WO2008071173A1 - Preparations for inhibiting prostaglandin e2 synthesis - Google Patents

Abstract

The present invention relates to preparations of acylphloroglucinols, in particular hyperforin and myrtucommulone and derivatives thereof, the provision of these preparations for the therapeutic treatment of inflammatory diseases, painful and feverous conditions and cancer diseases which are accompanied by increased activity of the inducible microsomal prostaglandin E<SUB>2</SUB> synthase-1 (mPGES-1) or increased prostaglandin E<SUB>2</SUB> synthesis, and also the appertaining methods.

Description

Preparations for the inhibition of prostaglandin E ₂ synthesis

description

The present invention relates to natural and synthetic acylphloroglucinols, in particular hyperforin and myrtucommulone and their structural derivatives as inhibitors of inducible microsomal prostaglandin E ₂ synthase-1 and their use for the treatment of prostaglandin E ₂ -mediated diseases.

Prostaglandin biosynthesis is initiated by the initial steps of converting arachidonic acid to prostaglandin (PG) H ₂ through cyclooxygenase (COX) -I or -2. Certain PGs, including PGE ₂ , are mediators in inflammation (especially rheumatoid arthritis), pain and fever, and are also involved in cancers (lung, colon, endometrium), while other PGs perform important physiological functions [1]. Inhibitors of COX-1 and -2 thus prevent the synthesis of all PGs and have considerable side effects due to the inhibition of physiologically important PGs (such as PGF _2α , PGI ₂ , PGD ₂ ) and due to the PGE ₂ inhibition in gastric mucosa (stomach, kidney, cardiovascular System) to [2]. The biosynthetic pathway of the prostaglandins is shown in FIG.

The inducible microsomal prostaglandin E ₂ synthase-1 (mPGES-1) is a member of the MAPEG family and catalyzes the conversion of PGH ₂ to PGE ₂ [3]. Besides mPGES-1, mPGES-2 and cytosolic (c) PGES are known as PGE ₂ synthases [4]. Interestingly, mPGES-1 is coupled to COX-2 activity and expression of both enzymes is inducible by inflammation-relevant stimuli (interleukin-1β, tumor necrosis factorα). In contrast, COX-1 provides PGH ₂ as a substrate for cPGES and both enzymes are constitutively expressed. In contrast to the physiologically necessary PGs, PGE ₂ , which is locally synthesized by COX-2 / mPGES-1, has pronounced pathophysiological properties (inflammation, pain, fever, cancers, angiogenesis). In contrast, PGE ₂ , which is protective for gastric mucosa, is produced by COX-1 / cPGES directly in the stomach.

Since the discovery of mPGES-1 in 1999, efforts have been made to develop potent and selective inhibitors of mPGES-1 in order to contribute to PGE ₂ synthesis to selectively inhibit inflammatory processes without suppressing the formation of the physiologically important PGs and the gastric protective PGE ₂ . Furthermore, in contrast to selective COX-2 inhibitors (so-called coxibs such as rofecoxib or celecoxib), the suppression of the vasodilatory PGI ₂ could be avoided by selective pharmacological attack on the mPGES-1. This makes the mPGES-1 a highly interesting drug target, especially in inflammatory diseases (eg rheumatoid arthritis), which are associated with pain or fever, but also in various cancers. However, so far no inhibitor of mPGES-1 is approved as a drug for therapy. The number of available inhibitors (such as MK-886) is still extremely low, which is currently still at the beginning of the clinical trial. The motivation of pharmaceutical research to find safe and selective inhibitors of mPGES-1 is enormous.

The object of the present invention is therefore to circumvent the disadvantages of the known processes (use of inhibitors of COX enzymes), and to identify active substances, in particular natural substances, and pharmaceutical preparations which are able to selectively inhibit mPGES-1 , These agents are to be provided for the therapeutic treatment of PGE ₂ -mediated diseases, especially rheumatoid arthritis, to have low side effects at a high efficiency.

This object is achieved by the use of Acylphloroglucinolen, in particular hyperforin and myrtucommulone and their structural derivatives, as described in claim 1. Preferred embodiments as well as the method for the therapeutic treatment are mentioned in the dependent claims 2 to 9.

Hyperforin is a naturally occurring, prenylated acylphloroglucinol from St. John's Wort (Hypericum perforatum L.) [5]. In common St. John's wort extracts commercially available in pharmacies or drug stores, hyperforin is up to 4%. St. John's wort extracts are mainly used to treat mild to moderate depression [6]. Hyperforin is regarded as an efficacy-determining ingredient and led in numerous cellular and animal studies to increase the concentration of various neurotransmitters in the central nervous system [7]. The exact mechanism of action is unknown. In addition, hyperforin has been reported to have apoptotic properties that appear to be particularly pronounced against cancer cells [8]. Hyperforin has also been used to treat atopic dermatitis [9]. As molecular targets for hyperforin the pregnane X receptor, 5-lipoxygenase and cyclooxygenase-1 have been described [10]. In the literature, studies of effects of hyperforin on the biosynthesis of PGE ₂ are unknown. Myrtucommulone is a non-prenylated acylphoroglucinol derivative that has so far only been detected in leaves (0.12% in dried leaves) of myrtle (Myrtus communis L.). Myrtle has been used for millennia as a spice and for the production of liqueurs. For myrtle or their characteristic ingredients only a few pharmacological effects have been detected so far. These include anti-hyperglycemic [11-13], antibacterial [14, 15] and analgesic effects [16]. In addition, weak antioxidant effects were observed [17-19]. It has also recently been shown that myrtucommulone suppresses proinflammatory properties of leukocytes [20]. Thus, myrtucommulone inhibits COX-1 moderately (IC ₅₀ = 17 μM), while COX-2 activity is not inhibited. Effects on the biosynthesis of PGE ₂ by preparations of myrtle or its characteristic ingredients (in particular myrtucommulone) are unknown. Likewise, toxic or undesirable effects on humans by application of myrtle preparations have not or hardly been observed. The structural formulas of acylphloroglucinols myrtucommulone and hyperforin as well as two other substances semi-myrtucommulone and isobutyrophenone nucleus are shown in FIG.

In the present invention, it has been possible for the first time to demonstrate that Hyperforin from St. John's wort, myrtle myrtle natural myrtucommulone and fully synthetic myrtucommulone inhibit the catalytic activity of human mPGES-1 (conversion of PGH ₂ to PGE ₂ ) (FIGS. 3 and 4). , Thus, acylphloroglucinols, in particular hyperforin and myrtucommulone and their derivatives are to be regarded as direct inhibitors of mPGES-1 or inhibitors of PGE ₂ synthesis. To date, neither hyperforin nor myrtucommulone has been described as inhibitors of PGE ₂ synthesis.

In one embodiment of the invention, extracts from all myrtle plants or parts thereof are used, in particular fresh or dried leaves of the myrtle which contain the highest concentration of the substance myrtucommulone being suitable. In addition, extracts from the flowers of St. John's wort are used. Particularly preferred are each lipophilic extracts, e.g. based on chloroform, acetone or the like, because they contain substantially more myrtucommulone or hyperforin compared to non-lipophilic extracts (e.g., to extracts in water or ethanol).

In a further embodiment of the invention, natural hyperforin from the flowers of St. John's wort, natural myrtucommulone from the leaves of myrtle, as well as fully synthetic myrtucommulone is used. In a preferred embodiment of the invention fully synthetic myrtucommulone is used. This can, for example, according to a method of Jauch et al. (not published). In another embodiment, structural derivatives of myrtucommulone or hyperforin are used which have a comparable or better inhibitory effect on mPGES activity.

The invention further comprises a pharmaceutical composition containing at least one active ingredient of St. John's Wort, myrtle or / and one of its derivatives and optionally a pharmaceutical carrier material.

The invention further comprises a pharmaceutical composition containing at least one other acylphloroglucinol and optionally a pharmaceutical carrier material.

For the therapeutic treatment of PGE ₂ -mediated diseases, plasma concentrations of about 3 μM myrtucommulone or hyperforin are desirable, which could be about the po dose of about 500-1000 mg / day.

The administration of acylphloroglucinols such as hyperforin or myrtucommulone and their derivatives or pharmaceutical compositions containing them for the treatment of diseases can be administered orally or parenterally.

The advantage of the present invention is that for the drug target mPGES-1 with acylphloroglucinols basic structures have been identified which lead to the inhibition of the activity of mPGES-1. Thus, the synthesis of PGE ₂ could be selectively inhibited by COX-2 / mPGES-1, without inhibiting the synthesis of other (physiologically important) PGs, as hitherto by inhibitors of COX-1 and -2. As a result, the therapy of PGE ₂ -mediated diseases by means of acylphloroglucinols should have fewer side effects compared to COX-1/2 inhibitors. Furthermore, by using acylphloroglucinols, the use or the dose of non-steroidal

Anti-inflammatory drugs (COX inhibitors) are reduced and shortened the duration of use, which leads to significant side effects because of their unspecific blockade of the synthesis of all PGs.

The invention can be used to treat all forms of diseases associated with increased production of PGE ₂ . These are primarily inflammatory diseases (especially rheumatoid arthritis), feverish and painful conditions, as well as cancers in which PGE ₂ plays a role. Further advantages, features and possible applications of the invention are described below with reference to the embodiments with reference to the drawings. The drawings show:

FIG. 1: Biosynthetic pathway of PGE ₂ ;

Figure 2: Structure of acylphloroglucinols myrtucommulone (MC), semi-myrtucommulone (S-MC), isobutyrophenone nucleus (IBP-C) and hyperforin (Hyp);

FIG. 3: Inhibition of the mPGES-1-mediated synthesis of PGE ₂ (percent activity versus control with DMSO as solvent) in the microsomal fraction of interleukin-1β-stimulated A549 cells (mean + standard error, n = 4). 1 = natural myrtucommulone, 2 = synthetic myrtucommulone, 3 = semi-myrtucommulone, 4 = isobutyrophenone nucleus;

FIG. 4: Inhibition of the mPGES-1-mediated synthesis of PGE ₂ (percent activity versus control with DMSO as solvent) in the microsomal fraction of interleukin-1β-stimulated A549 cells (mean + standard error, n = 4), Hyp = natural hyperforin.

embodiments

Influence of Acylphloroqlucinolen on the Activity of mPGES-1

A549 cells were incubated with interleukin-1β (1 ng / ml) for 72 hours. After harvesting and cell count determination, the pelleted cells were flash-frozen on dry ice / ethanol, thawed again by adding 1 ml of homogenization buffer (4 ^° C.) and homogenized by means of ultrasound. After centrifugation (10,000 g for 10 min at 4 ⁰ C) of the resulting supernatant at 174.000g and 4 ⁰ C was for 1 h hour centrifuged to obtain microsomes. The pellet (microsomes) was dissolved in Homogensierungspuffer and preincubated with the test substances (hyperforin, myrtucommulone and derivatives or DMSO) for 10 min at 4 ° C in 96-well plates. Then PGH ₂ was added as a substrate and the reaction after 1 min at 4 ⁰ C by means of stop solution (containing, inter alia, Fe ²⁺ , citric acid and 11-ß-PGE ₂ as standard) ended. An approach is added before the start of the reaction with the stop solution to determine the content of PGE ₂ in the PGH ₂ solution. After solid phase extraction (RP-18 columns and acetonitrile as eluent), the sample was analyzed by HPLC (RP-18, UV detection at 190 nm).

It has been shown that preincubation of the microsomal fraction of interleukin-1β-stimulated A549 cells results in a potent inhibition of mPGES-1 activity, By 3 μM myrtucommulone inhibits the PGE ₂ synthesis from PGH ₂ to about 74%, the IC 50 value is about 1 uM (Figure 3). Hyperforin inhibits the PGE ₂ synthesis from PGH ₂ at a concentration of 10 μM to 77%, the IC 50 value is about 2.8 μM (Figure 4).

literature

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Claims

claims 1) Use of preparations containing at least one Acylphloroglucinol and / or at least one Acylphloroglucinol derivative for inhibiting the PGE ₂ - synthesis, in particular for the inhibition of mPGES-1. 2) Use according to claim 1, characterized in that the Acylphloroglucinol is naturally occurring or artificially synthesized myrtucommulone or / and one of its derivatives. 3) Use according to claim 1, characterized in that the Acylphloroglucinol is naturally occurring or artificially synthesized hyperforin and / or one of its derivatives. 4) Use according to one of the preceding claims, characterized in that the Acylphloroglucinol by extraction, in particular by a lipophilic extraction, from myrtle (Myrtus communis) or / and St. John's Wort {Hypericum perforatum) are obtained. 5) Use according to one of the preceding claims, characterized in that the preparations containing Acylphloroglucinole further contains a pharmaceutical carrier material. 6) Use according to one of the preceding claims for the manufacture of a medicament for the treatment of PGE ₂ -mediated diseases. 7) Use according to claim 6, characterized in that the PGE ₂ - associated diseases are inflammatory diseases, in particular rheumatoid arthritis, feverish and painful conditions and / or cancers. 8) Use according to one of the preceding claims, characterized in that an effective amount of at least one Acylphloroglucinol and / or at least one Acylphloroglucinol derivative is applied. 9) Use according to claim 8, characterized in that the plasma concentrations of acylphloroglucinol or / and its derivatives after application is about 0.1 - 10 uM.