CA2323887A1

CA2323887A1 - Use of sphingosin-1-phosphate, sphingosin-1-phosphate derivatives and/or mixtures thereof for the treatment of inflammatory diseases of the skin

Info

Publication number: CA2323887A1
Application number: CA002323887A
Authority: CA
Inventors: Monika Schafer-Korting; Hans-Christian Korting; Burkhard Kleuser; Rolf Gerber
Original assignee: Individual
Current assignee: Dermapharm AG
Priority date: 1998-03-13
Filing date: 1999-03-11
Publication date: 1999-09-23
Also published as: ES2165233T3; EP1064002B1; WO1999047145A1; DE59900286D1; EP1064002A1; DE19810999A1; JP2002506825A; ATE206052T1; EP1064002B9

Abstract

The invention relates to a new use of sphingosin-1-phosphate, sphingosin-1-phosphate derivatives and/or mixtures of the above substances for the treatment of inflammatory diseases of the skin, i.e. especially psoriasis vulgaris, acne vulgaris or skin diseases comprising chronic light-damage of the skin, also known as dermatoheliosis.

Description

Use of sphingosine 1-phosphate, sphingosine 1-phosphate derivatives andlor their mixtures for the treatment of inflammatory dermatoses Description The invention relates to a new use of sphingosine 1-phosphate, sphingosine 1-phos-phate derivatives and/or mixtures of the aforementioned substances for the prepar-ation of a drug for the treatment of inflammatory dermatoses, viz., in particular of psoriasis vulgaris, acne vulgaris or of dermatoses implying a chronic phototoxis of the skin and which according to Kligman are also designated as dermatoheliosis.
The invention therefore concerns drugs for the treatment of the inflammatory derma-toses mentioned above which contain sphingosine 1-phosphate or sphingosine 1-phosphate derivatives and/or mixtures of the aforementioned substances as the active substance, as well as the usual supports, adjuvants, and diluents. It concerns drugs with novel supports such as liposomes and the so-called solid lipid nano-particles for the active substances. The invention preferably concerns remedies applicable topically for the treatment of psoriasis vulgaris, acne vulgaris or dermatoheliosis.
The glucocorticoids that have been administered for a long time in the external therapy of dermatoses, are as much as ever the most important antiinflammatory active substances. They are most widely used in the local therapy. Beyond that these substances have an antiphlogistic and antipruritic as well as an antiproliferative effect.
The former effects are the essential aspects of the major indication of the glucocorti-colds, that is, acute inflammatory reactions of the skin. The latter effect is important primarily in psoriasis.
Glucocorticoids have an inhibitory influence on the proliferation of keratinocytes and fibroblasts. This may be a desirable effect in the instance of inflammatory dermatoses having a hyperproliferative component. Thus, glucocorticoids are used as well against psoriasis. The effect exerted on the fibroblasts may lead to skin hypotrophy or derma-trophy, that is, to a very essential undesired local action.
Thus, psoriasis vulgaris is an important target affection. It is the most frequent dermatosis of fair-skinned people featuring polygenic heredity and multifunctional triggering. The essential characteristic of psoriasis vulgaris is a hastened epiderm formation (hyperproliferation), viz., a keratinocyte migration from the basal layerto the corneum within about four days (normally this takes 28 days). In addition a distinctly higher resorption and at the same time a more rapid excretion of foreign substances in urin was found in persons with psoriatic skin, and explained in terms of destruction of the barrier and reservoir function of the upper skin layers.
Acne vulgaris is a dermatosis occurring in puberty, rarely later but sometimes persist-ing up to the 30'" year of life, which through hyperplasia of the sebaceous glands and a disturbance of keratinization of the follicles leads to their blocking and to the for-mation of comedomes, that is, to the efflorescences typical of acne vulgaris.
A
disturbance of cell differentiation is a further peculiarity of acne vulgaris.
Ordinarily, the acne is treated keratolytically with benzoyl peroxide or tretinoin, or antimicrobially with erythromycin, clindamycin or azelaic acid. There are systemic possibilities to work antimicrobially with tetracyclins or antiseborrheically with antiandrogens or estrogens, or apply the systemic retinoid, isotretinoin.
More and more frequently a dermatosis is observed that must be regarded as chronic phototoxis of the skin, and which according to Kligman is designated as dermato-heliosis. An important affection among these forms is the actinic or solar keratosis, which is a preliminary stage of skin prickle cell carcinoma. Here the epiderm is affected by a serious perturbation of differentiation.
Sphingolipids cover a large class of biologically significant compounds. Such com-pounds are cell membrane components. An essential compound of the sphingolipids is sphingosine.

When present in cells, the sphingolipids are metabolized producing sphingosine as a metabolic intermediate. Sphingosine 1-phosphate is known as the product of sphin-gosine kinase (see, e.g., W. Stoffel, Hoppe-Seyler's Z. Physiol. Chem., 354, (1973)).
Sphingolipid degradation products such as ceramide, sphingosine and sphingosine 1-phosphate are discussed in the literature as new, so-called "second messengers".
Sphingosine and other degradation products of the sphingosine metabolism regulate, either apoptosis or mitogenetic effects, depending on the cell type and nature of the stimulus.
Apoptosis is an essential physiological process contributing to the regulation of cell numbers. In this connection, it is described in WO 96/18404 that sphingosine and N-methyl derivatives of sphingosine induce apoptosis in particular cell types.
The effect of sphingosine as an inhibitor of protein kinase C is described in this connection in WO 88/01869. An inhibition of protein kinase C leads to an inhibition of proliferation and to the induction of programmed cell death in a multitude of cells. This cell death occurring, for instance, at higher sphingosine concentrations with the keratinocytes is caused by a cytotoxic effect subsequent to inhibition of the protein kinase C.
In contrast to the effect described above, sphingosine acts so as to promote prolifer-ation in a number of cases, as for instance in fibroplasts. This proliferative effect can be induced stereospecifically, only by the D(+)-erythro enantiomer.
The inhibition of protein kinase C, on the other hand, is nonspecific and can be demonstrated for all enantiomers.
According to further discussions, sphingosine 1-phosphate promotes proliferation in Swiss 3T3 fibroblasts. A number of intracellular processes are influenced by sphin-gosine 1-phosphate. Thus, sphingosine 1-phosphate raises the intracellular CaZ+ ion concentration from intracellular storage sites in a way that is independent of IP3, and it lowers the intracellular concentration of cAMP. The increase in Ca2+
content is based on an intracellular action, but recently membrane-based receptors of sphingosine 1-phosphate have been detected as well. The inhibitor action exerted on the cAMP
content is based on a receptor effect.
From WO 95/21848, different synthetic procedures for sphingosine 1-phosphate and different derivatives of sphingosine 1-phosphate are known.
WO 93/19760 describes the effects of sphingosine 1-phosphate and its derivatives on cell mobility. The use of sphingosine 1-phosphate as a antitumor agent is also de-scribed in WO 93119760.
US-A-5 712 262 describes the therapeutic application of sphingosine 1-phosphate for the retardation of apoptosis in neurodegenerative affections and during the aging process.
US-A-5 391 800, US-A-5 663 404 and US-A-5 260 228 describe the application of sphingosine 1-phosphate (SPN-1-P) in inflammatory events, since SPN-1-P
antagon-izes the phenomenon of cell mobility that is essential for an inflammation.
US-A-5 627 171 describes the antiproliferative and antitumoral effect of sphingosine 1-phosphate and its derivatives.
Thus, it is the aim of the invention to furnish new active substances for the treatment of inflammatory dermatoses, and particularly of psoriasis vulgaris, acne vulgaris or dermatoheliosis. Further, a new drug containing the aforementioned new active sub-stances is to be provided. The drug is supposed to be applicable in particular topically, and a drug support free of surfactants is to be provided that is able to form a particle dispersion in an aqueous medium favouring absorption of the active substance into the skin.

The above-named aim is attained bythe novel use ofsphingosine 1-phosphate, sphin-gosine 1-phosphate derivatives of the general formula I, OH
O
CHI
R,O-P-O
E
ORz NHZ
where R, is an alkyl residue with 1 to 6 C atoms and RZ is hydrogen or ~o:
Advantageous embodiments of the invention are contained in the dependent claims.
It was surprisingly found by the inventors, in fact, that sphingosine 1-phosphate as well as derivatives of these substances inhibit the proliferation of keratinocytes but promote their differentiation, and they do so without any inhibiting effect on fibroblast .
proliferation.
It has been shown according to the invention that sphingosine 1-phosphate in parti-cular influences the growth behaviour and differentiation of keratinocytes and fibro-blasts, which are the most important skin cells. It could be shown for the first time that sphingosine 1-phosphate stimulates the proliferation of primary fibroblasts while the proliferation of keratinocytes strongly decreases. So far an inhibitory effect of sphingosine 1-phosphate on proliferation has not been shown for any other cell type.
Various sphingosine 1-phosphates of the above formula I have given similar results.
The results show unambiguously that the effect is specific to sphingosine 1-phos-phate, while other sphingosine derivatives display it in a somewhat weaker form. It is particularly important that the antiproliferative effect of sphingosine 1-phosphate is not based on a cytotoxic action such as present in antitumor agents, and that the differentiation profile ofthe cells is influenced. Thus, sphingosine 1-phosphate induces the differentiation of keratinocytes. It is for this reason that the use of sphingosine 1-phosphate, sphingosine 1-phosphate derivatives of the above formula I for the treatment of inflammatory, hyperproliferative dermatoses is not obvious.
Thus, the results according to the invention demonstrate that the action of sphingosine 1-phosphate is not based on an inhibition of protein kinase C. Sphingosine 1-phos-phate rather produces an antiproliferative effect not associated with an enhanced cyto-toxicity.
The differentiation of keratinocytes is also promoted by sphingosine 1-phosphate, a result that is related to the stimulation of protein kinase C and contrasts with an inhibition of protein kinase C.
Thus, it can be demonstrated according to the invention that sphingosine 1-phosphate is highly suitable for the treatment of hyperproliferative dermatoses.
It must further be pointed out that an activation of protein kinase C also leads to a protective effect with respect to apoptosis. Activators of protein kinase C
inhibit the programmed cell death. It could here be shown according to the invention that this protective effect is based at least in part on a stimulation of sphingosine kinase by protein kinase C. Sphingosine kinase is the decisive enzyme for the formation of sphingosine 1-phosphate. By analogy, sphingosine 1-phosphate also exerts a protect-ive effect in apoptosis.
Thus, in keratinocytes protein kinase C is a central enzyme participating in the induction of differentiation. A stimulation of the enzyme by phorbol ester is associated with influences promoting the differentiation. True, an antiproliferative effect in keratinocytes is attained by an inhibition of protein kinase C. But this effect is associated with cell death of the keratinocytes.
In contrast, the sphingosine 1-phosphate applied according to the invention has no _7_ toxic action in keratinocytes. In addition, an activation of protein kinase C
also induces a stimulation of sphingosine kinase and leads to enhanced levels of sphingosine 1-phosphate. Thus, the differentiation-promoting and antiproliferative effect of sphingosine 1-phosphate never is based on an inhibition of protein kinase C in the manner described for instance in WO 88/018169 for sphingosine.
Calcitriol (an active metabolite of vitamin D3) and calcipotriol have been shown to be effective therapeutics in the treatment of psoriasis vulgaris. However, their application is limited because of hypercalcemic side effects. These compounds produce an activation of protein kinase C in keratinocytes, so that an induction of differentiation is attained in this way.
It could now be demonstrated according to the invention that calcitriol and also calcipotriol exert a strongly activating influence on sphingosine kinase. The formation of sphingosine 1-phosphate leads to an antiproliferative effect and also to a promotion of differentiation in keratinocytes.
Therefore, according to the invention, calcitriol or calcipotriol can be added in the usual concentrations to the drug in a particular embodiment for the treatment of dermatoses, particularly of psoriasis vulgaris, acne vulgaris or dermatoheliosis.
Thus, the invention also relates to a drug for the treatment of inflammatory derma-toses, particularly of psoriasis vulgaris, acne vulgaris or dermatoheliosis.
The drug according to the invention contains one or several active substances selected from sphingosine 1-phosphate, sphingosine 1-phosphate derivatives of the above formula I and/or mixtures of the aforementioned substances and/or pharmaceutically acceptable salts of the aformentioned substances and a pharmaceutically acceptable support, diluent or excipient.
Possible supports are phospholipids such as soy and/or egg lecithins, natural and/or semisynthetic phospholipids in a concentration of 0.1 to 10 % by weight, preferably _$_ 1 to 5 % by weight. Further possibilities are glycosphingolipids in a concentration of 0.01 to 0.5 %, preferably 0.05 to 0.2 %. Further lipids that can be used in the drug support are phosphatidylcholine, such as phosphatidylcholine S100 (manufactured by Lipoid KG, Ludwigshafen), or cholesterol. Sphingosine 1-phospate is preferably used in the drug for the treatment of inflammatory dermatoses. The sphingosine 1-phosphate or its derivatives should contain essentially no L-threo isomer.
In a particularly preferred embodiment, the remedy according to the invention is encapsu-lated in liposomes, can be applied topically, and is present in particular as a hydrogel, oleogel, or a lotion. The active substance, and here sphingosine 1-phosphate in par-ticular, is present in the finished preparation in a concentration of 0.002 to 0.05 mg/g.
Particularly preferred is a concentration of 0.005 to 0.01 mg/g of the active substance in the finished preparation.
For the encapsulation of the active substances according to the invention, that is, of sphingosine 1-phosphate or sphingosine 1-phosphate derivatives or their mixtures, processes can be used which as such are well known to those skilled in the art. For instance, these active substances and the substances forming liposomes can be dis-solved in an organic solvent, the solution mixed with an aqueous phase and the solvent, if necessary after homogenizing, removed by distillation. Commonly, the substance or mixture of substances forming the liposomes is used in an amount of 100 to 500 000 times the weight of the active substance, for instance sphingosine 1-phosphate.
For the preparation of liposome suspensions, commonly 0.5 to 10 % by weight of the phospholipid or phospholipid mixture are employed relative to the aqueous phase.
Suitable mixtures may contain up to 60 % by weight of cholesterol and up to 30 % by weight of charge carriers. Alcohols are the preferred solvents.
Antioxidants etc. may be added since the lipids are sensitive to oxidizing action.

_g_ The encapsulation of sphingosine or sphingosine 1-phosphate in liposomes can be performed under the same conditions as described in processes of this kind in the prior art (see, for instance, EP 0 605 497 or DE 44 40 337).
The optimum concentration of the active substance in the finished drug preparations will of course be a function of the kind of active substance and galenic form, and must be determined in any given case by the usual preliminary experiments. It will suffice as a rule to apply pharmaceutical preparations containing 0.002 to 0.05 mg, and preferably 0.005 to 0.01 mg, of sphingosine, sphingosine 1-phosphate per gramme of the preparation.
The following examples and results serve for a more detailed explanation of the invention.
Influence on keratinocyte proliferation Keratinocyte proliferation was examined in the presence of 1 to 10 pM
sphingolipid derivatives. After a four-day treatment with different sphingolipids, incorporation of 1 S [3H]-thymidine into the DNA was measured in the logarithmic phase of keratinocyte growth.
Sphingosine 1-phosphate (SPP) had a strong inhibiting effect on proliferation in KGM
(keratinocyte growth medium), and a distinct concentration dependence was found for this effect. Inhibition ofproliferation already starts at a concentration of 1 ~M, while 10 ~M SPP
reduced the incorporation of thymidine by about 63.1 % (Fig. 1 ). With this effect it is just as potent as the calcipotriol D3 used against psoriasis vulgaris.
Sphingosin (SPH) also had an inhibitory effect on growth, but this effect was less pronounced than that of SPP. The cell vitality of SPP was examined with the MTT test in the presence of 1 to 25 ~M SPP; up to 10 ~M the substances had no toxic effect. Other sphingolipid derivatives such as dimethylsphingosin (DMS) and C2-ceramide (C2C) also inhibited the incorporation of thymidine, but this effect was solely due to the toxicity of the two substances. This was seen from microscopic studies, from the MTT test, as well as from flow cytometric studies in apoptosis and necrosis.

The tests were also performed with cultivated keratinocytes in a "keratinocyte basal medium" (KBM, without supplements). Here it could be established that SPP and SPH
have no mitogenetic effects on keratinocytes (Fig. 2). Both DMS and C2C proved to be toxic in this medium, too.
1. Studies concerning fibroblast proliferation The growth behaviour of human fibroblasts was examined in the presence of the test substances using nonstimulated cells cultivated in serum-free medium (Dulbecco-modified Earle medium (DMEM) without fetal calf serum (FCS)). The proliferation was strongly stimulated by SPH and particularly by SPP as a function of concentration (Fig. 3). The highest activity of SPP was measured at a concentration of 10 ~M
(approximately by a factor of 6.5), but even 1 g,M caused a perceptible increase in proliferation. The proliferative effect on fibroplasts thus is produced at the same concentrations as the antiproliferative effect on keratinocytes. Cell division of fibro-blasts was promoted as well by SPH, but again the effect was less strong than that of SPP. Toxic effects were induced already at a concentration of 10 ~M SPH.
DMS
and C2C had a very slight promoting effect on proliferation at low concentrations, while concentrations of 5 ~M and more exhibited pronounced toxicity.
Similar experiments were performed with cells cultivated in media containing serum while these cells were in their growth phase. As expected because of a superposition of the effects of serum components, the pro-proliferative effects were much less pronounced, but an antiproliferative effect could not be ascertained (Fig. 4).
2. Influence on keratinocyte differentiation When it had been shown that SPP inhibits the proliferation of keratinocytes without any associated, enhanced toxicity, the degree of differentiation of the cells after their exposure to the sphingolipid derivatives was then examined. It was seen when determining transglutaminase activity that only SPP and SPH promoted the diffe--rentiation of the keratinocytes, i.e., precisely those lipids which have an inhibiting effect of proliferation. For comparison the active metabolite of vitamin D3 which also promotes a differentiation was adduced. Thus, the increase in differentiation of keratinocytes produced by SPP and SPH is certainly comparable with that produced by calcitriol D3 (Fig. 5).
Example 1 Preparation of liposome suspensions containing sphingosine 1-phosphate In the following, the preparation of liposome suspensions by the film process and subsequent high-pressure homogenization is described.

7.5 g phosphatidylcholine S100 (manufacturer: Lipoid KG, Ludwigshafen), 0.75g cholesterol and 15 mg sphingosine 1-phosphate (D-erythro form) are dissolved in 5 ml ethanol (96 %). The solution is then gently evaporated to dryness with a rotary vaporizer, which leads to formation of a lipid film on the walls of the round-bottom flask. The lipid film is dissolved away with 141.7 g distilled water. The liposome suspension (MLV) obtained is then homogenized at 950 bar with a high-pressure homogenizer, and filtered through a 0.2 ~m filter. The resulting liposome suspension contains liposomes having a mean size of 35 nm. The phosphatidylcholine content is 48 mg/g; the concentration of active substance incorporated is 0.1 mg/g.
Preparation of a liposomal lipogel The liposomes prepared according to the above Example 1 are diluted 1:10 (by weight) with citrate-phosphate buffer of pH 5.5. Ketrol TF~ is sprinkled on and dispersed as a gel former. A slightly opalescent gel is obtained which as yet undergoes preservation. The concentration of the active substance is 10 ~g/g.

Summary The invention concerns a novel use of sphingosine 1-phosphate, sphingosine 1-phos-phate derivatives and/or mixtures of the aforementioned substances for the treatment of inflammatory dermatoses, viz., in particular of psoriasis vulgaris, acne vulgaris or of dermatoses implying a chronic phototoxis of the skin which according to Kligman are also designated as dermatoheliosis.

Claims

1. Use of sphingosine 1-phosphate and sphingosine 1-phosphate derivatives of the following formula I, where R1 is an alkyl residue with 1 to 6 C atoms and R2 is hydrogen or , and/or of pharmaceutically acceptable salts of the aforementioned substances and/or mixtures of the aforementioned substances for the preparation of a drug for the treatment of inflammatory dermatoses.

2. Use according to claim 1, characterized in that the inflammatory dermatoses are psoriasis vulgaris, acne vulgaris or dermatoheliosis.

3. Use according to claim 1 or claim 2, characterized in that the sphingosine 1-phosphate or the sphingosine 1-phosphate derivatives contain essentially no L-threo isomer.

4. Use according to one of the preceding claims 1 through 3, characterized in that the drug contains a pharmaceutically acceptable support, diluent or excipient.

5. Use according to claim 4, characterized in that the drug contains sphingosine as a diluent.

6. Use according to claim 4 or claim 5, characterized in that the drug contains sphingosine 1-phosphate and sphingosine 1-phosphate derivatives and/or their mixtures encapsulated in liposomes.

7. Use according to claims 4, 5 or 6, characterized in that the drug can be applied topically, and is present in the form of a hydrogel, oleogel, or lotion.

8. Use according to one of the preceding claims 1 to 7, characterized in that the drug contains a concentration of the active substance, particularly of sphingosine 1-phosphate, of 0.002 to 0.05 mg/g and particularly of 0.005 to 0.01 mg/g in the finished preparation.