Field of Art

The invention relates to a new immunobiological preparation suitable for the treatment of autoimmune skin diseases, particularly psoriasis, and to the use of this preparation in therapy. Background Art

Psoriasis is a non-contagious chronic inflammatory skin disease, which shows up through the dissemination of red or purple plastic, sharply demarcated plaques with a layer of silvery scales. Psoriasis is a so-called autoimmune disease, in which an individual's immune system attacks its own cells. The most common places of occurrence are elbows, knees, loins and the scalp area.

The first references to psoriasis have already been found in ancient civilizations, on Egyptian papyri. The word psoriasis comes from the Greek psora, i.e. itching, a scale. About 2 - 3 percent of the population suffer from psoriasis in Central Europe. Although psoriasis has already been reported in biblical times, treatment options for this skin disease, which used to discard patients from life and to cause disfigurement, have not improved until the second half of the 20 ^th century. However, a medicament has not been found yet for its cure, although patients now have a whole range of medicines destined for topical or systemic use or treatment based on ultraviolet radiation. These medicines can keep the disease under control and temporarily eliminate some of its symptoms. A new type of treatment for psoriasis, biological medicines, has been introduced in the 21 ^st century, which has brought new hope to people suffering from psoriasis all over the world.

The treatment of psoriasis can be divided into the following basic categories: use of topical medications (medications applied to the skin), phototherapy, or a combination of phototherapy and other medications, systemic therapy (medicines administered by injection or in tablets), biological treatment. A number of factors such as the type of psoriasis, the location of the affected area on the body, the severity of the symptoms, the patient's age and their anamnesis, the patient's response to a previous treatment, decide, which treatment is appropriate for the respective patient.

The topical therapy is usually used as the first option in the treatment of psoriasis. After the application the medicines act on a removal of lesions relatively quickly and are usually well tolerated. However, for the topical preparations to be active it is essential to use them repeatedly, because they are insufficient for the preservation of the temporary disappearance of psoriatic symptoms. They can also stain and their application can be lengthy, if psoriasis affects a larger part of the body. For the topical therapy, e.g., Dithranol (Anthralin) is used as a medicine, or in combination with UVB rays therapy. However, this medicine often causes skin irritation and burning. Additionally, everything that comes into contact with the medicine is stained.

Topically applied corticosteroids are medicines similar to naturally occurring hormones of the human body. They are available in many different efficacies and pharmaceutical formulation types such as creams, lotions, moisturisers, sprays, ointments and medicated plasters. Corticosteroids act in patients with psoriasis in such a way that they slow down the growth of skin cells and reduce inflammation of the lesions. Although corticosteroids can quickly remove the lesions, it is typical for them that the remission caused by them does not last for a long time, so that the lesions associated with psoriasis reappear after a short period of time. Side effects of long-term corticosteroids use are numerous and include skin atrophy, creation of telangiectasias (exerted dark red spots on the skin) and striae (scarring on the skin), after which there appear even scars on the skin.

Topically applied coal tar can be used alone or in combination with UVB. The coal tar usage is very restricted by its ability to stain everything around and especially by the fact that coal is a proven carcinogen.

Retinoids (Vitamin A derivatives) represent newly developed medicines for local (topical) treatment of psoriasis and are used to treat mild to moderate plaque psoriasis.

Calcipotriol, Vitamin D analogue, provided a certain alternative to topically applied steroids, tar, and Dithranol in the early nineties. Calcitriol and Tacalcitol substances were introduced later. Calcipotriol showed equal or better efficacy than other preparations and it is more acceptable from the cosmetic point of view and generally well tolerated.

Salicylic acid is a substance which helps to remove the scales from the lesions. This allows the application of topical preparations and their better penetration into the skin. It can be used in the form of a paste, a cream, an ointment or a solution.

For the treatment of psoriasis, for some individuals it is possible to try a variety of over-the-counter (OTC) medicines including moisturizers, bath oils, preparations with magnesium sulphate and oat flour with oil. They are not effective for everyone, but they are usually without any side effects.

For the patients with psoriasis unresponsive to topical therapy or where psoriasis is too widespread, phototherapy is suitable. Its principle is skin exposure to ultraviolet radiation, which has a therapeutic effect on psoriasis. The ultraviolet radiation can be either natural, as part of solar radiation, or artificial, from special lamps emitting radiation in a narrow band around the optimal wavelength of 31 1 nm.

The PUVA method, also called Photo-Chemotherapy, was developed in 1970s and is based on a combination of a preparation increasing the sensitivity to light (Psoralen) followed by irradiation with ultraviolet rays UVA (like UVB, UVA occurs in natural light). Psoralen induces a higher sensitivity of the skin and its stronger reaction to this type of ultraviolet radiation. A long-term PUVA therapy can lead to premature skin aging and also significantly increases the risk of skin cancer (basal cell carcinoma and carcinoma of superficial skin cells).

Laser therapy is a new method for the phototherapy of psoriasis. An excimer laser sends a beam of ultraviolet light at high intensity, aiming at selected areas of skin. Side effects of laser treatments include low risk of scar and bruise formation.

Patients who do not respond to phototherapy or cannot frequently undergo irradiation, which is necessary for the lesions to be removed, may be treated systemically. The concept of systemic therapy consists in the use of medicines acting throughout the body. These substances include Methotrexate, Cyclosporin and orally administered Retinoids. One of the biggest disadvantages of Methotrexate is the fact that its long-term use is associated with liver damage. In many countries it is therefore required that patients, who use Methotrexate for a long time, undergo a routine liver biopsy examination. It acts such a way that it binds to an enzyme involved in cell growth, and thereby slows down the growth of pathologically altered skin cells caused by psoriasis. However, Methotrexate also affects normal cells, including fetal cells, bone marrow and sperm cells. Cyclosporin slows down the growth of skin cells by inhibiting the immune system. Its long-term use is, however, associated with nephrotoxicity. The Retinoids affect cell behaviour including their growth rate and their separation from the skin surface. Orally administered Retinoids are very little effective as monotherapy, and the treatment is associated with numerous side effects such as hair loss and thinning of nails, and can also harm the fetus. The Retinoids are, however, very effective in small doses in combination with UVB phototherapy or PUVA, which allows them to substantially eliminate lesions at a low number of visits associated with phototherapy and at a low side effects.

The biological treatment of psoriasis represents a significant advance in the treatment of this disease. It is an effective treatment, long-term safe, comfortable and well- tolerated by patients. It is destined for a defined group of people suffering from psoriasis, who cannot be effectively treated with the current systemic therapy. These patients are therefore given a full personal life and position in society comparable to a healthy population. In the treatment of psoriasis the biological therapy offers a high efficacy and an improved safety because of using targeted medicines based on natural proteins, which interfere with certain steps in the pathogenesis of psoriasis. Biological preparations are targeted at the immune system, where they block the activity of certain immune system cells, which are of importance in the pathogenesis of psoriasis. While other treatment options for psoriasis, such as PUVA, Methotrexate and Cyclosporin, also affect the immune system, the effect of biological preparations is more specific and these preparations can be safer.

Two types of biological medicines are being used for the treatment of psoriasis - one type acts on the T-cells of the immune system and thus directly affects psoriatic inflammation, while the others are non-specific and affect Cytokines (TNFa) and influence the inflammation at many levels. The first group of medicines, developed specifically for psoriasis, include Efalizumab (Raptiva), which specifically affects T- cells and thus has a specific effect on the skin. Medicines from the other group (e. g. Etanercept (Enbrel), Infliximab (Remicade), Adalimumab (Humira)) affect Cytokines and are therefore suitable for the treatment of joints and skin. These medicines, as they act specifically against the aberrant immune response, have significantly less side effects. As with any progressive new medicines there arose concerns about the safety of their use.

15 - 25 percent of patients have moderate and severe psoriasis. These are the candidates for lifetime systemic treatment. Therefore, it is very important to be sure that the medicine has no serious side effects for them. Clinical observations showed a significant and long-term improvement in 73 percent of patients, when treating moderate and severe psoriasis, who used Efalizumab and for 40 percent of patients the symptoms disappeared completely. The side effects were comparable with the side effects when administering placebo.

It is therefore necessary to continue to explore more medicines and methods suitable for the biological treatment of psoriasis, which have sufficient efficacy and minimum side effects. Disclosure of the Invention

The object of the present invention is an immunobiological preparation, which contains at least one active ingredient selected from the group comprising of inactivated cells of Bacillus megaterium culture and Bacillus megaterium culture filtrate.

The term "inactivated cells of Bacillus megaterium culture" refers to cells, which are prepared by any of the known methods of inactivation, e.g. fractional sterilization. The Bacillus megaterium is preferably selected from the group consisting of Bacillus megaterium strains: 1/32, 2/37, 3/39, 4/58 and 5/74 according to the CNCTC (the Czech National Collection of Type Cultures), Catalogue of Strains - 5th Edition, p. 5.

The object of the invention is also a preparation according to the present invention for use in the treatment of an autoimmune skin disease, preferably in the treatment of psoriasis. The preparation according to the present invention is preferably administered in the form of a subcutaneous injection. The preparation can be administered again at an interval of 2 to 3 weeks, if the desired clinical effect fails to appear within one week after the first dose.

As shown below, administration of the preparation according to the present invention has proved to be very effective in treating psoriasis.

Overview of Illustrations on Drawings

Figure 1 shows a table for the calculation of the PASI score.

Figure 2 represents a chart showing changes in the average PASI score of monitored subjects according to Example 3 at time. Examples of Carrying Out the Invention

Example 1

Cultivation of Bacillus megaterium bacteria cultures, preparation of inactivated cells

Bacillus megaterium bacteria cultures from the Collection of the National Health Institute, CNCTC - the Czech National Collection of Type Cultures - Catalogue of Strains - 5 ^th Edition, page 5, were used in the present examples.

Bacillus megaterium de Bary (hereinafter referred to as Bacillus megaterium), the strains - 1/32, 2/37, 3/39, 4/58, 5/74.

6200 Bm 1/32 NIPH, "Mega I". (Medium 1 37C)

2/37 NIPH," Africa". (Medium 1 37C)

3/39 NIPH, "Bacillus petasites". (Medium 1 37C)

6756 4/58 I.Alf~ldi, Regional Hygiene Station, Szeged, Hungary,

"25". Producer of Megacine. (Medium 1 37C)

6213 5/74 ATCC, "13632". Non-spore-forming variant. (Medium 1 The culture from the lyophilisate is revived according to the following instructions:

1. The incised upper end of the vial is broken off.

2. The vial is incised by a file.

3. The surface of the vial is disinfected.

4. The incised vial is broken apart and the inner ampoule with the dry matter is taken out.

5. The dry matter is dissolved by adding a few drops of meat peptone broth (the preparation is described below)

6. This solution is aseptically poured into 1 litre of meat peptone broth, which is a multi-purpose medium for the cultivation of microorganisms. Then the incubation follows.

Preparation of meat peptone broth:

Ingredients: meat extract 10 g, pro bacto peptone 10 g, sodium chloride 5 g.

Procedure of preparation: The above ingredients are weighed into 1 ,000.0 ml of distilled water (into a sterile Erlenmeyer flask) and heated up to a complete dissolution. Then the flask and its contents are sterilized in an autoclave at 121 °C for 20 minutes.

pH: 7.2 ± 0.2 at 25 °C.

Incubation:

The flask and its contents is retained for 1 week at 37 °C - aerobically. The growth of the culture is manifested through the turbidity of the contents of the flask.

The intermittent, fractional sterilization:

The flask with its contents is heated to 100 °C for 30 minutes and then left for 24 hours at 37 °C. Under these conditions the possibly present spores germinate. The grown vegetative forms are destroyed by a triple repetition of this procedure.

Example 2

Preparation and administration of the immunobiological preparation The suspension of the inactivated cells prepared according to the procedure described in Example 1 is, after it has cooled to 25 °C, filtered with through a sterile S-PAK Millipore 0.22 μηι filter, and diluted 1 : 10 with water for the injection (Aqua pro injectione, Czech Pharmacopoeia 2003). It is possible to use filters from other manufacturers with similar characteristics.

Subsequently, an allergy test is carried out: The filtrate in a quantity of 0.1 ml is injected intradermally to the patient by a syringe. If the allergy test is positive, the filtrate must be further diluted (at 1 :20, 1 :30).

After a negative allergy test, 1 ml of the preparation is administered subcutaneously by an injection to the patient on the dorsolateral side of the arm.

Within 3 days induration appears at the injection site. The antipsoriatic therapeutic effect usually begins to show up in a week after the application of the preparation. If the therapeutic effect does not appear, the application is repeated within 2 to 3 weeks after the previous dose. This procedure can be repeated up to 3 times with the application into the second arm than in the one where the previous dose was administered.

Caution because of the possibility of allergization.

Example 3

Monitoring the effectiveness and safety of the immunobiological preparation

Psoriasis severity is assessed by the PASI score (Psoriasis Area and Severity Index - this is a scoring system, under which redness, induration and desquamation of the skin are assessed), where the PASI score 0 indicates the absence of psoriasis disease and the PASI score of 72 means the maximum skin involvement.

The table for calculating the PASI score is shown in Figure 1. The testing was conducted under the following conditions:

Entry criteria for the assessed subjects:

A/ PASI score > 22 B/ Men and women aged over 18 years

C/ No current treatment for psoriasis, excluding topical emollients (substances that soften, smoothen and hydrate the skin) Exclusion criteria for the assessed subjects:

A/ Systemic treatment by Acitretin, Cyclosporin, Methotrexate, Light Therapy or other systemic treatment of psoriasis within the last 3 months and throughout the study from the administration to the Day 180.

B/ Topical (local) treatment of psoriasis after Day -14 (the washout period)

CI Pregnancy or breast-feeding. Women of childbearing age were excluded from the participation if they did not use a reliable method of contraception.

D/ Acute ongoing infectious diseases

E/ Immunosuppressive Therapy

F/ Tumorous Diseases

Time Frame:

AJ A washout period of 2 weeks, when the studied subjects used only topical emollients to treat psoriasis.

B/ Evaluation intervals:

Day - 14, 0, 10, 14, 21, 28, 56, 90, 180.

Administration methodology:

After a negative allergy test, 1 ml of the filtrate is administered subcutaneously in the dorsolateral side of the arm. The antipsoriatic therapeutic effect usually begins to show up in 1 week after the application. If it fails to occur, the application is repeated 2 to 3 weeks after the previous dose. This procedure can be repeated 3 times with the application in the other arm than the previous dose was administered in.

Methodology of Effectiveness Evaluation:

The evaluation was based on an assessment of skin psoriasis infliction in categories: redness, induration, and desquamation of individual studied subjects. The PASI scores were calculated according to the table shown in Figure 1 on Days -14, 0, 10, 14, 21, 28, 56, 90 and 180. The overall effect of the treatment for the study subject was evaluated on Day 180 as follows: The deterioration was defined as the PASI score higher than the PASI score on Day 0.

No improvement was defined as a PASI score decrease of 0 to 25 percent in comparison with the PASI score on Day 0.

A slight improvement was defined as a PASI score decrease of 26 to 50 percent in comparison with the PASI score on Day 0.

An improvement or a positive clinical response was defined as a PASI score decrease of 51 to 75 percent in comparison with the PASI score on Day 0.

A highly significant improvement was defined as a PASI score decrease of 76 to 100 percent in comparison with the PASI score on Day 0.

The results are reported in Table 1 below.

Methodology of Safety Evaluation - Monitoring of Side Effects:

The incidence of side effects was observed and recorded from the time of administering the preparation (Day 0) to Day 180. The results are reported in Table 1 below.

Characteristics of the Evaluated Subjects:

Number of evaluated subjects: 25, including 7 women and 18 men

Average age: 47.4 years (19 - 68)

Average duration of psoriasis infliction: 15.4 years (2 - 36)

Average PASI score on Day 0: 45.3

Evaluation Results:

Table 1

Deterioration 0 (0 %)

No improvement 1 (4 %)

Slight improvement 2 (8 %)

Improvement 5 (20 %) Highly significant 17 (68 %)

improvement

Side effects lx inflammation at the injection site with the adjustment of status within one week

3x transient mild itching around the injection site

Need for a Repeated The application was repeated lx for 3 subjects on Application Day 14

The chart showing the change in the average PASI score at all times of subjects studied in the evaluation is shown in Figure 2. Conclusion:

The use of the preparation prepared and applied according to the above mentioned procedure was well tolerated and had a significant antipsoriatic therapeutic effect, which in 68 % of the subjects meant a highly significant improvement in their psoriatic skin condition during the period of evaluation. In 20 % of the subjects there was an improvement and in 8 % of the subjects there was a slight improvement during the monitored period. No improvement was noted in 4 % of the subjects and there was no deterioration of the psoriatic condition in the monitored period. The average PASI score for all subjects assessed on Day 180 was 9.1 , which corresponds with a significant antipsoriatic therapeutic effect, which was statistically significant at 5% level of significance. The side effects were mild and transient and did not enforce the withdrawal of any assessed subject from the evaluation. The product has proved itself very effective, well tolerated and suitable for the treatment of psoriasis with a long-term therapeutic effect.