DE4137093A1 - Use of avarol as an antioxidant - in the treatment of auto-immune diseases, dermatitis, psoriasis, rheumatic disorders etc. - Google Patents

Abstract

Use of avarol (I) (or a deriv.), as antioxidant and inhibitor of enzymes involved in synthesis of prostaglendins and leukotrienes, is new. USE/ADVANTAGE - (I) is described in DE3427383. Derivs. are described in US4946869. They can be used in treatment of inflammatory systemic disorders and skin disorders, esp. in treatment of rheumatic diseases, autoimmune diseases, Psoriasis vulgaris, atopic dermatitis, Crohn's disease, colitis ulcerosa and inflammation of skin, mucous membranes and the associated blood vessel systems.

Description

Basis of the invention is the finding that a number of inflammatory systemic and dermatological Diseases due to reactive oxygen species z. B. in a row a derailed arachidonic acid metabolism or as a consequence the deposition of immune complexes arise (Thiers, B .: Psoriasis. J. Am. Acad. Dermatol. 3: 101; 1980).

Metabolites of arachidonic acid, such as the prostaglandins as well as the leukotrienes and reactive oxygen species immunologically activated white blood cells known mediators of inflammatory processes. The mentioned substance classes become from the Arachidonsäure over synthesizes the enzymes cyclooxygenase and 5-lipoxygenase. An inhibition of these two enzymes leads to diminished Production of these inflammatory mediators (Bahr, T. et al., Agents and Actions, 32: 137-139, 1991; Grupe, R., Agents and Actions, 32: 79-81, 1991; Vane, J.R., TIPS, 8: 491-496, 1987; Bhattacherjee, P. et al., (Annals New York Academy of Sciences) Ann. N.Y. Acad. Sci., 524: 307-320, 1988).

It will now be described that the substances Avarol and Avaron and its derivatives are potent inhibitors of the 5- Lipoxygenase and cyclooxygenase are. The substances are in the published patent application DE 34 27 383 from 30. 01. 1986 described.

The subject of the present invention is the Description of the surprising finding that Avarol and his Represent derivatives of anti-inflammatory agents. In In vitro and in various animal models, they show good anti-inflammatory effects. Due to the following Data therefore it seems promising, the substances at  inflammatory systemic diseases, skin diseases and especially in diseases of the rheumatic type, in autoimmune diseases or psoriasis vulgaris To use people.

Avarol or its derivatives described in the USA Patent No. 49 46 869 are in suitable form in dosages from 0.01 to 100 mg / kg systemically applied or in Body cavities introduced. Suitable dosage forms are z. B. Combinations of the active substance with common pharmaceutical carriers and excipients in the form of Tablets, coated tablets, dragees, suppositories, semi-solid formulations, liposomes, emulsions, sprays, sterile solutions or suspensions for injection or Inhalation. Pharmaceutically acceptable carriers are z. B. Lactose, sucrose, sorbitol, talc, stearic acid, Magnesium stearate, lipids, gum arabic, cornstarch or Cellulose, combined with solvents such as water, Polyethylene glycol, etc., as well as conventional solubilizing Excipients.

The substances can also be applied in the form of ointments, sprays or other dermatics to the diseased parts of the skin. The preparation of the pharmaceutical agent is carried out by conventional methods, for example by emulsification in a hydrophilic base. The dosage depends primarily on the specific form of administration and the purpose of the therapy. The size of the single doses and the regimen of administration may best be determined by the physician on the basis of an individual assessment of the particular medical condition, taking into account the age, weight and condition of the recipient. In general, the daily dose of avarol or its derivatives is 1 gm of a 1-3% ointment per 10 cm ² body surface area. The duration of treatment depends on the type and severity of the disease. It generally extends over several weeks, for example 4-8 weeks.

The activity of the compounds used according to the invention is described below using the example of Avarol
1. Pharmacological in vitro as well
2. investigated in vivo test systems.

Examples example 1 Testing for malondialdehyde (circular reaction)

The test is based on the formation of a measurable at 532 nm Reaction product of thio-barbituric acid (TBA) with Malondialdehyde (MDA), the u. a. in the oxidative degradation of polyunsaturated fatty acids is formed (reference: H. Böhme, K. Hartke DAB7 1968, commentary, VAG Stuttgart, 2. 1971 edition, pp. 80-81).

Trilinolenin (Sigma, St. Louis) and Avarol are in DMSO solved.

500 μl trililonenin in 1 ml HCl for 1 hour at 37 ° C in the presence of DMSO as a control and the DMSO-containing Avarol solutions incubated.

To determine the total TBA-reactive material 1.0 ml of the incubation mixture and extracted with 1.5 ml of TBA Solution (0.7% in 0.05 M NaOH) and 1.5 ml of tri-chloroacetic acid (20%) mixed. After 60 minutes incubation in one boiling water bath, the samples are cooled and 10  Centrifuged at 2500 rpm for minutes. The optical density is determined at 532 nm. Fresh tetramethoxypropane, out which is produced under the experimental conditions MDA serves as Calibration standard. The results are reported as μmol MDA Equivalent calculated. There are triplicate provisions carried out.

Malondialdehyde arises by way of fat autoydation by converting atmospheric oxygen into radical creations reactive carbon atoms of fatty acid residues. Malondialdehyde condenses in acid solution with appropriate Reducing agents to polymethine dyes whose Concentration is determined photometrically. With this method lipophilic radical scavengers and antioxidants are recognized.

Table: Effect of Avarol in the antioxidant test [% inhibition (± SD)] the formation of TBA reactive material compared to Tetramethoxypropane control

Interestingly, Avarol is the formation of TBA-reactive Material inhibits what was previously unknown. The substance shows medium strength in the tested concentration range Effects with shallow dose-response relationship.  

Example 2 In vitro inhibition of cyclooxygenase

Cyclooxygenase activity was determined in monocytes in the presence or absence of the agonist lipopolysaccharide (LPS) (L-4130, Sigma, St. Louis). For this purpose, human monocytes were maintained at a density of 10 ⁶ cells per ml in RPMI 1640 medium for 24 h. Subsequently, 30 μM arachidonic acid were added to the cultures and the enzyme activity was measured 30 min later on the basis of the production rate of prostaglandin E2.

table

Effect of Avarol on Cyclooxygenase and 5- Lipoxygenase activity in human monocytes. As in the text described, the culture supernatants - 30 min after Addition of LPS - collected and with radioimmunoassays (Amersham) on the content of prostaglandin E2 (PEG 2) and Leukotriene B 4 (LTB 4). Set the values Mean values (± S.D.) of five independent results.

Result

Upon addition of LPS, the cyclooxygenase activity increases from 86 to 249 pg PGE 2 per 10 ⁶ cells x 30 min. Increasing concentrations of avarol increasingly increase the stimulating effect of LPS on cyclooxygenase. At a concentration of avarol of 0.71 ± 0.09 μg / ml, the induction of cyclooxygenase is reduced to 50%.

Example 3 Influence of avarol on 5-lipoxygenase activity

Monocytes were stimulated with LPS as described above and treated with 30 μM arachidonic acid [Fu, J.Y., Masferrer, J.L., Seibert, K., Raz, A., and Needleman, P. (1990) The induction and suppression of prostaglandin H2 synthase (cyclooxygenase) in human monocytes. The Journal of Biological Chemistry 265, 16,737 to 16,740]. After an incubation period of 30 min. Was the formation of leukotriene B4 (LTB 4) determined (see Table). The data showed that after stimulation of the cells with LPS the lipoxygenase activity in monocytes of 5.9 9.7 pg of LTB 4/106 cells × 30 min. Increases. A Avarol concentration of 0.67 ± 0.07 μg / ml reduces the induced lipoxygenase activity.

These results show that Avarol is a new Represents a substance class of anti-inflammatory substances, the lipoxygenase in a similar sensitive way as the pentadienone derivatives and cyclooxygenase such as Indomethacin inhibits.

Example 4

Mouse tail test test:
[Reference: PT Bladon et al. Arch. Dermatol. Res. 277: 121; 1985].
In vivo test system for the detection of antiporrosis activity of Avarol:

For these investigations, the established Mouse tail model used. The adult mouse tail model  is used as there are regions of orthotic and parakeratotic Stratum corneum has. In the orthokeratotic Scaly region is located in a granular position around the hair follicle; the stratum corneum of the mouse is histological built similar to that of the normal human epidermis. However, the dandruff region is thin or almost absent complete; the nucleated cells are visible in the stratum corneum. These regions of parakeratotic stratum corneum are microscopically similar to those found in the Stratum corneum of individuals with psoriasis vulgaris. Becomes when applying a test substance to the mouse tail one significant increase in the orthokeratosome zone can be measured to a potential antipsoriatic effect of the given Substance be closed.

experimental animals

Male NMRI mice (Zentralinstitut, Hannover) 26-29 g heavy and with Altromin standard food and water ad libidum fed.

substances

Avarol ointment, concentration 1%
Vehicle: Eucerin; anhydr.
Appl.-type: local (dermal) Appl. Vol: 0.1 ml per application

experimental arrangement

To a conversion of parakeratotic stratum corneum too To induce orthokeratotic, the mouse tails on proximal part each for 2 hours with the vehicle or the test substance brought into contact. During this Contact time becomes a plastic cylinder (blue eppendorf tips) slipped over the tail and fixed with Leukosilk. At the end the contact time is removed the plastic cylinder and the Washed off tail.  

After completion of the entire treatment (2 weeks) the Tails of the killed mice were histologically prepared: Fixation with 4% formalin, embedded in Paraplast. Then longitudinal sections are made (each 5 microns thick) and with Hematoxylin and eosin stained.

The sections are microscoped. It will be the percentage Proportion of orthokeratosis in each animal as well as the Thickness of the epidermis determined.

Measuring parameters: Orthokeratosis (%) instrument: microscope

experimental procedure

The substance or vehicle application takes place daily during 5 days, 2 days will be interrupted and for another 5 days continued.

2 hours after the last treatment the animals will be killed. After the histological preparation of the Mouse tails are microscopically stained for the stained sections.

Experimental design and evaluation

Mean value formation (± SD) of the two measured quantities orthokeratosis and epidermis thickness and respectively comparing these values of Vehicle and substance groups by t-test.

Number of animals per group: 8.

Result:
Control: 15.7 μm ± 4.3,
Test: 29.3 μm ± 4.9.

The test group differs highly significantly (P <0.001) by increasing formation of an orthokeratosis zone. As our result shows, the control animals have one Orthokeratose thickness of 15.7 microns, while those with Avarol treated animals an orthokeratosis layer of 29.3 microns respectively.

Example 5

Arachidonic acid induced edema (Reference: Jones, TR et al., Leukotrienes and Lipoxygenases, Rokach, J. ed., Elsevier, Amsterdam 1989)
Male NMRI mice (Zentralinstitut Hannover) were used for the experiments. The animals received a standard laboratory diet and tap water ad libitum. At baseline, they had an average body weight of 26-29 g. Arachidonic acid (Sigma, St. Louis) and Avarol were prepared fresh in 96% ethanol immediately before the experiment and 10 μl of solution used for the studies. The mice were randomized in groups of 10 animals and anesthetized with urethane (15%, 0.1 ml per 10 g mouse ip).

For edema induction, 4 mg arachidonic acid (AA) was added to the Applied inside of the left ear. The right ear served as a control and was treated only with ethanol. 15 minutes. before the edema induction by the AA Avarol was in the Dose range between 0.001 to 1 mg / ear applied.

60 min. After the edema induction, the animals were passed through Cervical killing killed. With a punch were discs of 9 mm diameter punched out of both ears and immediately weighed (averaging ± SD).  

Avarol has in this model a previously unknown distinct edema-inhibiting effect. The results point to this suggests that Avarol is also more inflammatory for therapy Skin diseases might be appropriate.

Example 6

Reference: Bhattacherjee, P. et al., (Anaal's New York Academy of Sciences) Ann. NY Acad. Sci., 524: 307-320, 1988
Effect of Avarol in a non-immunological model of edema after oral administration
Test animals are female Sprague Dawley rats (SPF, breeder Charles River, Sulzfeld, FRG) with a body weight of 180-230 g. At baseline, they are sober but have access to water ad libitum. The number of test animals per group is 10. The test substances are administered in logarithmic dosage in the range between 30 and 300 mg / kg in 0.05% carboxymethylcellulose as a vehicle. The application volume is 10 ml / kg.

Edema production is by subplantar injection of 0.1 ml kaolin suspension in a hind paw. The test substance or vehicle application is p.o. 60 min. before the Ödemauslösung. The paw volume will be before and 4 hours after the Edema release measured plethysmometrically.

Avarol surprisingly shows in this model after oral administration hitherto unknown anti-edematous effects.

Example 7

The effect of Avarol in an immunologically-induced Model, adjuvant arthritis.  

Reference: Mohr E. et al., Arzneim. Research / Drug Res. 26: 1860-1866, 1976.

By injecting Freund's complete adjuvant (FCA) intradermally into the rat paw becomes an immunological Reaction triggered. It comes after 9-12 days Periarthritis and arthritis of both hind paws. These arise as a result of deposition of immune complexes, Complement activation, leucocyte infiltrations, Release of lysosomal enzymes, thrombus formation and local Tissue and cartilage damage u. a. through reactive Oxygen species.

For the investigations were male Sprague Dawley Rats of 180-220 g body weight (Charles River Breeding Laboratories) used. The animals were before the attempt sober, but had water ad libitum. They became 10 each Animals randomized into 4 treatment groups for the experiments. Adjuvant arthritis was assessed by intradermal injection of 0.1 ml FCA triggered.

Avarol was given in doses of 30, 100 and 300 mg / kg 10 ml / kg suspension in 1% Tween 80 and 0.5% aqueous Carbomethoxymethylcellulose orally administered for 10 days.

The clinical picture of periarthritis and rheumatoid Arthritis of the Avarol-treated animals was on day 10 in the Comparison to control with the help of a score (see Mohr) assessed.

Result

Already at 100 mg / kg Avarol became oral after 10 days Give the severity (scores) of rheumatoid arthritis significantly inhibited by 20% (p <0.05). With 300 mg / kg rose this effect was significant at 33% (p <0.05).  

In summary, results from the results of studies described that avarol in mouse and rat in different models for both topical and after oral administration anti-inflammatory and anti-inflammatory Owns properties. Avarol inhibits inflammatory on the one hand pathological processes by non-immunological Mechanisms are triggered. On the other hand, but also immunologically or triggered by allergic mechanisms Inflammation inhibited by avarol.

Studies on the mechanism of action in vitro show that the antiphlogistic effect of Avarol on both anti-oxidative, radical-catching as well as on a the Cyclooxygenase and the 5-lipoxygenase inhibitory effect based. Antiproliferative properties were assessed on Mouse tail test proved in vivo.

The findings described suggest that Avarol is included in Illnesses with inflammatory symptoms could be useful. The good anti-inflammatory effect after topical application Avarol is beneficial in the treatment of dermatoses Expect effects, eg. In psoriatic skin diseases and other hyperproliferative dermatological disorders as well as in inflammatory changes of the skin and Mucous membranes as well as those supplying them venous or arterial blood vessel systems. In first therapy observations In humans, there were indications of transferability the experimental results on humans.

The also available after oral anti-inflammatory and Anti-edematous effects can be applied to the therapy use inflammatory systemic processes, as z. B. at Crohn's disease, ulcerative colitis, rheumatoid  Diseases or pathological autoimmune processes are observed.

The invention is illustrated by the following examples

The remedies include one for therapy from the above effective amounts of active substances described in optionally together with a pharmaceutical compatible carriers and excipients. For example contain such pharmaceutical agents 0.01 to 98 wt .-% at least one compound according to the invention together with a pharmaceutical carrier.

The method of treatment of the above Diseases involves the administration of a therapeutic effective amount of active substance to a patient of this Treatment is needed.

The dosage depends primarily on the specific Form of administration and the purpose of the therapy. The size single doses and the regimen can be administered on best based on an individual assessment of each Disease case be determined by the doctor, the Age, weight and condition of the recipient, who Route of administration and the nature and severity of the disease must be taken into account. In general, the Daily dose 1-20 mg / kg body weight.

The duration of the treatment depends on the type and severity the disease. It generally extends over several weeks, for example 4 to 8 weeks.  

If the agent is in the form of a unit dose, this preferably contains 10 to 500 mg of the invention used for connection.

The pharmaceutical agents may be for oral administration in solid form, for example as tablets, lozenges, Capsules, powders, or in liquid form, for example as aqueous or oily suspension, emulsions, liposomal Preparations, syrups, elixirs, solutions or liquids filled capsules are present.

Preferred oral agents are in the form of tablets or capsules and may be conventional carriers such as binders (For example, syrup, acacia, gelatin, sorbitol, tragacanth or Polyvinylpyrrolidone), fillers (eg lactose, sugar, Corn starch, potato starch, calcium phosphate, sorbitol or Glycine), lubricants (e.g., magnesium stearate, talc, Polyethylene glycol or silica), disintegrating agents (eg starch) and wetting agents (eg sodium lauryl sulphate), contain.

Agents for parenteral administration are generally in the form of a solution or suspension of the invention for Application coming connection together with usual pharmaceutical carriers, for example in the form of a aqueous solution for intravenous injection, a liposomal Preparation or an oily suspension for the intramuscular injection. For parenteral administration suitable agents are obtained by adding 0.1 to 10 Weight percent of the compound of the invention in water or a support made from an aliphatic polyalcohol, such as glycerol, propylene glycol or polyethylene glycols or a mixture thereof or other solubilizer Substances exists, dissolves. The polyethylene glycols consist of  a mixture of non-volatile, usually liquid Polyethylene glycols, both in water and in organic liquids are soluble and their Molecular weights ranging from 200 to 1500.

Pharmaceutical agents for rectal administration are in the form of suppositories, wherein the inventive Compounds of a suitable suppository base, such as Vaseline or cocoa butter, hydrogenated fats, poly waxes or Polyethylene glycols, in an amount of 1 to 10 Weight percent are incorporated.

Pharmaceutical agents for topical administration are in the form of hydrogels or lipogels, the compounds of a suitable basis according to the invention, like Vaseline or Vaseline, oils, poly waxes or Polyethylene glycols, in an amount of 1 to 10 Weight percent are incorporated.

The production of pharmaceutical agents takes place by conventional methods, for example by tableting, Incorporation of the present invention used Compounds in a suppository base, sterile filtration and filling in ampoules or dropper bottles of a solution of in accordance with the invention for use in compounds Injection water together with usual additives, such as Sodium chloride, sodium hydrogen phosphate, disodium EDTA (Ethylenediaminetetra-acetic acid disodium salt), benzyl alcohol or sodium hydroxide to adjust the pH.  

The following examples serve to explain the invention Examples of pharmaceutical agents

In the following formulation examples, as Active ingredient in each case one of the invention for use coming compounds individually or in admixture with a another compound of the invention or with another anti-viral agents are used.

Example a tablet formulation Active substance | 10 mg lactose 18 mg potato starch 38 mg gelatin 2 mg talc 2 mg magnesium stearate 0.1 mg

example b

tablet formulation Active substance | 10 mg potato starch 40 mg polyvinylpyrrolidone 5 mg

The tablets may contain a colored sugar layer be coated.

The dragee cover can consist of:

Sugar | 65.0 mg talcum 39.0 mg calcium carbonate 13.0 mg gum arabic 6.5 mg corn starch 3.7 mg shellac 1.1 mg Polyethylene glycol 6000 0.2 mg Magnesia usta 1.3 mg dye  0.2 mg 130.0 mg

Total drag weight at a core of 50 mg = 180 mg

example c

capsule formulation Active substance | 10 mg corn starch 90 mg lactose 50 mg talc 2 mg

This mixture is filled in gelatine capsules.

Example d Liquid oral formulation Active ingredient | 2 g sucrose 250 g glucose 300 g d-Sorbitol 150 g Agar Agar 0.15 g methylparaben 0.5 g Propylparaben 0.05 g Flavor (orange flavor) 10 g Tartrazine yellow solubilizer and purified water 1000 ml

Example e Liquid oral formulation Active ingredient | 2 g tragacanth 7 g glycerin 50 g sucrose 400 g Methylparabren 0.5 g Propylparabren 0.05 g Flavor (blackcurrant flavor) Red Dye No. 2C.E.184 0.02 g Purified water on 1000 ml

Example f Liquid oral formulation Active ingredient | 2.4 g sucrose 400 g Tincture of bitter orange peel 20 g Tincture of sweet orange peel 15 g Solubilizer and purified water 1000 ml

Example g ointment formulation Yellow wax | 6.5g spermaceti 8.0 g Peanut oil 60.0 g glycerol 0.5 g active substance 1.0 g Water, ad 100%

Example h ointment formulation Polyethylene glycol 300 | 49.0 g Polyethylene glycol 1500 49.0 g active substance 2.0 g

Example i ointment formulation Emulsifying cetylstearyl alcohol | 30.0 g Thick paraffin 35.0 g active substance 3.0 g White Vaseline, ad 100.0 g

Example k ointment formulation Wool wax alcohol ointment | 6.0 g Cetylstearylalkohol 0.5 g active substance 1.0 g White Vaseline, ad 100.0 g

Example 1 ointment formulation Wool wax alcohols | 50.0 g active substance 1.0 g Water, ad 100.0 g

example m injection

For the preparation of a 0.01% solution for infusion, 0.01% Active ingredient and 5% laevulose in bidistilled water with Help solved conventional solubilizing auxiliaries. The Solution is filtered through degermination filter, in 500 ml Infused bottles filled and sterilized.

The example refers to 50 mg of active ingredient per Single dose.

example n infusion

For the preparation of a 0.01% solution for infusion, 0.01% Active ingredient and 5% laevulose in bidistilled water with Help solved common solubilizing substances. The Solution is filtered through degermination filter, in 500 ml Infused bottles filled and sterilized.

The example refers to 50 mg of active ingredient per Single dose.

Claims (6)

1. Use of Avarol or its derivatives as Antioxidants and inhibitors of enzymes involved in the Synthesis of prostaglandins and leukotrienes are involved. 2. Use of Avarol or its derivatives for healing systemic inflammatory diseases. 3. Use of Avarol or its derivatives for healing inflammatory skin diseases. 4. Use of Avarol or its derivatives in the Treatment of the cytopathic effects of autoimmune diseases Diseases. 5. Use of Avarol or its derivatives in the Treatment of atopic dermatitis and psoriasis vulgaris. 6. Use of Avarol or its derivatives in the Treatment of rheumatoid diseases.