WO1999009835A1

WO1999009835A1 - Use of an anti-microbial composition

Info

Publication number: WO1999009835A1
Application number: PCT/SE1998/001510
Authority: WO
Inventors: Johan De Faire
Original assignee: Micro Active Protein In Sweden Ab
Priority date: 1997-08-27
Filing date: 1998-08-24
Publication date: 1999-03-04
Also published as: AU8895098A; SE9703085D0

Abstract

The present invention relates to the use of an anti-microbial composition comprising at least one anti-microbially active protein isolated from mussels and carbohydrates for the manufacturing of a product for treatment of tissues, body surfaces and cavities. In particular it relates to the use of an anti-microbial composition for the manufacturing of a product having an anti-microbial effect and a coagulative effect for anti-microbial treatment of microbially contaminated tissues, e.g. conective tissue, mucous membrane and intestinal tract and for treatment of bleeding and oozing tissue damages.

Description

USE OF AN ANTI-MICROBIAL COMPOSITION

Technical field

The present invention relates to the use of an anti-microbial composition comprising at least one anti-microbially active protein isolated from mussels and carbohydrates for the manufacturing of a product for treatment of tissues, body surfaces and cavities.

In particular, it relates to the use of an anti-microbial composition for the manufacturing of a product having an anti-microbial effect and a coagulative effect for anti-microbial treatment of microbially contaminated tissues, e.g. connective tissue, mucous membrane and intestinal tract and for treatment of bleeding and oozing tissue damages.

As used herein the term "anti-microbial treatment" means that the treatment results in neutralisation, inactivation or growth inhibition of at least part of the contaminating microbes and preferably of the pathogenic microbes. As used herein the term "microbe" includes viral structures and toxins. For simplicity reasons the term "cleaning" is in these specifications sometimes used instead of anti-microbial treatment and/or purification.

Background of invention

Infection Microbes, i.e. bacteria, protozoa, rickettsiae, fungi, viruses etc., are not recognised by human cells and tissues as natural or homologous but as foreign intruders that must be destroyed, neutralised and expelled from the tissues. A series of events is started as soon as the immune system receives enough chemotactic signals from the intruders, by damaged tissue cells and from the first responding immunocells, such as macrophages and leukocytes. The symptoms of an ongoing inflammation and/or infection are partly caused by the foreign intruders but also by some of the activated immunocells themselves, for example, cytokines from lymphocytes and contents from lysed cells. Normally, the immune system is successful in fighting foreign intruders, inflammations and infections but there are a number of reasons for an infection/inflammation going astray, such as temporary or chronic immune deficiencies, diseases, drugs and circulation insufficiencies. A common reason for an otherwise functional immune system of not responding is that the microbes may disguise themselves beyond recognition of the immunocells, e.g. they may fold back certain cell surface receptors which are the actual homing sites for immunocells, cover themselves with fibrin, excrete substances that destroy or paralyse the immunocells. In the case of viral infections, the formed antibodies are often unable to detect and to home with the viral structure since it is intracellular and the only opportunity the extracellular antibodies have to make contact is in the event the virus changes its host cell. An extremely severe situation is the case where an infection is treated with antibiotics because of a failing immune system and the microbial strain in question develops resistance to the drug - the consequences may easily become disastrous.

Microbial contamination in the clinical sense can roughly be divided into sub-clinical and clinical infections, respectively. Sub-clinical infection indicates that there is a microbial invasion present but not virulent enough to trigger the immune system into any greater action. Clinical infection, on the other hand, may show one or more of the symptoms of a responding immune system, e.g. inflammation, erythema, oedema, exudation, pus and soreness to different degrees.

Blood clotting The initial response to a vascular injury is a contraction of the vessel wall smooth muscle, which instantaneously diminishes blood flow from the rupture. Disruption of the endothehal hning of the vessel triggers the formation of a platelet plug, which is a loose aggregation of cells that needs to be reinforced by fibrin into a stable insoluble plug by stimulation of the coagulation cascade. Coagulation results from a series of sequential events involving clotting factors (enzymes), substrates, initiators and inhibitors. Most of the clotting factors exist as zymogens that need to be converted to their activated enzymatic forms during coagulation. Activated factor X is involved in one of the final steps of the coagulation by converting protrombin to trombin. There are two pathways that will generate activated Factor X, the extrinsic and the intrinsic pathways. The trombin formed cleaves fibrinogen, forming fibrin monomers. Factor XIII then catalyses the polymerisation of the monomeiic fibrin into highly cross-Unked and insoluble fibrin that holds the platelet plug firmly in place.

Several control mechanisms limit the coagulation process to the site of injury and maintain and restore the blood flow. The blood flow itself washes away and dilutes active coagulants and factors at the local site of injury and the liver removes the coagulant/factors from the circulation. Naturally occurring plasma inhibitors regulate the activity of activated factors. Fibrinolysis is initiated with the formation of a fibrin clot, in an effort to remove the clot and to restore blood flow. These control mechanisms help maintain fluid circulation during clotting to prevent a massive thrombosis which easily could be the result of an unopposed clotting process.

Tissue healing Tissue healing, e.g. following trauma, burn or post-surgical wound, sometimes lead to excessive scar formations, keloids and tissue adhesions. Especially when the healing process has gone astray, in for example acute inflammation, protracted infection or malfunctioning clotting process, abnormal tissue healing may occur. Acute inflammations and/or severe infections cause oedema and swelling of the affected area, which could cause temporary circulation deficiencies preventing the series of events of a healthy healing process. Necrotic tissue, pus and blood clots are also obstacles of the natural healing process that can lead to poor healing. The natural healing process requires a certain inflammatory response to trigger the mechanisms of the immune system and to initiate the series of events for a proper tissue healing. Disturbances of the process may lead to cosmetically and functionally poor results.

Prior Art

A polypepti.de fraction has been isolated from bivalve, Tridacna maxima, by

Baldo et al; Biochem. J. (1978), 175, p. 467-477 "Purification and Characterization of a Galactan-Reactive Agglutinin from The Clam Tridacna maxima (Rδding) and a Study of its Combining Site". This isolated fraction is said to have interesting immunological properties. No other use is disclosed or suggested.

EP-A-50 636 discloses a specific polypeptide fraction which has been isolated from Mytilus mussels and the use thereof as an anti-microbial drug. The fraction in question is characterised as capable of biospecifically binding at least one sialic acid in presence of calcium ions.

WO 88/04303 describes a lectin, which has been isolated from scallops, Placopecten magellanicus. The lectin binds to certain cell types, such as Gram- negative bacteria. The patent claims refer to ways of purification and identification of the lectin and no use is suggested for the product.

WO 85/05033 describes a crude aqueous extract from Green mussel, Perna canaliculus, which is suggested to be used as a nutritious protein supplement and as an antiphlogistic drug.

WO 94/04033 describes an anti-microbial composition comprising an antimicrobially active protein isolated from mussels.

Object of the invention

The object of the invention is to provide a product for treatment and cleaning of microbially contaminated human or animal tissues, body surfaces and cavities, including bleeding wounds, lesions and damaged tissues.

A further object of the invention is to provide a product having a coagulative effect resulting in decreased bleeding time of bleeding wounds and improved tissue healing. These and other objects of the invention are achieved by means of the specific features which are indicated in the appended claims and which will be explained further in the following.

Summary of the invention

The basic properties and features of an anti-microbial composition comprising an antimicrobially active protein isolated from mussels have been described in WO 94/04033.

The present invention relates to the use of such antimicrobial composition for the manufacturing of a product intended for treatment of bleeding wounds, lesions and damaged tissues wherein use is made of the previously known anti- microbial effect and in addition a coagulative effect.

The mechanism of action of the homing sequence between the proteins and a microbial cell is not yet completely elucidated. Macroscopically, the docking can be observed as a traditional agglutination reaction or as a precipitate. Microscopically, a docked cell is instantly paralysed/neutralised and is unable to multiply. Within two hours morphological changes can be observed and the cell collapses finally and within 36 hours it is completely lysed.

The docking mechanism between erythrocytes and the mussel protein seems to be identical to the one between microbes and the mussel protein, in macroscopic and microscopic perspectives. It has not yet been identified if the cell surface receptor(s) on erythrocytes is/ are identical to those on microbial cells. From the evolutionary point of view it is though likely that some basic functions may have been maintained when the haemo-lymphatic system was developed into two separate systems, blood and lymph systems, in higher species, and that is a hypothesis why the mussel protein reacts with cell types that are several hundred million years younger than the mussel itself.

In its present formulation, the mussel protein will have limited systemic function, i.e. direct contact with the blood system. In fact, administration into the blood stream could cause thromboses and emboli. Furthermore, it is not likely that the mussel protein, in high purity, will be very antigenic and cause formation of monoclonal antibodies since the binding to erythrocytes is instantaneous. It is more likely to expect that some interaction can be observed between the mussel protein and already existing antibodies.

According to the invention, the anti-microbial composition compiising at least one anti-microbially active protein isolated from mussels and carbohydrates, such as glycogen, is used for the manufacturing of a product which can be any product for which it is advantageous having an antimicrobial effect and/or a coagulative effect.

According to preferred embodiments of the invention, the anti-microbial compositon is used for the manufacturing of a product for external use such as a plaster, an adhesive dressing, a band aid, a bandage or a dressing.

According to other preferred embodiments of the invention the anti-microbial compositon is used for the manufacturing of a product for internal use such as a catheter, an implant, or a suture thread, or a biodegradable of the products mentioned above.

The following non-limitative examples illustrate the invention.

Example 1 Preparation of an anti-microbial protein fraction from Mvtilus edulis

One kg of fresh frozen mussels was ground to a fine texture and diluted with 3 kg of de-ionised water. The extract was pumped through a starch centrifuge with a 60 μm collecting cone to separate shell pieces and larger soft tissue pieces and particles from the aqueous extract. The extract was then adjusted to pH 3.0 with 6 M hydrochloric acid and left over night, >12 h. After the acidic hydrolysis the pH was adjusted to pH 7.0 with 5 M sodium hydroxide and the solution was centrifuged at 6,000 x g, for 30 min. The particle free yellowish extract was checked for conductivity and diluted with de-ionised water to a conductivity comparable to 0.10 M sodium chloride at 20 °C. The extract was pumped on to a DEAE-Sepharose CL6B column which had been equilibrated with 0.10 M NaCl + 50 mM Tris-HCl, pH 7.5, buffer. The column was washed with the buffer till UV reached baseline, about two bed volumes and then a gradient salt buffer was introduced, 0.20 - 1.0 M NaCl + 50 mM Tris-HCl, pH 7.5, over 20 bed volumes. All collected fractions were desalted using PD-10 columns and tested for haemo- agglutination with 1% sheep erythrocytes, heparinised. Noticeable, the early fractions from the gradient contained HA-activity as well as fraction collected from regenerating the DEAE- matrix with 4 M NaCl, pH 3. The HA-active fractions were correlated to their anti- microbial activity in a MIC-test using a strain of E. coli and all fractions showing HA-activity also showed growth inhibition of the E. coli strain.

The active protein obtained showed a molecular weight of approx. 18-20 kDa with a possible dimeric form at approx. 35-38 kDa, this under light reducing conditions, using SDS-PAGE electrophoresis. Under denatured conditions the protein showed a molecular weight of preferable 14-16 kDa and iso-electric focusing showed an i.p. at 4.3-4.8. The preparation contains carbohydrates, e.g. glycogen, necessary for the protein to keep its native properties without auto- agglutination. The yield of the protein was approx. 3 g lyophilised powder.

Example 2 Clotting time of normal whole blood

The lyophilised powder from example 1 was dissolved and diluted in 0.9% saline to final concentrations of 0.005, 0.05 and 0.5 μg/μl respectively.

Fresh blood from a healthy person was collected and immediately treated as follows: 200 μl of blood was pipetted onto clean and dry glass slides, as Control 1.

200 μl of blood + 20 μl of saline were pipetted onto identical glass slides, as Control 2.

200 μl of blood + 20 μl of test sample of the lowest concentration, 0.1 μg added, were pipetted onto identical glass slides, as Test 1.

200 μl of blood + 20 μl of the test sample of the middle concentration, 1.0 μg added, were pipetted onto identical glass slides, as Test 2.

200 μl of blood + 20 μl of the test sample of the highest concentration, 10 μg added, were pipetted onto identical glass slides, as Test 3.

The slides were tilted vertically every 15 seconds and the reaction was judged to be completed when a sohd drop had been formed that did not flow within 5 seconds in tilted position. These results are shown in Table 1 at the time from pipping to formation of a solid drop.

Table 1

Time to clotting, seconds

Example 3 Clotting time of heparinised whole blood

The lyophilised powder from example 1 was dissolved and diluted in 0.9% saline to final concentrations of 0.005, 0.05 and 0.5 μg/ μl respectively.

Fresh blood from a healthy person on heparin therapy after surgery, 10 U/kg/h, was collected and immediately treated as follows:

200 μl of blood was pipetted onto clean and dry glass slides, as Control 1.

200 μl of blood + 20 μl of test sample of the lowest concentration, 0.1 μg added, were pipetted onto identical glass slides, as Test 1. 200 μl of blood + 20 μl of the test sample of the middle concentration, 1.0 μg added, were pipetted onto identical glass slides, as Test 2.

The slides were tilted vertically every 15 seconds and the reaction was judged to be completed when a sohd drop had been formed that did not flow within 5 seconds in tilted position. These results are shown in Table 2 at the time from pipping to formation of a solid drop.

Table 2

Time to clotting, seconds

Example 4

A stock solution of the lyophilised powder from example 1 was prepared at a concentration of 1 mg/ml and dispensed in aliquots of 0.5 ml and frozen to -20 °C. The fabric pad on a soft plaster was soaked with one aliquot and the plaster was put on the side of the trunk of a person. The procedure was repeated twice daily for 21 days. No irritation, rash, inflammation or itching was observed though the skin in contact with the adhesive on the plaster from time to time showed mild maceration.

After 2 weeks the procedure above was then repeated for two days and again after 2 weeks for another two days. No signs of allergic reactions could be observed at either occasion.

Example 5 The identical aliquots from example 4 were used to treat a fresh cut on the index finger, 6 mm long and about 1 mm deep, heavily bleeding. Within 2 minutes after the accidental cut had happened, a soft plaster soaked with 0.5 ml of mussel protein was applied over the wound. The wound was redressed after 5 minutes with a new soft plaster soaked with the mussel protein. Thereafter, the wound was identically redressed three times a day.

At the first redressing the bleeding had stopped. The wound was completely re-epithelialised after 4 days. No signs of acute inflammation or clinical infection could be observed during the treatment though a certain soreness and swelling were reported. Two months after treatment a scar could be observed but no excessive fibrin or collagen formation. After six months no signs of the cut could be observed.

Claims

1. Use of an anti-microbial composition comprising at least one anti-micro- bially active protein isolated from mussels and carbohydrates, such as glycogen, characterized in that the composition is used for the manufacturing of a product having an anti-microbial effect and a coagulative effect, wherein the product is intended for: treatment and cleaning of microbially contaminated human or animal tissues, body surfaces and cavities, and/or treatment of bleeding wounds, lesions and damaged tissues.

2. The use according to claim 1, characterized in that the product is a product for external use such as an adhesive dressing, a band aid, a bandage, a dressing or the like.

3. The use according to claim 1, characterized in that the product is a product for internal use, such as a suture thread, a catheter or an implant or the like, optionally biodegradable.

4. The use according to any of the previous claims, characterized in that the product is intended for improved tissue healing.

5. The use according to any of the previous claims, characterized in that the product is intended for improved blood coagulation.

6. The use according to any of the previous claims, characterized in that the product is intended for the prevention of tissue adhesion.