WO1999049840A1

WO1999049840A1 - Skin treatment compositions and the use thereof

Info

Publication number: WO1999049840A1
Application number: PCT/NO1999/000111
Authority: WO
Inventors: Jan Wadstein
Original assignee: Jan Wadstein
Priority date: 1998-03-31
Filing date: 1999-03-31
Publication date: 1999-10-07
Also published as: EP1082096A1; AU3061199A

Abstract

The present invention relates to preparations comprising a lactoperoxidase system and/or antibodies (immunoglobulins) for the treatment and/or prophylaxis of skin conditions, such as acnes, as well as uses thereof.

Description

SKIN TREATMENT COMPOSITIONS AND THE USE THEREOF

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composi- tion comprising a lactoperoxidase system and/or antibodies (immunoglobulins) , as well as a two-component preparation comprising a composition containing a lactoperoxidase system and a composition containing antibodies (immunoglobulins) , as well as the use thereof for the inhibition and/or prophylaxis of bacterial growth and/or killing of bacteria on surfaces .

More specifically, the invention relates to the separate or combined use of the compositions for topical treatment and/or prophylaxis of surface-associated bacterial skin conditions, such as acnes.

Furthermore, the invention relates to the use of a lactoperoxidase system and/or antibodies (immunoglobulins) in the production of a medicament for the treatment and/or prophylaxis of surface-associated bacterial skin conditions, especially acnes.

BACKGROUND OF THE INVENTION Acne vulgaris is a chronic disorder of pilosebaceous units on the face, chest and back. Acne lesions are caused by a combination of bacteria normally found on the skin, including Propionibacterium acnes and coagulase-negative stap- hylococci and micrococci . The lesions are open comedones, which are dilated follicles with central dark, horny plugs (blackheads) ; closed comedones, which are small follicular papules without inflammatory changes (whiteheads) or with 2 inflammatory changes (red papules) ; superficial pustules, which are collections of pus, nodules, which are collections of pus deep in the dermis; and large, deep pustular lesions, which develop from nodules that break down ad- jacent tissue, enlarge, and form lakes of pus, sinuses and scars .

Acne problems is a common condition, especially among teenagers. A Swedish study among young people in the age-group 12-16 years showed that one third of these had acnes which required treatment . A British study has previously shown that even 5% woman and 3% men in the age-group 40-49 suffers from active acne ("Treatment of Acne - Recommendations from the Group" in Workshop Treatment of Acne, 1996, 3, Lakemedelsverket (Medical Products Agency) and Statens

Legemiddelkontroll (The Norwegian Medicines Control Authority) .

The skin's normal flora comprises a number of microorgan- isms which can be characterized either as transients or residents. The skin is an external organ which is continually being inoculated with transients, but virtually all of these are unable to multiply and will normally die. Residents are organisms which do not only survive on the skin surface, but are also able to multiply. The normal resident flora of the skin consists primarily of Gram-positive Bacteria restricted to a few groups, such as several species of Staphylococcus and a variety of both aerobic and anaerobic corynebacteria . Such organisms are normally not patho- genie, but can, especially in immunosuppressed hosts, incite or contribute to a number of skin conditions . 3

Staphylococcus aureus, a yellow-pigmented form of the Staphylococcus species, is normally present in the upper respiratory tract, especially nose, throat and surface of the skin. S. aureus can be associated with a number of pathological conditions, including a number of skin condition, such as purulent boils, blisters, pus forming infections, pimples and impetigo. Severe cases of rash caused by S. aureus may occur during toxic shock syndrome (TSS) . The corynebacteria Propionibacterium acnes will, on the other hand, cause acnes.

Gram-negative bacteria are almost always minor constituents of the normal flora, even though such intestinal organisms as Escherichia coli are being continually inoculated onto the surface of the skin by fecal contamination; Acineto- bacter is, however, an exception as this bacteria is commonly found on skin. The absence of Gram-negative bacteria on the skin is probably due to their inability to compete with Gram-positive organisms which are better adapted to the dry conditions of the skin. If Gram-positive microorganisms are eliminated by antibiotic treatment, the Gram- negative bacteria can flourish.

Skin conditions, such as acnes, can be serious problems because of damage to the skin, ulceration, pigmentation damage and associated psychological problems. Accordingly, there are many products on the market which aim to cure this condition.

Over the counter commercial preparations for the treatment of acne include i . a . Clearasil^®, Aco Acnelδsning, Scott Mathisens hudvann (skin water) etc. Such preparations are 4 primarily for the topical treatment of acne conditions and can be bought without a doctor's prescription. These preparations generally contain compounds such as salicylic acid, ichtammol, sulphur preparations, resorcinol, camphor, solutions of boric acid and ceriumbromide . However, these compounds have no documented effect, and the use of such preparations are not recommended by Statens Legemiddel- kontroll eller det svenske Lakemedelsverket ("Treatment of Acne - Recommendations from the Group" in Workshop Treat- ment of Acne, 1996, 3, Lakemedelsverket (Medical Products

Agency) and Statens Legemiddelkontroll (The Norwegian Medicines Control Authority) .

Traditionally, it has also been attempted to use iodine solutions and benzoyl peroxide-based compositions to treat different skin conditions. Iodine is a relatively cheap, conventional disinfectant agent, but is, however, toxic and can cause irritation of the skin, mucosa and eyes. Furthermore, the use of iodine can cause discoloration of the skin area in question. Benzoyl peroxide-based compositions may cause stinging pain, reddening or peeling of skin, and in some cases contact dermatitis.

Additionally, a number of medicaments for the treatments of skin conditions are available by prescription. For reviews of strategies for physician managed treatment of acne see e . g. Baur et al . , J. Oral Maxillofac. Surg. 56(5):651-55 (1998), Leyden, J^". Am. Acad. Dermatol . 38(4):S1-S4 (1998), Usatine et al . , Hosp. Pract . 33 (2) : 11-17 (1998) , and Weiss, Pediatr. Dermatol . 14 (6) :480-88 , all of which are incorporated herein by reference . 5

Prescription treatments are relatively effective, but their use must be monitored closely by a physician. Examples of such treatments are oral ( e. g. tetracyclines) and topical { e. g. chlindamycine) administration of antibiotics and azealic acid. A further disadvantage of such antibiotics treatment is also that sustained use of such medicaments can cause antibiotic resistency. For example, Staphylococcus aureus is a multiresistent organism; that is, it is resistent to a number of different antibiotics.

A number of clinical experiments performed in the late 1970 's and early 1980 's, demonstrated especially good results when retinoids, i.e. synthetical or naturally occurring derivatives of vitamin A ( e. g. retinol) , were used for therapeutic treatment of skin conditions. For example, Mobacken et al . (Mobacken H. , Hersle F., Michaels- son G. , Vaholquist A., Landegren J. , Rδnnerfalt L., et al . , Isotretinoin hjalp vid svar acne - men biverkningrana ar manga, Lakartidningen 1986, 83: 591-4) demonstrated that more than 90% of acne test subjects participating in their study responded to treatment, and 2 out of 3 test subjects were symptom-free a whole year after treatment. This study, and many others, has, however, shown extensive side effects including for example dry, scaly and cracked lips, bleeding from the nasal mucosa, hair loss, limb and joint pain, but also very serious side effects such as teratogenicity with embryonal malformations afflicting the central nervous system, the cardiovascular and the craniofacial bone systems as well as the ears; and upon sustained use, bone skeleton side effects in the form of hyperostoses, calcification of tendons and premature closure of the epiphyses; pseudo- tumour cerebri, as well as depression (Vahlquist C. & 6

Fyrand O., "Isotretinoin - Modes of Action Side Effects (clinical & laboratory) and Results (short- & long-term) " in Workshop Treatment of Acne, 1996, 3, Lakemedelsverket (Medical Products Agency) and Statens Legemiddelkontroll (The Norwegian Medicines Control Authority) .

The purpose of the present invention is, hence, to provide an effective, non-toxic, skin friendly preparation for topical treatment of skin conditions, which preparation is without the abovementioned side effects and available without consulting a doctor. Additionally, it is desirable that the preparation has a prophylactic ability; that is, is, at least to some extent, able to prevent or inhibit the skin conditions in question.

SUMMARY OF THE INVENTION

This invention relates to the treatment of acne. More particularly, this invention relates to the treatment of acne with compositions containing peroxidases, in parti- cular lactoperoxidases, and/or immunoglobulin (s) .

Some embodiments of the present invention provide a pharmaceutical composition containing a peroxidase system for the treatment and/or prophylaxis of skin conditions, in particular acne. Other embodiments of the present invention provide a pharmaeutical composition containing immunoglobulin (s) for the treatment and/or prophylaxis of skin conditions, in particular acne. Further embodiments of the present invention provide a two-component preparation for the treatment and/or prophylaxis of acne wherein the abovementioned compositions exist as two separate components of a kit or possibly combined in the same 7 composition.

In preferred embodiments, the present invention provides a pharmaceutical preparation or mixture for the treatment and/or prophylaxis of acne containing 10 to 60 % by weight, preferably about 30% by weight, of a peroxidase system. The ratio between the peroxidase and thiocyanate is from 1:1 to 1:10. In particularly preferred embodiments, the ratio is about 1:2,5. Preferably the peroxidase system is a lacto- peroxidase system, and the thiocyanate therein is preferably potassium thiocyanate.

Some embodiments of the present invention also provide a pharmaceutical preparation for the treatment and/or pro- phylaxis of acne. In some embodiments, this preparation contains from about 1 to 12% by weight, preferably from 4 to 5 % by weight immunoglobulins with a purity of 80% with respect to protein. In some preferred embodiments, the immunoglobulin are derived from colostrum, such as bovine colostrum.

In further embodiments, these pharmaceutical compositions may be packaged together as a kit in separate containers in the same container .

In still other embodiments of the present invention, the pharmaceutical compositions may be used in a method of treating or preventing acne. In some embodiments, a preparation containing a peroxidase system is applied to areas of the skin with acnes and allowed to dry. In other embodiments, a preparation containing immunoglobulin is applied to the skin and allowed to dry. In some preferred 8 embodiments, the peroxidase system preparation is applied first and allowed to dry, followed by application of the immunoglobulin preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a graph plotting the percentage of live bacteria present in an in vi tro system after treatment with varying amounts of lactoperoxidases .

Fig. 2 presents the results of an ELISA measuring immunoglobulin binding (1:2 dilution) to samples prepared from acne causing organisms.

Fig. 3 presents the results of an ELISA measuring immunog- lobulin binding (1:10 dilution) to samples prepared from acne causing organisms.

Fig. 4 presents the results of an ELISA measuring immunoglobulin binding (1:2 dilution, expressed as percent of ViM reference) to samples prepared from acne causing organisms.

Fig. 5 is a bar graph demonstrating the percent reduction of acnes after three weeks of treatment with the immunoglobulin containing gel formulation of the present inven- tion.

Fig. 6 is a bar graph demonstrating the percent reduction of acne after six weeks of treatment with the immunoglobulin containing gel formulation of the present inven- tion. 9

DEFINITIONS

To facilitate understanding of the invention, a number of terms are defined below.

As used herein, the term "peroxidase system" refers to a composition containing either natural or recombinant peroxidases ( e. g. lactoperoxidase, horse-radish peroxidase, myeloperoxidase, salivary peroxidase, thyroid peroxidase, or eosinophil peroxidase) and specific co-factors (hydrogen peroxide and, depending on the specificity of the peroxidases in question, thiocyanate, chloride, bromide or iodide) . In general, the peroxidase system of the present invention is sufficient to produce an oxidation product which oxidizes amino groups and/or thiols in enzymes which are essential for the metabolism of a bacteria (e.g. S. aureus eller P. actinus) .

In particular, when the peroxidase is lactoperoxidase and the co-factors are hydrogen peroxide and thiocyanate, the system is referred to as a lactoperoxidase system.

As used herein, the terms "immunoglobulin" and "antibody" refer to any full-length or fragmentary antigen binding protein produced either naturally or by recombinant means . The term "immunoglobulin" is further meant to refer to either a single immunoglobulin or mixtures of immunoglobulins. Examples of immunoglobulins include, but are not limited to: IgA, IgD, IgE, IgG, IgM, and fragments thereof ( e. g. Fab, Fc, and F(ab')₂ fragments), polyclonal anti- bodies, monoclonal antibodies, recombinant single-chain antigen binding proteins, and other recombinant immunoglobulins (e.gr. chimeric and humanized antibodies) produced 10 in non-human animals.

As used herein, the term "skin area affected by acne" refers to areas of the skin in which the signs and/or symptoms of acne are present , and includes multiple areas of infection. The term also encompasses areas in which bacteria associated with acne are present.

As used herein, the term "colostrum" refers to the immunoglobulin rich milk produced by mammals immediately following parturition. As used herein, the term "bovine colostrum" refers to colostrum produced by bovines . The term "colostrum" also encompasses various preparation of colostrum, including, but not limited to, freeze-dried colostrum, aqueous solutions of colostrum, and gel solutions of colostrum.

As used herein, the terms "anti-bacterial" , "bacterio- static", "antimicrobial", and "bactericidal" are used interchangeably in reference to any compound, substance or molecule capable of inhibiting growth of or of killing microorganisms. It is intended that the term be used in its broadest sense, and includes, but is not limited to compounds such as peroxidases and immunoglobulins which are produced naturally or synthetically. It is contemplated that compositions containing multiple compounds will find use in the present invention. For example, it is intended that the term encompass antimicrobial compositions in which peroxidases or immunoglobulins are included in addition to other compounds. It is not intended that the present invention be limited to any particular antimicrobial composition. 11

As used herein, the term "topically" means application to the surface of the skin. Both the peroxidase system preparation and immunoglobulin preparation may be applied topically.

The phrase "under conditions such that said acne is reduced" means a reduction in the number of or prevention of the recurrence of pustulae, papulae, whiteheads, black- heads, comedones or other physical manifestations of acne in a skin area affected by acne.

As used herein, the term "percentage by weight" of a given preparation, composition, or mixture means that the prepa- ration, composition or mixture contains a certain percentage of a compound based on its weight and is calculated by dividing the mass of the compounds by the mass of the preparation and multiplying by 100. For instance, a preparation which contains 5 grams immunoglobulins and has a total mass of 100 grams contains contains immunoglobulin which is 5% by weight of the preparation.

DESCRIPTION OF THE INVENTION

The object of obtaining an effective, non-toxic, skin friendly preparation for topical treatment of skin conditions, in particular acnes, is obtained by providing agents for the treatment of skin conditions, wherein the active substances are not foreign to the host organism, but are a part of the host ' s own defense against the present skin conditions.

Specifically, the present invention relates to the 12 treatment and/or the prophylaxis of acne with compositions containing peroxidases and/or immunoglobulins. The description discusses 1) natural host defense mechanisms and 2 ) acne treatment .

1. Host Defense Mechanisms

The natural defense in humans and animals can be divided into two categories, the non-spesific host defense includ- ing various enzymes and chemical substances which act as non-specific antimicrobial agents, and the specific host defense which is also known as the immune response or the immune system.

a. The Immune System

The immune system can be divided in the cellular, or cell- mediated (CMI) , and the humoral immune system (HMI) . HMI involves agents which are soluble, or at least non-cellular, such as antibodies or immunoglobulins.

In humans, immune response against pathogenic organisms are primarily produced by the white blood cells or leukocytes . Leukocytes develop from stem cells in the bone marrow and gives, via two different differentiating lines, phagocytes (the myeloid line) and lymfocytes (the lymfoid line) .

The phagocyting cells are a part of the cell-mediated immune system and act by enveloping and consuming the invading microorganisms. Certain bacteria are, however, able to defend themselves against phagocytosis. For instance, most strains of Staphylococcus aureus produce leukocidin which directly destroy the leukocytes, allowing the Staphylococ- 13 cus aureus cells to escape phagocytosis unharmed. Furthermore, Staphylococcus aureus produce an enzyme-like factor called coagulase that causes coagulation of blood and accumulation of fibrin around the bacterial cells and thus renders them resistant to phagocytosis. The formation of fibrin clots also results in the walling off of the infected area, making it difficult for host defense agents to come into contact with the bacteria. The use of medicaments based on phagocyting cells will therefore not be suf- ficiently effective to combat the bacteria in question.

Lymphocytes can be divided into two main groups; T cells, which belong to the cell-mediated immune system, and B cells, which are precursors of the antibodyforming cells, the AFC cells.

Antibodies are so-called antigen-reactive proteins; that is they are produced and secreted from the immune cells which are stimulated by the presence of a foreign agent in the body, a so-called antigen, such as pathogenic bacteria. Although cross-reactivity occurs, in general, the antibody binds specifically to the antigen which stimulated its forming. Antibodies are also called immunoglobulins and are divided into immunoglobulins A, D, E, G, M, which are ab- breviated IgA, IgD, IgE, IgG, and IgM.

b. Non-specific Host Defense Mechanism

Non-specific defense agents include various enzymes and chemical substances . The best known enzyme involved in defense mechanisms is lysozyme, which is present in tears, nasal secretion, saliva, mucosa and tissue fluids. Lysozyme acts as antibacterial agent by hydrolyzing the glycosidic 14 bond between the polysaccharide units in the peptidoglycane layer in the bacterial cell walls. This causes lysis of the cell walls and death.

Another example is the enzyme lactoferrin, which is present in tears, saliva, milk, mucosal secretions, etc. Lactoferrin prevents bacterial growth by binding iron and hence depriving the bacterial cells of a trace substance essential for growth.

Peroxidases, which are also present in tears, saliva, the small intestine, mucosa and milk, play a defensive role in bacterial invasion. Examples of peroxidases include: myeloperoxidase and horse-radish peroxidase (McCormick et al . , J. Biol . Chem. 273 (48) :32030-37 (1998); lactoperoxidase (Watanabe et al . , FEBS Letters 441, 476-79 (1998); and eosinophil peroxidase, thyroid peroxidase, and salivary peroxidase (Banerjee et al . , Mol . Cell . Biochem. 70, 21-29 (1986) . Peroxidases exhibit antibacterial action in con- junction with specific co-factors including hydrogen peroxide and, depending on the specificity of the peroxidase in question, thiocyanate, chloride, bromide or iodide. The reaction may be summarized as follows:

H₂0₂ + X^" → H₂0 + OX^"

wherein X is SCN, Cl, Br or I .

Bacterial activity is inhibited or eliminated when the oxi- dation product, OX^", oxidizes amino groups and/or thiols in enzymes which are essential for the metabolism of the bac- 15 teria .

When the peroxidase is lactoperoxidase and X is thiocyanate (SCN^") , the system is referred to as a lactoperoxidase sys- tem, LPS, and its components occur in milk, saliva and the gastrointestinal tract. Antibacterial inhibition by this system was first discovered in certain strains of milk producing streptococcus, whose growth, respiration and production of acid were inhibited. It is not intended that the present invention be limited to any particular mechanism of action. Indeed, an understanding of the mechanisms is not necessary to make and use the present invention. While not limiting this invention to any particular theory, it is generally thought that the oxidation product for the LPS- system, hypothiocyanite ions, OSCNJ oxidizes the bacteria ' s sulphhydryls and proteins to sulphenylthiocyanate and sulphonacid derivative, and also inactivates SH-depen- dent enzymes in the glycolysis.

2. Acne Treatment

According to the present invention, it has been found that topical application of a peroxidase system, in particular a lactoperoxidase system, and/or antibodies (called immunoglobulins below) effectively softens skin conditions such as the above.

Clinical experiments demonstrate that the treatment of skin conditions with medicaments including a lactoperoxidase system component and/or an immunoglobulin component not only reduced the outbreaks, but also reduced the number of recurring outbreaks, as well as reduced the severity of the outbreaks (see for instance Examples 1 and 5) . The lacto- 16 peroxidase system component and the immunoglobulins component can be applied separately, but clinical experiments indicate that combined application is advantageous (see Example 6) .

The effect of in vivo-use of the lactoperoxidase system has not been tested, but it is assumed that the effect of the two-component preparation has a synergistic effect.

Hence, the present invention provides a pharmaceutical composition containing a lactoperoxidase system and/or immunoglobulins and a two-component preparation comprising a composition containing a lactoperoxidase system and a composition containing immunoglobulins, packed separately or combined, and the use thereof to combat surface-associated bacterial skin conditions.

The lactoperoxidase system can exist as an aqueous solution or, preferably, as a gel, whereas the immunoglobulins com- ponent preferably is a gel containing bovine colostrum with a high concentration of immunoglobulins and a low concentration of lactoperoxidases .

The bovine colostrum can be provided as a freeze-dried pow- der. It can, however, be convenient to avoid freeze-drying because a freeze-drying process can inhibit the activity of the immunoglobulins.

In some embodiments of the present invention, the lacto- peroxidase composition is applied to the skin area to be treated first . This is because the lactoperoxidases may inhibit the activity of the immunoglobulins. Hence, it is 17 convenient to apply the lactoperoxidases first and let them act unhibited for 1-2 min before they dry out on the skin. In still further embodiments, the more specific immune system is activated by applying the immunoglobulin composition which is also allowed to dry on the skin. If desirable, moisturizer and other make-up can be applied thereafter.

Alternatively, but less preferred, the lactoperoxidase system and the immunoglobulins may be provided in the same preparation .

In another aspect , the present invention relates to the use of a lactoperoxidase system and/or immunoglobulins in the production of a medicament for the treatment and/or prophy- laxis of surface-associated bacterial skin conditions.

It will be recognized by those skilled in the art that the peroxidase system and immunoglobulins of the present invention may be formulated in a variety of compositions for topical use. These compositions include gels, creams, salves (e.g. an unctuous material), colloids, liquids and other suspensions or aqueous solutions . The formulations can comprise the active ingredients together with physiologically tolerable liquid, gel or solid carriers, di- luents, adjuvants and excipients. On the other hand, formulations may also contain such normally employed additives as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions typically contain 1-95% of active ingredient, preferably 2- 18

70%. The important is not the mode of topical application, but that the activity of the peroxidases and immunoglobulins be maintained.

Other possible applications of the present composition include cleaning/disinfecting of veterinary surfaces, such as udder during milking and the healing of superficial wounds.

In the following, the invention shall be described more de- tailed by way of examples. However, the examples are not intended to limit the scope of the invention in any way.

Example 1 Antibacterial activity of the lactoperoxidase system against the acne bacteria Staphylococcus and Propi - on i bacterium acnes - in vitro -experiment

Lactoperoxidase at concentrations of 1, 5, 30, 60, 120, 250, 500, 750, 1000 μg/ml was incubated with bacteria at a concentration of 5 x 10^s bacteria/ml in nutrient medium containing 0.25% glucose. The results are provided in

Figure 1. These results demonstrate a significant bactericidal effect of the lactoperoxidase system as all S. aureus bacteria were killed at a lactoperoxidase concentration of 60 μg/ml, and all P. acnes bacteria at a lactoperoxidase concentration of 120 μg/ml.

Example 2 Preparation of the gel formulation containing a lactoperoxidase system

A batch, i . e. approximately 500 kg, of a gel formulation containing lactoperoxidase was prepared in the following 19 way :

228 kg deionized water, 2 kg hydroxyethylcellulose and 15 kg glycerine were mixed and heated to 55°C. 0.75 kg Methyl- paraben and 0.75 kg propylene glycol were mixed and added to the reaction mixture. Then 250 kg of a dry powder containing 17.6 wt% lactoperoxidase, 44.1 wt% potassium thiocyanate, 29.4 wt% glucose pentaacetate, and 1.5 kg imi- dazolidinyl urea. The gel formulation was given a user- friendly scent by adding 1 kg concentrated extract of each of the plants Anthemis Nobilis and Hamamelis Virginiana .

The activity for the final gel formulation was :

Lactoperoxidase activity: 175000 ABTS units/g Glucooxidase activity: 23500 ABTS units/g.

The final gel formulation was filled into 200 ml squeeze- able bottles . Another volume and container can be chosen by the producer based on several factors, such as the duration of the product etc .

Example 3 In vi ro-activity of the immunoglobulin containing tablets against Staphylococcus aureus and Propionibac - te-rium acnes - comparative example

In this experiment, tablets containing bovine colostrum (15 mg/550 mg tablet) in accordance with the present invention, was evaluated with regards to antibodies against Staphylo- coccus aureus and Propionibacterium acnes with Enzyme- Linked Immunosorbent Assay, ELISA. The immunoglobulin con- 20 taining product ViM, a non-fractionated product from Jens Pedersens laboratory in Denmark, was used as a reference. The content of antibodies in this product is not given.

For both products, a tablet was dissolved in 15 ml phosphate buffered saline, which for the tablet of the present invention corresponds to 1 mg/ml colostrum protein (bovine immunoglobulines correspond to approximately 84 % of the total amount of protein) . Then the stock solutions were serially diluted by 10-fold dilutions.

The antigens were 3 different preparations of Staphylococcus aureus and Propionibacterium acnes . Table 1 describes the origin, type of antigen, product source and concentrations used for coating of microplates .

Table 1 Antigens

Origin Antigen Product source Coating concentration in

ELISA

S . Teichoic acid (cell Biosys inova 1 μg/ml aureus wall component)

" Alpha -toxin 2 , 5 μg/ml

P . acnes Filled whole bacterium chlin . Bact . 1 xlO⁸ bact . /ml GU

The antibody concentration was expressed as absorbance₄₀₅- values corresponding to 500 μg/ml and 1000 μg/ml colostrum protein solution; that is a dilution of 1 : 2 and 1 : 10 of the stock solution and antibody titers in % of tablet ViM equal to a dilution of 1 : 2 .

The results are given graphically in Figure 2 , 3 and 4 . 21

The amounts of antibodies for the different antigens cannot be directly compared as different concentrations are used for coating, and the antigens may vary with regards to their ability to bind to the plastic surface, which can result in different absorbancy values. However, Figure 2-4 clearly show that the colostrum tablet according to the present invention have a much better antibacterial effect than the ViM tablets .

Example 4 Preparation of the gel formulation containing im- munoglobulines

A batch, i.e. approximately 500 kg, of a gel formulation containing immunoglobulines was prepared in the following way: 426 kg deionized water, 3.75 kg hydroxyethylcellulose, 15 kg glycerine were mixed and heated to 80°C. 0.75 kg Met- hylparaben and 0.75 kg propylene glycol were mixed and ad- ded to the reaction mixture. 1.0 kg pharnesol, 1 kg drago- santol and 10 kg emulgator (E472C:E471, 2:1) were mixed and added to the reaction mixture while stirring. 2.5 kg pan- tenol and 22 kg immunoglobulin IgG (purity degree of 80% with respect to protein) were mixed and added to the reac- tion mixture while stirring. 1.5 kg imidazolidinyl urea and 15 kg deionized water were mixed and added to the reaction mixture. Finally 0.25 kg lactic acid and 0.25 sodium lac- tate (60 %) were mixed and added to the reaction mixture.

The finished gel was filled in 10 ml rollers. Another volume and container can be chosen by the producer on the ba- 22 sis of several factors, such as the duration of the product etc.

Example 5 In vivo-experiment with gel formulation contain- ing immunoglobulins

Twenty-one otherwise healthy test subjects at the Huslaker- mottagningen participated in this experiment. At the beginning of the experiment, age (13-39 years) , gender (13 women and 8 men) , duration of acne problems (1-240 months) , as well as previous treatment, use of oral anticonception agents and possible disease requiring treatment, were registered. Age, gender and duration of acne problems are given in Table 2 below.

23

Table 2 Age, gender and duration of the test subjects acne problems, as well as previous treatment

Test Duration of acnes Previous treatment subject Age Gender [months] no.

1 15 F 60 0

2 30 M 120 0

3 19 F 60 1

4 15 M 24 0

5 19 M 60 0

6 23 M 120 1

7 29 F 60 0

8 25 F 60 0

9 31 F 240 0

10 39 F 36 1

11 27 F 120 1

12 36 F 180 0

13 13 M 6 0

14 15 F 36 0

15 13 F 24 0

16 24 M 1 1

17 15 F 30 0

18 21 F 24 0

19 19 M 60 0

20 16 F 60 0

24

Test Duration of acnes Previous treatment subject Age Gender [months] no.

21*

* Test subject no. 21 withdrew shortly after the beginning of the experiment due to initial aggravation of the acne problem.

The test subjects were treated by opening pustula with a necrotic tip, washing the face with ACO's neutral facial water without alcohol or perfume, and the immunoglobulin composition in accordance with Example 5 was rolled directly on the area in question and the area in close vicinity to the former. The composition was applied in amounts sufficient to apparently cover the areas in question.

The test subjects were instructed to repeat the application procedure described above every morning and every night for 6 weeks .

The test subjects had 3 check-ups during the study; at the beginning, after 3 weeks and after 6 weeks. At every checkup, the number of papula and pustula and cysts were counted on the right and left side, as well as the front of the face. Individual results are given in Table 3 below.

The experiment was performed during a period of time with little sun (March - April) , and, hence, the beneficial effect of sunlight on the acnes can be excluded. 25

Furthermore, the antibacterial effect of the facial water can be excluded as this product does not contain any substances which are effective against acnes.

Table 3 Acne problems at the beginning of experiment, after 3 weeks treatment and after 6 weeks treatment with immunoglobulin containing gel formulation of the present inVO 00 vention

Test subAcne vulgariβ before treatment Acne vulgariB after 3 weeks Acne vulgaris after 6 weeks ject no . Papula Pustula Cysts Papula Pustula Cysts Papula Pustula Cysts

1 17 5 0 3 0 0 0 0 0

2 13 1 0 15 0 0 15 0 0

3 30 18 0 25 7 0 30 1 0

4 41 6 0 28 6 0 15 0 0

IV) en

5 25 0 0 22 0 0 8 7 0

6 15 2 1 6 1 0

7 15 0 0 11 0 0 13 0 0

8 21 7 0 15 1 0 3 0 0

9 10 0 0 9 0 0 10 2 0

10 16 0 0 7 0 0

11 10 1 0 4 0 0 2 0 0 o H

12 12 0 0 1 0 0 0 0 0 O

13 21 3 0 22 2 0 10 1 0 VO o

o

Test subAcne vulgariβ before treatment Acne vulgaris after 3 weeks Acne vulgaris after 6 weeks o vo ject no. Papula Pustula Cysts Papula Pustula Cysts Papula Pustula Cysts vo

VO 00

14 9 0 0 4 0 0 4 0 0

15 11 0 0 8 1 0 lacking lacking lacking

16 14 1 0 8 0 0 5 0 0

17 7 4 0 10 3 0 6 0 0

18 15 0 0 13 0 0 lacking lacking lacking

19 20 0 0 9 0 0 lacking lacking lacking

20 6 0 0 15 0 0 0 0 0

IV)

o

H

0 VO o

© o

28

It can be seen from Table 3 that the test subjects' acne problems were reduced upon treatment with the immunoglobulin composition according to the present invention.

Based on the data given in Table 3, p-values were calculated by using Student t-test. For papula, the values were found to be respectively p<0.00092 for reduction after 3 weeks treatment and p<0.0053 for reduction after 6 weeks. For pustula, the values were found to be, respectively, p<0.059 after 3 weeks reduction and p<0.025 after 6 weeks reduction.

In this experiment, p-values under 0.05, p<0.05, were regarded as significant. Data given in Table 3 therefore show that the reduction in the number of papula was significant already after 3 weeks treatment with the immunoglobulin composition in accordance with the invention, whereas the reduction in the number of pustula was significant after 6 weeks treatment .

The results for total number of papula and pustula, as given in Table 3 , are also shown graphically in Figure 5 and 6, respectively. The antibacterial effect of the composition is evident from these figures. Furthermore, Figure 5 and 6 show that the results are much better after 6 weeks treatment than after 3 weeks treatment. Therefore, a treatment period of 3 weeks seems insufficient . It is also fair to assume that further improvements could be observed after prolonged treatment .

The test subjects' condition was also evaluated, by a physician, on a descriptive scale from "aggravated" to "elimi- 29 nated" . The results of this evaluation are given in Table 4 below.

Table 4 Results of experiment as evaluated by physician

Change in problems After 6 weeks (number of test subjects) Aggravated

Unchanged 4

Somewhat improved 1

Improved 7

Much improved 3

Eliminated —--—~ — _-___ . -^--— ______

Acnes, which appeared during the treatment, are included in the values in Tables 3 and 4. However, all participants indicated that the new acnes were fewer, smaller and generally less aggressive. This change appeared during the sec- ond week of treatment and became more evident throughout the period of treatment .

Also, several of the participants recorded that the first week of treatment resulted in increased irritation and soreness in sensitive acnes, which implies that the effect of the antibodies set in shortly after application.

No other side effects were observed, which indicates that the tolerance of the product is high.

Furthermore, almost all test subjects stated that the roller was simple and practical to handle. 30

Example 6 Antibacterial activity of the two-component preparation - In vivo-experiment.

12 otherwise healthy test subjects in a private clinic in Sandefjord participated in this experiment. At the beginning of the experiment, age (16-33 years) , gender (10 women and 2 men) , duration of acne problems (3-48 months) were registered. The character of the acne problems varied from mild to moderate. All test subjects had tried a number of other products without satisfactory results. Age, gender and duration of acne problems for the test subjects are given in Table 5 below.

Table 5 Age, gender and duration of acne problems

Test subject Duration of acne

Age Gender no. [months]

1 18 M 36-48

2 17 F 3

3 19 F 30-40

4 26 F 20-25

5 33 F 24

6 22 F 15

7 16 F 24

8 25 F 26

9 16 F 12

10 19 F 36

11 20 F 36

12 27 M 20-25

The test subjects were instructed to apply the gel formulation containing the lactoperoxidase system to the sensi- 31 tive areas in amounts apparently sufficient to cover them. A small spatula was handed out for this purpose. After 1-2 min, the immunoglobulin composition was rolled on in such a manner that the acne appeared to be covered. The applica- 5 tion procedure was repeated every morning and night for 6 weeks .

The test subjects had 2 check-ups during the study; at the beginning and after 6 weeks treatment . At each control , the 10 number of papula on the left and right side, as well as front of the face was counted. The results of the experiment are given in Table 6 below.

Table 6 Acne problem at the beginning and end (6 weeks) 15 of the experiment

Test subAge Gender Acne vulgaris before Acne vulgaris after 6 ject no. treatment (# papula) weeks treatment

(# papula)

1 18 M 30-35 10-12

2 17 F 13-15 3-4

3 19 F 25-30 15

4 26 F 25-30 10-12

5 33 F 25-30 10-12

6 22 F 25-30 10-12

7 16 F 25-30 10-12

8 25 F 25-30 10-12

9 16 F 25-30 10-12

10 19 F 25-30 10-12

11 20 F 25-30 10-12

12 27 M 25-30 10-12

32

As is apparent from Table 6 , the number of papula was significantly reduced for all test subjects. The mean value for the number of papula was reduced from 25.2 ± 3.0 at the beginning to 8.4 ± 1.6 after 6 weeks. This represents an improvement of 67%. p<0.001.

The test subjects' condition was also evaluated, by a physician, on a descriptive scale from "aggravated" to "eliminated" . The results of this evaluation are given in Table 7 below.

Table 7 Results of experiment as evaluated by physician Change in problems After 6 weeks (number of test subjects) Aggravated Unchanged

Somewhat improved 3

Improved 5

Much improved 4

Eliminated

As given in Table 7 , the physician regarded the condition for all test subjects as significantly improved.

Furthermore, the individual test subject evaluated themselves in the same way once every week during the experi- ment. The subjects' own evaluations are summarized in Table 8 below. 33

Table 8 Result of experiment as evaluated by the test subjects

Weeks (number of test subjects)

Change in 1 2 3 4 5 6 acne problems

Aggravated 3

Unchanged 2 6 4 3 2 2

Somewhat im7 4 2 2 3 3 proved

Improved 2 4 4 4 3

Much improved 2 3 3 4

Eliminated

As given in Table 6, 10 out of 12 subjects, i.e. 83%, eva- luated their skin conditions as somewhat improved at the end of the experiment. 7 out of 12 subjects, i.e. 58%, characterized their skin conditions as "improved" or "much improved" after 6 weeks of treatment.

Also, Table 8 confirms that bacterial inhibition or treatment is a time-dependent function. Hence, it is likely that even better results would have been obtained if the experiment were to continue beyond the 6 weeks .

Furthermore, as only one subject reported some initially dry skin which disappeared upon continued treatment, user tolerance of the two-component preparation can be regarded as excellent. No other side-effects were reported or registered.

Compared to the retinoid based products, which are consid- 34 ered as some of the best on the market today, this is a good result, especially taken into consideration that the use of retinoid based products may result in rather serious side effects (Vahlquist C. & Fyrand O. , "Isotretinoin - Modes of Action Side Effects (clinical __ laboratory) and Results (short- & long-term) " in Workshop Treatment of Acne, 1996, 3, Lakemedelsverket (Medical Products Agency) and Statens Legemiddelkontroll (The Norwegian Medicines Control Authority) , which side-effect are absent when using the two-component preparation in accordance with the invention.

Furthermore, the results for the two-component preparation are better than the application of the immunoglobulincon- taining gel only, see Example 5, wherein the mean number of papula was reduced from 16.4 ± 6.0 to 8.1 ± 5.9, which represents a reduction of 51%.

Claims

35C l a i m s

1. A pharmaceutical composition, preferably as a gel, for topical treatment and/or prophylaxis of surface-associated skin conditions caused by bacteria, such as Staphylococcus aureus and Propionibacterium acnes, characterized in that it comprises from 10 to 60 wt%, preferably approximately 30 wt% of a peroxidase system.

2. The composition of claim 1, wherein the peroxidase system is a lactoperoxidase system.

3. The composition of claim 2, characterized in that the ratio between lactoperoxidase and thiocyanate is from 1:1 to 1:10, preferably approximately 1:2.5.

4. The composition of any of the preceding claims, characterized in that the thiocyanate is potassium cyanate .

5. A pharmaceutical composition, preferably as a gel, for topical treatment and/or prophylaxis of surface-associated skin conditions caused by bacteria, such as Staphylococcus aureus and Propionibacterium acnes, characterized in that it comprises from 1 to 12 wt%, pref- erably from 4 to 5 wt% immunoglobulins with a purity of 80 % with respect to protein.

6. The use of a peroxidase system in the production of a medicament for topical treatment and/or prophylaxis of surface-associated skin conditions caused by bacteria, such as Staphylococcus aureus and Propionibacterium acnes . 36

7. The use of the peroxidase composition in accordance with claims 1-4 in inhibiting bacteria such as Staphylococcus spp. and Propionibacteria spp., preferably Staphylococcus aureus and Propionibacterium acnes, on surfaces.

8. The use of immunoglobulines in the production of a medicament for topical treatment and/or prophylaxis of surface-associated skin conditions caused by bacteria, such as Staphylococcus aureus and Propionibacterium acnes .

9. The use of the immunoglobulin composition in accordance with claim 5 in inhibiting bacteria such as Staphylococcus spp. and Propionibacteria spp., preferably Staphylococcus aureus and Propionibacterium acnes, on surfaces.

10. Two-component preparation for topical treatment and/or prophylaxis of surface-associated skin conditions, characterized by comprising a pharmaceutical composition containing a peroxidase system and a pharmaceutical composition containing immunoglobulins.

11. The two-component preparation of claim 10, wherein the peroxidase system is a lactoperoxidase system.

12. The two-component preparation of claims 10 or 11, characterized in that the compositions are two separate components of a kit .

13. The two-component preparation of claims 10 or 11, characterized in that the lactoperoxidase system and the immunoglobulins are contained in the same composition. 37

14. The two-component preparation of claims 10-13, characterized in that the composition (s) are present as a gel .

15. The use of a lactoperoxidase system and immunoglobulins in accordance with claim 6 and 8 in the production of the two-component preparation of claims 9-13.