AU655008B2 - Peat-derived bioactive products and pharmaceutical and cosmetic compositions containing them - Google Patents

Description

X. s t-: OPI DATE 21/10/92 AOJP DPrE 26/11/92 APPLN. ID 13638 92 PCT NUMBER PCT/EP92/00491 INTERNATIONAL APPLICAU IUN rU15L13n n ui JPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 92/16216 A61K 35/10, 7/00, C07G 17/00 Al (43) International Publication Date: I October 1992 (01.10.92) (21) International Application Number: PCT/EP92/00491 (72) Inventors: TOLPA, Stanislaw Sudecka 124, 53-128 Wroclaw GERSZ, Tadeusz Zielonogorska 18 m 7, (22) International Filing Date: 4 March 1992 (04.03.92) 53-617 Wroclaw RITTER, Stanislawa Buska 4 m 15, 53-326 Wroclaw KUKLA, Ryszard Piastows- Priority data: ka 22 m 10, 50-864 Wroclaw SKRZYSZEWSKA, 91104098.8 16 March 1991 (16.03.91) EP Malgorzata enedyktynska 28 m 14, 50-350 Wroclaw (34) Countries for which the regional TOMKOW, Stanislaw Lotnicza 7a, 54-154 Wrocor international application law (PL).

wasfiled: DE et al.

P290283 17 May 1991 (17.05.91) PL (74) Agent: BUECHEL, Kurt, Bergstrasse 297, FL-9495 P290508 3 June 1991 (03.06.91) PL Triesen (LI).

P290509 3 June 1991 (03.06.91) PL P290510 3 June 1991 (03.06.91) PL (81) Designated States: AT, AT (European patent), AU, BB, BE P290606 10 June 1991 (10.06.91) PL (European patent), BF (OAPI patent), BG, BJ (OAPI P290607 10 June 1991 (10.06.91) PL patent), BR, CA, CF (OAPI patent), CG (OAPI patent), P290608 10 June 1991 (10.06.91) PL CH, CH (European patent), CI (OAPI patent), CM P290693 17 June 1991 (17.06.91) PL (OAPI patent), DE, DE (European patent), DK, DK P290694 17 June 1991 (17.06.91) PL (European patent), ES, ES (European patent), FI, FR P290695 17 June 1991 (17.06.91) PL (European patent), GA (OAPI patent), GB, GB (Euro- P291078 15 July 1991 (15.07.91) PL pean patent), GN (OAPI patent), GR (European pa- 91118269.9 26 October 1991 (26.10.91) EP tent), HU, IT (European patent), JP, KP, KR, LK, LU, (34) Countries for which the regional LU (European patent), MC (European patent), MG, ML or international application (OAPI patent), MR (OAPI patent), MW, NL, NL (Eurowasfiled: DE et al. pean patent), NO, PL, RO, RU, SD, SE, SE (European patent), SN (OAPI patent), TD (OAPI patent), TG (OA- (71) Applicant: TORF ESTABLISHMENT [LI/LI]; Stidtle 36, PI patent).

FL-9490 Vaduz (LI).

Published t\ With. ,n In, I aS 10 "7 Lwt e fc epyrt. V"' (54)Title: PEAT-DERIVED BIOACTIVE PRODUCTS AND PHARMACEUTICAL AND COSMETIC COMPOSITIONS CONTAINING THEM (57) Abstract The peat-derived bioactive product contains not more than 70 preferably not more than 60 by weight of inorganic salts, especially of sodium chloride, based on dry solids. It is obtainable by a process wherein a highly concentrated aqueous solution of inorganic salts, especially of sodium chloride containing peat-derived bioactive ingredients is diluted with demineralized water and subjected to reverse osmosis in order to desalinate the solution, inorganic salts being removed, and wherein the resulting solution is concentrated and clarified, and, optionally, in at least one further step, sterilized and/or spray-dried. A pharmaceutical formulation containing a peat-derived bioactive product, in the form of a gel, is prepared by combining a sterile alcoholic herb extract with sterile glycerol, a sterile aqueous solution of previously powdered peat-derived bioactive product and a sterile menthol solution; the resulting mixture is gradually combined with colloidal silica to convert the liquid composition into gel form, the weight ratio of liquid mixture to silica preferably being from 90:10 to 94:6. A cosmetic preparation such as a gel, ointbalm, shampoo, bath salt lotion etc. contains as active ingredient the instant peat-derived bioactive product in a quantity of 0.01-10 by weight, preferably 0.05-1 by weight, more preferably 0.05-0.1 by weight.

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r i c WO 92/16216 PCT/EP92/00491 1- PEAT-DERIVED BIOACTIVE PRODUCTS AND PHARMACEUTICAL AND COSMETIC COMPOSITIONS CONTAINING THEM The present invention relates primarily to novel peat-derived bioactive products and to a process for producing such products.

The invention also relates to pharmaceutical and cosmetic compositions containing these products and to processes for preparing pharmaceutical and/or cosmetic formulations containing said peat-derived bioactive products.

It is known to extract peat by various methods using various extraction media and to use such extracts, containing peatderived bioactive ingredients, for cosmetic and pharmaceutical purposes.

On of these known processes is described in Polish patent specification No. 124110 (Chemical Abstracts 101(10), 78854e).

According to this prior art process peat-derived bioactive products are obtained by primary and secondary alkaline hydrolysis of an air-dried raw peat material, followed by acidification of the thus obtained hydrolysate and separation of insoluble solid parts with subsequent second alkalization, acidification of the clear liquid phase and elimination of ballast substances by means of alcohol and ether extraction. In said process, the aqueous phase from the organic extraction is a liquid peat-derived bioactive product.

The known liquid product, being a solution of peat-derived active ingredients in a highly concentrated, nearly saturated aqueous solution of sodium chloride, obtained according to the above cited Polish patent specification, is unstable when stored for a long time and, moreover, contains regarding the biological activity of the composition a large excess of neutral inorganic substances. As a bulk product, it is hard to handle, store and process.

SUBSTITUTE SHEET WO 92/16216 PCT/EIP92/00491 -2- It is the main object of the present invention to provide a product which is not afflicted with these disadvantages, i.e. a product which is stable and which can easily be formulated into pharmaceutical and veterinary products as well as be introduced, either in solid state or in any suitable solution, into cosmetic preparations.

In particular, with respect to the use of a peat-derived bioactive product for pharmaceutical purposes, ie. for the production of pharmaceutical compositions, there was a strong need for providing a, solid form which would be well suited for this purpose. As previous attempts aiming at concentration of the known aqueous solution of peat-derived bioactive substances and desalination of the same failed to give a positive result due to the occlusion of active ingredients in a crystallising solid phase, causing decrease of biological activity of the composition, it was very hard to find a suitable process for converting the liquid composition into powder form.

Unexpectedly, it was found that a positive result is achievable if, prior to concentration, the liquid composition is first diluted several times, i.e. by water volumes several times the volume of the composition.

SAccordingly, the present invention provides a process by which a peat-derived bioactive product, featuring the above advantageous characteristics, is obtainable from a highly concentrated aqueous inorganic salt especially sodium chloride solution i containing peat-derived bioactive ingredients, by diluting said solution with demineralised water, e.g. distilled water, followed by reverse osmosis, concentration and clarification. A solution so obtained can easily be converted into a sterile and solid product, well suited to the intended purposes, by sterilization and spray-drying. Dilution is preferably effected with quantities of water several times, preferably 5 to 8 times, the quantity of the concentrated solution to be diluted.

SUBSTITUTE SHEET WO 92/16216 PC/EP92/00491 -3 The instant process, applied to the processing of a product obtained according to Polish patent specification No. 124110, consists in expelling residual organic solvents from a postextraction aqueous phase, separating insoluble parts by filtration under reduced pressure through a sintered ceramic material, diluting the permeate with several times the volume of distilled water and subjecting it to desalination by reverse ormosis to separate excessive mineral salts, mainly sodium chloride, as a permeate. Then, the desalinated solution is concentrated, clarified by centrifugation and sterilised by filtration through a membrane filter, e.g. a Millipore(R) filter. The resulting microbiologically clean solution may optionally be spray-dried. The sterilised product (liquid or solid) may be formulated into a cosmetic pharmaceutical or veterinary composition. Optionally, the concentrated and clarified solution may be used without sterilisation and spray-drying in any suitable dilution as a component in numerous cosmetic compositions.

Preferably, in a spray-drying step, the inlet temperature is set to about 180 0 C, while the outflow temperature is set to about 0

C.

While the process of the present invention is described above in combination with the process according to Polish patent specificaton No. 124110, its use is not restricted to such combination, but is applicable generally in the context of a process for obtaining a peat-derived bioactive product from a highly concentrated aqueous solution of inorganic salts, especially of sodium chloride, containing peat-derived bioactive ingredients.

The peat-derived bioactive products provided by the present invention do not contain more than 70% by weight, preferably not more than 60% by weight of inorganic salts, especially of sodium chloride. Since a sodium chloride concentration as low as possible would be desired for an optimal product, especially for SUBSTITUTE SHEET WO 92/16216 PCT/EP92/00491 -4pharmaceutical applications and such cosmetic applications where higher concentrations are required, i.e. for face care, lower sodium chloride concentrations, such as 55% and even lower, are most preferred, especially when obtainable by the steps of dilution and reverse osmosis.

Where the process is terminated with the concentration and clarification steps, the product is a concentrated (or thickened) solution. "Concentrated peat extract", referred to in this specification, is a dark-brown liquid of a density of 1,02 1.,09 g/ml and has a content of dry solids of not less than 5% by weight. The chloride ion content in dry solids, calculated as NaCl, is not higher than 70%, preferably not l:igher than and the pH value of a 1% aqueous solution is 5.0-6.5, generally about 6.0. The lack of a further sterilization step may not be detrimental in certain cases, e.g. for certain cosmetic uses of said concentrated peat extract.

On the other hand, the sterilization step will be mandatory, particularly when the product is intended for the preparation of pharmaceutical compositions. Particularly in such a case, the further step of spray-drying is most preferred, if not mandatory. The product resulting after such a spray-drying step is in powder form and thus particularly suited to the preparation of certain pharmaceutical compositions. A most preferred product of this type is the product commercialised under the designation "TOLPA(R) Torf Preparation", TOLPA(R) being a registered trade mark of Torf Corporation, ul. Mydlana 2, Wroclaw, Poland. The abbreviation TTP will be used in the course of this specification to designate said product.

The present invention also relates to pharmaceutical compositions containing as active ingredient a peat-derived bioactive product as hereinbefore described, particularly a product which contains not more than 70% by weight, preferably not more than by weight, of inorganic salts, especially sodium chloride, based on dry solids, together with a pharmaceutically acceptable SUBSTITUTE SHEET u WO 92/16216 PCT/EP92/00491 carrier. The peat-derived bioactive product contained in such pharmaceutical compositions is preferably TTP as defined above.

The pharmaceutical preparation contains the peat-derived bioactive product and the pharmaceutically acceptable carrier material, preferably in a weight ratio of between about 1:5 and 1:25, and most preferably between 1:9 and 1:19.

The present invention furthermore relates to a process for preparing a pharmaceutical formulation containing a peat-derived bioactive product, in the form of a gel. This process is characterised in that a sterile alcoholic herb extract is combined with sterile glycerol, a sterile aqueous preferably concentrated solution of previously powdered peat-derived bioactive product and a sterile menthol solution, and that the resulting mixture is gradually combined with colloidal silica to convert the liquid composition into gel form, the weight ratio of liquid mixture to silica being from 90:10 to 94:6. Preferably TTP is used as the powdered or concentrated peat-derived bioactive product.

The present invention also relates to a process for preparing a pharmaceutical formulation containing a peat-derived bioactive product, in the form of an ointment. This process is characterised in that a sterile herb extract is gradually combined with a sterile solution of powdered peat-derived bioactive product, I that the resulting mixture is gelled with the addition of colloidal silica and that the gel thus obtained is triturated with a previously sterilized mixture of fatty components, such as eucerine and petrolatum, preferably with a weight ratio of liquid components to silica of about 30:20 and of gel to fatty j composition of between 32:68 and 34:66. Also here, preferably TTP is used as the powdered or concentrated peat-derived bioactive product. Cosmetic preparations, which may comprise herbal extracts as well as other auxiliary and enriching components, fragrant compositions and carrier materials allowed for cosmetic use, SUBSTITUTE

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WO 92/16216 PCT/EP92/00491 6 contain the peat-derived bioactive product according to the present invention in an amount of 0.01-10% by weight, preferably 0.05-1.00% by weight, and most preferably 0.05-0.10% by weight.

Carrier materials may be aqueous solutions of alcohols, all types of emulsions, gels, foaming compositions and fatty carriers. Use of one specific carrier selected from the group of the above mentioned substances allows formulation of various types of cosmetic preparations according to the invention, such as tonics, creams, balsams, cleaning milks etc. for daily body care as well as shampoos, hair balms, foaming bath compositions, all with the addition of peat-derived bioactive products.

Peat-derived bioactive products (peat extracts in abbreviated form) stemming from original raw peat material, such as (among others) therapeutic mud, contain well balanced quantities of mineral and organic compounds, such as mineral salts of the following elements: B, Si, Ab, Fe, Mg, Mn, Cu, Sn, Ni, Ca, Ag and Na; organic compounds, such as aminoacids in free form and as salts; polysaccharides, partially degraded/reacted in the course of hydrolysis to desoxysaccharides and/or aminosaccharides. Peat, in particular therapeutic mud, is known and recognised as a material of biological plant and microorganism origin; due to its contents of nourishing and stimulating components it has beneficial effects on humans and mammals; therefore, peat-derived bioactive compositions contain the abovementioned substances in proportions characteristic for the living organisms; this is considered to be an explanation of the advantageous effects of cosmetic and pharmaceutical preparations containing peat-derived active products and compositions.

i" Particularly good effects of the new cosmetic compositions are observed when herb extracts are also present in the formulation.

Selection of a suitable herb extract is based on a known typical use of such extracts in cosmetics, modifying the activity of preparations and thus enabling the content of cosmetic preparations to be matched with demands and needs of individuals to be treated.

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WO 92/16216 PCr/EP92/00491 7 The present invention is better characterised and explained in the following examples.

Example 1: Starting with 1000 kg of air-dried raw peat material, following e.g. the known procedure according to Polish patent No. 124110, a solution of peat-derived bioactive ingredients in a saturated aqueous solution of sodium chloride was obtained in a quantity of about 10 liters. The solution was filtered through a sintered ceramic filter under reduced pressure in order to clarify the solution before desalination of the same. The clear solution thus obtained contains about 95% of NaCl in a dry mass. The dry mass constitutes about 32% by weight of the solution. The volume of this clear solution is about 7 liters.

The clear solution is diluted with 5 to 8 times the quantity of distilled water and in diluted form is subjected to a desalination step carried out by using a reverse osmosis technique using a DDS apparatus. Desalination was carried out for 3 to 4 hours, whereby the excess of mineral salts mainly NaCl is separated in the form of the permeate. The desalinated composition contained approx. 65-70% of sodium chloride in the solids.

The solution thus obtained, of 6-7 liters by volume, was concentrated 4-5 times in a Buechi rotating evaporator, so that a concentrated solution containing approx. 20% of dry mass was obtained. The resulting concentrated solution was clarified using a Biofuga-Heraeus centrifugal apparatus (flow separator) and then sterilised by filtering through a Millipore(R) filter.

The resulting microbiologically pure solution was spray-dried in an Anhydro dryer with the outflow temperature set at 90 0 C ana supply inlet temperature set at 180 0 C. The yield of dried powder was approx. 200 g.

_1 Example 2: SUBSTITUTE

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I j- I, p*- WO 92/16216 PCT/EP92/00491 8 The product obtained in Example 1 was used for preparing gel and ointment pharmaceutical compositions, containing also herb extracts synergistically improving the therapeutic effect with respect to certain diseases. For example, a gel and ointment against varicose ulcer of the shank was prepared as follows: g of hippocastanaceous extract, 10 g of calendula extract, g of glycerol, 0.1 g of TTP in the form of a powder obtained as described in Example 1 above, 0.1 g of salicylic acid, 1.0 g of distilled water and 8.8 g of Aerosil(R) (colloidal silica) were used in order to obtain a gel form of the preparation.

Liquid (non-volatile) ingredients were sterilised before use, by means of heating under reflux for two hours. Herb extracts were combined with glycerol and an aqueous solution of TTP and also with menthol, and silica was gradually added to the obtained mixture, under continuous stirring.

Similarly, in order to obtain an ointment composition, the following ingredients were used: 20 g of hippocastanaceous extract, 10 g of calendula extract, 0.1 g of salicylic acid, 0.1 g of TTP in powdered form obtained as described in Example 1 and g of Aerosil(R) (colloidal silica).

As fatty components, a mixture of the following substances was used: 22 g of eucerine and 45.8 g of petrolatum. Herb extracts were sterilised by heating under reflux for approx. 2 hours.

Eucerine and petrolatum were similarily sterilised. Liquid ingredients were carefully combined with silica to obtain a gel, which in turn was triturated with sterilised and fatty components cooled down to room temperature. A stable ointment was J obtained which did not separate when stored.

The gel and ointment obtained above were simultaneously applied in the treatment of varicose ulcer of the shank. Ulcers were treated with the gel preparation while the surrounding, nonaffected skin was treated with ointment. Addition of colloidal silica is believed to be responsible for prompt dessication SUBSTITUTE SHEET WO 92/16216 W92/16216 PCT/EP92/00491 -9while the herbal and peat-derived ingredients are believed to be responsible for the curing effect of the preparation. Fatty components helped to keep elastic the crust and the skin. The results obtained were compared with a control group of patients treated in a classic way. Those who received the new treatment were selected from a group of patients suffering from the disease for many months (sometimes years) without noticeable positive effects. Patients treated with compositions according to the invention showed better results already within the first few weeks than control patients.

Example 3: Products obtained in Example 1 were used to prepare pharmaceutical formulations in the form of tablets, or granules to be placed in capsules.

A sterile peat-derived bioactive composition in powdered form was combined with a carrier in a weight ratio of 1:9. As a carrier, MYVATEX(~)TL (tradename of Eastman-Kodak), a mixture of lactose and lubricating substances, was used in a weight ratio of 44:1. Lactose of 50 mesh particle size and MYVATEX(R)TL were finely disintegrated so that approx. 70% of its mass passed through a 100 mesh screen. A p~rt of the resulting mixture of active composition and carrier was formulated into tablets containing 5 mg of active ingredients. The total mass of each tablet was 50 mg. The other part of the same mixture of active composition and a carrier was granulated using q.s. of ethanol by volume). Granules were sieved and ground if necessary and then filled in capsules in such a quantity that each capsule A contained 5 mg of active ingredients by using TTP in mixture with a carrier at a ratio of 19:1.

The tablets obtained as above were tested in order to measure the time of their disintegration in an artificial gastric juice at 37 0 C 2 0 C using Erweka equipment. The artificial gastric juice was prepared as follows: 2.0 g of sodium chloride and 3.2g of pepsin were dissolved in 7 ml of hydrochloric acid and SUBSTITUTE SHEET -i WO 92/16216 92/16216 PCT/EP92/00491 10 distilled water was added up to a total volume of 1000 ml. The pH-value of the resulting solution was approx. 1.2.

Desintegration time of a tablet, having a diameter of 5.1 mm and a total mass of 0.0498 g, was 6 minutes.

Further examples relate to numerous cosmetic preparations according to the present invention, having different forms composition and being designed for different applications, containing the beneficial audition of bioactive ingredients derived from peat. Among others, preparations such as tonics, balms, creams, milks, shampoos, foaming bath compositions etc.

are described.

Example 4: A reaction vessel equipped with a stirrer was charged with 150 g of camomile extract obtained by the extraction of camomile inflorescence with a 1:1 ethanol:water solution, as well as 1 g of TTP as described above. 50.g of glycerol were added to the mixture obtained. The three substances were stirred to obtain a uniform mixture. Subsequently, a second mixture as previously formulated, was introduced into the same vessel. It comprised 340 g of a 95:5 ethanol:water solution, 1 g of salicylic acid and 0.5 g of menthol. The two mixtures were combined by stirring to form a uniform solution. 14ext, 3 g of a fragrant composition TILIANA H4308 were added. TILIANA H4308 is a product of Fabryka Syntetykow Zapachowych Pollena-Aroma (Synthetic Fragrance Works Pollena-Aroma), of Warsaw, Poland. The solution was then brought to a total volume of 1000 ml by adding 454.5 g of distilled water; stirring was continued until a homogeneous mixture was obtained.

In the above procedure, 86% glycerol, menthol and water according to the requirements of Polish Pharmacopea FP IV and ethanol in a concentration of 95% according to the Polish industrial standard BN-75/6193-01 were used.

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i i 1 c ~i WO 92/16216 PCT/EP92/00491 i j Si 11 The concentrated peat extract used was a dark-brown liquid of a density 1.020-1.090 g/ml and a content of dry solids not less than the pH value of a 1% aqueous solution was 5.0-6.5.

Camomile extract was a red-brown liquid of a density of 0.9160- 0.9503 g/ml, and ethanol content of 52-56% by volume.

The tonic Vreparation obtained above is suitable for all kinds of skin. It is a clear liquid without any solids. Its colour is yellow. The pH value is 4.28 and the ethanol content is 45.92% by volume. Total acidity calculated as salicylic acid content was not less than 0.1% by weight, namely 0.23% by weight. The preparation being stored for 12 months did not lose any of the above characteristic features.

Example The procedure described in Example 4 above was repeated, the only difference being that instead of camomile extract and the TILIANA H4308 fragrant composition, a marigold flowers extract and a composition FINUS H4625 (also a product of Fabryka Syntetykow Zapachowych Pollena-Aroma) were used in the same way and the same molar and volume ratios. The resulting tonic preparation is suitable for dry and fragile skin. Similarily it was a clear liquid without any solid particles. The pH value was 4.30, total ethanol content was 45.82% by volume and total acidity was 0.27% by weight. When stored, the preparation was unchanged after 12 months like the preparation obtained according to Example 4.

Example 6: The procedure of Example 4 was repeated, except that an extract of sage leaves was chosen instead of camomile extract, and the fragrance LELIA 90368 (Pollena-Aroma, Warsaw) was chosen instead of TILIANA H4308. The extract of sage leaves was obtained by extracting dried sage leaves with ethanol at 50 0 C and had a brownish colour, a characteristic sage odour and a density of 0.9160 to 0.9503; it contained 52 to 56% of ethanol. The resulting face care agent is particularly suitable for greasy skin. It is a SUBSTITUTE

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W 92/16216 PCT/EP92/00491 12 clear -,nd homogeneous liquid having a dark yellow colour; the pH value, the ethanol content and the acidity were comparable with the values of the care agent according to Example 4.

Example 7: The following composition proved to be a particularly effective gel for avoiding or treating periodontosis: 24.0 g camomile extract g sage leaf extract 0.3 g salicylic acid 0.2 g menthol 0.1 g TTP to 100.0 g commercial gel base Example 8: The following carefully weighed components were introduced into a reaction vessel of a volume of 2000 ml, equipped with a mechanical stirrer: 270 g of camomile extract obtained by extraction of camomile inflorescence with 50% ethanol; the extract was a red-brown liquid, having a density of 0.9160-0.9503 g/ml and an ethanol content of approx. 55% by volume 50 g of glycerol, 86%, according to the requirements of Polish Pharmacopea FP IV 30 g of a saponaria officinalis extract obtained by extracting saponaria officinalis roots with 70% ethanol; the extract was a red-brown liquid, the density was 0.9630-0.9810 g/ml, and the ethanol content approx. 75% by volume 1.0 g of the inventive concentrated peat extract, being a dark brown liquid having a density of 1.02-1.09 g/ml; not more than 2% chloride ions calculated as sodium chloride; the dry solids content was not less than the pH value of a 1% water solution of the extract was The ingredients listed above were mixed thoroughly. A previously prepared solution of 1 g of salicylic acid in 260 g of ethanol was added thereto. To the combined solution, 383 g of SUBSTITUTE SHEET 1 .I A f WO 92/16216 PCT/EP92/00491 13 distilled water and 5 g of fragrant composition TILIANA H3408 were added and stirred until a uniform solution was obtained.

The preparation was analysed and stored in retail size bottles of 200 ml volume. The resulting preparation was suitable as a hair care preparation. It was a clear, slightly opalescent liquid, containing approx. 45% by volume of ethanol; the pH value was 4.5; the total acidity calculated as salicylic acid was not less than 0.1% by weight. The preparation is suitable for blonde hair. During 12 months storing the preparation remained unchanged in its features.

Example 9: The procedure described in Example 8 was followed except that instead of camomile extract and TILIANA H4308 composition there were used in the same sequence and ratio: horsetail herb extract and the fragrant composition FINUS H4625. Horsetail herb extract was a green-brown liquid of a density of 0.9160-0.9503 g/ml and an ethanol content of 55% by volume.

The resulting preparation was suitable for all kinds of hair. It was a clear and transparent liquid without any solid parts, yellow-brown in colour. pH value, ethanol content, total acidity as well as stability after a 12 months storing period were the same as those of a preparation described in Example 6.

Example The procedure as described in Example 8 was repeated with comparable results. The only difference was that, instead of camomile extract and TILIANA H4308 composition, stinging nettle leaves extract and fragrant composition LELIA 90368 (product of the same Fabryka Syntetykow Zapachowych Pollena-Aroma) in the same sequence and ratio were used. The herb extract used was olive-green in colour, had a density of 0.9160-0.9503 g/ml and an ethanol content of approx. 55% by volume. The preparation was suitable for all kinds of hair.

SUBSTITUTE SHEET WO 92/16216 PCT/EP92/00491 14 Example 11: In general, cosmetic milks are dispersions of fatty substances acting in both chemical and mechanical ways on the skin. In fact, due to a convenient way of application and better interaction of the fluid and the skin, it is very appropriate to use liquid, more specifically emulsion creams. They can easily penetrate to deeper layers of the skin and thus prevent changes of the skin due to age. Cosmetic milks are used mainly to clean a dry and fragile skin. Accordingly, they must not contain any aggressive volatile oils, while frequently they contain suitable herb extracts like camomile extract or wheat germ extract. Addition of peat-derived bioactive products to such cosmetic milks further improves their positive effects. In particular, the new recipe is as follows: TTP 0.05 g aloe extract 20.00 g glycerol 3.00 g eucerine 2.00 g white paraffin oil 1.00 g triethylamine 1.00 g Aerosil(R) (colloidal silica)4.00 g Example 12: Improved regenerative results were observed when TTP and carefully selected fatty carriers were used in a classic nourishing and regenerative cream formula. TTP is used in an amount of 0.01 -1.00% by weight in combination with a herb extract (selection depends un the type of skin for which the cream is intended) in an amount of at least 0.05 1.00% by weight, antibacterial preparation in an amount of 0.05 1.00% .by weight, synthetic fragrant composition in an amount of 0.01 0.05% by weight and a fatty carrier in the form of a water emulsion, constituting 97.00 99.50% by weight of the whole composition. The fatty composition needs to be a good carrier for the active ingredients and to be well accepted by the skin.

Preferably, it is an emulsion of (all amounts in by weight) 35-45 eucerine, 8-14 petrolatum, 2.5-4 olive oil, 6-10 glycerol SUBSTITUTE SHEET WO 92/16216 ijij PCT/EP92/00491 15 and 35-40 water. Preferred herb extracts are marigold flower extract, camomile extract, thyme extract and the like.

Preferred recipes are as follows: 1. Eucerine 39.00 parts by weight Petrolatum 11.50 parts by weight Olive oil 3.13 parts by weight Glycerol 7.80 parts by weight Water 38.00 parts by weight NIPAGINA A (antibacterial preparation) 0.40 parts by weight TTP 0.05 parts by weight Marigold extract 0.10 parts by weight Synthetic fragrance 0.02 parts by weight total 100.00 parts by weight 2. Eucerine Petrolatum Olive oil Glycerol Water NIPAGINA A (antibacterial preparation)

TTP

Camomile extract Synthetic fragrance 42.00 parts by weight 8.50 parts by weight 3.08 parts by weight 7.90 parts by weight 38.00 parts by weight 0.40 parts by weight 0.05 parts by weight 0.02 parts by weight 0.05 parts by weight 100.00 parts by weight 4A Example 13: An after-shave preparation contains TTP as a peat extract in an amount of 0.01 1% by weight, herb extracts in an amount of 1by weight, glycerol in an amount of 1-8% by weight, salicylic acid and menthol in aqueous-alcohol solution.

Preferred herb extracts are: camomile, marigold, thyme, aloe extract and similar beneficial her extracts. Addition of glycerol is also beneficial due to its influence on the elasticity of the skin. It speeds up the spreading of the preparation on the face as well as the penetration into the SUBSTITUTE SHEET I

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I:r 1 WO 92/16216 PCT/EP92/00491 16 deeper layers of the skin, thus enhancing the beneficial effects of the active peat composition and herb extracts.

A preferred recipe is as follows: TTP 0.10 parts by weight Camomile extract 15.00 parts by weight Glycerol 5.00 parts by weight Menthol 0.10 parts by weight Salicylic acid 0.10 parts by weight Ethanol (conc.95%) 10.00 parts by weight Fragrant composition 0.30 parts by weight Distilled water ad 100.00 parts by weight Example 14: A shampoo composition was prepared according to the following recipe: Fuller's herb extract 15.00 g 7.50% by weight Stinging nettle leaves extract 20.00 g 10.00% by weight GAMAL SBS-11 (detergent) 30.00 g 15.00% by weight GAMAL NO-3 (detergent) 20.00 g 10.00% by weight Aseptina 0.40 g 0.20% by weight ethanol 1.60 g 0.80% by weight BRONOPOL (preservative) 0.04 g 0.02% by weight Sodium chloride 6.00 g 3.00% by weight Water 106.96 q 53.48% by weight total 200.00 g 100.00% by weight .C To 92 parts by weight of the above shampoo composition 8 parts by weight of TTP were added to obtain 100 parts by weight of a shampoo according to the invention. Other herb extracts can be used in place of stinging-nettle leaves extract.

Example The following shampoo composition was prepared: Horse chestnut extract 13.00 g Marigold extract 22.00 g GAMAL SBS-11 30.00 g GAMAL NO-3 20.00 g SUBSTITUTE SHEET 1 WO 92/16216 PCf/EP92/00491 17 Aseptina Ethanol

BRONOPOL

Sodium chloride Water 0.40 g 1.60 g 0.04 g 6.00 g 106.96 q 200.00 g total To 95 parts by weight of the above composition, 5 parts by weight of TTP were added to obtain 100 parts by weight of shampoo according to the present invention.

Example 16: A tooth paste contains TTP as a concentrated peat extract, in an amount of 0.01-0.10% by weight, etheral, oils or their compositions or else fruit essences in an amount of 1-10% by weight, glycerol in an amount of 5-10% by weight, herb extracts in an amount of 0.10-10% by weight and cleaning substances in an amount of 20-35% by weight dispersed in water in an amount of 45-60% by weight, and dyes and whitening components in an amount of 1-2% by weight.

Titanium dioxide may be used as a whitening component; TTP itself may be used as an anti-bacterial additive; sage leaves, camomile or marigold flowers extracts may be used as beneficial preferred herb extracts.

The preferred recipe is as follows: Precipitated calcium carbonate 150.00 g Magnesium carbonate 60.00 g Glycerol 70.00 g Herb extract 5.00 g TTP 0.50 g Titanium dioxide 10.00 g Etheral oils (or mint, lemon, etc essence) 5.00 g Water 400.00g Dye trace -c, SUBSTITUTE SHEET 1 WO 92/16216 PCT/EP92/00491 18 Example 17: Bath salt preparation: In the course of the process for obtaining a peat-derived bioactive product according to the present invention, in particular when converting a liquid form into a powdered one, there is the desalination step in which sodium chloride is separated as a by-product. In said byproduct, 95% constitutes sodium chloride, other mineral salts separated are calcium salts, magnesium salts, mainly chlorides and sulfates; these salty products also contain some organic peat-derived low molecular compounds occluded within the crystal structure of these inorganic salts. These organic compounds are components of TTP and are among others polysaccharides, aminoacids, fulvic acids and the like. The presence of these components in the salty by-products is beneficial when salt is used as a bath salt, because they may add additional beneficial effects to the standard activity of bath salt. Accordingly this by-product was tested for its chemical and physical properties in the Balneologic Institute in Poznan, Poland, to find out whether it can be used in cosmetic baths. Since the Institute has found no undesired entity in the salt, it was approved for cosmetic use.

The preferred recipe is as follows: Salt (NaC1) containing occluded TTP 97.00 g Pine etheral oil or etheral oils composition 3.00 g Example 18: A new hair balm contains TTP in an amount of 0.01-1% by weight, herb extracts in an amount of 0.01-10% by weight, anti-electrostatic components in an amount of 3-4% by weight, components preventing excessive drying of hair and skin in an amount of up to 2% by weight, glycerol in an amount or 1-5% by weight, preservative and stabilisers in an amount of 0.05-0.50% by weight and water to 100% by weight.

As an anti-electrostatic component, the present balm contains an alcoholic solution of trimethylamine and ammonium chloride salt, obtained from fatty animal-derived amines; as thickening agent SUBSTITUTE SHEET L Il 1

I

I a L WO 92/16216 PCT/EP92/00491 19 j1

K

acting also as stabilizing agent cosmetic alcohol; as agent preventing excessive dryness of hair and skin plant oils, acting simultaneously as co-emulsifying agents; and glycerol for easying spreading and peretration of the balm, in particular of its active ingredients TTP and herb extracts. As an acidic environment stops multiplication of bacteria, the balm according to the invention contains citric acid or fumaric acid in an amount of 0.1% as well as a preservative known as BRONOPOL and fragrant compositions.

The preferred recipe is as follows: Alcoholic solution of trimethylamine and ammonium chloride salts 3-4% by weight Cosmetic alcohols 3-4% by weight TTP 0.01-10% by weight Thickened herb extracts 0.01-10% by weight Glycerol 1.5% by weight Plant oils up to 2% by weight Citric or fumaric acid 0.1% by weight BRONOPOL 0.1% by weight Fragrant composition 0.3% by weight Distilled Water to 100% by weight Example 19: Cosmetic masks are well known cosmetic preparations serving many different purposes. As therapeutic mud has a known beneficial effect on the skin and body, it was believed that also postextraction peat obtained in the process of separation of bioactive peat-derived compositions from peat may be used in cosmetic applications. Post-extraction peat contains a solution of active bodies freed in the alkaline hydrolysis process due to extremely high sorptive properties of peat after neutralisation; It was therefore found to be a valuable component of cosmetic masks. To enrich the post-extraction peat with more of the valuable components, natural therapeutic mud and humic acid fractions were added which are present in natural peat and separated in a process for obtaining peat-derived bioactive compositions from the alkaline hydrolysate. Such a composition SUBSTITUTE SHEET i; ;i'A WO 92/16216 PCT/EP92/00491 20 was tested in the above-mentioned Balneologic Institute and was found suitable for cosmetic use.

The preferred recipe is as follows: Post-extraction peat 100.0 g Natural therapeutic mud or peat 20.0 g Humic acid fraction 10.0 g Magnesium carbonate 10.0 g Zinc oxide 5.0 g Citric acid or the like 0.1 g Herb extract or powdered plant material 5.0 g Distilled water q.s.

The following statements and explanations relate to the biological aspect of the products of the present invention, i.e. to the bioactive characteristics and to the compatibility of these products, particularly with a view to their usefulness as pharmaceuticals. The following abbreviations will be used below: TTP TOLPA(R) Torf Preparation (trademark of Torf Corporation, Wroclaw, ul. Mydlana 2, Poland) IFN Interferon exists as a ubiquitous cytokine (tissue hormone).

IFN genes are present in all cells. IFN is mainly induced by proteins or glyco-proteins. Substances stimulating the IFN genes for the production of IFN are called inducers. The process of the IFN induction is a highly regulated, sophisticated biochemical process; negative and positive regulatory genes controlling IFN production have been recognized.

Small amounts of IFN may be produced spontaneoulsy, without any detectable inducer. Such IFNs are sometimes named S"physiological IFNs". IFN exists in nature in three main molecular forms: IFN-a (or leukocyte IFN), IFN- (fibroblast IFN), and IFN-y (immune IFN).

IFN-a and IFN- are type I-IFNs, IFN-y is the type II-IFN.

SUBSTITUTE SHEET Example 2: SUBSTITUTE

SHEET

iK iV WO 92/16216 PCT/EP92/00491 21 The major biological activities of IFNs are antiviral, antiproliferative (anticancer), and immunomodulatory activities.

Various forms of IFN are produced commercially as the natural and recombinant preparations and are used as drugs for treatment of neoplastic, viral, and several other d- seases.

CTL NK cells cytotoxic T lymphocyte.

natural killer cell.

IL-1, IL-2 well known interleukins stimulating the proliferation of T cells and other lymphoid cells including B cells; RPMI 1640 tissue culture medium for the growth of human and other leukocytes (abbreviation of the Roswell Park Memorial Institute, Buffalo); FCS fetal calf serum (for assays with leukocytes it has to be pre-tested because it may contain mitogenic substances mimicking the action of interleukins); EMCV encephalomyocarditis virus, mouse picorna virus nonpathogenic for humans, it is often used as a challenge virus in the IFN bioassays; A-549 human adenocarcinoma cell line used in the IFN bioassays because of its high sensitivity for IFN-a,p, and y.

The line is recommended for such use by the WHO experts on IFN standardization; MTT 3-[4,5-dimethylthiazol-2-yll-2,5-diphenyltetrazolium bromide. Reagent used to measure the cell kill or cell growth in several bioassays, using the ELISA scanners (Hansen et al., J. Immunol. Meth. 1989, 119, 203-210); SUBSTITUTE SHEET

.I:

affected skin was treated with ointment. Addition of colloidal silica is believed to be responsible for prompt dessication SUBSTITUTE SHEET WO 92/16216 PCT/EP92/00491 22 L929 the mouse cell line, commonly used for assaying mouse IFN and human or mouse TNF; TNF tumor necrosis factor, cytokine, (relatively small protein, very susceptible to the proteolytic enzymes), produced by monocytes and macrophages (TNF-a known also as cachectin factor causing cachexia in humans and animals); produced after stimulation with LPS (lipopolysaccharides), viruses, bacteria, and many other agents, very toxic for many virus-infected and neoplastic cells; may also act as a growth factor for fibroblasts. Connected with inflammatory reactions. The related form TNF-P (lymphotoxin) is produced mainly by T cells and some other cells.

PBL peripheral blood leukocytes, normal human leukocytes from healthy blood donors, isolated from "buffy coats" (interphase between red cells and plasma). The responsiveness of PBL from individual donors to various cytokine inducers appears to depend on a genetic set-up of the donors.

The high responders and non-responders have been identified.

This refers also to the response of PBL to TTP.

The variation in the individual response to inducers of IFN or TNF is 'more visible when weak inducers are used than when very strong inducers, like viruses, are employed. This is due to the fact that the reaction to weak inducers is of the "all or none" type whereas viruses almost always induce detectable amounts of the cytokines.

Tolerance to inducer: Called also a hyporeactivity state. Occurs after administration of a single dose of an inducer, e.g.

after 20 h of exposure of PBL to an inducer virus); the cells stop further production of IFN. The hyporeactivity state lasts usually about seven days. It may be complete or partial. Such reactivity makes the therapeutic application of strong inducers difficult and/or ineffective. Weak IFN SUBSTITUTE

SHEET

2E WO 92/16216 PCT/EP92/00491 23 inducers either do not induce the hyporeactivity state or the tolerance is small.

Several natural drugs which are extracted from medicinal plants possess immunomodulating properties. TTP appears to be one of them. TTP alters many different immune functions in vitro and in vivo. It initiates balanced immunostimulation, with the capability of non-specifically activating all effector pathways (CD4 helper, weakly CD8 suppressor, CTL, NK cell, and activated macrophage) without cytotoxicity for normal tissues.

TTP has a restorative effect on normal wound healing. Low doses of TTP weakly stimulate IL-1 and IL-2 production. High doses may inhibit the cytokine synthesis.

The respective tests with TTP were carried out by the following methods: PBL from healthy blood donors were purified by ammonium chloride treatment. The culture medium was RPMI 1640 plus 10% FCS. Approximately 8 x 106 cells/ml were cultured for 20 h at 37°C,

CO

2 The antiviral activity of interferon was assayed by EMCV cytopathic effect inhibition in human A549 cells. The MTT method to measure cell kill was also used.

TNF activity was measured in L 929 cells. In order to define IFN type, the individual IFN samples were treated with different anti-IFN sera for 1 h. Their antiviral activities were compared with non-treated preparations.

The tests indicate T'P stimulates the production of endogenous interferons (IFNs) and tumor necrosis factor (TNF). The response is dose-related.

Seven assayed commercial batches of TTP had comparable biological activity as immunostimiUlant and the cytokine inducers.

SUBSTITUTE

SHEET

.i~i.M xL~ "il .:i WO 92/16216 PCT/EP92/00491 24 Considerable variation in the response to TTP of leukocytes of the individual blood donors have been observed. PBL of several donors were found to be unresponsive. This may reflect a genetic background.

Potent polyclonal antisera were used, such as anti-IFN-a (Cantell), anti-IFN-a Ly (Namalwa) from K. Fantes, and anti-IFNy (Cantell) to neutralize antiviral activity in the supernatants of PBL treated for 20 h with TTP.

The results of the neutralization assays were found to resemble the finger prints of individual blood donors. In other words, proportions of IFN types produced by the individuals varied considerably. The separation of PBL into adherent and non-adherent fractions may potentiate the induced cytokine synthesis.

The hyporeactivity (tolerance) to the induction of IFN by NDV observed 20 h after the initial stimulation of PBL with TTP was either minimal or absent.

All of the seven batches of TTP tested in human PBL as IFN and TNF inducers were found to be active in inducing IFN and/or TNF.

The optimal concentration of TTP for IFN induction was 30-100 ig/ml and for TNF induction 100-200 ug/ml. The dose of 200ug/ml may be subtoxic for PBL, but the synthesis of TNF occurs much faster than that of IFN and faster than the development of moderate cytotoxicity.

The conclusion of the foregoing is the experience that the active principle in TTP pharmaceuticals is an immunoactive fraction or an extract from peat containing organic, primarily bound sugars, aminoacids, uronic acids, humic acid substances and mineral salts including microelements. The LD 50 in animals is >2400 mg/kg per os.

I;

g _iI~ jCI SUBSTITUTE SHEET i;' i: 1 WO 92/16216 PCT/EP92/00491 25 No mutagenic, genotoxic, embryotoxic, teratogenic or carcinogenic activity of TTP was found. TTP shows no allergenic properties and had no topically irritating activity.

The therapeutic indications include chronic and recurring respiratory tract inflammations and lower leg varicose ulcers, supplementing the treatment of vaginal erosions, and periodontal diseases.

There are clinical observations suggesting that TTP may be useful as an immunomodulator in the supporting therapy of several neoplastic diseases. TTP is used orally (5 mg tablets) or topically.

Of particular relevance and importance appears to be the fact that PBL treated with TTP for 20 h at 37 C do not develop the hyporeactivity state because they retain the capacity to produce IFN after induction with NDV (Newcastle Disease Virus), a very potent IFN inducer.

The attached Tables 1-24 relate to various biological, e.g.

toxicological, hematological and immunological tests. These Tables appear to be self-explanatory and provide for the biologist pertinent information relating to compatibility and biological activity of TTP.

It is worth noting that the concentration of active ingredients in pharmaceuticals, as indicated in claim 13, may be higher than in cosmetic compositions (as indicated in claim 14) due to the following reasons: Pharmaceuticals are prepared in unit dosages, wherein the content of active ingredients is under strict control; e.g. in tablets, the concentration is adapted to the size of the tablet containing the effective daily dose (or a part of it) of the active ingredient.

SUBSTITUTE SHEET i r&~ 11 i

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i t i w WO 92/16216 PCT/EP92/00491 26- The concentration of the active ingredient, e.g. of TTP, in granules with which capsules are filled, preferably is only 5 by weight in order to achieve a sufficient tablet size and to allow an appropriate operation of the capsulating machine.

Another reason is that most pharmaceuticals are administered orally, and the active ingredient is distributed within the whole body. Even though it reaches the skin cells in a very low concentration, the therapeutic effect is remarkable.

Furthermore, cosmetic compositions contrary to pharmaceuticals are used in rather uncontrolled quantities, applied locally, with a different penetration rate to different cells.

Some compositions, such as a shampoo which is nearly immediately washed out, have short contact times with the body or hair, and may contain relatively more of the beneficial component; others are applied several times a day and therefore should have a lower content of the active ingredient.

Finally, as in the case of tooth paste, being in contact with the mucous membrane in the mouth, active ingredient penetration is much easier than through the skin, and the concentration of the active ingredient may be lower.

L SUBSTITUTE SHEET

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WO 92/16216 PCT/EP92/00491 27 TAB. 1 ACUTE TOXICITY. EFFECT OF ORALLY ADMINISTERED TTP ON THE BIOCHEMICAL PARAMETERS OF BLOOD OF RABBITS Sex Dose Day of Examined Parameters Sex Dose Day TTP Test Creatinine Total Protein y-Globulin g/kg 0.0 0 1.09 ±0.03 65.0 ±3.49 0.53 ±0.00 7 1.03 ±0.02 62.0.±0.33 0.56 ±0.01 F 2.0 0 1.06 ±0.01 58.0 ±1.46 0.50 ±0.0' 7 1.14 ±0.38 57.8 ±1.54 0.54 ±0.01 0 0.96 ±0.12 70.3 =3.52 0.55 ±0.00 7 0.94 ±0.13 60.0 ±2.34 0.57 ±0.00 0.0 0 0.80 ±0.11 73.8 ±3.23 0.72 =0.06 7 0.82 ±0.13 75.3 ±1.02 0.79 ±0.03 M 2.0 0 0.88 ±0.05 64.6 =2.86 0.62 :0.10 7 1.24 =0.34 64.6 ±2.03 0.67 :0.07 0 0.85 ±0.03 58.5 =3.19 0.57 =0.02 S7 1.04 ±0.12 57.6 ±4.68 0.56 ±0.01 TAB. 2 ACUTE TOXICITY. EFFECT OF ORALLY ADMINISTERED TTP ON THE ACTIVITY OF THE TRANSAMINASES IN THE SERUM OF RABBITS.

I Examined parameters Sex Dose Day of TTP Test Alanine Asparagine g/kg Aminotransferase Aminotransferase IU IU t. 0.0 0 8.67 ±1.20 22.33 :2.60 7 9.67 ±0.33 28.67 ±3.75 F 2.0 0 10.33 ±1.76 18.33 ±4.09 7 12.33 ±3.52 20.00 ±5.03 0 11.33 ±1.66 22.33 ±3.52 7 11.00 t3.05 20.67 ±4.66 0.0 0 10.33 ±1.76 23.00 ±3.05 7 12.00 ±2.64 25.67 ±4.17 M 2.0 0 9.00 ±1.00 18.00 ±1.52 7 9.00 ±0.57 20.66 ±3.52 0 8.00 ±1.00 18.00 ±1.00 7 11.33 ±1.66 23.00 ±1.99

I.

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WO 92/16216 TAB. 3 ACUTE PCT/EP92/00491 28 TOXICITY. EFFECT OF ORALLY ADMINISTERED N THE HEMATOLOGICAL PARAMETERS OF RABBITS Examined parameters Sex Dose Day of TTP Test Hemoglobin Hematocrit Leukocytes Erythrocytes g/kg x 10 x 0.0 0 12.4 ±0.44 38.3 ±1.85 7.32 ±0.43 5.21 ±0.20 7 11.8 ±0.63 37.7 ±1.20 6.30 ±0.66 5.11 ±0.11 F 2.0 0 11.5 =0.31 36.7 ±0.88 5.69 ±0.58 5.86 ±0.55 7 12.3 ±0.48 38.3 ±0.66 5.74 ±0.76 5.69 ±0.11 0 12.3 ±0.14 40.7 ±2.6 7.35 9 5.42 ±0.20 7 11.6 ±0.23 37.3 ±0.33 8.55 ±1.79 5.08 ±0.19 0.0 0 12.0 ±0.85 37.3 ±1.20 11.10 ±0.63 4.53 =0.26 7 11.9 ±0.61 35.7 ±1.85 10.80 ±0.941 4.54 =0.19 M 2.0 0 12.8 ±0.26 43.7 ±4.80 9.94 =2.00 4.46 ±0.65 7 11.8 ±0.39 34.7 ±2.02 11.06 ±1.08 5.37 ±0.56 0 12.7 ±0.63 38.7 t2.40 9.52 :1.361 4.59 =0.16 7 10.6 ±1.04 4.0 =2.08 8.87 =1.731 5.72 ±0.66 TAB. 4 CHRONIC TOXICITY. EFFECT OF ORALLY ADMINISTERED TTP ON THE MASS OF THE ORGANS OF RABBITS 4.

jA J Dose of TTP (mg/kg) 0.0 50 150 0.0 50 150 Sex F M Organ Mass of Organ in of Total Mass Lung 0.72 ±0.08 0.61 ±0.17 0.67 0.56 ±0.05 0.54 =0.09 0.71 ±0.14j Heart 0.23 ±0.01 0.22 ±0.02 0.25 ±0.03 0.24 ±0.02 0.23 ±0.01 0.23 ±0.02 Spleen 0.057±0.01 0.051±0.00 0.079±0.02 0.05 ±0.01 0.06 ±0.01 0.04 ±0.01 Liver 2.85 ±0.15 3.02 ±0.08 3.22 ±0.37 2.82 ±0.09 2.77 ±0.28 3.12 ±0.061 Kidney 0.59 ±0.03 0.52 ±0.02 0.50 ±0.01 0.48 ±0.03 0.55 =0.01 0.49 ±0.031 Adrenal Gland 0.016±0.01 0.015±0.01 0.020±0.01 0.013±0.01 0.013±0.02 0.012±0.0 Ovaries with Uterus 0.58 ±0.04 0.39 ±0.02 0.60 ±0.04 Testicles 0.30 ±0.01 0.32 ±0.03 0.36 ±0.021 SUBSTITUTE SHEET

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WO 92/16216 PCT/EP92/00491 29 TAB. 5 CHRONIC TOXICITY. EFFECT OF ORALLY ADMINISTERED TTP ON THE HEMATOLOGICAL PARAMETERS OF RABBITS Examined parameters Sex Dose Week TTP of Hemoglobin Hematocrit Leukocy es Erythroc~tes g/kg Test x 10 x 10 M 0.0 0 12.2 ±0.05 38.2 ±1.71 9.33 ±0.79 5.10 :0.56 7 12.3 ±0.15 40.7 ±0.33 9.39 ±1.18 6.34 ±0.23 6 3.0 ±0.35 41.7 ±1.30 9.58 ±1.12 7.32 ±0,70 12 13.5 ±0.19 40.7 ±0.67 11.23 ±1.10 6.30 :0.46 0 12.9 ±0.55 35.7 ±1.82 7.52 ±0.78 5.14 ±0.47 M 3 6 13.3 :0.35 41.0 ±2.10 8.08 11.17, 6.38 ±0.11 12 13.7 ±0.11 39.3 ±0.941 9.36 ±1.01 6.19 :0.19 150 0 14.20 ±0.21 36.7 ±1.81 8.19 ±0.16 5.34 ±0.16 M 3 11.05 ±0.26 38.5 ±1.53 8.83 ±0.78 5.68 ±0.40 6 12.40 ±0.63 37.3 ±1.45 8.18 =0.06 6.84 ±0.10 12 13.70 ±0.32 39.0 ±0.68 8.24 ±0.04 6.54 :0.54 0.0 0 12.6 ±0.27 40.2 ±0.83 7.07 :1.16 5.63 :0.17 F 3 12.1 ±0.23 39.8 ±0.72 8.16 :1.01 5.74 :0.12 6 12.4 ±0.86 42.3 ±1.20 9.78 ±1.69 5.86 :0.24 12 12.5 ±0.87 37.7 ±2.18 10.90 ±2.16 4.60 ±0.29 0 13.2 ±1.20 35.0 ±1.70 7.44 ±0.98 5.47 :0.60 F 3 6 13.0 ±0.55 39.3 ±1.20 7.25 ±0.96 7.08 ±0.94 12 12.8 =0.21 37.7 ±4.33 9.31 ±0.35 6.02 :0.32 150 0 15.2 ±1.15 38.3 ±1.401 8.50 =0.54 6.09 :0.08 F 3 13.1 ±1.10 47.0 ±2.80 8.10 ±0.76 5.85 :0.57 6 12.5 ±0.82 41.0 ±1.15 6.85 0.31 5.61 :0.30 12 13.1 ±0.70 41.0 ±1.00 8.15 ±1.

68 1 5.92 ±0.13 SUBSTITUTE SHEET

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WO 92/16216 PCT/EP92/00491 30 TAB. 6 EFFECT OF TTP ON THE PHAGOCYTIC ACTIVITY OF NEUTROPHILS IN THE PERIPHERAL BLOOD OF RABBITS Phagocytic Activity Neutrophiles Neutrophiles Group n (1000/1 mm NBT+ L.H L.h 0 3 6 0 3 6 0 3 6 0 3 6 Control 1 ml PBS 6 1.9 2.3 2.4 14.3 15.1 17.6 54.5 60.6 59.8 7.53 7.53 7.61 i.v. ±0.9 ±1.1 ±0.4 ±4.6 ±4.6 ±3.0 ±9.4 ±4.8 ±6.81±0.77 ±0.86 =0.4

TTP

(Test 1) *o *o *o mg/kg 15 2.0 1.2 1.7 18.4 26.8 27.2 59.2 69.3 74.9 8.75 10.3 9.88 i.v. =0.

9 :0.7 =7.1 =7.1 =6.7 :7.0 =6.7 =7.0 =7.8 :1.18 =1.25

TTP

(Test 2) 8o o *o *o o mg/kg 2.8 1.6 1.6 15.5 22.1 26.5 63.0 69.5 76.5 7.76 8.52 8.56 i.v. ±0.8 ±0.6 :1.0 ±4.9 ±7.0 ±9.1 =7.0 ±7.1 8.9 ±0.83 =1.04 Number of Test Animals phosphate buffer solution Before the administration of TTP 3 days after the administration of TTP 6 days after the administration of TTP Statistically significant variation at p<0.05 in relation to the value before administration of TTP o Statistically significant variation at p<0.05 in comparison to the control group 4A SUBSTITUTE SHEET ''4 -iic-'i, St 27 WO 92/16216 PCT/EP92/00491 31 TAB. 7 EFFECT OF TTP ON THE NUMBER OF LYMPHOCYTES AND ON THE PERCENTAGE OF THE T-LYMPHOCYTES (E-ROSETTES) AND OF THE B-LYMPHOCYTES (EAC-ROSETTES) IN THE PERIPHERAL BLOOD OF RABBITS.

Lymphqcyt s E-Rosettes EAC-Rosettes x 10- /mmI Group n 0 6 0 6 0 6 Control 1 ml PBS 6 6.6 6.0 19.0 20.1 42.1 42.3 i.v. t2.0 ±1.9 ±5.7 ±5.5 ±6.3 ±2.05 TTP (Test 1) *o mg/kg 15 6.6 5.4 19.2 27.1 43.7 47.7 i.v. ±1.5 ±1.4 ±4.1 ±3.2 ±2.6 ±4.9 TTP (Test 2) 8 *o mg/kg 6.0 6.8 17.0 24.8 41.6 41.0 i.v. ±1.0 ±0.6 ±1.9 -2.9 =1.3 =2.1 n Number of Test Animals PBS phosphate buffer solution 0 Before the administration of TTP 6 6 days after the administration of TTP Statistically significant variation at p<0.05 in relation to the value before administration of TTP o Statistically significant variation at p<0.05 in comparison to the control group TAB. 8 NUMBER OF SPLENOCYTES AFTER LONG-TERM THERAPY WITH TTP

A

<Sb! d.

Control TTP 10 mg/kg.day TTP 50 mg/kg.day n 10 n Time of PFC/10 6 Splenocytes TTP Doses in Weeks 4th Day 7th Day 4th Day 7th Day 4th Day 7th Day 3 367 ±157 156 ±41 1120 ±328* 336 ±240*1 772 ±236w 151 ±57 5 572 ±134 138 ±47 1528 ±346* 292 ±62* 1239 ±280* 232 7 518 ±45 133 ±40 699 ±136* 159 ±44 722 ±258* 141 ±63 9 466 ±185 175 ±75 395 ±94 132 ±36 412 ±138 167 ±62 12 287 ±18 153 ±30 287 ±131 156 ±39 376 ±130 167 ±69 Statistically significant variation at p<0.05 in comparison to the control group SUBSTITUTE SHEET fc*_ -ti 1-1.1y .LaIIjJjjt-, CL1 CX1ILLILPII.L UMLL 4.LJ u obtained from fatty animal-derived amines; as thickening agent- SUBSTI7TUTE SHEET M WO 92/16216 PCT/EP92/00491 32 CONCENTRATION OF THE ANTI-SRBC (19S+7S) ANTIBODIES AT LONG-TERM THERAPY WITH TTP TAB. 9 Control TTP 10 mg/kg.day TTP 50 mg/kg.day n =10 n Time of Hemagglutinin, -10g2 of Titer

TTP

Doses1 in1I Weeks 4th Day 7th Day 4th Day 7th Day 4th Day 7th Day 3 3.75±1.78 7.12±1.36 8.80±0.97* 9.20±1.46* 18.20±0.40* 6.90±1.37 5.82±1.40 6.50±1.00 7.70±0.84* 9.10±2.02*[7.70±0.64* 9.33±2.90* 1-60±0. 48 5.20±.7 4 83 3 .50±0.8 3 3.55±1.21 13. 20±1 .40 4.66±1.24 3.50±1.28 4.00±0.81 2.90±1.22 6.33±1.52J 12 14.80 ±0.74 4.40±1.011]5.11±1.09 5.50±1.51 5.11±1.19 6.10±1.743 Statistically signiificant variation at p<~0.05 in comparison to the control group TAB. 10 CONCENTRATION OF THE ANTI-SRBC (7S) ANTIBODIES AT LONG-TERM THERAPY WITH TTP Control TTP 10 mg/k TTP 50 mg/kg Tim In 10 n of Hemagglutinin, -10g2 of Titer

TTP

Doses Weeks 4th Day 7th Day 4th Day 7th Day 4th Day 7th Day i 3 1.75±0.60 6.37±0.85 3.20±0.87* 5.90±1.70 13.80±0.87* 4.90±0.74*j 2.50±0.70 6.12±1.16 2.20±0.97 5.70±1.10 12.30±1.41 5.33±0.74*1 7 1.00±0.89 3.80±1.09 0.60±0.80 2 .50±1,11* 0.55±0.83 '2.55±0.68* 9 0.40±0.48 3.66±1.24j 0.30±0.64 3.25±0.96 0.00 4.33±1.24 12 0.80t0.97 3.80±0.7631.11±0.99 4.40±1.35 1.55±1.49 4.30±1.26

I

Statistically significant variation at p<~0.05 in comparison to the control group -SUBSTITUTE SHEET separatea in a proize.5 zuir ptLaLd±L1JA1vj-" A.U.J.V6.compositions from the alkaline hydrolysate. Such a composition SUBSTITUTE

SHEET

PC.-/EP92/00491 WO 92/16216 TAB. 11 IMMUNOMODULATORY 33 EFFECT OF TTP (±SD) Dose I n PFC/10 6 -Rosettes Hemagglutinin* }emagglutinin* TTP n Splenocytes Type 19S 7S Type 7S mg/kg 4th Day 4th Day 4th Day 7th Day Control 30 469 13.6 5.4 8.2 ±111 ±3.2 ±1.1 ±1.8 x 10 997 15.6 6.8 8.4 ±139 ±1.4 ±1.0 ±1.1 x x x x 10 839 20.3 6.9 9.7 ±177 ±4.1 ±1.0 ±1.4 x x x x 10 746 23.2 7.2 9.8 ±t129 ±4.7 ±0.6 4 10.0 10 795 18.4 8.3 1.

±129 ±4.0 ±1.8 ±1.

x x 25.0 10 560 16.3 6.1 9.3 ±145 ±2.5 ±1.1 ±1.1 50.0 10 400 14.7 5.3 6.7 57 ±4.0 ±1.0 ±1.9 x x x 100.0 10 375 11.3 5.1 5.7 67 ±2.2 ±0.8 ±1.6 x x 200 10 305 9.5 4.8 5 .3 67 ±2.0 ±1.1 ±1 .7 Statistically significant (Student t-Test) -1092 Of titer variation at total comparison 0.05 Sc SUBSTITUTE

SHEET

WO 92/16216 PCT/EP92/00491 34 TAB. 12 TTP ADMINISTERED DAILY IN 3 DOSES 4th Day After Immunization with SRBC 10th Day After Immunization with SRBC Dose TTP Control 1 x 0.3 ml E- PFC/1 0 6 Rosettes SpJlenocytes Hemagglutinin -10o92 of titer Hemagglutinin -1092 Of titer I v ID 0 13.7 0 469 ±125 5.7 ±1.3 0.8 ±1 .0 Control I 15 15.3 579 6.1 0.6 3 x 0.1 ml ±4.0 ±143 ±1.1 ±0.9 mg/kg 15 19.4* 864* 7.4- 1.1 1 x 0.3 ml ±3.8 ±190 ±1.1 ±1.1 mg/kg 15 19.0* 769* 7.1w 3 x 0.1 ml ±5.0 132 j±0.8 ±0.8 mg/kg 10 15 19.3* 795* 7.0* 0.8 1 x 0.3 ml ±102 ±0.7 mg/kg 15 17.5 656 7.3* 1.3 3 x 0.1 ml 1:3.7 ±128 ±t1.4 ±1.2 SUBSTITUTE SHEET WO 92/16216 PCr/EP92/00491 35 TAB. 13 EFFECT OF TTP ADMINISTERED ORALLY FOR 12 WEEKS ON ThE IMMUNOLOGICAL RESPONSE OF MICE IMMUJNIZED WITH SRBC 4th Day After l0th Day After 6 Hemagglutinin PFC/10 6 Hemagglutinin Weeks E Splerio- -1092 Of titer 's Spleno- 1092 of titer ___Rosettes cytes Rosett75s cytes 15+7 S 15.2 565 6.0 1.6 14.7 256 8.7 7.9 3 ±3.8 ±56 ±0.9 ±1.3 ±3.4 ±67 ±1.0 23.7* 1035* 7.5* 2 9* 23.5* 406* 10.4* 9.7* ±7.8 ±177 ±0:5 ±0.2 ±3.7 1±88 ±1.2 ±1.0 13.8 422 5.1 0 12.7 209 7.1 6.7 ±2.4 ±63 ±0.9 0 t2.1 ±41 ±1.4 ±1.6 23.0* 933* 7.6* 2.7* 20.5* 430* 11 .01, 11.0* =6.1 ±248 ±1.5 =1.5 ±2.7 ±86 ±1.2 :1 .0 12.8 495 4.6 0.5 14.0 245 8.8 7.9 7 ±2.9 ±85 ±1.1 ±0.9 ±5.1 ±48 ±1.6 ±1.6 20.5* 1120* 79* 2.1w 26 437* 11 .3K 10.3* ±5.3 ±214 ±0.6 ±1.5 ±7.7 ±117 ±1.0 ±0.4 9 15.0 5311 6.2 0.5 14.1 294 8.7 8.4, 18.6* 1049* 7,7* 1 1.9 18.2* 515 1.9* 0.

±2.8 ±184 ±0.7 ±1.2 ±3.4 t530 ±0.8 ±105 15.0 573 7.2 1 .4 16.5 266 7.5 7.1 12 ±2.6 ±143 ±1.0 ±1 .2 ±2.6 ±58 ±1.1 ±0.8 14.1 770w 7.2 1 .7 19.0 294 11.8w 10.6* =2.7 -±132 ±1.4 ±1 .7 ±4.1 ±65 ±0.4 Statistically significant variation at p=0.05 in comparison to the control group. in each group, there were 40 animals.

~F

SUBSTITUTE SHEET WO 92/16216 PCT/EP92/00491 36 TAB. 14 INFLUENCE OF THE STORAGE CONDITIONS ON THE ACTIVITY OF TTP IN VIEW OF THE ABILITY OF FORMING E-ROSETTES E-Rosettes after two months storage Dose n Starting Activity Temperature At Room At Room 4 0 C Temperature Temperature Light Admitted in the Dark Control 8 13.6 ±2.4 12.8 ±2.3 12.8 ±2.3 12.8 ±2.3 0.1 mg/kg 8 15.8 ±1.7 16.1 ±3.04 15.1 ±2.9 16.0 1 mg/kg 8 18.0 19.8 15.3 ±2.8 19.8 ±2.7* mg/kg 8 20.4 18.0 16.8 16.9 S Statistically significant variation at a 0.5 in comparison to the control group TAB. 15 INFLUENCE OF THE STORAGE CONDITIONS ON TE ACTIVITY OF TTP IN VIEW OF THE NUMBER OF CELLS PRODUCING ANTIBODIES OF THE TYPE 19S PFC/106 Splenocytes after two months storage Dose n Starting Activity Temperature At Room At Room +4 0 C Temperature Temperature Light Admitted in the Dark Control 8 571 ±69 514 ±128 514 ±1:28 514 :128 0.1 mg/kg 8 747 ±144 1039 ±326* 718 ±135 1002 :210* 1 mg/kg 8 1204 ±155* 1026 ±314* 793 ±186* 1046 ±331* mg/kg 8 1075 ±232* 1070 ±249* 869 ±160* 848 ±137* I Statistically control group significant variation at a 0.5 in comparison to the SUBSTITUTE

SHEET

WO 92/16216 WO 9216216PCr/EP92/00491 37 TAB. 16 INFLUENCE OF THE STORAGE CONDITIONS ON THE ACTIVITY OF TTP IN VIEW OF INFLUENCING THE FORMATION OF ANTIBODIES OF THE TYPE ANTI-SRBC 19S+7S II after two months storage Starting Activity Temperature +4'C Dose At Room Temperature Light Admitted At Room Temperature in the Dark 4 1 0th 4th 10th 1 0th 4th 4.

Control 0. 1 mg/kg 1 mg/kg mg/kg 5.6 9.8 ±0.9 ±0.6 7.4 11.4 iQ,4* 5.0 1 1 7.8 +1 o0w 6.8 ±6.3* 6.4 2 9. 5 ±7.1 10.5 ±1 .7* 13.8 1* 12.0 ±t2.3* ±1.

5.4 6.2 3 4.8 ±-1.3 9.5 ±2.1 10.8 ±0.7* 13.2 4* 10,6 t2. 4* 12.2 6.7 11.0 6.2 ±0.6 0.5 7* 6.5 ±1 .3* 6.0 ±0.5 5.0 9.6 ±1 .1 ±2.1 12.6 ±2,2* 13.1 ±1 .3* 13.6 ±1 .3*

I

Statistically significant variation at a 0.5 in comparison~ to the control group TLAB. 17 INFLUENCE OF THE STORAGE CONDITIONS ON THE ACTIVITY OF TTP IN VIEW OF INFLUENCING THE FORMATION OF ANTIBODIES OF THE TYPE ANTI-SRBC 7S Dose Statingafter two months storage Activity Temperature At Room At Room +4 0 C Temperature ITemperature Light Admitted~ in the Dark Day I4th 10th 4th 10th 4th 10th I 4th Control 8 0.4 9.2 0.3 8.1 0.3 8.1 0.3 8,1 i0.7 ±0.7 ±1.4 ±0.7 ±1,4 ±0.7 ±0.4 0.1 mg/kg 8 0 10.8 0.7 9.8 0.8 10.0 0.7 11.3 ±0.9 ±1,4* 1 mg/kg PF 0.8 10,6 0.3 11.5 0.7 10.3 0.7 11.0 ±0.9 ±0.i7 ±0.9 mg/kg 8 0 10.2 0.3 9.8 0.3 10.7 0.7 10,3 ±1.0 I±0.7 ±L2.0* ±0.9 ±0,9*1 Statistically significant control group variation at a 0. 5 in comparison to the SUBSTITUTE SHEET

V

I

VO 92/16216 PCI/EP92/00491 TAB. 18 HEMATOLOGICAL-PARAMETERS FOR HEALTHY VOLUNTEERS AFTER TWO-WEEK ADMINISTRATIONS OF TTP AT~ A DOSE 1Placebo n=5 TTP BEFORE AND OF 1 MG/DAY n= jaaer-er 14 days 14 days Eryt rocytes x 4.44 ±0.46 4.32 ±0.45 4.32 ±0.32 4,18 ±0.32 Hemoglobin g/611 12.4 ±2.5 11.9 ±2.8 12.8 ±0.8 11.8 ±0.6 Hematocri t 38.1 ±6.7 38 ±6.9 38.7 2.1 38.1 ±1.6 Leuk~cytes I 5 .25 ±0.59 5.27 ±0.8 5.23 4.86 ±0.6 Net-pis 52.6 ±12 57.6 1 54.2 ±2.5 55 ±3.6 Lymphocytes 26 ±7.5 30.4 ±8.3 34 ±4.1 32.4 t3.,3 Thro~bocytes /pi. 220 4 0 .4 214 t35.8 1221 ±38 214 t±39.6 Blood Sediment mm/lh 7.6 ±9 5.6 ±1.9 j 7.8 =2.7 6.8 :3i1

,I

SUBSTITUTE SHEET WO 92/16216 WO 9216216PCFIEP92/00491 39 TAB. 19 BIOCHEMICAL PARAMETERS FOR HEALTHY VOLUNTEERS BEFORE AND AFTER TWO-WEEK ADMINISTRATIONS OF TTP AT A DOSE OF 1 MG/DAY Placebo n=5 TTP Paramne ter 0 14 days 0 14 days Total Protein g/l 72.8 ±1.76 68.42 ±3.2 73.8 ±1.92 70 Albumine 63.2 ±2.5 63.8 ±2.3 63.6 ±1.8 64.4 ±2.1 Globulin al 3.1 ±0.4 3 ±0.5 2.8 ±0,5 2.8 0 .451 Globulin a2 5.95 ±1.9 6.8 ±1.5 6.93 ±0.9 06.9 ±0.9 Globulin 14.4 ±2.2 14.2 ±1.5 13.3 ±1.9 13.2 ±1.3 Globulin y 5 J2.2 ±1.5 12.1 ±1.3 113.7 ±0.9 12.3 ±1 IgG g/l 110.3 ±1l. 1 10.2 ±1 11.7 ±1.6 11.1 ±1.4 IgA g/1 1.8 ±0.6 1.9 ±0.5 1.8 ±0.5 1.9 0 .51 figm g/l 1.4 ±0.3 1.4 ±0.2 J1.1 =0.2 1.1 ±0.4 Complement C3 g/l 1.1 ±0.1 1.1 =0.1 1.1 ±0.2 0.2 ±0.2 Complement C4 g/l 0.2 ±0.5 9.3 ±0 .041 0.2 ±0.0 0.2 ±0 .03 Alanine U/1 J34.2 ±0.05 0.3 ±0.04130.2 t8.2 31.2 ±3.2 {Asparagine U/1 32.8 ±4.4 40.2 ±6.6 130.4 t7.1 39,6 t6.6

N

if 9.

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SUBSTITUTE SHEET i i j Ir.

-L:fn WO 92/16216 PCIP/EP92/00491 40 TAB. 20 COMPARISON OF THE FREQUENCY OF ACUTE INFECTIONS OF THE RESPIRATORY TRACTS DURING THE LAST QUARTERS OF THE YEARS 1989 AND 1990 IN RESPECT OF TWO GROUPS OF PATIENTS TREATED WITH TTP AND PLACEBO RESP. DURING OCTOBER 1990

TTP

applied during the 4th quarter 1990 n 4th quarter 4th quarter statistical 1989 1990 significance (no TTP) (with TTP) "p" average average number of number of infections infections per patient per patient Cold 20 4.0 ±1.3 1.0 ±0.5 0.01 Sore 20 3.3 ±1.3 0.8 ±0.6 0.01 throat Fever blisters 20 2.1 ±2.2 0.3 ±0.4 0.01 Cough 20 1.4 ±1.1 0.3 ±0.6 0.01 Bronchitis 20 0.3 ±0.5 0 Pneumonia 2 0 0.2 ±0.9 0 Placebo applied during the 4th quarter 1990 n 4th quarter 4th quarter statistical 1989 1990 evaluation of (no TTP) (with TTP) difference"p" average average number of number of infections infections per patient per patient Cold 20 3.9 ±1.4 3.1 ±1.3 0.05 Sore 20 2.3 ±2.0 2.6 ±1.7 0.05 throat Fever blisters 20 2.3 ±1.8 1.7 ±2.1 0.05 Cough 20 1.5 ±1.5 1.9 ±2.0 0.05 Bronchitis 20 0.1 ±0.3 0.1 ±0.3 Pneumonia 20 0.1 ±0.3 0.1 ±0.3 SUBSTITUTE

SHEET

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WO 921 'issl-~oaR~~~, r .:I:-CYlr V ^-jr

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JI~L~ i? o 1 j- 1 /16216 PCT/EP92/00491 41 TAB. 21 EFFECT OF INTRADERMAL INJECTION OF BACTERIAL ANTIGENS OR PHA (PHYTOHE'. GGLUTININ) IN PATIENTS TREATED WITH TTP Tuber- Stretio- Staphylo- PHA NaCl Patient culin lycin cocci/ 0.9 Ser. (Init- RT 23 0 anatoxin No. ials) pre 3 pre 3 pre 3 pre 3 pre 3 1. B.T. 2.1 P.E. -I 3. M.H. 4.1 W.H.

W.H.

6.1 Z.N. 1 I 7.1 P.J. 8.1 S.M. I+ pre pretreatment 3 after 3 weeks TAB. 22 EFFECT OF INTRADERMAL INJECTION OF BACTERIAL ANTIGENS OR PHA IN UNTREATED (PLACEBO) PATIENTS Tuber- Strepto- Staphylo- PHA NaCJ Patient culin lycin cocci, 0.9 Ser. (Init- RT 2 3 0 anatoxin No. ials) pre 3 pre 3 pre 3 pre 3 1pre 3 1. M.M. 2. O.T. t- 3. O.H. 4. W.J. I K.C. 6. M.J. 7. B.W. I +I 8. K.T. I+ I+I I 19u pre 3 pretreatment after 3 weeks SUBSTITUTE

SHEET

WO9/61 PCT/EP92/00491 42 TAB. 23 IMMUNOGLOBULINS IN THE SERUM OF PATIENTS WITH ULCUS CRURIS TREATED WITH TTP n[ Test Group Imnxunoglobulin in mg before Treatment after Treatment 1 IgG 1732.87 ±207.52 7 IgG 1732.87 ±20-7.52j 8 A 2 IgA 304.25 t47.501 8 IgA 1283.0,7 ±44.10 IgM] 202.87 71.12 9 f gM 1189.75 z75.151 41 EgG 1924.37 ±246.25 10 IgG 11972.00 :-239.33 8 B 5~ IgA 796.50 ±59.84 11 IgA 285.50 56.76 6 gM 242.12 56.95 12 IgM 232.87 t 65.45 1 vs 7 P> 0.05 4 vs 10 p 0.05 Statistical 2 vs 8 p 0 .05 5 vs 11 p)>0.05 Evaluation of 3 vs 9 p >0.05 6 vs 12 p )0.05 Difrec vs 4 pO> .05 7 vs 10 pO> 2 vs 5 p 0.05 8 V s 11 p 0.05 3 vs 6 p 0.05 9 vs 12 p 0.05

I

I

SUBSTITUTE

SHEET

Claims (16)

1. A peat-derived bioactive product, characterised contains not more than 70%, preferably not more weight of inorganic salts, especially of sodium based on dry solids. in that it than 60% by chloride,

2. A peat-derived bioactive product obtainable by a process wherein a highly concentrated aqueous solution of inorganic salts, especially of sodium chloride containing peat-derived bioactive ingredients is diluted with demineralized water and subjected to reverse osmosis in order to desalinate the solution, inorganic salts being removed, and wherein the resulting solution is concentrated and clarified, and, optionally, in at least one further step, sterilized and/or spray-dried.

3. A peat-derived bioactive product obtainable by a process wherein a highly concentrated aqueous sodium chloride solution containing peat-derived bioactive ingredients, said solution being obtainable by primary and secondary alkaline hydrolysis of air-dried raw peat material, followed by acidification of the hydrolysate, separation of insoluble parts and elimination of ballast substances by extraction with organic solvents, and removal of the residual organic solvents from the post-extraction aqueous phase, is filtered through a sintered ceramic filter under reduced pressure, wherein the clear filtrate thus obtained is diluted with demineralized water and subjected to reverse osmosis in order to desalinate the solution, inorganic salts being removed, and wherein the resulting solution is concentrated and clarified, and, optionally, sterilised and/or spray-dried. SUBSTITUTE SHEET e -If lI 1 i a ,u _i WO 92/16216 PC/EP92/00491 44 1/ tP

4. A process for producing peat-derived bioactive products from a highly concentrated solution of inorganic salts, especially of sodium chloride, containing peat-derived bioactive ingredients, characterised in that said solution is diluted with demineralized water, the dilution being effected with water volumes several times, preferably 5 to 8 times, the volume of the solution to be diluted, that the diluted solution is subjected to reverse osmosis in order to desalinate the solution, inorganic salts being removed, whereafter the desalinated solution is concentrated and clarified, and that, optionally, the concentrated and clarified solution is sterilized and/or spray-dried.

A novel process for producing peat-derived bioactive products from a highly concentrated aqueous sodium chloride solution containing peat-derived bioactive ingredients, as 4- claimed in claim-6-, said solution being obLainable by primary and secondary alkaline hydrolysis of air-dried raw peat material, followed by acidification of the hydrolysate, separation of insoluble parts and elimination of ballast substances by extraction with organic solvents, characterised in that residual organic solvents are expelled from the post-extraction aqueous phase, the remaining solution being filtered through a sintered ceramic filter under reduced pressure, the clear filtrate thus obtained being diluted with demineralized, e.g. distilled, water, said dilution being effected with a water volume several times, preferably 5-8 times, the volume of the solution to be diluted, whereafter the diluted solution is subjected to reverse osmosis in order to desalinate the solution, inorganic salts being removed, and the desalinated solution is concentrated and clarified and, optionally, sterilized and/or spray-dried.

6. A process as claimed in claim 5, characterised in that the solution is sterilized by means of a membrane filter, e.g. a Millipore(R) filter. SUBSTITUTE SHEET WO 92/16216 PC/EP92/00491 45

7. A process as claimed in claim 5 or 6, characterised in that the solution is spray-dried with an inlet temperature of about 180 0 C and an outflow temperature of about 90 0 C.

8. A peat-derived bioactive product, obtained by the process of either of claims 5 and 6, said product being in the form of a concentrated solution.

9. A peat-derived bioactive product, obtained by the process of any one of claims 5 toad& said product being in powder form.

A process for preparing a pharmaceutical formulation containing a peat-derived bioactive product, in the form of a gel, characterised in that a sterile alcoholic herb extract is combined with a sterile aqueous solution of previously powdered peat-derived bioactive product, a sterile solution of a flavouring compound preferably selected from the group comprising menthol, thymol, mint, lemon and eucalyptus, and with a gel-forming composition such as sterile glycerol with colloidal silica to convert the liquid composition into gel form, the weight ratio of liquid mixture to silica preferably being from 90:10 to 94:6.

11. A process for preparing a pharmaceutical formulation containing a peat-derived bioactive product, in the form of an ointment, characterised in that a sterile herb extract is gradually combined with a sterile solution of powdered peat- derived bioactive product, that the resulting mixture is gelled with the addition of a gel-forming ingredient such as colloidal silica and that the gel thus obtained is triturated with a previously sterilized mixture of fatty components, such as eucerine and petrolatum, preferably in a weight ratio of liquid components to silica of about 30:20, and of gel to fatty composition of between 32:68 and 34:66. SUBSTITUTE SHEET L 1- I :i ii i x r -il I WO 92/16216 PCr/EP92/00491 46

12. A process as claimed in claim 10 or 11, characterised in that the peat-derived bioactive product is as claimed in any one of claims 1 to 3, 8 and 9, preferably the "TOLPA(R)Torf Preparation" as described herein.

13. A pharmaceutical composition containing as active ingredient a peat-derived bioactive product as claimed in any one of claims 1 to 3, 8 and 9 and a pharmaceutically acceptable carrier material, preferably in a weight ratio of between about 1:5 and 1:25, and most preferably between 1:9 and 1:19.

14. A cosmetic preparation containing as active ingredient a peat-derived bioactive product as claimed in any one of claims 1 to 3, 8 and 9 in a quantity of 0.01-10% by weight, preferably 0.05-1% by weight, more preferably 0.05-0.1% by weight.

A cosmetic preparation as claimed in claim 14, containing, in addition, at least one herb extract and/or at least one fragrance.

16. A cosmetic preparation as claimed in claim 4 or 5, contain- ing as the peat-derived bioactive product a "concentrated peat extract" as described herein. -J 4.iC CrlCI- SUBSTITUTE SHEET J TM -4, INTERNATIONAL SEARCH REPORT International Applicatdon No PCT/EP 92/00491 1. CLASSIFICATION OF SUBJECT MATrER (if several classification symbols apply, Indicate all)' According to international Patent Classification (IPC) or to both National Classification and IPC Int. Cl 5 A61K35/10; A61K7/00; C07G17/00 aI. FYELDS SEARCH{ED Mi1nimum Documentation Starchedl Classificationl System Clasification Symbols Int.Cl. 5 A61K Documentation Searched other than Minimum Dccumnentation to the Extent that such Documents are Included in the Fields Searchedl MI. DOCUMIENTS CONSIDERED TO BE RELEVANT 9 Category Citation of Doctumnt, It with indication, where appropriate, of the relevant passages 12 Pelevant to Clm No. 1 3 A DE,A,2 846 482 (AKADEMIA ROLNICZA WE WROCLAWIU) 1-16 26 April 1979 cited in the application see the whole document A EP,A,0 281 679 (RUTGERSWERKE 14 September 1-16 1988 see claims; example 1 A EP,A,0 083 285 (LE THERMOGENE) 6 July 1983 1-16 see page 4, line 11 line 33; claims Special categories of cited documents t10 'T latt-r document published after the international filing date A doumet dfinng he eneil sateof he rt hic isnotor priority date and not in conflict with the application hut 'A douetdfnn h eea stt fh r whic is no died to understand the principle or theory underlying the consderd t be f p~cuar rlevnceInvention earlier dlocument but published on or after the International docuent of particular relevance; the claimed Invention filing date cannot be considered novel or cannot be considered to 'V document which may throw doubts on priority claim(s) or Involve an inventive step which is tited to estahlish the publication date of another document of particular relevance; the claimed invention citation or other specIal reason (a~s specified) cannot be considered to involve ino inventive step when the 0' document referring to an oral disclosure, use, exihition or document Is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled 'I document published prior to the international filing date but In the ami later than th priority date claimed document member of the same patent family IV. CRTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 26 MAY 1992 0 3 JUN 1992 International Searching Authority Signature of Authorized Officer EUROPEAN PATENT OFFICE RYCKEBOSCH A 0-C PeruPCT/LSA2104*= taea e I Janer 1"51 ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENTr APPLICATION No. EP 9200491 SA 56904 Thir annex lists the patent family members: relating to the patent documents cited in tbe above-mentioned iriernational search report. The members are as contained in the European Patent Office EDP file on The Europan Patent Office is in no way liable for these particulars which ame rwerey given for the purpose of informoation. 26/05/9 2 Patent document Publication Patent famly Publication cited in search report date member(s) dat DE-A-2846482 26-04-79 CH-A- 639279 15-11-83 EP-A-0281679 14-09-88 DE-A- 3707909 22-09-88 CA-A- 1293717 31-12-91 JP-A- 63238096 04-10-88 US-A- 4918059 17-04-90 EP-A-0083285 06-07-83 FR-A- 2518404 24-06-83 I A SFor more details about this annex we Official Journal of the European Patent Office No. I2S