WO2010079209A2 - Compositions for treating wounds and skin conditions - Google Patents

Abstract

The invention is the composition and the use of the composition substantially consisting of a siliceous sorbent and a medicinal agent immobilized thereon, wherein the siliceous sorbent is aerosil and the medicinal agent is serrathiopeptidase, and where the composition may be used to particular advantage for treating and promoting the healing of acute and chronic, open and closed wounds, burns, skin infections, for reducing bleeding, for wound cleansing, for removal of necrotic tissue, for reducing scar-formation, for treating skin conditions, and for cosmetic skin cleansing by applying the composition to the wound, the tissue or the skin.

Description

Compositions for treating wounds and skin conditions

Background of the Invention

This invention relates to methods of treating and healing wounds, skin infections, and skin conditions and for reducing wound bleeding and scar formation.

Maheshwari et al . (AAPS Pharm Sci Tech 2006;7(3), Article 76) disclose a pluronic gel comprising tetracycline. Serrathiopepti- dase or Aerosil 200 can be present in minor amounts as additive,

Summary of the Invention

The present invention is based on the priority application US 61/143178 of 8 January 2009 which is incorporated herein in its entirety.

The invention is a composition substantially consisting of a siliceous sorbent and a medicinal agent immobilized thereon, wherein the medicinal agent is serrathiopeptidase . The sorbent in one particular embodiment may be aerosil. The composition may be used to particular advantage for the treatment of wounds, burns, infections, peritonitis, sepsis, for reducing wound haemorrhage, for reducing scar-formation, for treating skin conditions such as psoriasis, rashes, eczema, and lupus, and for cosmetic purposes, and it may have particular advantage as an antimicrobial agent, for improving frequency of wound closure, for accelerating wound healing and for reducing wound pain, inflammation and oedema. The present invention is defined in the claims .

In particular the present invention relates to a composition comprising a siliceous sorbent and a medicinal agent immobilized thereon, wherein the siliceous sorbent is aerosil and the medicinal agent is serrathiopeptidase, where aerosil is fine pyro- genic silica that consists of hydrated globules with an average silica particle radius of between 1 and 50 nm or aerogel of polysilicic acid SiO2 x H2O, the water content being up to 20% by weight, and where serrathiopeptidase (CAS number 37312-62-2) is a proteolytic enzyme isolating from the nonpathogenic intes- tinal bacterium Serratia E15.

Detailed Description of the Invention

Definitions and Abbreviations

The following definitions are given for the purpose of facili^¬ tating understanding of the inventions disclosed herein. Any terms not specifically defined should be interpreted according to the common meaning of the term in the art.

As used herein, the term "aerosil" shall refer to ci_iccr ciox- _Jo in the forms of fumed silica, pyrogenic silica, a;iυrphυup art), V" rυi is

1 uzanes α,/1 aerogel of polysilicic acid.

As UoGO

£c∑c£3 LO any typo of oil- ,^r,d qe^'c, 'ric]"iLno e,o, q"l_o

si/.d, -^e^oge., parous ^i . ice, p ^■) . ynethy J si 5Xs'e B-=, we J st> thei ' oe~ rive L iVGS ourh cs o±lylcLod funoo oilicc ^;.d J^spo_s^J s_l^ca. As used herein, the term "wound" shall refer to an injury to the body, which may result from a disease or a disease-associated condition, burns or frost, infections, radiation, chemicals, violence, accident, or surgery that typically involves laceration or breaking of a membrane, including but not limited to the skin, and may involve damage to underlying or surrounding tissues. The term "wound" shall include acute wounds and chronic wounds, where chronic wounds include but are not limited to diabetic foot ulcers, venous and arterial leg ulcers, pressure ulcers and wounds caused by ischemia or radiation. The term "wound" shall also include both internal and external wounds. The term "wound" shall also include open and closed wounds. As used herein, the terms "individual" and "patient" shall refer to animals and humans.

As used herein, the term "animal" has its ordinary meaning, and include, but are not limited to non-human primates, canine, feline, rodent (racine, murine, lupine, etc.), equine, bovine, ovine, caprine, and porcine species.

As used herein, the terms "treatment" and "treating" have their ordinary meaning and shall include the meanings: prophylactic, curative, prevention, healing, and managing.

As used herein, the terms "reduce", "improve", "promote", "facilitate" and similar comparative statements is meant to compare the condition where the patient is treated with the invention to the untreated state.

As used herein, by the term "therapeutically effective dose" is meant a dose that produces the desired effect for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques.

Compositions

The composition substantially consists of a siliceous sorbent and a medicinal agent immobilized thereon, wherein the siliceous sorbent in one preferred embodiment is aerosil ("fumed silica" or "pyrogenic silica" or aerogel of polysilicic acid SiO2* x H2O) and the medicinal agent is serrathiopeptidase .

Silica or silicon dioxide is a natural component of bone and connective tissue, it is present in most food items, it is often recommended as a food supplement, and it has been used by the pharmaceutical industry as an excipient for solid dosage forms for many years. Ingested silica rapidly penetrates the intestinal wall and becomes distributed throughout the whole extracellular fluid. In a review of a number of toxicological studies on the effects of ingested silica on man, the WHO (1974) concluded that the acceptable daily intake for man of silicon dioxide is not limited.

One form of Aerosil is pyrogenic or fumed silica, which is prepared by vapour-phase hydrolysis of a silicon compound, such as silicon tetrachloride. The product itself is usually a submicron, fluffy, light, loose, bluish-white, odorless and tasteless amorphous powder which is commercially available from a number of sources, including for example Cabot Corporation (under the trade name Cab-O-Sil) ; Evonik-Degussa, Inc. (under the trade name Aerosil); Huber Engineered Materials (Huber GLlOO and GL200); Wacker (Wacker HDK®; and E.I. DuPont & Co. Pyrogenic silica is also known as fumed silica, silicon dioxide fumed, colloidal silica, colloidal silicon dioxide, light anhydrous silicic acid, silicic anhydride, among others. A variety of commercial grades of pyrogenic silica are produced by varying the manufacturing process. The relationship between the radius - A - of the silica particle and the surface area can be described by this formula, which is included for informative purposes only: Specific surface area (Ssp) = 3 x 1000/2.3R (m2/g) , where R is the radius of the particle in nm.

A second form of Aerosil is aerogel of polysilicic acid SiO2* x H2O, where aerogel essentially is the dry, low-density, porous, solid framework of a gel. Aerogels are open-porous and typically have pores in the range of <1 to 100 nanometers in diameter and usually <20 nm.

Serrathiopeptidase, also named serratiopeptidase, Serathiopepti- dase, serratiapeptase, serratia peptidase, serrapeptidase or CAS 37312-62-2, is a proteolytic enzyme isolated from the nonpathogenic enterobacteria Serratia E15 found in silkworms. It has a history of homeopathic use in Japan and Europe and is available in many countries as prescription or OTC medicine for the treatment of pain and inflammation. In the US it is sold as a dietary supplement. Serrathiopeptidase can be given orally as tablets and is absorbed in the gut. Side-effects associated with oral intake can be gastric discomfort and nausea.

In one embodiment, Aerosil is fine pyrogenic silica that consists of hydrated globules of an average silica particle radius of between 1 and 50 nm, where 1 nm corresponds to an average surface area of 1300 sq. meters per gram and 50 nm corresponds to 26 sq. meters per gram. Non-limiting examples of forms of pyrogenic silica that are readily commercially available and that may be used in SertaSil, are hydrophilic pyrogenic silica (also called fumed silica or snorrnouε anhyirc_^s collciial sili:cn ii ^■■ with surface areas ranging from 90 to 600 sq.m/g, e.g. an average surface area of at least 90 sq.m/g, an average surface area of at least 150 sq.m/g, an average surface area of at least or greater than 200 sq.m/g; preferably about 300 sq.m/g and up to 600 sq.m/g. In a preferred embodiment the pyrogenic silica has an average radius of between 3 nm, corresponding to 435 sq.m/g and 15 nm, corresponding to 86 sq.m/g (e.g. Aerosil 90, Aerosil 150, Aerosil 200, Aerosil 300, Aerosil 380 or Aerosil 400, where the number indicate the average sq. meter surface area per gram) . In a still further preferred embodiment, the py- rogenic silica has an average radius of approximately 4.35 nm corresponding to an average surface area of 300 sq. meter per gram.

In further embodiments the aerosil component of the inventive composition comprises derivatives of pyrogenic silica such as silylated fumed silica and dispersed fumed silica. E.g. sily- lated fumed silica is obtained by the sililation of initial fumed silica (e.g. reaction with silanes: methyl or dimethyl alkoxy or chloro silanes) . Dispersed fumed silica can be obtained in a similar manner by processing of normal fumed pyrogenic silica.

In further embodiments, Aerosil consists of aerogel of polysilicic acid SiO2* x H2O with the water content being up to 20% by weight and with a preferred embodiment of the water content not exceeding 10%. In the production of the aerogel, additive may be added to produce a flexible pad that can adapt to the wound surface.

For these embodiments, the preferred ratio of Aerosil to serra- thiopeptidase is within the following amounts, % by weight: aerosil 99.9 to 90 serrathiopeptidase 0.1 to 10, or it may be up to the amount bindable by the sorbent (absorption limit) . However, lower or higher amounts of serrathiopeptidase can also be used depending upon the clinical need.

In an alternative embodiment, the siliceous sorbent is silica gel that for example may be based on pyrogenic silica, aerogel, porous silica, polymethylsiloxane, as well as their derivatives such as silylated fumed silica and dispersed silica.

For this embodiment, as in any inventive combination of the silica sorbent and serrathiopeptidase, the preferred ratio of silica gel to serrathiopeptidase is within the following amounts, % by weight: silica gel 99.9 to 90 serrathiopeptidase 0.1 to 10, or it may be up to the amount bindable by the sorbent (absorp- tion limit) . However, lower or higher amounts of serrathiopepti- dase can also be used depending upon the clinical need.

In an alternative embodiment, the siliceous sorbent comprises porous silica and their derivatives, silica micro- or nano- spheres and their derivatives, or silica nanotubes and their derivatives, where the size of the pores of porous silica or the diameter of the nanotubes are preferably between 1 nm and 50 nm, but other diameters can also be used.

For this embodiment, the preferred ratio of sorbent to serra- thiopeptidase is also within the following amounts, % by weight: Sorbent 99.9 to 90 serrathiopeptidase 0.1 to 10, or it may be up to the amount bindable by the sorbent (absorption limit) . However, lower or higher amounts of serrathiopeptidase can also be used depending upon the clinical need.

Further equivalent compositions of the present invention are disclosed in the claims which all fall in to the same inventive concept of using a siliceous sorbent together with serrathiopeptidase. All of the present inventive compositions can be used for the treatment wounds and skin diseases etc., e.g. as disclosed in any one of the claims for any specific composition.

Mode of Action

Table 1 outlines the main phases of the wound healing process and is only included as a useful reference for the description below, and should be in any way be limiting for the invention. Both ingredients of the inventive composition have beneficial effects on wound healing and their ratio is optimised to act in synergy and to complement each other to promote wound cleansing and healing. Based on the known actions of the ingredients, the mode-of-action of the composition appears to be the following:

• It creates a physical barrier over the wound that reduces adhesion of dressings as well as the penetration of micro organisms and other contaminants into the wound.

• It activates local haemostatic factors producing a haemo static effect.

It breaks down serotonin, bradykinin and histamine; this:

• produces an anti-inflammatory effect.

• reduces vasodilatation and capillary permeability, which reduces and prevents oedema.

• reduces pain by inhibiting the pain stimulating effects of bradykinin and histamine.

It stimulates monocyte and lymphocyte migration into the wound to facilitate wound healing.

It has proteolytic activity that lyses necrotic tissue and breaks down exudates and proteins.

It sorbs proteins from the wound, e.g. pus, toxins from micro-organisms and material from lysed exudates, necrotic tissue, and fibrin.

It reduces the viscosity and the rarefaction of exudates from the wound. This improves the conditions for passive wound drainage. As a result this ensures that the correct level of moisture at the surface of the wound is maintained and that maceration of the tissues surrounding the wound is prevented. Clinical experience shows that the composition does not cause over-drying of the wound.

It binds microorganisms, e.g. bacteria, protozoa, fungus and viruses and exerts an anti-microbial effect that effectively can treat infections. All bacteria are bound without selectivity and it will therefore be effective against antibiotic resistant strains and it will be difficult for bacteria to develop resistance.

It inhibits plasmin inactivators, whereby it increases the fibrinolytic activity of plasmin, resulting in the breakdown of fibrin clots. Plasminogen, the precursor of plasmin, is only released by endothelia cells after bleeding is con trolled and the activation of plasmin will therefore not interfere with coagulation.

Inflammatory Phase (Immediate to 2-5 days)

Haemostasis (bleeding is stopped by the formation of blood clots)

Migration of leucocytes into wound area (the invasion of monocytes and lymphocytes into the wound is essential for healing) Inflammation and oedema

Debridement (necrotic, i.e. nonDviable tissue is re moved)

Infections (infections are fought by the immune sys tern)

Pain (the wound becomes sore due to the inflammatory process and the release of bradykinin and histamine)

Proliferative Phase (2 days to 3 weeks)

Granulation (fibroblasts lay bed of collagen and new capil laries are produced)

Contraction (wound edges pull together to reduce defect) Epithelialization (new skin cells migrate across the wound to form the new skin)

Remodelling Phase (3 weeks to 2 years)

Collagen remodelling (new collagen forms which increases tensile strength to wounds)

Table 1. Phases of Wound Healing

Clinical Profile

Based on observations made during the treatment of 150 patients

SertaSil has been found to have the following properties.

SertaSil accelerates one or more stages of the wound healing process and reduces the time to wound closure:

In a 266 patients study SertaSil was shown to accelerate the time to wound healing for a wide range of acute and chronic wounds and burns, including for example acute wounds (post operative and traumatic) , chronic wounds (venous and arterial leg, diabetic foot, and pressure ulcers), thermal burns (e.g. Ill and IV degree) and infected wounds (acute and chronic wounds, burns and skin infections e.g. carbuncles, abscesses, furuncles, phlegmons, necrotic erysipelas, purulent paraproctitis) .

SertaSil increases the incidence of complete wound closure:

SertaSil has been shown to increase the number of chronic wounds closures. All wounds treated with SertaSil have reached complete closure, including wounds that had been treated for a long time with a range of other wound care products without closing. SertaSil improves the quality of healing:

An improved wound healing process will lead to reduced scar- formation and in several patients that were treated with SertaSil it has been observed that scar-formation was less than anticipated. SertaSil may therefore be used to reduce scarring associated with a normal wound healing process as well as hypertrophic scarring and keloidal scarring.

SertaSil can treat wound infection :

SertaSil has been shown to effectively treat a broad range of bacterial infections in acute and chronic wound, burns and skin conditions, including bacterial strains that were resistant to antibiotic treatment.

SertaSil effectively debrides a wound:

SertaSil has been shown to perform an effective mild debridement, i.e. removal of necrotic tissue, of necrotic tissue on wounds without causing tissue irritation. It can remain on the wound until complete closure.

SertaSil reduces wound pain:

SertaSil has in patients and animals been observed to reduce pain associated with the wound, probably due to its breakdown of bradykinin and histamine as well as to its anti-inflammatory and anti-oedematous properties.

SertaSil controls wounds exudates:

SertaSil lyses and sorbs proteins from the wound, e.g. pus, bacterial toxins, exudates, necrotic tissue, and fibrin and has been observed to reduce and control this release of exudates.

SertaSil controls the wound moisture balance:

SertaSil reduces the viscosity of exudates and facilitating passive wound drainage. These effects have been observed to avoid a wet wound surface area and to avoid maceration of surrounding tissue, thereby ensuring an optimal wound moisture balance.

SertaSil reduces wound inflammation and oedema:

SertaSil has been observed to reduce wound inflammation and oedema rapidly upon administration and thereby to reduce patient discomfort and to assist wound healing.

SertaSil reduces bleeding:

Upon application to the wound, it has been observed that SertaSil reduces bleeding.

Figures and Examples

The present invention is further illustrated by the following examples and figures.

Figures :

Fig. 1 Effects of treatment for 24 hours with SertaSil on venous leg wounds in patients;

Fig. 2 Faster wound closure following treatment with SertaSil as shown by observations of wound surface area (sq.cm) for the first 10 days following start of treatment. Each group included 30 patients and the distribution of wound types was 80% carbuncles, abscesses, and cysts and 20% infected wounds for all three treatment groups. Treatment groups were SertaSil, Gentamicin and Ioddicerin. ***:P<0.001 relative to Ioddicerin;

Fig. 3 Acceleration of the wound healing process following treatment with SertaSil. The time to reach defined stages in the wound healing process was recorded. Each group included 30 patients and the distribution of wound types was 80% carbuncles, abscesses, and cysts and 20% infected wounds for all three treatment groups. Wound surface area measurements for the same group of patients are included for comparison. Treatment groups were SertaSil, Gentamicin and Ioddicerin. ***:P<0.001 relative to Ioddicerin;

Fig. 4 Acceleration of the wound healing process following treatment with SertaSil. The groups include a broad range of acute and chronic wounds and burns. The wounds were treated for 3 days with SertaSil (n=88), Gentamicin (n=90) or Ioddicerin (n=88) . The time to reach defined stages in the wound healing process was recorded, and shows that the time to reach Wound Cleansing; Onset of Granulation and Epithelialization was significantly shorter for SertaSil compared to Gentamicin and Iod- dicerin. ***:P<0.001 relative to Ioddicerin;

Fig. 5 Acceleration of the wound healing process in patients with diabetic foot ulcers following treatment with SertaSil. The patients represent a subgroup of the patients in Fig. 4. The groups include SertaSil (n=10), Gentamicin (n=13) or Ioddicerin (n=13) . ***:P<0.001 relative to Ioddicerin;

Fig. 6 Acceleration of the wound healing process in patients with venous leg ulcers following treatment with SertaSil. The patients represent a subgroup of the patients in Fig. 4. The groups include SertaSil (n=20), Gentamicin (n=18) or Ioddicerin (n=16) . ***: P<0.001 relative to Ioddicerin;

Fig. 7 Aseptic wound was modelled by administering 1 ml 10% calcium chloride solution into the subcutaneous layer in the rat. The images show at the time course and process of healing following treatment with SertaSil vs. Gentamicin;

Fig. 8 Aseptic wound was modelled by administering 1 ml 10% calcium chloride solution into the subcutaneous layer in the rat, and the days to reach four stages in the wound healing process following treatment with SertaSil, Gentamicin, and Control (no treatment) was recorded. N= 15 per group. In the SertaSil group wound cleansing was achieved in 3 days compared to 7 days for the Gentamicin group and 10 days for the Control group. The bottom graph "Days to complete wound closure" shows that the SertaSil group demonstrated a 25% faster wound closure compared to the other groups;

Fig. 9 Wound surface area in rats Day 13 following opening of the abscess for the same study as Fig. 8. N= 15 per group. The surface areas of the wounds were similar at Day 1 following the opening of the abscess, but at Day 13 the wounds treated with SertaSil were significantly smaller compared to the other groups and were almost completely closed.

Fig. 10 Body weight in rats at Day 1 and Day 24 for the same study as Fig. 8. N= 15 per group. Rats receiving SertaSil showed a significant increase in body weight and this indicates that only this group returned to their normal growth curve. The study is conducted in young, growing animals and a return to normal growth is a strong indicator of good health status.

Examples :

The composition of the invention hereinafter referred to as Ser- taSil can be obtained in the following way.

Example 1: The method of obtaining SertaSil.

The method of obtaining consists in the formation of a solution or a suspension of serrathiopeptidase in a proper solvent in a predetermined proportion, that is added to a solid adsorbent (aerosil, polymethylsiloxane and so on) by means of impregnation, and subsequent lyophilic, vacuum or air drying at temperatures below 40-45⁰C (e.g. 30⁰C) until obtaining a light air-dry white powder or as a result of mechanical dispersion of an active substance on the adsorbent surface.

Further examples of preparing SertaSil are set forth below.

Example 2: Additional method of obtaining SertaSil

100 mg of serrathiopeptidase are dissolved in 100 ml of distilled water and stirred for 30 to 60 minutes at room temperature. Then Aerosil A-300 in the amount of 10 g is added into the solution while the stirring is continued. The mixture thus obtained is dried in vacuum at a temperature of 30⁰C. A granular white powder is formed.

Example 3: Additional method of obtaining SertaSil

16 mg of serrathiopeptidase are dissolved in 10 ml of distilled water and stirred for 30 to 60 minutes at room temperature. Then 1 g of aerosil A-300 is added into the solution while the stirring is continued. The mixture thus obtained is dried in vacuum at a temperature of 40⁰C. A granular white powder is formed.

A particular preferred embodiment is that solutions of serrathiopeptidase containing no more than 0.5% of active substance should be used for making the preparation SertaSil. Example 4: Possible routes of administration of SertaSil The administration of SertaSil and other embodiments of the present invention are in one preferred embodiment topical application of SertaSil or the composition to the internal or external wound, body area or area of the skin in need of treatment, cleansing, healing, reduction of bleeding or reduction of scar- formation. For topical application, preferred embodiments will be as a powder, gels, cremes, ointments, solutions or sprays or combinations of these with bandages, dressings, sutures, gauze, cellular matrixes and other wound treatment products. For topical application, SertaSil or other embodiments of the invention may in one embodiment be applied as a layer, where the layer in thickness can range from a very thin, e.g. 0.001 mm to thick, e.g. 10 mm, but other configurations may be used depending upon the location of area. The duration of applying SertaSil and other embodiments of the invention can range from a single treatment to continuous treatment until complete healing of the wound, elimination of the infection or symptomatic or curative treatment of a skin condition.

In other preferred embodiments SertaSil and other compositions can be applied as liposome/aerosol, intraocular, parenteral, oral, intraventricular, intratecal, intranasal, intravenous, in- tra-arterial, intramuscular, subcutaneous, intratumal, or by any other suitable means, including by pro-drugs. The dosage administered is dependent upon the age, clinical stage and extent of the disease or genetic predisposition of the individual, location, weight, kind of concurrent treatment, if any, and nature of the pathological condition. The dose may vary from 0.001 mg per individual to 20 grams or more per administration. The effective delivery system useful in the method of the present invention for administrations other than topical application may be employed in such forms as liposomal aerosol, capsules, tablets, liquid solutions, suspensions, or elixirs, for oral administration, or sterile liquid forms such as solutions, suspensions or emulsions for injection. The duration of applying SertaSil and other embodiments of the invention can range from a single treatment to continuous treatment until complete cure or remission of the condition. Example 5: Treatment of traumatic wound with SertaSil The patient M., male, born in 1964, was admitted to a surgical department with a two week old gun bullet wound complicated with inflammatory infiltration of the anterior abdominal wall. On the examination an infiltration 11.0 x 6.5 cm in the left iliac area was found. In the infiltration centre there was a bullet hole 1.5 x 0.7 cm, with a wound channel located from the outside inside, about 3.5 cm long. Tissues around the wound channel were necrotized, puffed up, dark. At the bottom of the wound channel a bullet was found. The patient was operated - the bullet was removed, necrectomy was performed. In the postoperative period he received the following medicines: cephtriaxon 1.0 g x 2 per day, intramuscularly, during 5 days and nights, sodium diclofenac 3.0 ml intramuscularly x 1 per day, during 3 days and nights, lidase 64 units x 1 per day, intramuscularly, UHF on the infiltration area during 7 days and nights, daily bandaging with ioddycerin, dioxysole. The performed treatment had no substantial effect: the infiltration retained its previous sizes, and no evidence of wound cleansing and healing was observed. The patient rejected outright the proposed surgical treatment of excising the infiltration. It was decided to cure using SertaSil by means of administering it into the wound channel after pre- treatments with solution of 3% hydrogen peroxide. Over the next 48 hours the wound channel was completely cleansed from necrotic masses, over 7 - 8 days and nights after the treatment began the infiltration became 3.0 x 2.5 cm, the wound channel got superficial, the wound was filled up with granulations and was actively epithelializing. The wound healed completely and the infiltration resolved over 14 days and nights after the beginning of treatment with SertaSil. The patient was inspected after one month - a surface scar about 1.0 x 0.4 cm was found in the area of the former wound and infiltration. The patient made no complaints .

Example 6: Treatment of thermal burn wound with SertaSil The patient B., male born 1948, was hospitalized with a two week old thermal burn of the right thigh and shank, 4%, III B-degree. The state of the patient is moderate, body temperature is 38.5°C, the whole surface of the skin and subcutaneous basis in the affected area is necrotized with dense scab. Operative ne- crotomy was performed. SertaSil was applied to the wound surface. After 2 hours the temperature decreased. A complete wound cleansing from necrotic masses took place over 48 hours. Besides SertaSil, the patient received only anaesthetics and vascular agents, because he was allergic to antibiotics of the most commonly used groups. SertaSil was applied for a total of 3 days. Henceforth bandages with methyluracil ointment were applied every 48 hours until complete healing. The wounds healed completely over 17 days.

Example 7: Treatment of venous leg ulcer with SertaSil The patient K., born in 1935, was hospitalized according to the diagnosis - chronic venous insufficiency of lower extremities, complicated with trophic ulcer, covered here and there with necrotic tissues. B. Fragilis, E. CoIi 108 B Ig. tissue was obtained in the microbiological examination. After two bandages with SertaSil no microbes were obtained, the ulcer was completely cleansed over 3 days and nights. Henceforth methylu- racilic ointment was used for bandages until the complete healing, which came after 17 days and nights.

Example 8: Treatment of Fouriner's Phlegmon with antibiotic re- sistant bacteria by SertaSil

Patient K., male, 59 years old, was hospitalized 13 days after becoming ill. Upon arrival, it was discovered that he suffered from diabetes. The clinical diagnosis of his illness was acute putrefactive pelviorectal periproctitis; acute rotting (decomposing) of tissue around the rectum and in the pelvic region; Fournier's phlegmon with acute gangrene in the genitalia and toxaemia from anaerobe bacterial infection.

The patient was immediately put on i.v. metronidazol and gati- floxacyn (antibiotics) and underwent surgery to aerate the infected area and to disinfect it. The wound was treated with "Plason", a medical device for the exogenous application of nitric oxide (NO) . Nitric oxide disinfects open wounds from bacteria, viruses, and mould.

An inoculation, made during surgery of the infected area, showed the presence of Staphylococcus aureus. It did not respond to any antibiotics, including metronidazol and gatifloxacyn . It was therefore decided to maintain the patient on i.v. treatment with metonidazol and gatifloxacyn to prevent follow-on infections, but not to include other antibiotics as they were ineffective anyway.

On Days 1 and 2 after surgery, the patient is continuously receiving i.v. antibiotics, and the wound is cleaned daily with hydrogen peroxide and nitric oxide. However, none of this can combat the infection and the patient is deteriorating. On Day 4, it is decided to treat with SertaSil and by Day 7 the infection is gone and the tissue and wound edges are healthy and are able to begin a normal healing process. The patient continuously receives SertaSil until Day 17 and is discharged from the hospital Day 66. The healing process was prolonged because the patient suffered from diabetes.

Example 9: Treatment of non-healing venous leg ulcer with Serta- SiI.

A male patient, born 1952, suffered from varicose disease of the lower extremities, post trombophlebitis syndrome in both lower extremities, and an indurative ulcer. He had chronic venous insufficiency of lower extremities and had developed trophic ulcers on the left (lower) leg, which would not heal.

Over a two year period doctors at another hospital had unsuccessfully tried a number of different treatments to heal the wounds. The first group of treatments were topical application of antibiotics or antiseptics for various durations of time to promote wound cleansing followed by methyluracil or actogegin to promote epithelialization .

• Ioddicerin (polyvinylpyrrolidone with iodine water solu tion, an antiseptics)

• Dioxizole (gel-like preparation consisting of a sorbent with the antibiotic agent dixoine (hydroxymethylquinoxilindiox ide) and the local anaesthetic agent Trimecaine)

• Gentamicin in a polymethylsiloxane gel (antibiotic)

• Boric acid (an antiseptics) For the second and third phases of the wound process the following drugs were used:

• Methyluracil ointment (accelerates processes of cellular re generation by stimulating cellular and humour factors of im munity, provides anti inflammation effect by inhibiting the activity of proteolytic enzymes; possesses anabolic and pho toprotective effect.)

• Actovegingel (deproteinized hemoderivative of calf blood that is used for treating disturbances of peripheral (arter ial, venous) blood flow and sequels resulting from these disturbances)

• Miramistin (quaternary ammonium antiseptic for disinfection of skin and mucous membranes)

These treatments resulted in wound cleansing and granulation, but only limited epithelialization took place and the wounds did not heal .

Next, the following treatments were used:

• Phlebotonics (detralex 1000 mg/day for 2-3 month) (purified flavonoidic fraction from plants, which are recommended for treating venous leg ulcers)

• Lyoton 1000 gel 3 times per day (drug for the treatment of venous leg ulcers)

• Trental (pentoxifylline, reduces blood viscosity and improve blood flow by altering the rheology of red blood cells) and Vasonit (naphazoline, a vasoconstrictor to reduce swelling)

• Elastic compression combined with dimedrol (a histamine Hl antagonist used for dermatoses and pruritus)

• Cardiomagnil (antiaggregant therapy)

• Thiotriazoline (antioxidant with antiDischaemic and membrane stabilising properties)

• Ketanov (NSAID for the treatment of inflammation and acute pain)

These treatments were also unsuccessful, where after magnetotherapy, phonophoresis (ultrasound) , and laser therapy were tried, but again these did not lead to wound closure.

The patient was transferred to the Inventor' s hospital and treatment with SertaSil was initiated November 20, 2008. The wounds were cleaned and SertaSil was applied as a 3 mm topical layer of powder on the wound and covered by dressings. Dressings were replaced daily. The patient received SertaSil daily for the first 4-5 days to promote wound cleansing and to initiate intensive epithelialization followed by 2 weeks with one treatment every 2-3 days. At the start of treatment (Nov. 20, 2008), the wound area was 23.57 sq.cm and by Day 50 (Jan. 8, 2009), it was 0.36 sq.cm. By Day 83 the wounds were completely healed for the first time in 2 years, demonstrating that SertaSil is effective in the treatment of non-healing wounds.

Example 10: Comparative study of SertaSil, Gentamicin and Iod- dicerin on wound healing in patients

Summary of Findings: The effects of SertaSil on wound healing were compared to Gentamicin and Ioddicerin in a clinical study involving 266 patients. The wounds were primarily trophic ulcers caused by pancreatic diabetes and venous insufficiency of lower extremities, carbuncles, infected heat burns of degree II B - III, and infiltrations of postoperative wounds. SertaSil was applied as a 3 mm layer of powder to the wound for the first 3 days and nights. Gentamicin (gentamicin in a polymethylsiloxane gel) was applied as a 3 mm layer of gel over the wound for the first 3 days and nights. Control treatment consisted of the antiseptic Ioddicerin (polyvinylpyrrolidone with iodine water solution) for the first 3 days and nights. In case further treatments were needed, bandages with methyluracilic ointment were applied every 24 hours either until full healing or (more often) until the formation of active granulations and marginal epi- thelization (i.e. till active tendency to healing) .

SertaSil demonstrated a clear and significantly superior wound healing effects compared to Gentamicin and Ioddicerin. The number of days to wound cleansing was 3.01 ± 0.87 for SertaSil compared to 7.00 ± 1.13 and 7.95 ± 1.14 for Gentamicin and Ioddicerin, respectively. Time period to onset of granulation were for SertaSil, Gentamicin and Ioddicerin: 4.45 ± 0.83, 9.19 ± 1.42 and 10.33 ± 1.55 days, respectively and number of days to epithelialization, i.e. active healing were: 7.82 ± 1.06, 14.12 ± 1.92 and 16.44 ± 2.69, respectively.

SertaSil did not cause any severe adverse reactions. In 3 patients with wide open trophic ulcers located on top of nerve fascicle (nerve bundles) treatment had to be discontinued due to painful symptoms. No other adverse reactions were noticed.

SertaSil demonstrated pronounced anti-microbial effect and effectively treated 11 carbuncles. Samples for bacteriological analysis were taken from the carbuncles, and seven of these tested positive for Staphylococcus aureus, one for Pseudomonas aeruginosa and for the remaining three the infective agent was not identified.

The findings demonstrate that SertaSil compared to Gentamicin and Ioddicerin for a wide range of acute and chronic wounds and burns accelerates several stages of the wound healing process, reduces the time to wound closure, can effectively treat wound infections and does not cause any serious adverse effects. SertaSil is therefore a promising new treatment for wound healing of both infected and non-infected wounds in humans and animals.

Example 10 continued: Full Study Report

The purpose of the study was to compare the effects of SertaSil on the wound healing process in patients with acute and chronic wounds and burns to the antibiotic Gentamicin and the antiseptic Ioddicerin. These reference treatments were chosen because most of the wounds were also infected.

Method Description

Pa tien t popula ti on

The study included a total of 266 patients. Patients receiving SertaSil were randomly selected as they entered the hospital. The Gentamicin and the Ioddicerin groups were formed in retrospect from hospital files that had been established to allow such comparative studies. The patients were selected based on primary pathology, concomitant diseases and age with the primary pathology as the principal criterion. Patients with advanced diabetic foot ulcers or venous leg ulcers that required surgery were not included. Patient demographics are shown in Table 2.

Table 2. Patient demographics

Table 3 lists the main pathologies in each of the three treatment groups. These were primarily trophic ulcers caused by venous insufficiency of lower extremities and diabetes, carbuncles, phlegmons and infected heat burns of degree II B - III, and infiltrations of postoperative wounds. Table 4 lists concomitant pathologies.

Treatments

Dressings were replaced daily. Wounds were cleaned with an antiseptic (mostly 3% hydrogen peroxide) and necrotomy was performed if needed. SertaSil was applied as a 3 mm layer of powder to the wound for the first 3 days and nights. Gentamicin (trade name Gentaxane, consisting of gentamicin in a polymethylsiloxane gel) was applied as a 3 mm layer of gel over the wound for the first 3 days and nights. loddicerin treatment consisted of bandages with loddicerin (polyvinylpyrrolidone with iodine water solution) for the first 3 days and nights. After the initial treatment period of 3 days, further care, if required, consisted of bandages with methyluracilic ointment that were applied every 24 hours either until full healing or (more often) until the formation of active granulations and marginal epithelization (i.e. active tendency to heal) .

Wound measurements

Published clinical studies have shown that the time to complete the early phases of wound healing is predictive of time to wound closure, and it was therefore decided to measure the number of days it took for a wound to reach three well-defined stages in the early wound healing process. These measures would be inde- pendent of initial wound size and avoid entry criteria based on wound size. In order to validate these measures, it was decided for a subgroup of wounds of similar pathology and initial wound size to measure the wound surface area over several days to enable comparison between the measures.

Therefore, in all patients the number of days to reach 1) "Wound Cleaning" defined as the wound being free from pus and fibrino- genous thickenings; 2) "Onset of Granulation" defined as the appearance of active granulations; and 3) "Epithelialization" defined as "full wound cleaning and stabile progressive epitheli- zation with active reducing of wound" were recorded. In a subgroup of patients the area of the wound defect, its perimeter and average diameter were determined by applying PE film on wound and surface. The wound circumference was copied onto the film and the lengths at all sides were measured in mm. The data was entered into the computer program SigmaScanPro and calculated. For the Gentamicin and Ioddicerin groups, the data was obtained from their hospital file. For this subgroup, each treatment group included 30 patients and the distribution of wound types was 80% carbuncles, abscesses, and cysts and 20% infected wounds for all three treatment groups.

Bacteriological measurements

For patients with carbuncles samples were taken and analysed at the hospital laboratory.

Table 3. Types of wound pathologies treated

Table 4. Concomitant pathology of patients. Some patients expressed several additional pathologies, which explain why the total number of pathologies can exceed the total number of patients .

Statistical analysis

Data were analysed statistically by two-way ANOVA with Fischer' s LSD post-hoc test using the program package Systat 8.0.

Ethical permissions

The study was approved by the Hospital Ethical Board in accordance with the Laws of Ukraine.

Results

Wound Surface Area

In a subgroup of patients with comparable wound sizes, the wound surface area was measured at 4 time points (Table 5 and Fig. 2) . Each group included 30 patients and the distribution of wound types was 80% carbuncles, abscesses, and cysts and 20% infected wounds for all three treatment groups. At Day 1 upon start of treatment, there were no differences between the treatment groups (F (2, 87) =2.4 ; P=NS), but at Day 5 (F (2, 87) =229.9; P<0.001); Day 7 (F (2, 87) =40.3; P<0.001) and Day 10 (F (2, 87) =129.6; P<0.001), it was found that the group receiving SertaSil had highly significantly smaller wound surfaces, and that these patients reached wound closure much faster than patients treated with Gentamicin or Ioddicerin.

Table 5. Wound surface (sq.cm) at Days 1 to 10 following start of treatment. N=30 per group.

Fig. 3 shows for the same group of patients the healing process as measured by the day when the patients reach each of three pre-defined wound healing stages. There is a significant difference for all three measurements: Wound Cleansing: (F (2, 87)=158.5; P<0.001); Onset of Granulation: (F(2, 87)=189.7; P<0.001); and Epithelialization: (F(2, 87)=179.0; P<0.001), and for all three stages the SertaSil group reaches this significantly faster than Gentamicin and loddicerin.

A comparison of the wound surface measurements and the Wound Stage measurements in Fig. 3 shows that the stage Wound Cleansing is reached just before the wound undergoes a reduction in size and that the stage Epithelialization is only reached after the wound is in stable progress towards closure, e.g. for SertaSil the wound surface is only 0.84 sq.cm, when it reaches the stage of Epithelialization. For larger wounds, the difference will be more pronounced, but the point to notice is that Epithelialization is a conservative measure indicating that a wound is stably progressing towards closure.

Wound Healing Stages

Table 6 and Figure 4 compare the effects of treatment with SertaSil, Gentamicin and loddicerin on wound healing in all the patients included in the study. There was a significant difference between the treatment groups for Wound Cleansing (F (2, 263)=546.0; P<0.001), for Onset of Granulation (F(2, 263)=507.8; P<0.001) and Epithelialization (F(2, 263) =441.3; P<0.001) with SertaSil being significantly faster than the other groups for all parameters.

Figure 5 and 6 shows the wound healing process for a subgroup of patients with diabetic foot (Figure 5) or venous leg (Figure 6) ulcers, respectively. The pattern seen in these subgroups reflects the overall effects seen in Figure 4, indicating that SertaSil has a generalised beneficial effect on wound healing.

Table 6. Wound parameters for each of the three treatment groups. SertaSil (n=88), Gentamicin (n=90) or loddicerin (n=88) .

Adverse Reactions

Treatment in 3 patients with wide open trophic ulcers located on top of nerve fascicle (nerve bundles) had to be discontinued due to painful symptoms; no other no adverse reactions were noticed.

An ti -microbial Effects

The anti-microbial effect of SertaSil was pronounced and was demonstrated to be effective by the treatment of 11 carbuncles. After opening of the carbuncle, seven of these were tested positive for Staphylococcus aureus, one for Pseudomonas aeruginosa and for three the infective agent was not identified.

Current use of SertaSil

Initially, SertaSil was (as a rule) applied to a wound for the first 2-3 days and nights. There was usually no need to use the preparation any longer, since a wound normally would cleansed therein. Moreover, the preparation was not used for longer periods due to fear of over-drying a wound and causing death of granulations. For further cure bandages with methyluracilic ointment were applied every 24 hours either until full healing or (more often) until the formation of active granulations and marginal epithelialization, i.e. till active tendency to healing. Further treatment was provided in the outpatient setting, and recommendations were given to continue with ointment dressings every 24 hours until full healing. However, the use of me- thyluracil almost always caused pronounced mucous thickenings, which had to be removed at every dressing and this caused some pain. In these cases, it was decided to apply SertaSil in a smaller quantity, and surprisingly not only did it not lead to over-drying of the wound, quite the contrary a wound healed up more rapidly. Following these observations, it has become standard practice to use SertaSil for both the first and the second phases of wound healing.

Conclusion

The study has compared the effects of SertaSil, Gentamicin and Ioddicerin on wound healing in patients. SertaSil was found to be clearly superior to both Gentamicin and Ioddicerin in terms of promoting wound cleaning, active granulation and epitheliali- zation, and to accelerate the wound healing process. Also, the study has shown that SertaSil has strong antimicrobial effects and does not cause any serious adverse reactions. Subsequent use of SertaSil in the hospital has demonstrated that SertaSil can be used not only in the first phase of the wound formation process but also in the second, and lately this has become standard practice at the hospital.

SertaSil contains the enzyme serrathiopeptidase, which is a proteolytic enzyme isolating from the nonpathogenic intestinal bacterium Serratia E15, which has fibrinolytic, antiinflammatory and anti-oedematous activity. This makes it possible to remove necrotizing tissues from the wound more rapidly. In spite of assurances of the producers of Gentamicin (a preparation, which is based upon imosgent (polymethylsiloxane with adsorbed gentamy- cin) and widely used in practice) , that the preparation does not result in formation of a pellicle upon the wound surface, this pellicle is formed nevertheless and hampers the outflow of wound exudate and acts as a specific «thermostat», under the conditions of which micro-organisms do rather well. This effect is not observed in case of SertaSil. The fact, that the preparation does not contain an antibiotic, confers a substantial advantage rather than a disadvantage. This «contradiction» can be explained by the fact that one should not kill but refine the microflora in a wound, because an absolutely sterile wound does not heal .

The mode-of-action SertaSil can be attributed to several factors :

1. A direct antimicrobial and haemostatic effect.

2. Stimulation of leucocyte migration into a wound. 3. Lysis of necrotizing tissues and fibrinogenous furs.

4. Decrease of adhesion of microorganisms and bandages to a wound.

5. An osmotic and sorption action weakens diffusion of toxins and microorganisms deep into tissues, thus reducing intoxic ation and infection of a wound surface.

6. Decrease of viscosity and rarefaction of exudation makes conditions for passive wound drainage better.

7. Osmo-hydrophilic coefficient is 0,53, so that the preparation may work under conditions of pyoinflammatory process and do not overdry a wound surface (it sorbs mainly proteins, pus, toxins of microorganisms, that are also proteins in their nature) .

8. An active sorption of pathogenic microorganisms, in absence of selectivity, what makes it possible to avoid selection of pathogenic strains in a wound.

9. Stimulation of fibrinolytic activity of wound exudation (due to serrathiopeptidase) .

In conclusion, the study has demonstrated a superior clinical profile for SertaSil for the accelerated healing of a broad range of acute and chronic wounds, including venous and pressure leg wounds and diabetic foot ulcers with pronounced fibrinous thickenings and necrosis; infected wounds; carbuncles; heat burns of degree II A - II B - III; post-operative wounds and traumatic wounds.

Example 11: A comparative study of the effects of SertaSil on the acceleration of wound healing and wound closure in the rat

Summary of Findings:

The purpose of the study was to evaluate the novel drug SertaSil for its ability to accelerate wound healing and to reduce the time to wound closure in a pre-clinical animal model. An aseptic wound was modelled in the rat by administering 1 ml 10% calcium chloride solution into the subcutaneous layer under local anaesthetic conditions. Following opening of the abscess, the wound was treated every 24 hours with SertaSil, Gentamicin (gentamicin in a polymethylsiloxane gel) , or Control (no treatment) until wound cleansing, i.e. free of pus and fibrinogenous thickenings. Rats receiving SertaSil reached the stage of full wound cleansing in 3 days compared to 7 days for Gentamicin and 10 days for Control. The time to complete wound closure was 14 days for SertaSil, 19 days for Gentamicin, and 23 days for Control.

The surface area of the wound was measured Day 1 and Day 13. At Day 1, the wound surface was similar in all three groups, but at Day 13 the SertaSil group had a much smaller wound surface area compared to the Gentamicin and Control groups.

The study was conducted in young rats that are still growing and gaining weight. At the start of the study the average weight was similar in all three treatment groups, but at Day 24 after start of treatment, it was only the rats receiving SertaSil that exhibited a weight increase during the study. In contrast, animals receiving Gentamicin did not increase in weight and animals in the Control group had lost weight. This indicates that rats receiving SertaSil more rapidly recovered.

Evaluations of smears from the wounds during the first three days of treatment showed that the microflora of the wounds normalised itself more rapidly in the SertaSil and the Control group compared to the Gentamicin group, and immunological cell counts showed a stronger and more rapid increase in the number of monocytes and lymphocytes in the wound for the SertaSil group compared to the Gentamicin and the Control groups. It is known that a normal wound microflora and the presence of monocytes and lymphocytes in a wound are required for wound healing.

The study has demonstrated that SertaSil can accelerate the wound healing process for an acute wound in young healthy rats compared to rats receiving Gentamicin or no treatment (Control) and that SertaSil treatment leads to faster wound closure. These findings suggest that SertaSil may be a valuable therapeutic for the acceleration of wound healing and wound closure in patients with wounds .

Example 12 continued: Full Study Report.

The purpose of the study was to determine if the drug SertaSil can accelerate the wound healing process and can reduce the time to wound closure compared to treatments with the antibiotic Gen- tamicin (gentamicin in a polymethylsiloxane gel) and an unassisted healing process, i.e. Control.

Methods Animals

Forty-five white sexually mature rats of both sexes with an average weight of 230-250 g were used for the experiments. The animals were kept in a vivarium at constant temperature, pressure and humidity. Food and drink were accessible ad libitum. The light cycle was 12:12. All rats were kept under equal conditions. The animals were kept in individual cages to avoid that any stimulatory effect of the saliva components from other rats could influence the wound healing process (Hutson et al . 1979) . According to the generally accepted ethic standards, all painful manipulations with the animals were carried out under local anaesthesia of 0,5% Novocaine solution.

Inducti on of wound

Aseptic inflammation was modelled by the procedure of S. A. Shalimov and co-workers (1989) . The fur of the laboratory animals was shaved off on the back area, the back was treated with iodine alcoholic solution, and 1 ml 10% calcium chloride solution was administered into the subcutaneous layer.

Treatment

Following surgical opening of the abscess that had formed under the skin, the preparations were applied as a near 3 mm thick layer on top of the wound every 24 hours. Each animal was treated until the wound cleansed, i.e. until the wound was free of pus and fibrinogenous thickenings.

Experimental groups

The experiment included three treatment groups, each with 15 rats. The first group received the preparation SertaSil; the second group received the preparation Gentamicin (gentamicin in a polymethylsiloxane gel) ; and the third was Control, where no treatment was provided. Measurements

The wounds were studied clinically, planimetrically, cytologi- cally, and bacteriologically .

The animals were inspected daily and it was recorded which day they reached each of four stages in the wound healing process: 1) Wound cleansing, i.e. free of pus and fibrinogenous thickenings; 2) Second scab shedding from abscess opening; 3) Beginning of marginal and islet epithelization; and 4) Complete wound closure, i.e. full epithelialization .

The average surface area of the wound was measured on the first day after abscess opening and on Day 13 of treatment. The area of the wound defect, its perimeter, and average diameter were determined by applying PE film over the wound. The wound circumference was copied onto the film and the lengths at all sides were measured in mm. The data was entered into the computer program package SigmaScanPro and the surface area was calculated.

The body weight of the rats was measured on Day 0 and Day 19. The change in an animal body mass can be used as a non-specific index of how an organism is affected by various stress factors.

Samples of wound excretions were taken by gauze swab from the wound area. Native (naturally occurring) smears obtained from the gauze swabs were dried and imbued by Romanowsky-Gimza (in: Fenchin, K.N. Wound healing. Kiev 1979) . Cytological measurements were taken 1, 2 and 3 days after wound initiation. Cytological evaluations of microflora and immune response in the wound were made by studying smear-imprints using the method of Pokrovska and Makarova (Datsenko et al . Theory and practice of topical [local] treatment of purulent wounds. Kiev 1995) . Measurements were made 6, 12, 24 and 48 h after opening of abscess. Bacteriological measurements were made by qualitative estimation of bacterial inoculation from wound excretions during the first day and at 24 hours.

Statistical analysis

Data were analysed statistically with use of the program package

Systat 8.0 using ANOVA with Fischer's LSD post-hoc test. Results

Following the injection of 1 ml 10% calcium chloride solution into the subcutaneous layer of the rat, a purulo-necrotic process developed on average in 3 to 4 days. After lancing of the abscess, it was treated with SertaSil, Gentamicin, or Control.

Figure 7 shows the time course of healing for two rats that have been treated with SertaSil or Gentamicin, respectively. The pictures demonstrate a much faster and much improved recovery process for the SertaSil-treated rats compared to Gentamicin.

Figure 8 and Table 7 summarises the qualitative outcome measures for each of the three treatment groups. There were significant difference between the three groups for days to wound cleansing

(F(2,42)=1295, 0; P<0.001); days to scab removal (F (2, 42) =1317.0 ; P<0.001); days to beginning of marginal and islet epithelization

(F (2, 42) =408.15; P<0.001); and days to wound closure

(F(2,42)=1567.21; P<0.001) .

The SertaSil group reached the wound cleansing stage after only 3 days compared to 7 days for Gentamicin and 10 days for Control. Animals were treated until wound cleansing and the SertaSil group was therefore only treated for 3 days, whereas the Gentamicin group was treated for 7 days. The stronger wound healing effects of SertaSil compared to Gentamicin can therefore not be attributed to a longer duration of treatment.

The SertaSil group reached complete wound closure after 14 days compared to 19 days for Gentamicin and 23 days for Control. In spite of the shorter duration of treatment, the SertaSil group displayed accelerated wound closure compared to Gentamicin and Control. As expected the Gentamicin group showed faster wound closure than the Control group.

The effects of SertaSil on Days to wound closure are also reflected in the wound surface area. At Day 1 following opening of the abscess the wound surface area was similar for all groups (F (2, 42) =0.07; P=NS), but as shown in Figure 9 at Day 13 there was a significant difference (F (2, 42) =708.15; P<0.001) with the SertaSil group having the smallest wound surface followed by the Gentamicin group. At Day 13 all the wounds in the SertaSil group were almost closed.

SertaSil also had beneficial effects on the general health status of the animals. The study was conducted in young rats that are still growing and gaining weight. Figure 10 shows that on Day 1, the three groups had the same weight (F (2, 42) =0, 08 ; P=NS), but at Day 19 (F (2, 42) =29.62 ; P<0.001) it was only the SertaSil group that had gained weight whereas the Gentamicin group had remained on its starting weight and the Control group had lost weight. Changes in body weight is generally a very strong indicator of excess stressors on an organism and these data show that it was only the SertaSil group that was able to maintain a normal development.

Table 7. Summary of wound healing data.

Swabs of wound exudates were taken following opening of the abscess and start of treatment. An analysis of the microflora in the wound (Table 8) demonstrated that in the SertaSil and the Control group the microflora normalised itself very rapidly towards the normal state. In contrast, in the Gentamicin group, the microflora was inhibited by the antibiotic effects of gen- tamicin and in this group a pathogenic bacterial strain was identified.

Table 9 shows the presence of neutrophils, monocytes and lymphocytes in the wound at 4 time points following opening of the abscess. It is noteworthy that the levels of monocytes and lymphocytes are highly elevated in the SertaSil group compared to the Gentamicin and the Control groups, indicating a much more robust wound healing process.

Table 8. Microflora in the wound at 3 different time points following opening of the abscess.

Table 9. Presence of neutrophils, monocytes and lymphocytes in the wound at 4 time points following opening of the abscess.

Conclusion

The study has compared the treatment effects of SertaSil to Gen- tamicin and Control treatment on wound healing and time to wound closure in rats. The data has shown that SertaSil was able to induce wound cleansing in 3 days compared to 7 and 10 days for Gentamicin and Control, respectively, and that it led to complete wound closure in only 14 days compared to 19 and 23 days for Gentamicin and Control, respectively. Measurements of wound surface area Days 1 and 13 demonstrated the same trends. The data therefore show that SertaSil can significantly accelerate the wound healing process in rats and leads to much faster complete wound closure.

SertaSil treatment also led to an overall better health status, because it was only the SertaSil group that demonstrated an increase in body weight during the study period, whereas the Gentamicin group did not gain weight and the Control group actually lost weight.

The analysis of wound smears showed that SertaSil affects the underlying physiology of wound healing. The microflora of the wound normalised itself rapidly and the immunological cell counts showed increased invasion of monocytes and lymphocytes into the wound compared to Gentamicin and Control. Both of these cell types are known to be necessary for a normal wound healing process and their increased presence in the SertaSil group support the hypothesis that SertaSil accelerates the wound healing process itself. In conclusion, these data strongly indicate that SertaSil has therapeutic value for the acceleration of wound healing in patients .

References

Experimental methods in: Shalimov S. A., Radzihovskiy A. P., Keis- evich L. V. Handbook for experimental surgery. Moskow: Medicine,

1989, p. 121-123

Hutson, J. M., Niall M., Evans D., Fowler R. Effect of salivary glands on wound contraction in mice. Nature, 1979, vol. 279, pp.

793-795.

K. Schmidt-Nielsen. Why is size so important? (Translation by

Kulikov V.F., Poletaeva 1.1., Moskow, "Mir" . -1987. -p .29-34) .

Example 12: Treatment of moist eczema in dogs with SertaSil. Two Labrador Retrievers, age 10 years, suffered regularly from moist eczema (also called Summer Sores or Hot Spots) . Moist eczema is a very painful and itchy condition that can seemingly appear spontaneously anywhere on a dog's body. This moist, raw skin disorder has a variety of causes but the most consistent factor is bacteria. The infection goes into the deep layers of the skin. Normally several types of bacteria can be cultured from a hot spot and the very same moisture oozing slightly from the sore itself provides nutrients for bacterial growth and makes the sore grow with a velocity that allows even an untrained eye to notice the expansion hour by hour. A hot spot can under the cover of the fur easily grow from a tiny dot to an 8 x 8 cm exudating wound in just 24 hours.

The normally recommended treatment for moist eczema is: An area well beyond the margins of the lesion should be shaved followed by treatment with topical hydrogen peroxide every two hours, systemic antibiotics to combat the deep skin infection and a single, short acting corticosteroid to stop the inflammatory reaction. Oral antibiotics and topical medication are continued for at least a week, and two weeks is even better. It is difficult to stop the continuing deterioration of the area, and to obtain a wound with an active healing process may easily take two weeks from commencement of the treatment. A normally effective treatment for the two Labradors in question is to shave the area over and 1.5 cm beyond the margins of the lesion to allow drying. It is washed with topical hydrogen peroxide every four hours, dried and topical antibiotic in powder is applied. Within 36 hours the expansion is normally controlled. In some cases the crust created on the top of the wound by the powder is so thick that it allows a different bacterial growth underneath it. The crust is then removed, the wound is cleaned thoroughly and the treatment is continued. After 60-72 hours the cleaning can be limited to twice a day and the antibiotic powder can be changed into a chlorhexidine powder to keep the wound clean and dry. After additional 12 hours the healing process is clearly noticeable in the edges of the wound, and the wound heals at its natural pace.

SertaSil was used for the treatment of several cases of moist eczema in these dogs, and because their usual response to standard treatment is known, it is possible to identify any beneficial effects of SertaSil.

Case 1: First, the area was shaved well beyond the margins of the lesion. It was washed with hydrogen peroxide, dried and SertaSil was applied. After four hours the wound was inspected and was found to be nice and dry, so no further washes with hydrogen peroxide were made. In places, where the wound was no longer white from powder, additional SertaSil was applied. 12 hours after initiation of SertaSil treatment the wound was inspected. The edges were showing clear signs of healing and the wound was dry and covered with a thin not too hard crust. There was no bacterial growth under the crust. After additional 12 hours the wound was inspected again, and the healing was clearly in progress. The wound was inspected every 12 hours, but no further treatments were made. During the following days the crust fell off in small pieces revealing completely healed skin underneath, resulting in a fully closed wound.

Case 2: The same dog had a second case of moist eczema and to evaluate the usefulness of SertaSil as a first-aid, the wound was not shaved. Only hair on top of wound that normally is loose was removed. The wound was washed with hydrogen peroxide and SertaSil was applied. After eight hours the wound was inspected and healing was clearly in progress. SertaSil was applied as necessary two more times within a period of 24 hours. After a week, the wound was completely healed. The same procedure was applied for the treatment of the other dog, and the outcome was identical .

Case 3: To further evaluate the usefulness of SertaSil as a first-aid two cases of moist eczema in the same dogs were evaluated and in these cases the wounds were not cleaned or disinfected before the application of SertaSil. Only lose hair on the wounds was mechanically removed, where after SertaSil was applied to cover the wound surface. After one hour the wound was inspected. The SertaSil was moist and therefore a new thin layer of SertaSil was applied. Three and a half hours later the wound had stopped growing in size, but again it was moist. It received a thin layer of SertaSil. Four hours later the edges of the wound were well defined and showing unambiguous signs of pulling towards the center. The wound was covered with a porous white layer, like a dry bendable crust, that still allowed the wound to breathe. The wounds were inspected every two hours, but they did not need further treatment. After additional six hours the wound was visibly smaller in contour and still covered with the described layer.

In conclusion, SertaSil is able to treat moist eczema and the infection associated with this condition and to initiate a normal healing process in the wound in only a matter of hours, whereas these wounds normally require 1-2 weeks for treatment to reach a healthy healing process. Also, cases of moist eczema are normally very itchy and painful. It was obvious from the behavior of the dogs, e.g. relaxation in muscles and breathing pattern, that in 2-3 minutes after applying SertaSil that the pain was relieved, indicating a potent pain-relieving effect of SertaSil.

Claims

Claims :

Claim 1: A composition comprising a siliceous sorbent and a medicinal agent immobilized thereon, wherein the siliceous sorbent in one preferred embodiment is aerosil and the medicinal agent is serrathiopeptidase, where aerosil is fine pyrogenic silica that consists of hydrated globules of an average silica particle radius of between 1 and 50 nm or aerogel of polysilicic acid SiO2* x H2O, the water content being up to 20% by weight and where serrathiopeptidase (CAS number 37312-62-2) is a proteolytic enzyme isolating from the nonpathogenic intestinal bacterium Serratia E15.

Claim 2: The composition of claim 1 wherein the serrathiopeptidase amount is up to the amount bindable by aerosil, or preferably wherein aerosil and serrathiopeptidase are both present in the following amounts, % by weight: aerosil 99.9 to 90 serrathiopeptidase 0.1 to 10.

Claim 3: The composition of claim 1 or 2, where fine pyrogenic silica consists of hydrated globules with an average silica particle radius between 3 and 15 nm, preferably about 4.35 nm, in particular preferred where the water content of aerosil is not exceeding 10 %.

Claim 4: The composition of any one of claims 1 to 3, where the composition or serrathiopeptidase itself is attached, mixed with, applied as a layer to or coated upon bandages, dressing, gauze, sutures, artificial skin, gels, collagen matrixes, or other devices that are used for treating, protecting or covering wounds or skin conditions.

Claim 5: The process of manufacturing the composition of claims 1 to 4, where the solution of serrathiopeptidase used in the preparation of the composition contains no more than 1% of active substance and where the solution of serrathiopeptidase is prepared by stirring the solution containing serrathiopeptidase for 10 min or more before adding the sorbent aerosil.

Claim 6: The use of the composition of Claims 1 to 4 for the treatment of festering wounds, trophic ulcers and burns, and infiltrations with marked necrotic and exudative components.

Claim 7: A method for treating festering wounds, trophic ulcers and burns, infiltrations with marked necrotic and exudative components by applying the antimicrobial composition of Claims 1 to 4 topically to the surface of a wound.

Claim 8: The use of the composition of Claims 1 to 4 for the treatment of chronic wounds, including but not limited to diabetic foot ulcers, venous and arterial leg ulcers, pressure ulcers, cancer wounds, wounds caused by ischemia or radiation, wounds caused by a disease or a disease-associated condition and where the wound or the ulcer may be infected.

Claim 9: The invention of Claim 8, where the wound is caused by a parasite.

Claim 10: The use of the composition of Claims 1 to 4 to reduce the time, compared to the untreated condition, for a wound to reach the stages of wound cleansing, granulation, epithelializa- tion, wound closure, or regrowth of tissues and membranes in an individual .

Claim 11: The use of Claim 10, where the wound is a chronic wound, including but not limited to diabetic foot ulcers, venous and arterial leg ulcers, pressure ulcers, cancer wounds, and wounds caused by ischemia or radiation.

Claim 12: The use of Claim 10, where the wound is an acute wound that for example may be caused by a disease or disease- associated condition, burns or frost, infections, radiation, chemicals, violence, accident, or surgery.

Claim 13: The use of Claim 10, where the wound is a closed or an internal wound.

Claim 14: The use of Claim 10, where the wound is caused by a parasite .

Claim 15: The use of the composition of Claims 1 to 4 to stop or reduce haemorrhage from a wound in an individual .

Claim 16: The use of the composition of Claims 1 to 4 to reduce scar-formation during wound closure in an individual, including but not limited to scarring resulting from normal wound healing and abnormal scarring including but not limited to hypertrophic and keloidal scarring.

Claim 17: The use of the composition of Claims 1 to 4 for the topical treatment of skin, tissue, organ, and wound infections caused by parasites, virus, prions, protozoa, fungus and bacteria, including but not limited to gram-positive and gram- negative bacteria and antibiotic resistant bacterial strains such as MRSA in an individual .

Claim 18: The use of Claim 17, where the wound is a chronic wound with an infection, including but not limited to diabetic foot ulcers, venous and arterial leg ulcers, pressure ulcers, cancer wounds, and wounds caused by ischemia or radiation.

Claim 19: The use of Claim 17, where the wound is an acute wound with an infection and where the wound for example may be caused by a disease or disease-associated condition, burns or frost, infections, MRSA-induced tissue and flesh wounds, radiation, chemicals, violence, accident, or surgery.

Claim 20: The use of Claim 17, where the wound is a closed or an internal wound.

Claim 21: The use of Claim 17, where the wound is caused by a parasite .

Claim 22: The use of the composition of Claims 1 to 4 for symptomatic or curative treatment of acute and chronic skin conditions, including but not limited to rashes, allergies, psoriasis, lupus, Acne (Pimples) , Alopecia Areata, Atopic Dermatitis, Boils, Cellulite, Cuts, Scrapes, and Punture Wounds, Cysts, Dandruff, Dupuytren Contracture, Eczema, Edema, Ehlers-Danlos Syndrome, Eosinophilic Fascitis, Erythema Nodosum, Hives, Impetigo, Itch, Jaundice, Keloid, Keratosis Pilaris, Klippel-Trenaunay- Weber Syndrome, Lichen Planus, Lichen Sclerosus, Marfan Syndrome, Mycobacterium Marinum, Poison Ivy, Oak & Sumac, Insect or Spider bites, Psoriasis, Pseudoxanthoma Elasticum (PXE), Raynaud's Phenomenon, Reflex Sympathetic Dystrophy (RSDS), Rosacea, Rothmund-Thomson Syndrome , Scabies, Scars, Scleroderma, Shingles, Skin Tag, Sporotrichosis, Bacterial skin infection, Stretch Marks, Sunburn and Sun Poisoning, Thrush and Other Yeast Infections, Tinea Versicolor, Vitiligo, Weber-Christian Disease, Wrinkles, and Yaws, in an individual.

Claim 23: The topical use of the composition of Claims 1 to 4 for cosmetic skin cleansing and for reducing allergic reactions and skin blemishes.

Claim 24: The use of the composition of Claims 1 to 4 for gastrointestinal and cardiovascular surgery where the composition is applied topically to wound edges to prevent undesired attachments to nearby membranes or tissues.

Claim 25: The use of the composition of Claims 1 to 4 for treating peritonitis, including local peritonitis in the peritoneal cavity by applying the composition locally to the area infected or by applying it widely to the peritoneal cavity, for example by rinsing the cavity with the composition dissolved in water, saline or another solvent.

Claim 26: The use of the composition of Claims 1 to 4 for improving the outcome of skin grafts, organ transplantation and prosthetic surgery such as implantation of artificial hips and knees by facilitating the healing process or by reducing infections or by reducing tissue rejection of the transplant by applying the composition topically to the area in need of treatment or by administering it systemically or by injecting it locally.

Claim 27: The use of the composition of claims 1 to 4 for emergency treatment of wounds to reduce haemorrhage, to reduce infections of the wound and to facilitate wound cleansing and to initiate the processes of wound granulation, epithelialization and wound closure.

Claim 28: The use of Claim 27, where the wound is caused by mechanical damage such as knives, bullets, fragments and any other types of devices designed to inflict damage and harm to individuals .

Claim 29: The use of the composition of Claims 1 to 4 to remove necrotizing tissues and fibrinogenous furs in wounds.

Claim 30: The use of the composition of Claims 1 to 4 for treating or promoting the healing of a wound or a skin infection or a skin condition by exerting combinations of the following actions: antimicrobial effects, haemostatic effects, stimulating lymphocyte migration into area, normalisation of wound flora, lysis of necrotizing tissues and fibrinogenous furs, antiinflammatory effects, decreasing the adhesion of microorganisms and bandage to a wound; exerting an osmotic and sorption action that weakens diffusion of toxins and microorganisms into tissues, decreasing the viscosity and rarefaction of exudation, and stimulating fibrinolytic activity of wound exudation.

Claim 31: The use of the composition of Claims 1 to 4 for the treatment of sepsis in an individual in need of treatment.

Claim 32: The use of the composition of Claims 1 to 4 to improve the outcome of surgical procedures and to prevent infections by applying the composition topically to individual tissue layers during surgery and when the individual tissue layers are sutured, clamped or closed by other means during the process of wound closure.

Claim 33: The use of the composition of Claims 1 to 4 for the treatment of blood, organ or tissue infections, including but not limited to infections caused by bacteria, protozoa, prions, viruses and parasites by administering the composition systemi- cally, for example intravenously or intramuscularly or subcuta- neously, to an individual in need of treatment.

Claim 34: The use of the composition of Claim 1 to 4 for the treatment of gastrointestinal inflammations by administering it to an individual in need of treatment by a method that prevents the degeneration of the composition by the acid in the stomach.

Claim 35: The invention in Claim 34, where the composition of Claim 1 to 4 is administered as an entero-coated tablet or in a capsule .

Claim 36: The use of the composition of Claims 1 to 4 for the treatment of local inflammations by administering the composition topically or locally to the area in need of treatment or for the treatment of global inflammations by administering the composition systemically .

Claim 37: The use of the composition of Claims 1 to 4 for the cleaning and disinfection of wounds, exposed tissue surfaces, skin surfaces and non-living surfaces including but not limited to floors, furniture, tables, instruments, operation theatres, hospital wards, and sick wards by applying it topically to the area in need of disinfection.

Claim 38: The use of Claim 37, where the composition of claims 1 to 4 is present at a concentration of 0.1% to 20% by weight and where it is dissolved in a solution consisting of 50% or more of water .

Claim 39: The use of the enzyme Serrathiopeptidase for the treatment of festering wounds, trophic ulcers and burns, and infiltrations with marked necrotic and exudative components by applying it topically to the area in need of treatment.

Claim 40: The use of Claim 39, where Serrathiopeptidase is applied in a concentration of between 0.1 and 10% by weight.

Claim 41: The use of Claim 39 or 40, where Serrathiopeptidase is attached, mixed with, applied as a layer to or coated upon bandages, dressing, gauze, sutures, artificial skin, gels, collagen matrixes, or other devices that are used for treating, protecting or covering wounds or skin conditions.

Claim 42: The use of Claim 39, where Serrathiopeptidase is applied or present in a concentration of between 0.1 and 10% by weight and the sorbent is silica gel, preferably silica gel fibers .

Claim 43: The use of the enzyme Serrathiopeptidase for the cleansing of wounds by applying it topically in a concentration of between 0.1% and 10% by weight to the area in need of treatment .

Claim 44: The use of the enzyme Serrathiopeptidase for reducing scar formation during the healing of a wound by applying it topically in a concentration of between 0.1% and 10% by weight to the area in need of treatment.

Claim 45: The use of fine pyrogenic silica that consists of hy- drated globules of an average radius of between 1 and 50 nm, or aerogel of polysilicic acid SiO2* x H2O for creating a protective barrier over a wound in order to decrease the penetration of microorganisms into a wound and to decrease the adherence of bandages or dressings to a wound.

Claim 46: The use of Claim 45, where the silica has an average radius between 3 and 15 nm, preferably about 4.35 nm.

Claim 47: The use of Claim 45 or 46, where the silica is attached, mixed with, applied as a layer to or coated upon bandages, dressing, gauze, sutures, artificial skin, gels, collagen matrixes, or other devices that are used for treating, protecting or covering wounds or skin conditions.

Claim 48: The use of fine pyrogenic silica that consists of hy- drated globules of an average silica particle radius of between 1 and 50 nm or aerogel of polysilicic acid SiO2* x H2O for the cleansing of wounds, for improving granulation, for improving epithelialization, and for reducing scar formation during the healing of the wound by applying the silica topically to the area in need of treatment.

Claim 49: The use of Claim 48, wherein the silica has an average radius between 3 and 15 nm, preferably about 4.35 nm.

Claim 50: The use of Claim 48 or 49, where the silica is attached, mixed with, applied as a layer to or coated upon bandages, dressing, gauze, sutures, artificial skin, gels, collagen matrixes, or other devices that are used for treating, protecting or covering wounds or skin conditions. Claim 51: The use of Claims 48 to 50 for the treatment of festering wounds, trophic ulcers and burns, and infiltrations with marked necrotic and exudative components by applying it topically to the area in need of treatment.

Claim 52: A composition comprising siliceous gel, preferably silica gel fibers, and a medicinal agent, wherein the medicinal agent is serrathiopeptidase that is preferably present in the following amounts, % by weight: serrathiopeptidase 0.1 to 10.

Claim 53: The use of the composition of Claim 52 for the treatment of festering wounds, trophic ulcers and burns, wound infections, preferably bacterial infections, and infiltrations with marked necrotic and exudative components.

Claim 54: A composition comprising a siliceous sorbent and a medicinal agent immobilized thereon, wherein the siliceous sorbent is porous silica, derivatives of porous silica, silica micro- or nanospheres, derivatives of silica micro- or nanospheres, silica nanotubes or derivatives of silica nanotubes and where serrathiopeptidase (CAS number 37312-62-2) is a proteolytic enzyme isolating from the nonpathogenic intestinal bacterium Serratia E15.

Claim 55: The composition of claim 54 wherein the serrathiopeptidase amount is up to the amount bindable by the siliceous sorbent, or preferably wherein the sorbent and serrathiopeptidase are both present in the following amounts, % by weight: Sorbent 99.9 to 90 serrathiopeptidase 0.1 to 10, or it may be up to the amount bindable by the sorbent (absorption limit) .

Claim 56: The use of the composition of Claims 54 or 55 for the treatment, including acceleration of wound closure and reduction of scar formation, of festering wounds, trophic ulcers and burns, wound infections, and infiltrations with marked necrotic and exudative components, and of chronic wounds, including but not limited to diabetic foot ulcers, venous and arterial leg ulcers, pressure ulcers, cancer wounds, wounds caused by ischemia or radiation, wounds caused by a disease or a disease-associated condition, and of acute wounds including but not limited to wounds caused by a disease or disease-associated condition, burns or frost, infections, MRSA-induced tissue and flesh wounds, radiation, chemicals, violence, accident, or surgery.