EP1434591A1

EP1434591A1 - Use of fly larvae extracts for the treatment of wounds

Info

Publication number: EP1434591A1
Application number: EP02794527A
Authority: EP
Inventors: Karl-Heinz Nietsch; Rainer Pooth; Heinz Mehlhorn; T. Ruzicka; Helger Stege
Original assignee: Aventis Pharma Deutschland GmbH
Current assignee: Sanofi Aventis Deutschland GmbH
Priority date: 2001-08-10
Filing date: 2002-07-31
Publication date: 2004-07-07
Also published as: CA2456814A1; IL160274A0; JP2005501078A; WO2003013557A1; MXPA04000298A; WO2003013557A8

Abstract

The invention relates to the topical application of fly larvae extracts obtained from fly larvae which are killed and extracted in an aqueous medium under cooling conditions or in dissolvents and freed from non-dissolved components. Different types of fly larvae extracts are suitable for the treatment of superficial or deep chronic and acute wounds of any kind. Said fly larvae extracts have a wound-healing effect and can be obtained from, for instance, the genera Sarcophaga or Lucilia.

Description

Use of fly larvae extracts for wound treatment

The invention relates to the topical application of fly larvae extracts of various species, which belong for example to the genera Sarcophaga or Lucilia, for the treatment of superficial or deep chronic and acute wounds of any origin.

Wound healing is a complex process in which multiple target cells and target structures must interlock in an orderly sequence of processes. These processes take place regardless of the type of wound (chronic / acute), while the duration of individual phases is variable. There are generally three main phases, the exudative phase, the proliferative phase and the epithelialization or repair phase.

In the exudative phase, the focus is on the acute traumatization of the hemostaseological and vasoconstrictive reactions. Clinically, wound edema and wound pain are impressive. The resulting vascular defect is closed under the influence of the platelets. These release platelets with a chemotactic effect. Immigration of macrophages, neutrophils and lymphocytes occurs. This builds up a phagocytosis system that is highly effective for tissue debridement.

After cell debris has been removed, the proliferative phase begins due to the migration of fibroblasts and vascular endothelial cells. The cell content increases massively due to the immigrated fibroblasts and endothelial cells. At the same time, cytokines and growth factors are released, which in turn stimulate new vascularization and cell proliferation. In addition, a matrix conversion takes place in this phase. This takes place through the formation and conversion of collagen type III into collagen type I. This creates a well-capillaryized, macrophage, fibroblast and mast cell-rich granulation tissue. The epithelization and repair phase follows this proliferative phase. In this final phase there is wound contraction and migration of marginal keratinocytes into the wound. Neoangiogenesis and capillary density decrease. The collagen content, however, increases. This process is relevant for the mechanical strength of the resulting scar tissue.

Delays in wound healing can occur if these complex interactions are disturbed. Depending on the genesis of the affected processes, one speaks of chronic after a wound that has existed for 6-8 weeks

Wound healing disorders. These occur in a variety of immunologically related diseases, varicose veins, arterial occlusive disease, after infections and, for example, diabetes mellitus. Measures to promote wound healing serve the accelerated or regular sequence of the processes described above. Wound cleansing procedures, in addition to granulation-promoting processes, are to be mentioned here. Epithelization can also be promoted by modern "wound dressings". One of the main causes of delayed wound healing is the lack of formation of granulation tissue. This can either be caused by reduced endogenous wound debridement, or it can result from infections, circulatory disorders, immunological diseases an excessive formation of cell and tissue detritus.

Therapeutically, wound cleansing measures are used. The aim of cleaning is to create a clean wound bed. For this purpose, ointment residues and crusts must first be removed and any existing necrosis removed. The latter is done either surgically with a sharp spoon (curettage) or with tweezers and scissors. Alternatively, one can use enzymatic ointments that preferentially break down denatured protein. They contain enzymes such as trypsin, chymotrypsin, enzymes from bovine material such as pancreatic enzymes, or collagenase, fibrolysin, streptokinase or calf blood dialysates. At the same time, regular disinfection is carried out, for example with potassium permanganate or with Rivanol® baths. Disinfectant measures can also be carried out with preparations containing silver or iodine

However, the enzymatic preparations often show only limited effectiveness on the patient. The dosage of the enzyme is often very low and the half-life of the known enzyme preparations is 6 to 12 hours. For this reason, daily, even repeated dressing changes are necessary. Some of the preparations are combination preparations with topical antibiotics. The disadvantage of combination products is the risk of epicutaneous sensitization. Almost 60-70% of patients with chronic leg ulcers suffer from one or more sensitizations based on ointments or other components of topical externals.

Therefore, the use of local antibiotics should be avoided, because on the one hand the development of resistance and on the other hand the sensitization rate is high.

The creation of larvae (maggots) of the species Lucilia sericata on wounds is also known. This therapy is based on ancient folk medicine and partly on war medicine findings and observations about the contamination of war injuries with fly maggots. The maggots of this species feed exclusively on necrotic tissue. This material is, as it were, digested by secretion of saliva and only then absorbed by the maggot. The maggots of L. sericata do not actively chew or bite. This ensures that the maggots cannot penetrate other parts of the body or unaffected body cavities. The treatment with the maggot shows very high therapeutic effectiveness. However, the current treatment method is extremely complicated, expensive and requires a high logistical effort. For use in humans, the maggots should be grown under controlled conditions. Asepticism must be guaranteed both for rearing and for transport from the laboratory to the patient. The therapeutic effectiveness cannot be dosed. It is possible to determine the number of maggots that are applied to the wound, but not the enzymatic activity they produce. The maggots themselves succumb a biological development process, at the end of which is the metamorphosis of the larva to the pupa and then to the fly. For this reason, the application of the larvae must be repeated closely. A high level of compliance is required for patients and medical service providers to carry out the therapy, since cultural and civilizational boundaries have to be overcome psychologically. In addition, hooking the larval mouth hooks can be quite painful.

Patent application WO 01/31033 describes a protein secreted to the outside by living maggots of the species Lucilia sericata, for which a wound healing property is assumed. However, physiological experiments on wounds that could support this assumption are missing. The application WO 01/31033 describes the isolation of a very small amount of the secreted protein, but an economical method for obtaining marketable amounts of protein would still have to be developed. Furthermore, an oily formulation of a powder from dried fly larvae in oil is known, which is or has been used for wound treatment in China (Yang Zheng .: China Science: House fly yields medicine, expert say. In: Inter Press Service; 03.09. 1997, 97: 314231 NLDB). A disadvantage of the oil is that it can cause allergies as a side effect. The oily galenics of the Chinese product conflict with the state of the art for wound treatment in medicine in western countries: according to current and general opinion, modern wound dressings should have a hydrophilic environment, in which better healing is observed than when using wound-covering hydrophobic formulations ( Pontieri-Lewis V. (1999) Principles for selecting the right wound dressing. Medsurg Nurs 8: 267-70; Casey G (2001) Wound dressings. Pediatric Nurs 13: 39-42; Casey G (2000) Modem wound dressings. Nurs Stand 15: 47-51; Ruszczak Z, Schwarz RA (2000) Modern aspects of wound healing: an update. Dermatol Surg 26: 219-229; Probst W (2000) Local treatment of chronic wounds. Pharm Ztg 145: 3907-3920; Strobel HG (2000) Primer for Wounds; Quality standards for wound treatment at the University Clinic Essen. Hospital Pharmacy 21: 350-361). Disadvantages of oily galenics exist especially in that they inhibit the migration and function of immune cells and disrupt the proliferation of newly formed cells.

In an effort to find effective treatment methods for superficial, deep, chronic or acute wounds of any genesis, it has now been found that the extracts according to the invention from fresh fly larvae of various species, for example of the genera Sarcophaga or Lucilia, can remedy the disadvantages mentioned.

The fly larvae extracts according to the invention represent a clear one

Further development of the maggot therapy in terms of application and dosage. It is also possible to use the extracts in modern, hydrophilic wound dressings. The therapy can be better controlled by standardizing the manufacturing process. As a finished product, the fly larvae extracts have a continuous performance that does not depend on the maggot development cycle.

The invention therefore relates to fly larvae extracts obtainable from fly larvae, the fly larvae first being cooled and then homogenized and the homogenate obtained finally being freed from undissolved components of the fly larvae.

If necessary, an extraction medium can be added before homogenization.

The extraction medium contains water or is an organic solvent. Furthermore, the soluble components can be preserved or applied topically to the wounds immediately. When applied topically, the extract according to the invention has a wound healing effect in the case of superficial, deep, chronic or acute wounds of any origin.

Suitable fly larvae come, for example, from the genera Sarcophaga, Lucilia, Musca, Calliphora and Stomoxys. Mixtures of Fly larvae from the genera mentioned are used in the process according to the invention.

Suitable species from the genera mentioned are, for example, Lucilia sericata, Lucilia caesar, Lucilia cuprina, Sarcophaga carnaria, Sarcophaga agyrostoma, Musca domestica, Calliphora erythrocephala, Calliphora vicina or Stomoxys caicitrans. The genera Sarcophaga and Lucilia are, for example, ubiquitous and a person skilled in the art can easily find these insects, for example by laying out fresh meat.

The fly larvae extracts according to the invention are produced, for example, by keeping eggs or larvae of the species Lucilia sericata and / or Sarcophaga carnaria on fresh meat. The larvae grow and thrive on the meat and are harvested shortly before entering the pupation stage. It is advantageous to harvest the larvae from day 5 to day 8 after hatching from the egg.

The larvae are killed and processed about 5 to 8 days after the larvae hatch from the egg, but each time before pupation. The killed larvae are cooled before and during the further processing to the fly larvae extract. Possible cooling temperatures are temperatures below 0 ° C, i.e. in the frozen state, for example at temperatures from 0 ° C to - 80 ° C. However, it is also possible to work at temperatures from 0 ° C. to 15 ° C., preferably from 0 ° C. to 10 ° C., in particular from 2 ° C. to 6 ° C. The larvae can also be frozen for homogenization or for further processing, or crushed and homogenized in the frozen state.

To this end, the larvae are first made largely sterile on the outside and freed of possible secretions and excretions (SE) that could adhere to the body of the female. This is done by several washing steps in aseptic solutions in decreasing concentration. Sterilized NaCI solution is used in the last washing steps, thereby ensuring that the larvae are largely sterile. Further all secretions and excretions of the maggots are washed off and the maggots are preserved on ice.

The larvae are homogenized, for example, by mechanical comminution or ultrasound. The fly larvae can be homogenized as such or preferably with the addition of an extraction medium. 0.1 ml to 500 ml of extraction solution, preferably 0.5 ml to 100 ml, very preferably 1 to 5 ml of extraction solution, are added per gram of wet weight of fly larvae. Sterile extraction solutions, for example aqua purificata, physiological salt solutions, buffers, electrolyte, sugar or protein solutions and aqueous emulsions and organic solvents are particularly suitable. The addition of extraction media can also be dispensed with entirely and only the liquid components of the maggots can be squeezed. The extract can also be prepared in such a way that the active ingredients are precipitated by adding organic solvents and then extracted. The homogenate is separated into solid and soluble constituents, for example by filtration or centrifugation. The fly larvae extracts may be preserved by freezing or freeze-drying. Furthermore, other known agents for stabilizing active molecules can be used, for example protease inhibitors, trehalose, ectoin or buffers.

After the homogeneous liquid has been obtained, the extract is filtered, for example sterile filtered, using a filter which has a pore diameter of 0.1 μm to 0.4 μm. In the last step, the extract is aliquoted and frozen in liquid nitrogen. Permanent storage takes place at a temperature of around -21 ° C to -80 ° C or in liquid nitrogen. The sterile-filtered extracts obtained can also be lyophilized.

The extracts according to the invention can also be further purified or fractionated using conventional purification methods, such as by selective precipitation steps or chromatographic or electrophoretic processes. The invention also relates to pharmaceuticals, characterized by an effective content of the fly larvae extracts according to the invention together with a pharmaceutically suitable and physiologically compatible carrier, additive and / or other active substances and auxiliaries.

Due to the pharmacological properties, the fly larvae extracts according to the invention are suitable for the therapy of superficial or deep chronic and acute wounds of any origin.

The term "chronic and acute wounds of any genesis" is understood to mean, for example, wounds such as surgical wounds which are intended to heal secondarily or unintentionally, cuts, stab wounds, abrasions, bites, burns or bullet wounds, and other wounds which are not can be treated primarily with surgical sutures or primary wound closure. Furthermore, the term acute wounds means all wounds which cannot be healed primarily by superinfection and all wounds whose manifestation is 4 weeks and less. Chronic wounds are all injuries associated with the loss of epithelial integrity that are manifest for more than 4 weeks. In particular, this means poorly healing wounds based on diabetes mellitus, varicose vein or venous thrombosis, rheumatic disease, vasculitis, arterial occlusive disease, suffering from the lymphatic vessels, haematological diseases and under or after infections of the wounds.

The invention also relates to a method for producing a medicament, which is characterized in that the fly larvae extracts according to the invention are brought into a suitable dosage form with a pharmaceutically suitable and physiologically tolerable carrier and, if appropriate, other suitable active ingredients, additives or auxiliaries. The invention also relates to the use of the fly larvae extracts according to the invention for the production of medicaments for the therapy of superficial or deep chronic and acute wounds of any origin.

The medicaments according to the invention are generally applied topically.

Suitable pharmaceutical compositions for topical application on the skin are preferably in the form of a solution, suspension, powder, liposomal formulations, gel, lotion, paste, spray or aerosol. Polyethylene glycols, alcohols and combinations of two or more of these substances can also be used as carriers. The list can in no way be interpreted as restrictive. Depending on the extraction conditions, the fly larvae extracts according to the invention are present in a concentration of 0.1% by weight to 100% by weight of the composition, for example from 1.0% by weight to 60% by weight.

Transdermal administration is also possible. Suitable pharmaceutical compositions for transdermal applications can be presented as individual patches which are suitable for long-term close contact with the patient's epidermis. Such plasters suitably contain the fly larvae extracts according to the invention in an optionally buffered aqueous solution, dissolved and / or dispersed in an adhesive or dispersed in a polymer. A suitable active ingredient concentration is from about 0.1% by weight to 75% by weight, preferably from 1% by weight to 70% by weight. As a special possibility, the active ingredient can be released by electrotransport or iontophoresis, as described, for example, in Pharmaceutical Research, 2 (6): 318 (1986).

The fly larvae extracts according to the invention can also be applied to the wound by wound dressings made of gauze, from alginates, from hydrocolloid materials, foams and silicone dressings which have been coated, soaked or treated with these fly larvae extracts and are therefore able to place the fly larvae extracts in or on the Deliver the wound or wound surface. Suitable solid or galenical forms of preparation are, for example, granules, powders, solutions, suspensions, emulsions or drops, as well as preparations with a protracted release of active ingredient, in the preparation of which conventional auxiliaries or carriers are used. Magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, cellulose and its derivatives, polyethylene glycol and solvents such as sterile water and mono- or polyhydric alcohols such as glycerin are mentioned as frequently used auxiliaries.

Furthermore, the fly larvae extracts according to the invention can also be used in pharmaceutical preparations which contain the fly larvae extracts in inactive form and which are then applied to or on the wound and are activated by adding specific substances. Simple examples are the use of a powder or of lyophilisates, which are dissolved with physiological solutions (e.g. 0.9% NaCl). If the stability is sufficient, the pharmaceutical preparation can also be a solution.

The appropriate pharmaceutical compositions are applied after mechanical wound cleaning. Mechanical wound cleaning is carried out, for example, by bathing or flushing the wound with Ringer's lactate. After application of the fly larvae extracts according to the invention, the wound is optionally covered by means of hydrocolloid wound dressings or by means of a self-adhesive surgical film. Dressing changes with new administration of the fly larvae extracts according to the invention take place daily. example 1

Production of the fly larvae extract according to the invention

Larvae of the species Lucilia sericata and / or Sarcophaga carnaria were kept on fresh and not or only slightly contaminated horse meat and harvested shortly before entering the pupation stage. In a number of washing steps in aseptic solutions in decreasing concentration and in the last washing steps in sterilized NaCl solution, the larvae were largely sterile. The larvae were then decapitated, i.e. the front third was separated from the rest of the larval body. Both larval parts were immediately preserved separately on ice in a carrier medium. The larvae were then homogenized. This was done in several steps by mechanical comminution and homogenization using ultrasound. Attention was paid to constant cooling at about 4 "Celsius. After obtaining a homogeneous liquid, the extract was sterile filtered (Millipore filter). In the last processing step, the extract was aliquoted and frozen in liquid nitrogen. The permanent storage took place at about -21 ° C to -80 ° C.

Example 2

wound treatment

In an 82-year-old patient who has suffered from chronic recurrent ulcer curum for years, the fly larvae extract prepared according to Example 1, made from equal parts by weight maggots and physiological saline solution, was applied with 2 ml each. The ulcers are of venous origin and were influenced by the use of painkillers in the sense of vasculitis. At the beginning of the treatment, there were several ulcers, some of which were fibrinous, on both lower legs. After consultation with the patient, the treatment began with systemic administration of steroids and simultaneous local application of the fly larvae extract prepared according to Example 1 to promote debridement. It became comparative Fibrolan® ointment and aqueous solutions of the extract according to the invention are used. Fibrolan® ointment is a preparation on the red list containing plasmin from bovine plasma and deoxyribonuclease from bovine pancreas. In the case of the extracts from the larvae, a distinction was made between the extracts from the front part and the rear part of the larvae. The assignment was made at the beginning of the therapy and was maintained over the entire duration of the treatment.

It was shown that the occlusive application of the extracts according to the invention was clearly superior to the application with Fibrolan® ointment with regard to the debriding effect and the speed of wound closure. The success of the treatment was documented using color photographs.

The fly larvae extract prepared according to Example 1 was used in an 87-year-old patient who had been suffering from ulcera curum vasculitic genesis for years. No further ulcerations occurred after systemic steroid use. Local therapy was compared with Fibrolan® ointment and the extracts according to the invention. The ulcer that was treated with the extracts according to the invention initially showed a stronger necrotic and fibrinous coverage than the comparative ulcer that was treated with Fibrolan® ointment. After 8 days of treatment, there was a significantly faster debridement of the ulcer, which was treated with the extracts according to the invention, in comparison with the Fibrolan® ointment. The success of the treatment was documented using color photographs.

Claims

claims:

1 . Fly larva extract, obtainable from fly larvae, whereby the fly larvae are cooled and then homogenized and finally the undissolved components of the fly larvae are separated from the homogenate obtained.

2. Fly larvae extract according to claim 1, characterized in that an extraction medium is added before the homogenization.

3. fly larvae extract according to claims 1 or 2, obtainable from fly larvae of the genera Sarcophaga, Lucilia, Musca, Calliphora and / or Stomoxys or mixtures of representatives of these genera or from the species Lucilia sericata, Lucilia caesar, Lucilia cuprina, Sarcophaga carnaria,

Sarcophaga agyrostoma, Musca domestica, Calliphora erythrocephala, Calliphora vicina or Stomoxys caicitrans.

4. Fly larvae extract according to one or more of claims 1 to 3, characterized in that the fly larvae are 5 to 8 days old.

5. fly larvae extract according to one or more of claims 1 to 4, characterized in that the homogenization of the fly larvae in the frozen state at temperatures below 0 ° C or the homogenization and separation of insoluble constituents at temperatures from 0 ° C to

15 ° C, preferably from 0 ° C to 10 ° C, in particular from 2 ° C to 6 ° C.

6. fly larvae extract according to one or more of claims 1 to 5, characterized in that the homogenization is carried out by mechanical comminution or by ultrasound.

7. Fly larvae extract according to one or more of claims 1 to 6, characterized in that water, physiological salt solutions, buffers, electrolyte, sugar or protein solutions, emulsions or organic solvents are used as the extraction medium.

5

8. fly larvae extract according to one or more of claims 1 to 6, characterized in that an organic solvent is used as the extraction medium for the precipitation and subsequent extraction of the active ingredients.

10

9. fly larvae extract according to one or more of claims 2 to 8, characterized in that 0.1 gram to 500 ml of extraction medium, preferably 0.5 ml to 100 ml, very preferably 1 to 5 ml of extraction medium, are added per gram of wet weight of fly larvae.

15

10. fly larvae extract according to one or more of claims 1 to 9, characterized in that the separation of insoluble constituents takes place in the homogenate by centrifugation or filtration.

20 11. Fly larvae extract according to one or more of claims 1 to 10, characterized in that the fly larvae extract is preserved by freezing or lyophilization.

12. A method for producing the fly larvae extract according to one or

25 several of claims 1 to 11, characterized in that fly larvae are killed before pupation and homogenized with cooling and components which are not dissolved are separated from the homogenate obtained.

13. The method according to claim 12, characterized in that fly larvae 30 of the genera Sarcophaga, Lucilia, Musca, Calliphora and / or Stomoxys or

Mixtures of the genera are used or the species Lucilia sericata, Lucilia caesar, Lucilia cuprina, Sarcophaga carnaria, Sarcophaga agyrostoma, Musca domestica, Calliphora erythrocephala, Calliphora vicina or Stomoxys caicitrans can be used.

14. The method according to claims 12 or 13, characterized in that the fly larvae before the homogenization of secreted secretions and

Excreta of the larvae can be freed by washing.

15. The method according to one or more of claims 12 to 14, characterized in that an extraction medium is added before the homogenization and the homogenization is carried out mechanically or by ultrasound.

16. The method according to one or more of claims 12 to 15, characterized in that water, physiological salt solutions, buffers, electrolyte, sugar or protein solutions, emulsions or organic solvents are used as the extraction medium

17. The method according to one or more of claims 12 to 16, characterized in that the homogenization at temperatures below 0 ° C or at temperatures from 0 ° C to 15 ° C, preferably from 0 ° C to 10 ° C, in particular from 2 ° C to 6 ° C.

18. The method according to one or more of claims 12 to 17, characterized in that the separation of insoluble constituents is carried out by centrifugation or filtration.

19. Medicament, characterized by an effective content of the fly larvae extract according to claims 1 to 11 together with a pharmaceutically suitable and physiologically compatible carrier.

20. Use of the fly larvae extract according to claims 1 to 1 1 for the manufacture of medicaments for the therapy of superficial or deep chronic and acute wounds of any genesis.

5 21. Use according to claim 20, characterized in that a galenic composition is used for topical application on the skin, for example as a solution, suspension, powder, cream, liposomal formulations, gel, lotion, paste, spray or aerosol.

22. Use according to claims 20 or 21, characterized in that the fly larvae extract is used in a concentration of 0.1% by weight to 100% by weight in the pharmaceutical composition.

23. Use according to claim 21, characterized in that a

15 galenic composition for transdermal applications is available as a plaster.

24. Use according to claim 21, characterized in that the fly larvae extract according to claims 1 to 11 in a wound dressing made from 0 gauze, from alginates, from hydrocolloidal materials, foams or

There are silicone coatings, each coated or soaked with the fly larvae extract mentioned.

25. Use according to claim 21, characterized in that the 5 fly larvae extract according to claims 1 to 1 1 in galenic

Preparations are used in which the fly larvae extract is contained in inactive form and then apply the preparation to the wound and the fly larvae extract is activated by adding specific substances. 0