Methods for treating or preventing tissue adhesions

Abstract

The present invention relates to the treatment of tissue adhesions, e.g. tissue adhesions that occur after surgical interventions. More specifically, the invention refers to an enzyme having DNAse activity for use in a method of treating or preventing tissue adhesions. The invention also relates to a pharmaceutical composition that comprises an enzyme having DNAse activity for use in a method of treating or preventing tissue adhesions.

Claims

1. A method of treating or preventing tissue adhesion in a subject undergoing abdominal surgery, comprising administering an enzyme having a deoxyribonuclease (DNase) activity to the subject as a solution one or more times during surgery directly to tissue or organ surfaces injured during the surgery that are at risk of forming adhesions, wherein the enzyme is a human DNase-1-like 3, wherein said human DNase-1-like 3 comprises the amino acid sequence of SEQ ID NO: 6, or a sequence having at least 95% sequence identity thereto, wherein said method does not involve the administration of plasmin.

2. The method of claim 1, wherein said method comprises the administration of between 1,000 to 10,000 Units (U) of the enzyme to the subject.

3. The method of claim 1, wherein said method comprises the administration of the enzyme between 0.15 mg per kg body weight of the subject to 200 mg per kg body weight.

4. The method of claim 1, wherein the enzyme is a recombinantly produced enzyme.

5. The method of claim 1, wherein the surgery is hernia repair.

6. The method of claim 1, wherein the surgery involves intestinal anastomosis.

7. The method of claim 1, wherein the surgery is cesarean section.

8. The method of claim 1, wherein the surgical intervention involves damaging of a serous membrane.

9. The method of claim 8, wherein the serous membrane is a peritoneal membrane.

10. The method of claim 1, wherein the subject suffers from a thermal lesion in a serous membrane.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the peritoneal adhesion as measured by the Leach, Kraemer and Nair adhesion score 7 days after making incisions of 1.5 cm and 3 cm, respectively. Differences were calculated by the Mann-Whitney-U-Test.

(2) FIG. 2 shows the time course of the peritoneal adhesion as measured by the Leach, Kraemer and Nair adhesion score. Differences were calculated by the Mann-Whitney-U-Test.

(3) FIG. 3 shows the peritoneal adhesion after 72 hours accessed by the Leach, Kraemer and Nair adhesion score. The figure shows a comparison of the treatment with DNAse1, controls (no treatment) and sham animals (laparotomy only, no peritoneal incision or suture). Differences were calculated by the Mann-Whitney-U-Test.

(4) FIG. 4 shows the peritoneal adhesion after 21 days evaluated by the Leach, Kraemer and Nair adhesion score. Differences were calculated by the Mann-Whitney-U-Test.

(5) FIG. 5 shows the amino acid sequence of the mature human DNAse 1 after cleavage of the signal peptide.

(6) FIG. 6 shows adhesion that was induced by (A) deserositation, (B) intestinal anastomosis, and (C) heat treatment accessed by the Leach and Nair adhesion score.

EXAMPLES

(7) The experiments referred to below were approved by the Hamburg State Administration for animal research. All environmental parameters within the animal facility were in compliance with the German Law for the Care and Use of Laboratory animals.

Example 1: Induction of Peritoneal Adhesion

(8) The mice were randomly divided into groups comprising the same number of animals. The mice were anesthetized with 5% isoflurane (Baxter, Unterschleißeim, Germany) and maintained with 2.5% isoflurane that was delivered through a facemask. Preoperative antisepsis was performed with betaisadona (Mundipharma GmbH, Limburg, Germany). All mice received 0.02 mg/kg buprenorphine (Reckitt Benckiser, Mannheim, Germany) for analgesia and 10 mg/kg enrofloxacin (Bayer, Leverkusen, Germany) as prophylactic antibiotic therapy subcutaneously.

(9) Adhesions were induced using bipolar electrocoagulation. Standardized lesions were inflicted in an area of 0.5 cm×1.5 cm by sweeping bipolar electrocoagulation forceps over the abdominal peritoneum for 2 seconds. The current was delivered using the following settings: Bipolar Soft, Effect 4, 40 W. The soft coagulation delivers a sinusoidal current of more than 200V ensuring a slow and deep hemostasis without adhering to the tissue.

(10) The defects were subsequently closed using two interrupted sutures (6/0 Vicryl, Ethicon, Norderstedt, Germany) so as to induce an ischemic field around the traumatized area. The sutures were placed equidistantly from each other over the defect (distance: 5 mm). All stitches were made 1 mm from the wound edge. For better standardization, all operations were performed by one operator.

(11) The animals were treated as follows: In the controls, only adhesion induction, but no other intervention was performed. In the lavage control group, the abdomen of the mice was washed before closure with 1 ml NaCl 0.9%. In the lavage group, the abdomen of the mice was washed before closure with 1 ml NaCl 0.9% comprising 10 mg/kg bodyweight DNasel (Pulmozyme, Roche, Grenzach, Germany). In the DNasel control group, 100 μl NaCl 0.9% was injected i.p. after closure every 24 hours for three days. In the DNasel group, 10 mg/kg bodyweight DNasel (Pulmozyme, Roche, Grenzach, Germany) was injected i.p. after closure every 24 hours for three days.

(12) The abdomen war closed using a 6x0 Ethilon (Ethicon Norderstedt, Germany) running suture.

Example 2: Induction of Adhesion by Deserositation

(13) The mice were randomly divided into groups comprising the same number of animals. The mice were anesthetized with 5% isoflurane (Baxter, Unterschleißheim, Germany) and maintained with 2.5% isoflurane that was delivered through a facemask. Preoperative antisepsis was performed with betaisadona (Mundipharma GmbH, Limburg, Germany). All mice received 0.02 mg/kg buprenorphine (Reckitt Benckiser, Mannheim, Germany) for analgesia subcutaneously. Deserositation was induced by rubbing a mini-prep on the small intestinal wall (distal ileum). No further manipulation was performed.

(14) The animals were treated as follows: In the controls, only adhesion induction, but no other intervention was performed. In the DNasel multi group, the abdomen of the mice was washed before closure with 1 ml NaCl 0.9% comprising 10 mg/kg bodyweight DNasel (Pulmozyme, Roche, Grenzach, Germany). Additionally, 100 μl NaCl 0.9% was injected i.p. after 24 and 48 hours.

(15) The abdomen war closed using a 6x0 Ethilon (Ethicon Norderstedt, Germany) running suture.

Example 3: Induction of Adhesion by Intestinal Anastomosis

(16) The mice were randomly divided into groups comprising the same number of animals. The mice were anesthetized with 5% isoflurane (Baxter, Unterschleißheim, Germany) and maintained with 2.5% isoflurane that was delivered through a facemask. Preoperative antisepsis was performed with betaisadona (Mundipharma GmbH, Limburg, Germany). All mice received 0.02 mg/kg buprenorphine (Reckitt Benckiser, Mannheim, Germany) for analgesia subcutaneously. Intestinal anastomosis was performed with 8x0 Vicryl (Ethicon Norderstedt, Germany) continuous suture after dissection of the small intestine (distal ileum).

(17) The animals were treated as follows: In the controls, only adhesion induction, but no other intervention was performed. In the DNasel multi group, the abdomen of the mice was washed before closure with 1 ml NaCl 0.9% comprising 10 mg/kg bodyweight DNasel (Pulmozyme, Roche, Grenzach, Germany). Additionally, 100 μl NaCl 0.9% was injected i.p. after 24 and 48 hours.

(18) The abdomen war closed using a 6x0 Ethilon (Ethicon Norderstedt, Germany) running suture.

Example 4: Induction of Adhesion by Heat Treatment

(19) The mice were randomly divided into groups comprising the same number of animals. The mice were anesthetized with 5% isoflurane (Baxter, Unterschleißheim, Germany) and maintained with 2.5% isoflurane that was delivered through a facemask. Preoperative antisepsis was performed with betaisadona (Nundipharma GmbH, Limburg, Germany). All mice received 0.02 mg/kg buprenorphine (Reckitt Benckiser, Mannheim, Germany) for analgesia subcutaneously.

(20) After median laparotomy the thermic lesions were induced by heat exposure after the viscera (especially the intestine) using a red lamp with a distance of 1 meter for 10 minutes. No further manipulation was performed.

(21) The animals were treated as follows: In the controls, only adhesion induction, but no other intervention was performed. In the DNasel multi group, the abdomen of the mice was washed before closure with 1 ml NaCl 0.9% comprising 10 mg/kg bodyweight DNasel (Pulmozyme, Roche, Grenzach, Germany). Additionally, 100 μl NaCl 0.9% was injected i.p. after 24 and 48 hours.

(22) The abdomen war closed using a 6x0 Ethilon (Ethicon Norderstedt, Germany) running suture.

Example 5: Adhesion Assessment

(23) All adhesions induced in Example 1-4 were evaluated after relaparotomy and immediately documented with digital photography in a standardized fashion for later blinded evaluation. Macroscopic grading was assessed by two surgeons using the modification tool of Leach grade and Nair grade (Leach et al. (1998), Fertil. Steril. 69: 415-8; Nair et al. (1974), Arch. Surg. 108: 849-53). The Leach grade was originally designed to score adhesion to the uterine horn and was thus modified to evaluate peritoneal adhesions. The Leach score consists of three factors: severity of adhesion (0=no adhesion, 1=filmy avascular, 2=vascular or opaque, 3=cohesive attachment), degree of adhesion (0=no adhesion, 1=adhesion separated with gentle traction, 2=adhesion separated with moderate traction, 3=requiring sharp dissection) and extent of adhesion (0=no adhesion, 1=1-25%, 2=26-50%, 3=51-75%, =76-100%). See Leach et al. (1998), Fertil. Steril. 69: 415−8; Nair et al. (1974), Arch. Surg. 108: 849-53.

(24) Moreover, the Kraemer grade was accessed (Kraemer et al. (2014), Biomed Res Int 2014; 435056). The Kraemer grade is used as follows: 0=no adhesion, 1=avascular adhesion, 2=filmy vascular adhesion, 3=dense, vascular adhesion. The adhesion quality was considered “filmy” if the adhesion was see through. Otherwise, it was considered “dense”.

(25) The Nair score consist of two factors (Nair et al. (1974), Arch. Surg. 108: 849-53): macroscopic adhesion (0=no adhesion, 1=single band of adhesion between viscera to abdominal wall, 2=two bands wither between viscera to abdominal wall, 3=more than two bands to abdominal wall) and microscopic adhesion (0=no fibrosis, 1=fibrosis with thin collagen bundle, 2=tissue with wider and less vascularized collagen fibrosis, 3=tissue with thick collagen bundle).

(26) Microscopic Assessment

(27) All specimens were washed in PBS and fixed in 10% buffered formalin. The specimens were embedded in paraffin, cut in sections having a thickness of approximately 5 μm, stained by using hematoxylin and eosin (H&E) and examined by light microscopy by a pathologist blinded to the particular animal groups.

(28) All specimens were histologically evaluated. The adhesive fibrous tissue was dissected with the continuity of the transition zone to the macroscopic normal peritoneal wall. All specimens were fixed in buffered formalin (4%) and embedded in Paraffin according to standardized methods. Serial sections were stained with haematoxylin and eosin and evaluated for the grade of adhesion and fibrosis using light microscopy. All evaluations were performed by a single pathologist who was blinded to the methods and groups.

(29) The degree of fibrosis was assessed by the percentage of extent adhesion area, the grade of fibrosis and the fibrosis depth. The extent adhesion area was defined as the percentage of fibrotic area on the most severe adhesion field. The grade of fibrosis was divided into four grades according to the density of collagen fibers (0: none, 1: thin, 2: moderate, 3: thick). The depth of fibrosis was evaluated by penetration depth of fibrosis from serosa to mucosa (0: none, 1: serosa, 2: muscle, 3: submucosa, 4: mucosa). The inflammation degree was evaluated by the number of inflammatory cells (neutrophils, macrophages, lymphocytes, giant cells and mast cell) under the 10 high power fields (HPFs).

(30) Results:

(31) The results of the macroscopic and microscopic evaluation revealed were as follows. In a first series of experiments, the incision breadth and number of threads was evaluated which reliably induces an abdominal adhesion that imposes no significant burden on the animals. FIG. 1 shows that a more homogeneous adhesion was observed with an incision length of 1.5 cm compared to a 3 cm incision. In both cases a Vicryl thread was inserted 0.5 cm from the margin.

(32) In a second set of experiments, the time course of adhesion was examined, and the animals were removed from the experiment at different points of time. FIG. 2 depicts shows that adhesions having a score of 5/10 were observed after 6 hours and initially dissolved easily. The adhesions became more distinct with time and increasingly more difficult to dissolve. After 21 days the adhesion reached a maximum.

(33) In a third set of experiments, the systemic or local treatment with DNasel was examined after 72 hours and 21 days, respectively. As can be seen in FIG. 4, an effective inhibition of the adhesion was achieved both with systemic and local DNasel. Compared to the control group, all differences were significant.

(34) The results are shown in FIG. 6. As can be seen in FIG. 6A, Dnasel treatment after deserositation significantly reduced adhesion. In addition, Dnasel treatment after deserositation reduced the mortality relative to the control (deserosation 77.8% vs. 70.6%, p>0.05).

(35) Similarly, as can be seen in FIG. 6B, Dnasel treatment after intestinal anastomosis significantly reduced adhesion. Compared to a control, the mortality was also significantly reduced by Dnasel treatment (100% vs. 90%, p>0.05).

(36) Finally, as shown in FIG. 6C, Dnasel addition after heat treatment likewise reduced adhesion.