WO1988003029A1

WO1988003029A1 - Composition for the stimulation of wound healing containing an immunostimulant bacterial exotoxin and use thereof

Info

Publication number: WO1988003029A1
Application number: PCT/SE1987/000502
Authority: WO
Inventors: Juha Niinikoski; Matti Laato; Bengt Gösta GERDIN
Original assignee: Pharmacia Ab
Priority date: 1986-10-28
Filing date: 1987-10-28
Publication date: 1988-05-05
Also published as: EP0288525A1; JPH01501065A; SE8604587D0

Abstract

Composition stimulating the healing of wounds and containing an effective wound healing amount of an immunostimulant bacterial exotoxin in substantially pure form. The preferred exotoxins are the staphylococcal enterotoxins. Methods for stimulating wound healing and for production of the composition are also disclosed.

Description

Composition for the stimulation of wound healing containing an immunostimulant bacterial exotoxin and use thereof

The invention relates to a formulation for stimulating the healing of wounds in mammals (including man) , and furthermore relates to a method for using this drug as well as to a method for producing it.

The term "stimulating the healing of wounds" in the present context refers to the taking of steps by means of which wounds showing no tendency to heal are induced to start the healing process, and wounds that have started to heal are induced to heal more quickly; or to the taking of steps which are conducive to a cosmetic improvement of the func¬ tional result during the healing or after the completion thereof. Stimulation of wound healing is particularly important when natural healing is slow or is rendered difficult by a number of negative factors like e.g. infection of the wound, impeded blood flow, medical treatments with cell poisons or with steroids of various kinds, or in cases where patients suffer from chronical disorders with concomitant impairment of normal wound healing, e.g. when they are bedridden for prolonged periods of time or suffering from old age, cancer, diseases giving rise to serious nutritional deficiencies of such types as e.g. chronical inflammatory conditions of the intestine, conditions caused by extensive bodily injuries (so-called polytrauma patients) , and comparable types of conditions.

Factors stimulating the healing of wounds have been de¬ scribed earlier; examples are the epidermal growth factor (EGF) , platelet derived growth factor (PDGF) (Grotendorst, J. Clin. Invest. 76:2323- , 1985) and bacterial endo- toxins (Hunt et al., Surgery 96:48- , 1984). These factors are known to produce an increased cell division in organ cultures. EGF was thought at the outset to be a promising healing stimulant when tested in experimental studies of its effects in vivo. However, in experiments with established wound models it was found that comparatively large doses are required for obtaining an effect of stimulated wound healing. Bacley et al. (Proc. Nat. Acad. Sci. USA 82:7340-7344, 1985) have shown that a dose of 5-10 .ug EGF/day will stimulate wound healing. Laato et al. (Ann. Surg. 203:379-81, 1986) have shown that more than 0.2 ,ug/day of EGF is required if a stimulation of the healing process is to be obtained. In cell cultures, on the other hand, both PDGF and EGF are active in concentrations of 1-10 ng/ml. Recently Barbul et al. (J. Surg. Res. 40:315-9, 1986) have shown that a systematic treatment with interleukin (IL 2) may be ben¬ eficial in order to promote wound healing. It is well-known, thus, that certain factors can be administered to wounds so as to stimulate healing. If the drug is administered locally, relatively large amounts are required for obtaining an effect.

It is also known that small amounts of viable bacteria, e.g. of the genus Staphylococcus, Escherichia and Pseudomonas, may have a beneficial effect on wound healing in that they are conducive to an enhanced formation of collagen (Carrel A. , J. Exp. Med. 34:425- , 1921; Oloumi M. , et al., Surg. Gynecol. Obstetr. 145:702- , 1977; and Laato M. et al., Acta Chir. Scand. 151:313-8, 1985} . It has also been known that different complex compositions of bacterial origin and containing bacterial toxins may have similar effects (e.g. filtrates of culture medium see Beck et al., Acta Biol. Med. Ger. 13:343-50, 1964; and Ficai, Chemical Abstracts 43

($.949) 8434a). In US-A-4,604,234 experiments in vitro has demonstrated that cholera toxi may have similar effects on epidermal cell growth (compare also Green, Cell 15:801-11, 1978) . It has not been known heretofore which particular bacterial substance(s) may be required or how such substances may be able to cooperate so as to give the beneficial effect actually obtained. The present invention proposes that bacterial exotoxins having an immunostimulant capacity can act as wound healing stimulants; and the text of the present patent application will demonstrate that these substances may be significantly more potent than EGF - the substance which was believed to be one of the most promising.

Results indicating the use of exotoxins as drugs have been presented (HlavayovS. and Sved, Neoplasma 30:667-80, 1983; Mueller et al., Zentralbl Bakteriol Mikrobiol Hyg, Abt 1 Orig A 252:74-82, 1982; Japanese patent abstract JP 56-29518; US-A-4,285,931; and US-A-3,882,233) .

The main objects of this invention are to provide improved ways (methods) and means (compositions) for promoting wound healing, especially for local treatment of and administration to wounds. As regards the functional result, the main object is to obtain an enhanced growth of connective tissue.

The composition of this invention contains as active sub¬ stance an immunostimulant bacterial exotoxin (IBE) (including fragments thereof having this property) in an amount that stimulates the healing of wounds but is non-toxic. In its broadest sense, the invention involves using IBE for any type of wound healing stimulation. The term employed for the amount as being one that "stimulates the healing of wounds but is non-toxic" means that the amount of IBE is such that the toxic effects are acceptable in their relation to the required wound healing stimulant effect.

In a composition of the invention the exotoxin contemplated exists in substantially pure form with respect to other proteins originating from the source of raw material, i.e. substantially pure from other proteins of the bacteria that produced the exotoxin. Normally, substantially pure means that the exotoxin in question amounts to 90 % (w/w) or more with respect to such proteins. The exotoxins contemplated within the framework of this invention are immunostimulants in that sense that they are capable of inducing the formation of lymphokines in the mammal to be treated, such as gamma-interferon and IL 2 and/or capable of inducing cell division (i.e. are mitogenic) . The formation of lymphokines is essential for the wound healing effect. They always have a peptide structure to which optionally may be bound carbohydrate and/or lipid structures. Their molecular weight as a rule exceeds 5000 daltons. The IBEs which are most suitable for the invention, on the basis of the data currently available, are of the type having both immunostimulant and toxic properties - although it will very probably be found that fragments thereof which are non-toxic but immunostimulant in character are actually preferable. Molecularly, the preferred exotoxins either are constituted by one polypeptide chain forming a loop by means of an intramolecular disulfide bond, or are composed of two polypeptide chains which are held together by an intermolecular disulfide bond. (In this latter case, the two chains may conceivably have been formed as a consequence of proteolytic cleavage of one or more peptide bonds in the aforesaid loop.)

Among preferred exotoxins may be mentioned such exotoxins as will affect the intestine, i.e. enterotoxins, especially such toxins from Micrococcus bacteria, e.g. Staphylococcal enterotoxins A, B, C, D and E (SEA, SEB, SEC, SED, SEE) and exotoxins stemming from other bacteria and having similar properties, e.g. exotoxins from Streptococci. For a survey of various bacterial exotoxins, with special emphasis on enterotoxins, see Bergdoll ϋ G (The Staphylococci, Wiley - Interscience, New York, 301-331, 1972). It is a well-known fact that the aforesaid exotoxins can be fragmented and derivatized in a manner such as to give fragments (and derivatives) possessing only the immuno¬ stimulant property (see also Spero L and collaborators in J. Immunol. 122:1285-9, 1979 and J. Biol. Chem. 250:5026-32, 1985; and Noskova V P et al.. Int. J. Biochem. 16:201.6, 1984) . The invention, because it is based on the concept of obtaining improved wound healing by means of immunostimu- lation, comprises also the immunostimulant fragments and derivatives of the exotoxins.

For deciding whether or not a given exotoxin or its fragment is useful in the context of this invention the exotoxin or fragment may be studied in various models adapted for studies of mitogenicity, induction of lymphokines like IL 2 and interferon, and wound healing stimulation. Mitogenicity can be assayed by way of DNA-thymidine uptake in cultured lymphocytes that have been exposed to the exotoxin under^' examination; whereas induction of interferon and IL 2 may be assayed by way of interferon activity and IL 2 activity in the supernatants from cultured lymphocytes similarly exposed. (See for example Noskova V P et al., Int. J. Biochem. 16:201-6, 1984.) With the aid of standardized experimental wound models, like e.g. the one set forth in our exemp¬ lification, it is an easy matter for a man skilled in the art to evaluate the wound healing stimulant effect obtain¬ able with a particular exotoxin or its fragments for a particular mammal.

Among the exotoxins that are potentially of importance in the context of the present invention, there are several which are commercially available but are rather expensive. SEA, SEB, SEC, SED and SEE can be purchased from Toxin Technology, Madison, Wi, USA. Possibilities of large-scale production of the toxins and their wound-healing stimulant fragments have been much improved since the introduction of recombinant DNA techniques. The exotoxins as here contemplated are often highly toxic. Therefore, when they are administered within the context of this invention it is imperative that the composition employed is of a type such that the wound can be maintained in contact with the active substance (preferably for a prolonged period of time) without the toxic effects becoming too obtrusive. In order to facilitate a correct dosage the active substance is usually incorporated, and thus diluted, in a suitable vehicle. This vehicle may be in the form of for instance a physiological aqueous solution buffered to a pH that is suitable for the wound healing process; that is, pH 4.5-8, preferably pH 5-8.0. The active substance may also be incorporated in a vehicle that is soluble or insoluble in water. Suitable vehicles of this type are e.g. various hydrophilic macromolecular materials which are capable of absorbing water. Examples are gauze bandages, compresses and various water-absorbing types of layers useful and used for treating injured tissues (see e.g. GB-A-2,048,292) . A type of very suitable materials are water-insoluble macro¬ molecular compounds in a particulate form which when contacted with water become capable of a limited degree of swelling so as to form discrete gel particles. These types of materials have been employed earlier not only for wound dressing (GB-A-1,454,055) but also as carrier phases in gel and affinity chromatography. These hydrophilic particles consist usually of an a ine- and/or hydroxyl-containing polymer, for example a polysaccharide in an insoluble form such as an insoluble dextran derivative, cellulose, starch, agarose etc; a polymerized mono-, di-, or oligosaccharide in an insoluble form; or a sugar alcohol similarly polymerized. Optionally the polymer may be crosslinked or be provided with covalently bound ion-exchanging or hydrophobic or hydrophilic groups. Such a type of derivatization may be effective for bestowing on a given basal polymer some particular physical and chemical properties that are desirable for the purpose contemplated. The active substance may also be incorporated in a liposomal form. Among vehicles of special interest may be mentioned vehicles based on water- soluble polymers, like for example hyaluronic acid, which form highly viscous solutions with water.

Conveniently, the insoluble vehicle employed may be one permitting the toxin to be released slowly or in a delayed mode. The toxin thus may be bound covalently to the vehicle via bonds that are capable of being cleaved by means of enzymes present in the wound, e.g. hydrolases. Some types of insoluble vehicles are resorbable, for example those that are composed of polylactate, polyglycolate and starch, so as to slowly release bound toxin as a result of such resorption, Also, the active substance may be enclosed physically in a manner such that its diffusion from the vehicle is thwarted. Thus for instance crosslinking or other suitable types of derivatizations of insoluble polymers are well-known ex¬ pedients for achieving a decreased diffusion of a substance occluded within the polymer.

The compositions employed in accordance with the invention may be in a dry state, for example in the form of a dry free-flowing powder, dry sponge or dry compress. Particulate vehicles may be suspended in various known per se suspension- type ointment bases (for example oil-in-water, water-in-oil and fatty ointments) .

The compositions of the invention are always sterile. Sterility is obtainable either by manufacturing the compo¬ sition aseptically from sterile starting materials or by sterilizing it after manufacture.

In the process according to the invention for manufacturing the composition, the IBE is mixed with, bound to, enclosed within, or in some other manner incorporated into one of the aforesaid vehicles, whereupon the resultant product is if required transformed into a suitable form as set forth above. As will be apparent from the explanations given above an important feature in the context of this invention may reside in an ability of the composition to present the active substance (IBE) in a soluble form to the immune system of the patient.

The amount of IBE in the composition of the invention may vary with the particular IBE employed and with the type of wound for which it is to be used. Two important properties to be taken into consideration are (1) the wound-healing potency of the IBE - for as a rule high potency implies that lower concentrations can be used - and (2) the toxicity of the IBE - inasmuch as a high degree of toxicity of course means that only small amounts can be used and such highly toxic IBEs therefore must be present in a very diluted state in the composition or in a form of very well-defined release. Normally the composition of the invention contains a healing stimulant IBE in a concentration that is lower than about 5 % (w/w) .

According to the process of stimulating wound healing in conformity with this invention, the wound is contacted in a manner known per se with an effective amount of the par¬ ticular IBE chosen, the IBE being preferably incorporated in a vehicle of the aforesaid type that is suitable for the purpose contemplated, whereupon after a sufficient period of time excess amounts of IBE (plus residual vehicle) are removed. The treatment may be repeated if required.

The invention will now be illustrated by way of non-limi¬ tative patent examples.

Example 1

Production of compositions for treatment

A. 10 ,ug of SEA (Toxin Technology Inc., Madison, Wi, USA) are dissolved aseptically in 10 ml of sterile, phosphate buffered saline according to Dulbecco, containing 0.5 % (w/w) of bovine serum albumin. The solution may be divided into minor aliquot portions but can also be used as a stock solution.

B. 0.21 ,ug of SEA is dissolved as in (A) in 20 ml of Healon (1 % w/w hyaluronic acid having a molecular weight of 3.5+;0.5 x 10 daltons, Pharmacia AB, Sweden) This solution too may either be divided into minor aliquot portions, e.g. in disposable syringes, or be used as a stock solution.

Example 2

The effect of wound healing stimulation was studied in a standardized experimental wound model in rats as described by Niinikoski, Heughan and Hunt (Surg. Gynecol. Obstet. 1971; 133: 1003-1007). A short description of this model is as follows:

A. Treatment of the wound with an aqueous solution of SEA

Cellulose sponge was employed as an inductive matrix for repaired tissue. The material was cut into cylindrical pieces of 40 mm length and 10 mm diameter, a tunnel of 3 mm diameter then being made through the center of the sponge. Discs of silicone rubber having a diameter of 10 mm and a thickness of 2 mm were sewn onto both ends of the sponge to create a stable void volume. The cylinders were sterilized by being boiled for 30 minutes in physiological saline, and subcutaneous implants were made using a strictly aseptic technique. Male Sprague- Dwaley rats weighing 230-250 g were anesthesized with ether, and a 4 cm incision was made on the lower back portion in the central line thereof. Each rat received a sponge cylinder which was implanted longitudinally under the skin. All in all 24 rats were studied which were divided into 4 groups. A control group which was only given solvent (0.1 % bovine serum albumin in phosphate buffered saline, 0.05 ml/day in the central tunnel) ; one group was given 50 ng of Staphylococcal enteoroxin A in the same solvent; one group was given 10 ng of Staphylococcal enterotoxin A per day; and one group was given 2 ng of Staphylococcal enterotoxin A per day. Seven days after the implantation the rats were sacrificed. Two of the rats that had been given 2 ng/day of Staphylococcal enterotoxin A died during the operation, so this group then contained only 4 animals. The implants were dissected free from sur¬ rounding tissue and the silicone rubber discs were removed. The amount of connective tissue was evaluated as the amount of hydroxyproline.

In a similar manner the effect of EGF was studied at three concentration levels.

Results

mg of hydroxyproline relative stimul

Control 1.16 + 0.26 (n=6) 100 % 22 %

SEA, 50ι ng/day 3.02 + 0.67 (n=6) 260 + 58 %

SEA, 10t ng/day 4.37 + 0.70 (n=6) 377 + 58 %

SEA, 2 ng/day 3.46 + 0.55 (n=4) 298 + 33 %

EGF, 0. 2 _;ug 135 + 5 %

To sum up, the results show that wound healing is greatly stimulated by Staphylococcal enterotoxin A in doses of from 2 to 50 ng per day. The potency of Staphylococcal enterotoxin is therefore about 3 powers of ten higher than that of EGF which in itself is a healing stimulant which once had sent expectations soaring high. B. Treatment of the wound with SEA incorporated in a highly viscous vehicle

In a second series of experiments, using the same wound model, 10 animals were treated with either 10, 50 or

100 ng of SEA dissolved in 1.0 ml of 1 % (w/w) hyaluronic acid (Healon ®, average molecular weight 4x106 daltons,

Pharmacia AB, Uppsala, Sweden) . Controls were given the same amount of Healon without SEA or physiological saline. Only one treatment was performed after implan¬ tation. Seven days later the animals were sacrificed and the sponge was taken out for microscopic examination. It was found that the animals that had been treated with SEA showed more widespread scar tissue formation in the sponge than the controls.

Example 3

Inhibition of the wound healing effect by neutralizing gamma-interferon

In order to show that i munostimulation was an obligate prerequisite to obtain the effect found for an exotoxin, 21 rats were examined in the above-mentioned experimental model. 7 animals were treated with saline, 7 animals with SEA (10 ng daily during 7 days) and the last 7 animals with SEA (10 ng daily during 7 days) and during the first 4 days with in anti-rat gamma-interferon monoclonal antibody (40 ,ug locally and daily) .

Compared to the animals treated with saline, those having been treated with only 10 ng SEA showed a wound healing of 160 %, while those having also been given anti-gamma- interferon showed a wound healing of 94 %. This result shows that the formation of a lymphokine is essential for our invention. The characteristic features of the invention will be evident from the claims which form an integral part of this speci¬ fication.

Claims

1. Composition stimulating the healing of wounds charac¬ terized in that it contains an effective amount for wound healing of an immunostimulant bacterial exotoxin in substantial pure form.

2. Composition according to claim 1, characterized in that the exotoxin is an enterotoxin.

3. Composition according to claim 1 or 2, characterized in that the exotoxin is a Staphylococcal enterotoxin such as SEA.

4. Method of stimulating wound healing in mammals, charac¬ terized by administering, in a manner as known per se in wound healing treatments, an immunostimulant bacterial exotoxin in substantially pure form and in an amount that is wound-healing-stimulant but is non-toxic.

5. Method according to claim 4, characterized in that the exotoxin is an enterotoxin.

6. Method according to claim 4 or 5, characterized in that the exotoxin is a Staphylococcal enterotoxin.

7. Method according to any of claims 4-6, characterized in that the enterotoxin is SEA.

8. Immunostimulant bacterial exotoxin for being used as a factor stimulating the healing of wounds.

9. Use of an immunostimulant bacterial exotoxin for producing a drug composition to be employed for the therapeutic treatment of wounds.