dilution is 1:8 (70 mg/ml:9=7.7 mg/ml) and complete depletion of calcium ions (Table 9 sample 10).

[0489] The results also show that the clots (with fibrinogen diluted both 1:4 and 1:8) enable to retain the highest amount of water (88.5% and 90%) when albumin concentration or total protein is low (Group 4 vs. Group 2 or Group 3 vs. Group 1).

[0490] The results also show that the presence of EDTA (or absence of free calcium) did not change the clot capability to retain the water when albumin concentration or total protein is low, i.e. the trend of the water loss was the same like in Groups 1-4 (with the presence of free calcium).

[0491] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0492] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.