EP3917549A1 - Procedure for the preparation of an amniotic membrane homogenate based antimicrobial agent - Google Patents

Abstract

The present invention fits in the field of medical or veterinary science, and relates in general to a procedure for the preparation of mammalian amniotic membrane homogenate that can be used as an antimicrobial agent. More specifically, the present invention relates to a procedure for the preparation of an antimicrobial agent characterized in that a homogenate of whole mammalian amniotic membrane is used. The present invention also relates to a procedure for the preparation of a homogenate derived from whole mammalian amniotic membrane. The present invention also relates to the homogenate and antimicrobial agent obtainable by the procedures of the present invention.

Description

PROCEDURE FOR THE PREPARATION OF AN AMNIOTIC MEMBRANE HOMOGENATE

BASED ANTIMICROBIAL AGENT

Field of the invention

The invention fits in the field of medical or veterinary science, and relates in general to procedures including the preparation of mammalian amniotic membrane homogenate that can be used as an antimicrobial agent. More specifically, the present invention relates to a procedure for the preparation of an antimicrobial agent characterized in that a homogenate of whole mammalian amniotic membrane is used. The present invention also relates to a procedure for the preparation of a homogenate derived from whole mammalian amniotic membrane. The present invention also relates to the homogenate and antimicrobial agent obtainable by the procedures of the present invention.

Background of the invention

During the years 2000 and 2015, the use of antibiotics worldwide increased for 65% (Klein et al, Proc Natl Acad Sci USA, 1 15(15), 2018). Epidemiological studies showed the correlation between the increasing use of antibiotics and the emergence and spreading of bacteria, resistant to antibiotics (Zaman et al, Cureus, 9(6), 2017; Ventola, P T, 40(4), 2015). In the European Union 25.000 people die each year due to infections with microorganisms, resistant to antibiotics (ECDC/EMEA Technical report - The bacterial challenge: time to react; 2009). Due to emergence and spread of microorganisms, resistant to antibiotics, there is a great need for discovery and development of novel antimicrobials.

Amniotic membrane is the extraembryonic membrane that ensures the protection of the embryo against the external mechanical forces and dehydration and also ensures the suitable environment for its development (Hilmy et al, Anatomy and Histology of Amnion, World Scientific Publishing 2017; Cirman et al, Cell Tissue Bank, 2014). It is composed of the monolayer of amniotic epithelial cells, basal lamina and stromal tissue (Rocha et al, Amniotic membrane, Springer Netherlands, 2015). Amniotic membrane is suitable for clinical use, since it i) provides an extracellular matrix, which enables the attachment and proliferation of cells (Cornwell et al, Clin Podiatr Med Surg, 26(4), 2009), ii) promotes epithelization and inhibits fibrosis (Fukuda et al, Cornea, 18(1), 1999; Jerman et al, Tissue Eng Part C Methods, 20(4), 2014); Koizumi et al, Invest Ophthalmol Vis Sci, 41 (9), 2000), iii) has low immunogenicity (Cornwell et al, Clin Podiatr Med Surg, 26(4), 2009; Szekeres-Bartho, Int Rev Immunol, 21 (6), 2002), iv) has anti-inflammatory activity (Insausti et al, Stem Cells Cloning, 7, 2014), v) anti- cancer activity (Niknejad et al, Cell Tissue Res, 363, 2016; Niknejad et al, Med Hypotheses, 81 (5), 2013; Jiao et al, Mol Biol Rep, 39(1), 2012; Mamede et al, Med Oncol, 32(12), 2015) and also vi) antimicrobial activity (Mao et al, Sci Rep, 7(1), 2017; Mao et al, J Diabetes Complications, 8(2), 2016; Yadav et al, Front Microbiol, 8, 2017; Tehrani et al, Sci Rep, 7(1), 2017; King et al, Placenta, 28(2-3), 2007). In the clinical setting human amniotic membrane is most commonly used in ophthalmology, in cornea and conjunctiva reconstruction procedures (Costa et al, Amniotic membrane, Springer Netherlands, 2015), and it is also being used in dermatology for treatment of burns and chronic ulcers (Lo et al, Int J Dermatol, 48(9), 2009), in abdominal surgery for prevention of peritoneal adhesions and treatment of gastroschisis (Kubanyi, Br Med J, 2(4514), 1947; Gharib et al, Pediatr Surg Int, 1 1 (2-3), 1996) and in vagina and urinary tract reconstruction surgeries (Silini et al, Front Bioeng Biotechnol, 3, 2015; Koziak et al, Int J Urol, 14(7), 2007). Currently there are 155 clinical trials in running (Clinicaltrials.gov), in which amniotic membrane is being used for treatment of diabetic foot, chronic wounds, burns, osteoarthritis etc. Antimicrobial activity of amniotic membrane has been proven against several bacteria ( Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumonia, Staphylococcus saprophyticus itd.) and fungi ( Blastomyces albicans, Fusarium solani, Aspergillus fumigatus, Aspergillus niger, Aspergillus nidulans) (Mao et al, Sci Rep, 7(1), 2017; Mao et al, J Diabetes Complications, 8(2), 2016; Yadav et al, Front Microbiol, 8, 2017; Tehrani et al, Sci Rep, 7(1), 2017; Talmi et al, Placenta, 12(3), 1991 ; Kjaergaard et al, Eur J Obstet Gynecol Reprod Biol, 94(2), 2001 ; Tehrani et al, Cryobiology, 67(3), 2013; Parthasarathy et al, J Academia and Industrial Research, 2(10), 2014; Wang et al, African J Microbiology Res, 6(21), 2012; Zare-Bidaki et al, J Reprod Infertil, 18(2), 2017; Mao et al, J Funct Biomater, 9(1), 2018).

Patent application no. CN106978389 describes the procedure for preparation of human amniotic membrane homogenate to be used as a component of the culture medium for culturing amniotic epithelial cells. First, the deepithelization of human amniotic membrane was performed by incubation of human amniotic membrane in 0.25% solution of trypsin (2 hours), followed by freezing in liquid nitrogen and pulverization by mortar and pestle. Then DMEM and F12 culture media were added, the mixture was sonicated (30 cycles), filtered and sterilized.

Patent application no. US20150010610A1 describes the procedure of preparation of pig / cow / horse / human amniotic membrane homogenate. First, amniotic membrane was stored at 4°C in phosphate-buffered saline (PBS), which contained penicillin (1 ,000 U/ml) and streptomycin (20 mg/ml). Afterwards, amniotic membrane was sonicated and amniotic membrane homogenate was centrifuged for 10 minutes at 4,000 rpm at 4°C. Then the supernatant alone was centrifuged for 5 minutes at 14,000 rpm and therefore the amniotic membrane extract was produced. The latter was frozen and stored at -80°C until lyophilization, which was carried out for 24 hours at 4°C. Before use the amniotic membrane homogenate was diluted in the suitable volume of balanced salt solution (BSS), stored at 4°C and used e.g. for treatment of eye pemphigoid (8 mg amniotic membrane per ml BSS).

Patent application no. US20040057938A1 describes the procedure of preparation of mammal amniotic membrane extract (pig, cow, horse, human). Pieces of amniotic membrane were weighed and the correct volume of neutral buffer solution was selected to ensure the ratio 0.3 of amniotic membrane (g) against neutral buffer solution (ml). Pieces or amniotic membrane were homogenized (3 times 3 minutes; ultrasound homogenizer Branson 250) and centrifuged for 10 minutes at 4,000 rpm at 4°C and then only the supernatant was centrifuged again for 5 minutes at 14,000 rpm. Afterwards, the proteins in the homogenate were quantified and the homogenate was filtered through a 0.8 pm filter under a sterile hood, protecting the compound from light and overheating and maintaining the homogenate temperature at 4°C. Aliquots containing 300-350 mg of amniotic membrane per ml of solution, were frozen in 100% ethanol on dry ice and stored at -80°C until lyophilization. The amniotic membrane homogenate was lyophilized for 24 hours and stored for 6 months at -20°C. Before use, lyophilized amniotic membrane homogenate was diluted in the correct volume of BSS and stored at 4°C; e.g. for high concentrations of the preparation, the solution was prepared in ratio 8 mg of amniotic membrane per ml BSS.

To examine the antimicrobial properties of amniotic membrane, Yadav et al. (Front Microbiol, 8, 2017) prepared an extract from human amniotic membrane. Only women who did not have gestational diabetes, preeclampsia or infectious diseases and who gave birth by Caesarean section, were eligible to participate in the study. After the separation of amnion from chorion, the amniotic membrane was washed three times with PBS, cut into small pieces, followed by freezing in liquid nitrogen and pulverization by mortar and pestle. Amniotic membrane was then mixed with PBS in 1 : 1 ratio, homogenized on ice (1 hour) and centrifuged twice at 12,000 rpm at 4°C for 10 minutes. The supernatant was then sterilized by filtration through a 0.22 pm filter and the extract was stored at -80°C. Yadav et al. (Front Microbiol, 8, 2017) showed that the extract of human amniotic membrane inhibits the growth of bacteria Streptococcus pneumoniae, grown in planktonic form or in biofilms. Furthermore, treatment of the infected rat middle ear with human amniotic membrane extract also significantly decreased the concentration of bacteria in the middle ear.

Mao et al. (Sci Rep, 7(1), 2017) used cryopreserved viable human amniotic membrane (Osiris Therapeutics, Inc.). First, the amniotic membrane, which was stored at -80°C, was thawed by incubation in a water bath at room temperature (3-5 minutes) and washed with sterile PBS. To obtain conditioned medium, human amniotic membrane was incubated in a tube containing Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen; 1 ml / 4 cm² human amniotic membrane) and fetal bovine serum (FBS, Atlanta Biologicals; 10%). The tube was incubated on a shaker in a C0 incubator at 37°C, 5% carbon dioxide, 95% humidity. Conditioned medium was obtained after 6 hours / 22 hours / 24 hours of incubation. The conditioned medium was used at once or stored at -80°C. Their results showed that conditioned medium from amniotic membrane has antimicrobial activity against bacteria Pseudomonas aeruginosa, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. In another study, Mao et al. (J Funct Biomater, 9(1), 2018) prepared the conditioned medium of cryopreserved viable human amniotic membrane, obtained after 24 hours of incubation in DMEM culture medium and FBS (10%), on a shaker at 37°C, 5% C0₂, 95% humidity. Their results show that bacteria P. aeruginosa form statistically significantly less biofilm when grown in culture medium containing the extract of amniotic membrane than when grown in culture medium alone.

Tehrani et al. (Sci Rep, 7(1), 2017) used fresh human amniotic membrane. Namely, they cut the pieces of human amniotic membrane (1 x1 cm²) into smaller pieces and added the equivalent volume of PBS. They obtained the extract by sonication (on ice, 10 minutes, 80 W, 0.5 second cycles) and the remnants of amniotic membrane were removed by centrifugation (4 minutes, 800 rpm). Their results showed that human amniotic membrane extract has antimicrobial effect on bacteria S. aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and two clinical isolates of E. coli, T3 and T4.

Wang et al. (African J Microbiology Res, 6(21), 2012) used fresh human amniotic membrane, which was first washed three times in PBS (0.01 mol/L; 4°C), weighed and cut into smaller pieces. The PBS (0.01 mol/L) was added to amniotic membrane in 1 : 1 ratio and the mixture was homogenized on ice in aseptic conditions. Then the mixture was further processed with ultrasound homogenizer and afterwards double volume of PBS (0.01 mol/L) was added to the suspension. The latter was centrifuged (2,000 rpm, 10 minutes, 4°C) and the supernatant was stored at 4°C and at -20°C until use (i.e. intact amniotic membrane supernatant). Using the same procedure, they prepared also supernatant from fresh human amniotic membrane, from which the amniotic epithelial cells were removed (i.e. deepithelized amniotic membrane supernatant). Briefly, they added a suspension of 0.25% trypsin and 0.06% EDTA to the amniotic membrane and incubated it for 15 minutes at 37°C. After the incubation, epithelial cells were removed with a cell scraper. Their results showed that the supernatant of deepithelized human amniotic membrane had no antimicrobial effect on bacteria Staphylococcus aureus (ATCC 25923), whereas the intact amniotic membrane supernatant had antimicrobial effect on bacteria Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus epidermidis, Proteus mirabilis, Streptococcus pneumoniae, Enterococcus faecalis and fungi Fusarium solani, Blastomyces albicans and Aspergillus fumigatus. On the other hand, the intact human amniotic membrane supernatant had no antimicrobial effect on clinically-isolated multidrug- resistant bacteria Enterobacter cloacae and E. coli and methicillin-resistant S. aureus (MRSA).

The largest shortcoming of the aforementioned procedures is their length. To prepare the conditioned medium according to the protocols, developed by Mao et al. (Sci Rep, 7(1), 2017; J Fund Biomater, 9(1), 2018), the incubation of amniotic membrane alone takes 6 to 24 hours. Similarly, the patent applications no. CN106978389 and US20040057938A1 describe protocols, where the lyophilization alone takes 24 hours.

A further shortcoming of procedures for preparation of amniotic membrane homogenate and supernatant, described, e.g., by Wang et al. (African J Microbiology Res, 6(21), 2012) and patent application no. CN106978389, is the removal of amniotic epithelial cells from amniotic membrane. This results in the loss of several antimicrobial molecules, e.g. human a- and b- defensins and SLPI (secretory leukocyte protease inhibitor), produced by amniotic epithelial cells (King et al, Placenta, 28(2-3), 2007; Svinarich et al, Am J Reprod Immunol, 38(4), 1997; Zhang et al, Mol Hum Reprod, 7(6), 2001 ; Buhimschi et al, Am J Obstet Gynecol, 191 (5), 2004).

Mao et al. (Sci Rep, 7(1), 2017; J Funct Biomater, 9(1), 2018), Yadav et al. (Front Microbiol, 8, 2017) and Tehrani et al. (Sci Rep, 7(1), 2017) describe the procedures for preparation of amniotic membrane conditioned medium, supernatant and extract. These procedures enable the preparation of amniotic membrane, which includes only some of the active ingredients of amniotic membrane. Namely, the amniotic membrane conditioned medium contains only the active ingredients that amniotic cells secrete in culture medium; the amniotic membrane supernatant contains only the active ingredients that have been preserved after the homogenization and centrifugation of the amniotic membrane, the rest of the components were disposed of and similarly, when preparing the amniotic membrane extract, only the active ingredients, that were kept in supernatant, have been preserved, while the rest in the sediment fraction was discarded.

Accordingly, the remains a need to provide improved procedures which are time efficient as well as prevent the loss of active antimicrobial molecules. Summary of the invention

The present invention addresses this need by providing procedures which use whole mammalian amniotic membrane. The use of whole amniotic membrane, rather than fractions thereof, ensures that only minimum amounts of the active antimicrobial molecules found in the amniotic membrane are lost during the preparation. As will be demonstrate further below, the procedures of the present invention result in products shown to be effective against a great number of microbial strains, including important multidrug-resistant bacteria. The final product of the present invention can thus be used as an antimicrobial agent.

The present invention can be summarized as follows:

1. A procedure for the preparation of an antimicrobial agent characterized in that a homogenate of whole mammalian amniotic membrane is used.

2. The procedure according to item 1 , wherein the homogenate is used without being further processed by sonication, centrifugation and/or lyophilisation.

3. The procedure according to item 1 or 2, comprising the step of homogenizing the mammalian amniotic membrane in a homogenizer.

4. The procedure according to item 3, wherein the mammalian amniotic membrane is washed at least one time, such as two times or three times, in an aqueous solution prior to being homogenized.

5. The procedure according to item 4, wherein the aqueous solution is selected from saline, balanced salt solution or cell culture medium.

6. The procedure according to item 4, wherein the aqueous solution is saline or a balanced salt solution.

7. The procedure according to item 5 or 6, wherein the balanced salt solution is selected from phosphate buffered saline (PBS), TRIS buffered saline (TBS), Alsever’s solution, Earle’s balanced salt solution (EBSS), Gey’s balanced salt solution (GBSS), Hanks’ balanced salt solution (HBSS), Puck’s balanced salt solution, Ringer’s balanced salt solution (RBSS), Simm’s balanced salt solution (SBSS) and Tyrode’s balanced salt solution (TBSS). 8. The procedure according to item 5 or 7, wherein the balanced salt solution is phosphate buffered saline (PBS).

9. The procedure according to any one of items 5 to 8, wherein the pH of the balanced salt solution or cell culture medium is in the range of from about 7.00 to about 7.50, such as from about 7.15 to about 7.45.

10. The procedure according to any one of items 5 to 8, wherein the pH of the balanced salt solution or cell culture medium is in the range of from about 7.20 to about 7.40.

1 1. The procedure according to any one of items 1 to 10, comprising the steps of cutting the mammalian amniotic membrane into pieces and homogenizing the pieces in a homogenizer.

12. The procedure according to item 1 1 , comprising adding an aqueous solution to the pieces of mammalian amniotic membrane to form a mixture followed by homogenizing the mixture in a homogenizer.

13. The procedure according to item 12, wherein the aqueous solution is selected from saline, balanced salt solution or cell culture medium.

14. The procedure according to item 13, wherein the aqueous solution is saline or a balanced salt solution.

15. The procedure according to item 13 or 14, wherein the balanced salt solution is selected from phosphate buffered saline (PBS), TRIS buffered saline (TBS), Alsever’s solution, Earle’s balanced salt solution (EBSS), Gey’s balanced salt solution (GBSS), Hanks’ balanced salt solution (HBSS), Puck’s balanced salt solution, Ringer’s balanced salt solution (RBSS), Simm’s balanced salt solution (SBSS) and Tyrode’s balanced salt solution (TBSS).

16. The procedure according to item 13 or 14, wherein the balanced salt solution is phosphate buffered saline (PBS).

17. The procedure according to any one of items 13 to 16, wherein the pH of the balanced salt solution or cell culture medium is in the range of from about 7.00 to about 7.50, such as from about 7.15 to about 7.45. 18. The procedure according to any one of items 13 to 17, wherein the pH of the balanced salt solution or cell culture medium is in the range of from about 7.20 to about 7.40.

19. The procedure according to any one of items 3 to 18, comprising filtering the homogenate.

20. The procedure according to any one of items 3 to 19, comprising filtering the homogenate through a mesh filter, preferably having a pore size of 1 mm or below.

21. The procedure according to item 20, wherein the mesh filter has a pore size from 0.5 mm to 1 mm, such as a pore size selected from 0.5 mm, 0.55 mm, 0.6 mm, 0.65 mm, 0.7 mm, 0.75 mm., 0.8 mm, 0.85 mm, 0.9 mm, 0.95 mm or 1 mm.

22. The procedure according to any one of items 1 to 21 , wherein the homogenate is directly formulated as antimicrobial agent or is stored under cool conditions, such as at a temperature ranging from -80°C to +4°C, before being formulated as antimicrobial agent.

23. The procedure according to any one of items 1 to 22, comprising the steps of: a) washing an mammalian amniotic membrane in an aqueous solution selected from saline, balanced salt solution and cell culture medium; b) cutting the mammalian amniotic membrane into pieces and mixing the pieces with an aqueous solution selected from saline, balanced salt solution and cell culture medium to obtain a mixture; c) homogenizing the mixture obtained in step b) in a homogenizer; d) optionally, filtering the homogenate through a mesh filter, preferably having a pore size of 1 mm or below; and e) formulating the homogenate obtained in step c) or d) as antimicrobial agent or storing the homogenate obtained in step c) or d) under cool conditions before formulating same as antimicrobial agent. 24. The procedure according to any one of claims 1 1 to 23, wherein the mammalian amniotic membrane is cut into pieces ranging from 2x2 cm to 5x5 cm, such as from 3x3 to 5x5 cm.

25. The procedure according to any one of items 1 1 to 24, wherein the pieces of mammalian amniotic membrane are mixed with the aqueous solution in a ratio of at least 1 :3 (1 part amniotic membrane pieces, 3 parts aqueous solution), such as 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 :1 1 , 1 : 12, 1 :13, 1 :14, 1 :15 or 1 :16.

26. The procedure according to any one of items 1 1 to 25, wherein the pieces of mammalian amniotic membrane are mixed with the aqueous solution in a ratio ranging from 1 :3 to 1 : 16, such as from 1 :3 to 1 :12.

27. The procedure according to any one of items 3 to 26, wherein the homogenizer is a homogenizer having a motor speed in the range of from 400 to 800W, such as 600W.

28. The procedure according to any one of items 3 to 27, wherein the homogenizer is a 600W homogenizer.

29. The procedure according to any one of items 3 to 28, wherein the homogenizer is operated at a rotational speed of from about 5,000 to about 24,000 rpm, such as from about 18,000 to 24,000 rpm, such as at about 22,400 rpm.

30. The procedure according to any one of items 1 1 to 29, wherein the pieces of mammalian amniotic membrane or mixture comprising same are/is homogenized at a rotational speed of from about 5,000 to about 24,000 rpm, such as from about 18,000 to 24,000 rpm, such as at about 22,400 rpm.

31. The procedure according to any one of items 3 to 30, wherein the homogenizer is operated for 3 to 8 minutes, preferably 3 to 5 minutes.

32. The procedure according to any one of items 3 to 31 , wherein the pieces of mammalian amniotic membrane or mixture comprising same are homogenized for 3 to 8 minutes, preferably 3 to 5 minutes. 33. The procedure according to any one of items 1 to 32, wherein the mammalian amniotic membrane has been obtained from a mammal selected from human, monkey, pig, cow, horse, cat, dog, sheep and goat.

34. The procedure according to any one of items 1 to 32, wherein the mammalian amniotic membrane is a non-human amniotic membrane.

35. The procedure according to any one of items 1 to 32, wherein the mammalian amniotic membrane is a human amniotic membrane.

36. The procedure according to any one of items 1 to 35, wherein the mammalian amniotic membrane has been separated from chorion after elective Caesarean section.

37. The procedure according to any one of items 1 to 36, wherein the mammalian amniotic membrane has been separated from chorion within 120 minutes, such as within 15, 30, 45, 60, 75, 90 or 105 minutes, after elective Caesarean section.

38. The procedure according to any one of items 1 to 37, wherein the mammalian amniotic membrane has been separated from chorion 10 to 120 minutes, such as 15 to 60 minute, after elective Caesarean section.

39. An antimicrobial agent obtainable by the procedure according to any one of items 1 to 38.

40. The antimicrobial agent according to item 39 for use in the treatment of a bacterial infection.

41. A procedure for the preparation of a homogenate derived from whole mammalian amniotic membrane.

42. The procedure according to item 41 , wherein the homogenate is prepared without being processed by sonication, centrifugation and/or lyophilisation.

43. The procedure according to item 41 or 42, comprising the step of homogenizing the mammalian amniotic membrane in a homogenizer. 44. The procedure according to 43, wherein the mammalian amniotic membrane is washed at least one time, such as two times or three times, in an aqueous solution prior to being homogenized.

45. The procedure according to item 44, wherein the aqueous solution is selected from saline, balanced salt solution or cell culture medium.

46. The procedure according to item 45, wherein the aqueous solution is saline or a balanced salt solution.

47. The procedure according to item 45 or 46, wherein the balanced salt solution is selected from phosphate buffered saline (PBS), TRIS buffered saline (TBS), Alsever’s solution, Earle’s balanced salt solution (EBSS), Gey’s balanced salt solution (GBSS), Hanks’ balanced salt solution (HBSS), Puck’s balanced salt solution, Ringer’s balanced salt solution (RBSS), Simm’s balanced salt solution (SBSS) and Tyrode’s balanced salt solution (TBSS).

48. The procedure according to item 45 or 46, wherein the balanced salt solution is phosphate buffered saline (PBS).

49. The procedure according to any one of items 45 to 48, wherein the pH of the balanced salt solution or cell culture medium is in the range of from about 7.00 to about 7.50, such as from about 7.15 to about 7.45.

50. The procedure according to any one of items 45 to 48, wherein the pH of the balanced salt solution or cell culture medium is in the range of from about 7.20 to about 7.40.

51. The procedure according to any one of items 41 to 50, comprising the steps of cutting the mammalian amniotic membrane into pieces and homogenizing the pieces in a homogenizer.

52. The procedure according to item 51 , comprising adding an aqueous solution to the pieces of mammalian amniotic membrane to form a mixture followed by homogenizing the mixture in a homogenizer.

53. The procedure according to item 52, wherein the aqueous solution is selected from saline, balanced salt solution and cell culture medium. 54. The procedure according to item 53, wherein the aqueous solution is saline or a balanced salt solution.

55. The procedure according to item 53 or 54, wherein the balanced salt solution is selected from phosphate buffered saline (PBS), TRIS buffered saline (TBS), Alsever’s solution, Earle’s balanced salt solution (EBSS), Gey’s balanced salt solution (GBSS), Hanks’ balanced salt solution (HBSS), Puck’s balanced salt solution, Ringer’s balanced salt solution (RBSS), Simm’s balanced salt solution (SBSS) and Tyrode’s balanced salt solution (TBSS).

56. The procedure according to item 53 or 54, wherein the balanced salt solution is phosphate buffered saline (PBS).

57. The procedure according to any one of items 53 to 56, wherein the pH of the balanced salt solution or cell culture medium is in the range of from about 7.00 to about 7.50, such as from about 7.15 to about 7.45.

58. The procedure according to any one of items 53 to 56, wherein the pH of the balanced salt solution or cell culture medium is in the range of from about 7.20 to about 7.40.

59. The procedure according to any one of items 43 to 58, comprising filtering the homogenate.

60. The procedure according to any one of items 43 to 59, comprising filtering the homogenate through a mesh filter, preferably having a pore size of 1 mm or below.

61. The procedure according to item 60, wherein the mesh filter has a pore size from 0.5 mm to 1 mm, such as a pore size selected from 0.5 mm, 0.55 mm, 0.6 mm, 0.65 mm, 0.7 mm, 0.75 mm., 0.8 mm, 0.85 mm, 0.9 mm, 0.95 mm or 1 mm.

62. The procedure according to any one of items 41 to 61 , wherein the homogenate is stored under cool conditions.

63. The procedure according to any one of items 41 to 62, comprising the steps of: a) washing an mammalian amniotic membrane in an aqueous solution selected from saline, balanced salt solution and cell culture medium; b) cutting the mammalian amniotic membrane into pieces and mixing the pieces with an aqueous solution selected from saline, balanced salt solution and cell culture medium to obtain a mixture; c) homogenizing the mixture obtained in step b) in a homogenizer; d) optionally, filtering the homogenate through a mesh filter, preferably having a pore size of 1 mm or below; and e) optionally, storing the homogenate obtained in step c) or d) under cool conditions, such as at a temperature ranging from -80°C to +4°C.

64. The procedure according to any one of claims 51 to 63, wherein the mammalian amniotic membrane is cut into pieces ranging from 2x2 cm to 5x5 cm, such as from 3x3 to 5x5 cm.

65. The procedure according to any one of items 51 to 64, wherein the pieces of mammalian amniotic membrane are mixed with the aqueous solution in a ratio of at least 1 :3 (1 part amniotic membrane pieces, 3 parts aqueous solution), such as 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 :1 1 , 1 :12, 1 :13, 1 :14, 1 :15 or 1 :16.

66. The procedure according to any one of items 51 to 65, wherein the pieces of mammalian amniotic membrane are mixed with the aqueous solution in a ratio ranging from 1 :3 to 1 : 16, such as from 1 :3 to 1 :12.

67. The procedure according to any one of items 43 to 66, wherein the homogenizer is a homogenizer having a motor speed in the range of from 400 to 800W, such as 600W.

68. The procedure according to any one of items 43 to 67, wherein the homogenizer is a 600W homogenizer.

69. The procedure according to any one of items 43 to 68, wherein the homogenizer is operated at a rotational speed of from about 5,000 to about 24,000 rpm, such as from about 18,000 to 24,000 rpm, such as at about 22,400 rpm.

70. The procedure according to any one of items 51 to 69, wherein the pieces of mammalian amniotic membrane or mixture comprising same are/is homogenized at a rotational speed from about 5,000 to about 24,000 rpm, such as from about 18,000 to about 24,000 rpm, such as at about 22,400 rpm.

71. The procedure according to any one of items 43 to 70, wherein the homogenizer is operated for 3 to 8 minutes, preferably 3 to 5 minutes.

72. The procedure according to any one of items 51 to 71 , wherein the pieces of mammalian amniotic membrane or mixture comprising same are homogenized for 3 to 8 minutes, preferably 3 to 5 minutes.

73. The procedure according to any one of items 41 to 72, wherein the mammalian amniotic membrane has been obtained from a mammal selected from human, monkey, pig, cow, horse, cat, dog, sheep and goat.

74. The procedure according to any one of items 42 to 72, wherein the mammalian amniotic membrane is a non-human amniotic membrane.

75. The procedure according to any one of items 41 to 72, wherein the mammalian amniotic membrane is a human amniotic membrane.

76. The procedure according to any one of items 41 to 75, wherein the mammalian amniotic membrane has been separated from chorion after elective Ceasarean section.

77. The procedure according to any one of items 41 to 76, wherein the mammalian amniotic membrane has been separated from chorion within 120 minutes, such as within 15, 30, 45, 60, 75, 90 or 105 minutes, after elective Ceasarean section.

78. The procedure according to any one of items 41 to 77, wherein the mammalian amniotic membrane has been separated from chorion 10 to 120 minutes, such as 15 to 60 minutes, after elective Ceasarean section.

79. A homogenate obtainable by the procedure according to any one of items 41 to 78.

80. The homogenate according to item 79 for use as an antimicrobial agent.

81. The homogenate according to item 79 for use in the treatment of a bacterial infection. Brief description of drawings

Figure 1 : Antimicrobial effect of fresh (fAM) and cryopreserved amniotic membrane (cAM) homogenates on Staphylococcus aureus, uropathogenic Escherichia coli (clinical strain DL94) and Enterobacter sp.

Figure 2: Antimicrobial effect of fresh (fAM) and cryopreserved amniotic membrane (cAM) homogenates on various uropathogenic strains

Figure 3: Antimicrobial effect of fresh (fAM) and cryopreserved amniotic membrane (cAM) homogenates on multidrug-resistant bacteria, namely standard and clinical strain of methicillin- resistant Staphylococcus aureus (MRSA) and Acinetobacter baumanni

Figure 4: Antimicrobial effect of fresh (fAM) and cryopreserved amniotic membrane (cAM) homogenates on various multidrug-resistant bacteria, namely standard and clinical strains of methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumanni, and bacterial strains that express extended spectrum b-lactamases (ESBL), namely standard strain of Klebsiella pneumoniae and a clinical strain of Escherichia coli.

Figure 5: Comparison of the antimicrobial effect of amniotic membrane against several strains of E. coli, prepared according to our protocol and prepared by Tehrani et al, Cryobiology, 67(3), 2013.

Figure 6: Comparison of the effect of (A) cryopreserved amniotic membrane homogenate, prepared according to our protocol, (B) the patches of human cryopreserved viable amniotic membrane (hCVAM), as described by Mao et al. (J Diabetes Complications, 8(2), 2016), and (C) the hCVAM conditioned medium and the air-dried devitalized amniotic membrane (dhCVAM) conditioned medium against Staphylococcus aureus as described by Mao et al (Sci Rep, 7(1), 2017).

Detailed description of the invention

The present invention relates in general to procedures involving the preparation of mammalian amniotic membrane homogenate that can be used as an antimicrobial agent.

An important feature of the present invention is the use of the whole amniotic membrane, which ensures that only a minimum amount of the active antimicrobial molecules are lost during preparation of the amniotic membrane homogenate. With“whole” amniotic membrane” is meant the intact amniotic membrane including amniotic epithelial and mesenchymal cells and extracellular matrix. Importantly, the mammalian amniotic membrane homogenate, prepared according to the procedures of the present invention, is not further processed, e.g. by centrifugation, sonication and/or lyophilisation, and thus contains all molecules found in the intact amniotic membrane including markers characteristic for amniotic epithelial and mesenchymal cells and extracellular matrix. A detailed list of markers characteristic for each of the amniotic membrane's components is provided in the table below (see also Ramuta and Kreft, Cell Transplant, 27(1), 2018; Ramuta et al, Slov Med J, 87(9-10), 2018). Each of these markers can be found in the mammalian amniotic membrane homogenate of the invention and can be detected using known detection means such as antibody technology.

As demonstrated in the example section, the amniotic membrane homogenate, prepared according to the present invention, have shown to be effective against a great number of microbial strains, including important multidrug-resistant bacteria. These results are surprising since multiple research articles showed ambivalent results regarding the antimicrobial activity of amniotic membrane. For example, Wang et al, 2012, showed that amniotic membrane preparation has no antimicrobial effect against methicilin-resistant Staphylococcus aureus, while Mao et al, 2017 and 2018, demonstrated that amniotic membrane preparation has potent antimicrobial effect on methicilin-resistant Staphylococcus aureus. Conversely, the results provide by the present inventors are consistent, also in the case of methicilin-resistant Staphylococcus aureus. The amniotic membrane homogenate had always significant bacteriostatic effect on Staphylococcus aureus.

Accordingly, the procedures of the present invention involving the use of whole amniotic membrane, rather than fractions thereof, prevent the loss of active antimicrobial molecules, which are otherwise lost when preparing amniotic membrane conditioned medium, supernatant or extract in accordance with the prior art procedures.

The present invention thus provides in one aspect a procedure for the preparation of an antimicrobial agent characterized in that a homogenate of whole mammalian amniotic membrane is used. Particularly, the procedure is characterized in that the homogenate is used without being further processed by sonication, centrifugation and/or lyophilisation.

The present invention provides in another aspect a procedure for the preparation of a homogenate derived from whole mammalian amniotic membrane. Particularly, the procedure is characterized in that the homogenate is prepared without being processed by sonication, centrifugation and/or lyophilisation. Suitably, the procedures of the invention comprise the step of homogenizing the mammalian amniotic membrane in a homogenizer.

A homogenizer or blender (which terms can be used interchangeably) employed in accordance with the present invention may be any piece of hardware used for the homogenization, i.e. mechanical disruption, of tissue, and typically involves the use of rotating blades. These blades work to grind and disperse cells, and are most effective at homogenizing tissues. Non-limiting examples of homogenizer include commercially available laboratory or kitchen blenders (e.g., offered by Russell Hobbs, Waring, Phillips, etc.). Such blenders usually have a motor speed in the range of from 400 to 800W, such as 600W, and a maximum rotational speed of up to 24,000 rounds per minute (rpm).

Thus, according to certain embodiments, the homogenizer is operated at a rotational speed of from about 5,000 to about 24,000 rpm. According to some embodiments, the homogenizer is operated at a rotational speed of from about 10,000 to about 24,000 rpm. According to some embodiments, the homogenizer is operated at a rotational speed of from about 16,000 to about 24,000 rpm. According to some embodiments, the homogenizer is operated at a rotational speed of from about 18,000 to about 24,000 rpm. According to some embodiments, the homogenizer is operated at a rotational speed of from about 20,000 to about 24,000 rpm. According to some embodiments, the homogenizer is operated at a rotational speed of from about 21 ,000 to about 23,000 rpm. According to some embodiments, the homogenizer is operated at a rotational speed of from about 22,000 to about 23,000 rpm, such as at 22,400 rpm.

According to certain embodiments, the homogenizer is a homogenizer having a motor speed in the range of from 400 to 800W, such as 600W. Preferably, the homogenizer is a 600W homogenizer.

Generally, good results are achieved if the homogenization takes about 3 to about 8 minutes, while about 3 to about 5 minutes are considered optimal. If the time range is shorter, the release of antimicrobial molecules is less sufficient and therefore the antimicrobial effect is diminished. On the other hand, if the time range is longer, that can increase the temperature of the suspension and the mechanical shear forces, which can altogether lead to the damage of the antimicrobial molecules. Thus, according to certain embodiments, the homogenizer is operated for about 3 to about 8 minutes, preferably about 3 to about 5 minutes. According to some embodiments, the mammalian amniotic membrane is washed at least one time, such as two times or three times, in an aqueous solution prior to being homogenized. During the washing remnants of bloods and small lipid deposits may also be removed.

The aqueous solution may be any suitable aqueous solution known in the art. Non-limiting examples of suitable aqueous solution include saline (e.g., 0,9% sodium chloride) or balanced salt solution or neutral buffer solution such as phosphate buffered saline (PBS), TRIS buffered saline (TBS), Alsever’s solution, Earle’s balanced salt solution (EBSS), Gey’s balanced salt solution (GBSS), Hanks’ balanced salt solution (HBSS), Puck’s balanced salt solution, Ringer’s balanced salt solution (RBSS), Simm’s balanced salt solution (SBSS) and Tyrode’s balanced salt solution (TBSS). Preferably, the aqueous solution is saline or phosphate buffered saline (PBS).

Furthermore, when using the amniotic membrane homogenate for cell culturing, the homogenate can also be prepared in various culture media suitable also for culturing of different prokaryotic or eukaryotic cell types (without antibiotics).

The pH of the balanced salt solution or cell culture medium is usually in the range of from about 7.00 to about 7.50, such as from about 7.10 to about 7.45. According to certain embodiments, the pH of the balanced salt solution or cell culture medium is in the range of from about 7.15 to about 7.45. According to certain embodiments, the pH of the balanced salt solution or cell culture medium is in the range of from about 7.20 to about 7.40.

The procedures of the present invention may further comprise the step of cutting the mammalian amniotic membrane into pieces prior to homogenizing in a homogenizer. The size of the pieces may be any suitable size allowing the processing in the homogenizer, but typically ranges from 2x2 cm to 5x5 cm, such as from 3x3 to 5x5 cm.

The pieces of mammalian amniotic membrane may be mixed with an aqueous solution to form a mixture which is then homogenized in the homogenizer. The aqueous solution may be any of the aqueous solution as discussed above, including saline, balanced salt solution or cell culture medium. According to certain embodiments, the aqueous solution is saline or a balanced salt solution, such as such as phosphate buffered saline (PBS), TRIS buffered saline (TBS), Alsever’s solution, Earle’s balanced salt solution (EBSS), Gey’s balanced salt solution (GBSS), Hanks’ balanced salt solution (HBSS), Puck’s balanced salt solution, Ringer’s balanced salt solution (RBSS), Simm’s balanced salt solution (SBSS) and Tyrode’s balanced salt solution (TBSS). Preferably, the aqueous solution is saline or phosphate buffered saline (PBS).

The pieces of mammalian amniotic membrane and the aqueous solution may be mixed in any suitable ratio, such as 1 :1 , 1 :2 or 1 :3. Advantageously, however, the pieces of mammalian amniotic membrane are mixed with the aqueous solution in a ratio of at least 1 :3 (1 part amniotic membrane pieces, 3 parts aqueous solution), such as 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 :1 1 , 1 :12, 1 :13, 1 :14, 1 :15 or 1 :16. According to certain embodiments, the pieces of mammalian amniotic membrane are mixed with the aqueous solution in a ratio ranging from 1 :3 to 1 :16, such as from 1 :3 to 1 :12. According to certain embodiments, the pieces of mammalian amniotic membrane are mixed with the aqueous solution in a ratio ranging from 1 :4 to 1 : 16, such as from 1 :4 to 1 :12.

The volume of the mixture subject to homogenization may be any desirable volume, but generally ranges from about 40 ml to about 500 ml, such as from about 40 ml to about 250 ml, about 40 ml to about 200 ml, about 40 ml to about 150 ml, about 40 ml to about 100 ml, 40 ml to about 50 ml, about 50 ml to about 250 ml, 50 to about 200 ml, 50 to about 150 ml or 50 to about 100 ml.

The procedures of the present invention may further comprise filtering the homogenate. The homogenate may be filtered using any suitable filtering means, such as a mesh filter, preferably having a pore size of 1 mm or below. According to certain embodiments, the pore size of the filter is in the range from about 0.5 mm to about 1 mm, such as a pore size selected from about 0.50 mm, about 0.55 mm, about 0.60 mm, about 0.65 mm, about 0.70 mm, about 0.75 mm, about 0.80 mm, about 0.85 mm, about 0.90 mm and about 0.95 mm. Once having passed the filter, the diameter of the particles of the filtered homogenate is 1 mm or below.

As a result of the above described procedures, a homogenate is obtained with at least 70% of the particles having a diameter between 0.5 mm and 1 mm (as measure e.g., by ocular micrometer using light microscopy or scanning electron microscopy).

The homogenate obtained according to the present invention can be directly used, and hence formulated, as antimicrobial agent or can be stored under cool conditions, such as at a temperature ranging from -80°C to +4°C, such as at -20°C, before being formulated as antimicrobial agent. According to certain embodiments, the homogenate is cryopreserved. Generally, the homogenate may be stored until use at 4° C up to one week, stored until use at -20 °C or at -80°C up to 1 year. According to particular embodiments, the procedure for the preparation of an antimicrobial agent according to the present invention comprises the steps of: a) washing a mammalian amniotic membrane in an aqueous solution selected from saline, balanced salt solution and cell culture medium; b) cutting the mammalian amniotic membrane into pieces and mixing the pieces with an aqueous solution selected from saline, balanced salt solution and cell culture medium to obtain a mixture; c) homogenizing the mixture obtained in step b) in a homogenizer; d) optionally, filtering the homogenate through a mesh filter, preferably having a pore size of 1 mm or below; and e) formulating the homogenate obtained in step c) or d) as antimicrobial agent or storing the homogenate obtained in step c) or d) under cool conditions before formulating same as antimicrobial agent.

According to particular embodiments, the procedure for the preparation of a homogenate according to the present invention comprises the steps of: a) washing a mammalian amniotic membrane in an aqueous solution selected from saline, balanced salt solution or cell culture medium; b) cutting the mammalian amniotic membrane into pieces and mixing the pieces with an aqueous solution selected from saline, balanced salt solution and cell culture medium to obtain a mixture; c) homogenizing the mixture obtained in step b) in a homogenizer; d) optionally, filtering the homogenate through a mesh filter, preferably having a pore size of 1 mm or below; and e) optionally, storing the homogenate obtained in step c) or d) under cool conditions. The mammalian amniotic membrane used in accordance with the present invention may be obtained from any mammal of interest, but preferably has been obtained from a mammal selected from human, monkey, pig, cow, horse, cat, dog, sheep and goat. According to some embodiments, the mammalian amniotic membrane is a non-human amniotic membrane. According to other embodiments, the mammalian amniotic membrane is a human amniotic membrane.

The mammalian amniotic membrane used in accordance with the present invention may be one which has been separated from chorion within 120 minutes, such as within 15, 30, 45, 60, 75, 90 or 105 minutes, after elective Caesarean section.

According to certain embodiments, the mammalian amniotic membrane used in accordance with the present invention may be one which has been separated from chorion 10-120 minutes after elective Caesarean section. According to certain embodiments, the mammalian amniotic membrane used in accordance with the present invention may be one which has been separated from chorion 10-60 minutes after elective Caesarean section.

The present invention further provides as further aspects a homogenate, respectively antimicrobial agent obtainable by the procedures described above. The homogenate, respectively the antimicrobial agent may be for use in the treatment of a bacterial infection.

The present invention further provides a method for treating a patient in need thereof, e.g., a patient suffering from a bacterial infection, the method comprising preparing a homogenate as described above and administering same to said patient.

Examples

The present invention is explained in more detail in the following non-limiting example on human amniotic membrane.

Example 1 - Preparation of amniotic membrane homogenate

The approval for the use of human amniotic membrane was given by the National Medical Ethics Committee, University Clinical Centre Ljubljana, in sessions on 25.10.2010 (decree no. 43/12/09) and 16.4.2018 (decree no. 0120-179/2018/5). Preferred volunteers were 25-35 years old, gestational age 37-40 weeks. Only healthy volunteers, who tested negative for syphilis, HIV, hepatitis B and C, and had healthy newborns, were included in this study. Volunteers were excluded from this study in the case of bacterial or viral infection of the genital tract, infection of the amnion, gestational diabetes, meconium in the amniotic fluid, congenital anomalies of the newborn, premature rupture of membranes. Amniotic membranes were obtained with written informed consent from volunteers who underwent Caesarean section. Fresh human amniotic membranes, which were separated from chorion 10-120 minutes after elective Caesarean section, were washed three times in sterile PBS (pH 7.2-7.4). The latter was prepared from 100 ml of stock solution (1 ,000 ml of stock solution contains 4 g KH P0 , 23 g Na₂HP0₄, 4 g KCI, 160 g NaCI, 810 ml distilled water) and 1 ,900 ml of distilled water (pH 7.2-7.4). During the washing also the remnants of blood were removed and afterwards all parts of human amniotic membrane, containing the remnants of blood or small lipid deposits were removed by scalpel, washed again in sterile PBS (pH 7.2-7.4), and cut into pieces (3x3- 5x5 cm). The pieces of amniotic membrane were transferred into a sterile test tube with graduation and the volume of pieces was measured. Afterwards, sterile PBS (pH 7.2-7.4) was added to the pieces of amniotic membrane in 1 :3 ratio (1 part amniotic membrane, 3 parts sterile PBS). Then the mixture of pieces of amniotic membrane and sterile PBS was transported into a homogenizer (600 W) and homogenized for 3-5 minutes (volumes 20-50 ml) or 5-8 minutes (volumes 50-150 ml). Afterwards, the amniotic membrane homogenate was a) used at once, b) stored until use at 4°C up to one week, c) stored until use at -20°C or at -80°C up to 1 year.

The final product can be used as an antimicrobial agent against several clinically important bacteria, including multidrug-resistant bacteria.

Example 2 - Antimicrobial efficiency assay

Bacteria were inoculated in Luria-Bertani broth and grown with aeration (180 rpm) overnight at 37°C. Soft agar Muller-Hinton was first cooked, cooled to 48°C and then inoculated with 100 pi of overnight culture and poured over the Muller-Hinton agar plate. After 15 minutes of incubation at room temperature, 3-times of 5 mI and 3-times of 10 m I of homogenate were placed on the agar plate. Plates were incubated at 37°C for 24 hours and then the mean diameters of the antimicrobial zones were measured. The results are shown in Figures 1 to 4.

Figure 1 shows that both fresh (fAM) and cryopreserved amniotic membrane (cAM) homogenates have antimicrobial effect against various tested uropathogenic strains. (A-F) Homogenates of fAM and cAM have antimicrobial effect on S. aureus, UPEC DL94 and Enterobacter sp. The range of antimicrobial effect of fAM and cAM (1 week at -80°C) homogenates varies, namely between strong (S. aureus) / moderate (UPEC DL94) / minor ( Enterobacter sp.) antimicrobial effect. The quantity of fAM and cAM (1 week at -80°C) homogenates used was 5 mI (upper rows) and 10 pi (lower rows). Scale bars: 10 mm.

Figure 2 shows the antimicrobial effect caused by fresh (fAM) and cryopreserved amniotic membrane (cAM) homogenates on various uropathogenic strains. The antimicrobial effect is seen for both fAM and cAM homogenates on all tested strains. fAM homogenate has the largest antimicrobial effect, followed by cAM (1 week at -80°C) and cAM (10 weeks at -20°C) homogenates, while the cAM (10 weeks at -80°C) homogenate has the smallest antimicrobial effect.). Bars in red represent the mean diameter of the antimicrobial zone ± standard error (mm) of all tested strains.

Figure 3 shows that fresh (fAM) and cryopreserved amniotic membrane (cAM) homogenates have an antimicrobial effect on tested multidrug-resistant bacteria, namely standard and clinical strain of methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumanni. (A-H) Homogenates of fAM and cAM (1 week at -80°C) have larger antimicrobial effect on standard and clinical strains of MRSA than on Acinetobacter baumanni. The quantity of fAM and cAM (1 week at -80°C) homogenates used was 5 mI (upper rows) and 10 mI (lower rows). Scale bars: 10 mm.

Figure 4 shows the antimicrobial effect caused by fresh (fAM) and cryopreserved amniotic membrane (cAM) homogenates on various multidrug-resistant bacteria, namely standard and clinical strains of methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumanni, Klebsiella pneumoniae, which expresses extended spectrum b-lactamases (ESBL) and a clinical strain of Escherichia coli, which expresses ESBL. Larger volumes of homogenates (10 mI) have greater antimicrobial effect than smaller volumes (5 mI). fAM homogenate has a larger antimicrobial effect than cAM (1 week at -80°C) homogenate. Bars in red represent the mean diameter of the antimicrobial zone ± standard error (mm) of all tested strains.

In conclusion, the amniotic membrane homogenate prepared according to the present invention is effective against a great number of microbial strains, including important multidrug- resistant bacteria. These results are surprising since multiple research articles showed ambivalent results regarding the antimicrobial activity of amniotic membrane.

Example 3 - Comparison of the antimicrobial effect of amniotic membrane on various strains of E. coli We inoculated six bacterial strains of Escherichia coli (five DL clinical strains and one laboratory strain E. coli DH5a) in Luria-Bertani broth and grew it with aeration (180 rpm) overnight at 37°C. Then soft agar Muller-Hinton was first cooked, cooled to 48°C and inoculated with 100 pi of overnight culture and poured over the Muller-Hinton agar plate. After 15 minutes of incubation at room temperature, 3-times of 5 mI and 3-times of 10 mI of amniotic membrane homogenate, prepared according to our procedure, were placed on the agar plate. Plates were incubated at 37°C for 24 hours and then the mean diameters of the antimicrobial zones were measured. The results are shown in Figure 5.

We compared our results with the results published by Tehrani et al (Cryobiology, 67(3), 2013). They tested the antimicrobial effect of human amniotic membrane patches. Namely, amnion was separated from chorion, rinsed three times with cold PBS and cut into pieces. To perform the antimicrobial efficiency assay, bacterial strains E. coli (standard strain, ATCC25922) and two clinical strains of E. coli (T3, T4) were cultured on the blood agar or EMB agar and incubated at 35±2°C overnight. Then some isolated bacterial colonies were harvested from plates and suspended in the sterile normal saline (0,5 McFarland) and subsequently cultured on the Muller-Hinton agar plates. Then the patches of fresh amniotic membrane were put on the cultured Muller-Hinton agar plate and incubated at 35±2°C overnight. Afterward the inhibition zone was measured. For freeze-drying, the fresh amniotic membrane was pre-frozen for 30 minutes and lyophilized in a freeze-dryer at -55°C for 24 hours (freeze-dried amniotic membrane). Then freeze-dried amniotic membrane was put into the PBS for 2 hours to rehydrate and afterwards patches of the rehydrated freeze-dried amniotic membrane were applied on the Muller-Hinton agar plates, inoculated with bacterial strains, and incubated overnight, as described previously. For cryopreservation, fresh amniotic membrane was placed in sterile PBS containing 10% dimethylsulphoxide, 10% Dulbecco’s modified Eagle medium (DMEM)/F12, 10% FBS with 8 minutes equilibrium time and stored rapidly at -80°C for 6 months. Before use, patches of cryopreserved amniotic membrane were thawed at room temperature and rinsed three times in PBS. Then patches of cryopreserved amniotic membrane were applied on the Muller-Hinton agar plates, inoculated with bacterial strains, and incubated overnight, as described previously. The results are shown in Figure 5.

Figure 5 shows the antimicrobial effect of fresh and cryopreserved (1 week at -80°C / 10 weeks at -80°C / 10 weeks at -20°C) amniotic membrane homogenate (10 pi). Results by Tehrani et al. show the antimicrobial effect of patches of fresh / cryopreserved / freeze-dried amniotic membrane. All results are shown as mean diameter of the antimicrobial zone ± standard error (mm). To conclude, our results show the importance of the manner of preparation of amniotic membrane to obtain the optimal antimicrobial effect. Namely, when using the whole amniotic membrane as a patch, the antimicrobial effect is not optimal, since most of the antimicrobial molecules are still stored in the amniotic epithelial cells and extracellular matrix. Further processing (e.g. preparation of the amniotic membrane homogenate) allows the optimal release of the antimicrobials in the mixture, which can be used as an antimicrobial agent.

Example 4 - Comparison of the antimicrobial effect of amniotic membrane on S. aureus

We inoculated bacterial strain Staphylococcus aureus in Muller-Hinton broth and grew it with aeration (180 rpm) overnight at 37°C. The overnight culture was then diluted 500-times and transferred to a 96-well plate. Then we added three different dilutions of amniotic membrane, namely 1 :4, 1 :8 or 1 :16 (diluted in PBS) and incubated the plate at 37°C for 7 hours. At time point 0 the concentration of bacterial culture was 3x10^® CFU/ml. We sampled all conditions each hour and after serially diluting bacterial cultures, we plated them on Muller-Hinton agar. The CFUs were counted after overnight incubation at 37°C. The results are shown in Figure 6.

We compared our results with the results published by Mao et al. (J Diabetes Complications, 8(2), 2016). They used human cryopreserved viable amniotic membrane (hCVAM; Osiris Therapeutics, Inc.), which was stored at -80°C. They thawed cryopreserved hCVAM by incubation in a water bath at room temperature (3-5 minutes) and washed with sterile PBS. hCVAM patches of 3 cm² were used in the following experiments. For antimicrobial assays, patches of hCVAM or control samples (positive control - Acticoat 7, negative control - Puracol Plus) were placed into tubes with 1 ml of assay medium containing approximately 100 CFU of S. aureus and were incubated at 37°C with shaking for 24 hours. After incubation the bacterial cultures were serially diluted and plated onto TSB agar plates. The CFUs were counted after overnight incubation at 37°C. The results are shown in Figure 6.

Further, we compared our results also with the results published by Mao et al (Sci Rep, 7(1), 2017). Human cryopreserved viable amniotic membrane (hCVAM; Osiris Therapeutics, Inc.), which was stored at -80°C, was thawed by incubation in a water bath at room temperature (3-5 minutes) and washed with sterile PBS. To obtain conditioned medium, human amniotic membrane was incubated in a tube containing Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen; 1 ml / 4 cm² human amniotic membrane) and fetal bovine serum (FBS, Atlanta Biologicals; 10%). The tube was incubated on a shaker in a C0 incubator at 37°C, 5% carbon dioxide, 95% humidity. Conditioned medium was obtained after 24 hours of incubation. The conditioned medium was used at once or stored at -80°C. Bacterial strain S. aureus (ATCC 25923) was cultured in tryptic soy broth at 37 °C with shaking until the absorbance optical densities measured in the range of 0.2 to 0.6 at a wavelength of 600 nm. The number of colony forming units (CFUs) for was estimated based on an OD₆oo = 1 .0, which corresponds to 10⁹ CFU/ml. To prepare the inoculum for hCVAM antimicrobial assays, the bacterial stock solution was diluted with either assay medium or conditioned medium to approximately 100 CFU/ml of bacteria. These 100 CFU S. aureus were cultured with 1 ml of assay medium or conditioned medium and incubated at 37°C with shaking for 24 h. Serial dilutions were then prepared of each culture and plated onto tryptic soy broth agar plates. CFUs were counted after overnight incubation at 37°C. The results are shown in Figure 6.

Figure 6 shows comparison of the effect of (A) cryopreserved amniotic membrane homogenate, prepared according to our protocol, (B) the patches of human cryopreserved viable amniotic membrane (hCVAM), as described by Mao et al. (J Diabetes Complications, 8(2), 2016), and (C) the hCVAM conditioned medium and the air-dried devitalized amniotic membrane (dhCVAM) conditioned medium against Staphylococcus aureus as described by Mao et al (Sci Rep, 7(1), 2017). We used three different dilutions of AM homogenate, namely 1 :4, 1 :8, 1 :16 (diluted in sterile PBS). The concentration of bacteria at the start of the experiment (treatment with a selected amniotic membrane preparation) was 3.6x10^® CFU/ml (equivalent to 6.55 CFU (log/ml)) for our experiments, while in experiments performed by Mao et al (2016, 2017), the concentration of bacteria at the start of the experiment was only 100 CFU/ml (equivalent to 2 CFU (log/ml)). We measured the antimicrobial effect of AM homogenates after 7 hours of incubation, while Mao et al (2016, 2017) measured the antimicrobial effect after 24 hours of incubation. These results show that AM homogenate has higher antimicrobial efficiency against S. aureus than hCVAM patch or hCVAM and dhCVAM conditioned media. Since we started the experiment with the concentration of bacteria, which is 360.000-times higher than the concertation used by Mao et al, 2017, we demonstrate that the AM homogenate is significantly more antimicrobial efficient than hCVAM patches, and conditioned media of hCVAM and dhCVAM.

To conclude, also in the case of S. aureus, amniotic membrane homogenate produced more potent antimicrobial effect than the patches of human cryopreserved viable amniotic membrane (hCVAM) or the human cryopreserved viable amniotic membrane (hCVAM) conditioned medium and the air-dried devitalized amniotic membrane (dhCVAM) conditioned medium (Mao et al, 2016 and 2017). When comparing our results with the antimicrobial effect of hCVAM conditioned medium on S. aureus, the CFU/ml values were similar. However, it is important to point out that Mao et al. (2017) inoculated only 100 CFU/ml (equivalent to 2 CFU (log/ml)) at time 0, while our initial concentration of bacteria was 3.6^X10^® CFU/ml (equivalent to 6.55 CFU (log/ml)). Moreover, that shows that hCVAM conditioned medium only decreased the proliferation of bacteria (initial concentration was 2 CFU (log/ml) and in 24 hours it increased to approximately 4.5 CFU (log/ml), while the amniotic membrane homogenate killed bacteria, since the bacterial load decreased only after 7 hours of incubation from 6.55 CFU (log/ml) at the start of the experiment to 5.75 CFU (log/ml) when using AM homogenate 1 ; 5.45 CFU (log/ml) when using AM homogenate 2 and 4, .6 CFU (log/ml) when using AM homogenate 3;. Therefore, we unequivocally show that the AM homogenate is significantly more antimicrobial efficient than hCVAM patches, hCVAM conditioned medium and dhCVAM conditioned medium.

Claims

1. A procedure for the preparation of an antimicrobial agent characterized in that a homogenate of whole mammalian amniotic membrane is used without being further processed by sonication, centrifugation and/or lyophilisation.

2. The procedure according to claim 1 , wherein the homogenate is directly formulated as antimicrobial agent or is stored under cool conditions before being formulated as antimicrobial agent.

3. The procedure according to claim 1 or 2, comprising the steps of cutting the mammalian amniotic membrane into pieces and homogenizing the pieces in a homogenizer.

4. The procedure according to claim 3, comprising adding an aqueous solution selected from saline, balanced salt solution or cell culture medium to the pieces of mammalian amniotic membrane to form a mixture followed by homogenizing the mixture in a homogenizer.

5. The procedure according to claim 3 or 4, comprising filtering the homogenate through a mesh filter, preferably having a pore size of 1 mm or below.

6. The process according to claim 3 or 4, comprising filtering the homogenate through a mesh filter having a pore size from 0.5 mm to 1 mm.

7. The procedure according to any one of claims 1 to 6, comprising the steps of: a) washing an mammalian amniotic membrane in an aqueous solution selected from saline, balanced salt solution and cell culture medium; b) cutting the mammalian amniotic membrane into pieces and mixing the pieces with an aqueous solution selected from saline, balanced salt solution and cell culture medium to obtain a mixture; c) homogenizing the mixture obtained in step b) in a homogenizer; d) optionally, filtering the homogenate through a mesh filter, preferably having a pore size of 1 mm or below; and e) formulating the homogenate obtained in step c) or d) as antimicrobial agent or storing the homogenate obtained in step c) or d) under cool conditions before formulating same as antimicrobial agent.

8. The procedure according to any one of claims 1 to 6, comprising the steps of: a) washing an mammalian amniotic membrane in an aqueous solution selected from saline, balanced salt solution and cell culture medium; b) cutting the mammalian amniotic membrane into pieces and mixing the pieces with an aqueous solution selected from saline, balanced salt solution and cell culture medium to obtain a mixture; c) homogenizing the mixture obtained in step b) in a homogenizer; d) filtering the homogenate through a mesh filter having a pore size from 0.5 mm to 1 mm; and e) formulating the homogenate obtained in step c) or d) as antimicrobial agent or storing the homogenate obtained in step c) or d) under cool conditions before formulating same as antimicrobial agent.

9. The procedure according to any one of claims 3 to 8, wherein the mammalian amniotic membrane is cut into pieces ranging from 2x2 cm to 5x5 cm, such as from 3x3 to 5x5 cm.

10. The procedure according to any one of claims 4 to 9, wherein the pieces of mammalian amniotic membrane are mixed with the aqueous solution in a ratio of at least 1 :3 (1 part amniotic membrane pieces, 3 parts aqueous solution), such as 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 : 1 1 , 1 :12, 1 :13, 1 :14, 1 :15 or 1 :16.

1 1. The procedure according to any one of claims 4 to 10, wherein the pieces of mammalian amniotic membrane are mixed with the aqueous solution in a ratio ranging from 1 :3 to 1 :16, such as from 1 :3 to 1 :12.

12. The procedure according to any one of claims 3 to 1 1 , wherein the homogenizer is a homogenizer having a motor speed in the range of from 400 to 800W, such as 600W.

13. The procedure according to any one of claims 3 to 12, wherein the homogenizer is a 600W homogenizer.

14. The procedure according to any one of claims 3 to 13, wherein the homogenizer is operated at a rotational speed of from about 5,000 to about 24,000 rpm, such as from about 18,000 to about 24,000 rpm, such as at about 22,400 rpm.

15. The procedure according to any one of claims 3 to 14, wherein the pieces of mammalian amniotic membrane or mixture comprising same are homogenized for about 3 to about 8 minutes.

16. The process according to any one of claims 3 to 15, wherein the pieces of mammalian amniotic membrane or mixture comprising same are homogenized for about 3 to about 5 minutes.

17. The process according to any one of claims 3 to 15, wherein the pieces of mammalian amniotic membrane or mixture comprising same are homogenized for about 3 to about

5 minutes with the homogenizer being operated at a rotational speed of from about 5,000 to about 24,000 rpm.

18. An antimicrobial agent obtainable by the procedure according to any one of claims 1 to 17 for use in the treatment of a bacterial infection.