A new therapeutic approach to tre

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					C L I N I C A L A N D LA B O R A T O R Y I N V E S T I G A T I O N S

DOI 10.1111/j.1365-2133.2005.06811.x

A new therapeutic approach to treat psoriasis by inhibition of fatty acid oxidation by Etomoxir
´ F. Caspary, G. Elliott,* B.T. Nave, P. Verzaal,  M. Rohrbach, P.K. Das,à L. Nagelkerken  and J.D. Nieland
Medigene AG, Lochhamerstrasse 11, 82152 Martinsried, Germany *Derphartox, PO Box 609, 2600 AP Delft, the Netherlands  TNO Pharma, PO Box 2215, 2301 CE Leiden, the Netherlands àDepartment of Pathology, UvA-AMC, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands

Summary
Correspondence
J.D. Nieland. E-mail: jdnieland@web.de

Accepted for publication
30 March 2005

Key words:
autoimmune disease, BNX transplanted skin model, carnitine palmitoyltransferase-1, Etomoxir, fat metabolism, psoriasis

Conflicts of interest:
F.C., B.T.N., M.R. and J.D.N. developed for Medigene this tested medication to be used in psoriasis.

Background The dogma in psoriasis is that due to pathogen-induced inflammatory responses, an autoreactive immune response is induced that leads to tissue destruction. However, this model might be too simplistic. Literature data suggest that the expression of enzymes crucial for fatty acid oxidation is upregulated in the skin of patients with psoriasis compared with healthy individuals. Objectives To examine the influence of fatty acid oxidation on psoriasis with regard to expression and activity of the key enzyme in fatty acid oxidation, carnitine palmitoyltransferase-1 (CPT-1) and the effect of the CPT-1 inhibitor, Etomoxir. Methods Experiments were performed with homogenates of lesional and healthy skin, fibroblast cultures and a model of human psoriatic skin transplanted on immune-deficient BNX mice. Results CPT-1 was highly active in lesional skin. Etomoxir was able to block CPT1 activity in skin, implying that this antagonist may have the potential to suppress psoriasis when administered topically. In the mouse model, Etomoxir had an antipsoriatic effect that was at least as good as that of betamethasone, as evidenced by reduction of epidermal thickness, keratinocyte proliferation and differentiation. Conclusions We conclude that fatty acid metabolism and in particular CPT-1 may be an excellent target for treatment of psoriasis.
acid oxidation may be required. Accordingly, fatty acids have the potential to increase the growth rate of fibroblasts in vitro.10 Evidence for the shift to fatty acid oxidation in psoriatic lesions comes from reports showing that markers such as epidermal fatty acid binding protein (E-FABP), also known as psoriasis-associated fatty acid binding protein, are strongly increased in psoriatic lesions.11,12 Therefore, inhibition of fatty acid oxidation in the skin might result in the blockade of an important energy source required for cell proliferation and lead to a deceleration of cell growth. In addition, such an approach might ameliorate the flaky and dry phenotype of the psoriatic skin because normalized levels of fatty acids may restore barrier formation. A potential target to inhibit fatty acid oxidation would be carnitine palmitoyltransferase-1 (CPT-1), because it is the rate-limiting enzyme in the transport of long-chain fatty acids into the mitochondria, where fatty acid oxidation takes place. In vitro, CPT-1 is specifically and irreversibly inhibited by Etomoxir,13 which therefore might be a novel drug for the treatment of psoriasis.
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Skin cells have several functions. One of their most important functions is to protect the host from invasion by pathogens. This is achieved by three main mechanisms: (i) barrier formation by dead keratinocytes; (ii) immune defence by immune cells, keratinocytes and skin fibroblasts responding to tissue damage and infection by producing proinflammatory cytokines, chemokines and prostaglandins; and (iii) production and secretion of fatty acids to the skin surface to form a protective barrier against pathogens and water loss.1 The current understanding of the pathogenesis of psoriasis is that a bacterial or viral infection (e.g. Staphylococcus aureus, Coxsackie B virus, Streptococcus sp., varicella-zoster virus2–6) takes place by accident or by wounding. Consequently, an inflammatory response is initiated which in turn induces enhanced proliferation of keratinocytes. Especially interleukin (IL)-1, IL-6 and prostaglandin-2 are key mediators in this respect.7–9 Energy is needed for this excessive cell growth. Normally the energy source of the skin cells is glucose. However, to meet the increased energy demand, a switch to fatty

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938 Etomoxir treatment of psoriasis, F. Caspary et al.

Currently, therapies for treatment of psoriasis are mainly focused on the suppression of the immune response. The most commonly used drugs in this field are immunosuppressive corticosteroids. Also, novel therapies such as blockers of tumour necrosis factor, intercellular adhesion molecule-1 and very late activation antigen mediate downregulation of the immune response.14 Treatments that inhibit the immune response seem not to affect the skin metabolism. After antiinflammatory therapy the skin looks normal but the lesions usually reappear within a few weeks. Apparently, continuous suppression of the immune system or elimination of an as yet unidentified trigger is required to inhibit disease activity. The purpose of this study was to demonstrate that CPT-1 is active in psoriatic skin and that blockade of CPT-1 activity in the skin by Etomoxir inhibits the development of psoriasis in a model of human psoriatic skin transplanted on immunedeficient BNX (beige ⁄nude ⁄xid) mice. These data assume that excessive fatty acid oxidation may generate a trigger signal for immune over-reaction in the skin and that fatty acid oxidation thus represents a novel therapeutic target.

resuspended in 200 lL buffer A. The final total skin extracts were kept on ice for further analysis. Concentrations of total protein in the samples were determined with the Bradford assay (Bio-Rad, Munich, Germany) as recommended by the manufacturer. Determination of carnitine palmitoyltransferase-1 activity in vitro Skin and cell culture samples with fixed amounts of total protein were made up to 100 lL with buffer A and were preincubated for exactly 5 min at 30 °C (background was buffer A only). Then 100 lmol L)1 of reaction mix [220 mmol L)1 sucrose, 50 mmol L)1 KCl, 10 mmol L)1 Tris–HCl pH 7Æ4, 4 mmol L)1 EGTA pH 7Æ4, 1% fatty acid-free bovine serum albumin (BSA), 1 mmol L)1 dithiothreitol, 0Æ2 mmol L)1 palmitoyl-coenzyme A (CoA), 1Æ2 mmol L)1 L-carnitine and 0Æ6 lL L-[methyl-3H]carnitine hydrochloride (Amersham, Freiburg, Germany)] were added and samples were again incubated for exactly 5 min at 30 °C. Reactions were stopped by adding 800 lL of 6% perchloric acid. Suspensions were centrifuged and pellets were washed with 6% perchloric acid before they were dissolved in 600 lL of double-distilled water. Butanol extraction was performed with 400 lL of water-saturated butanol, 150 lL of 6% perchloric acid and 150 lL of saturated ammonium sulphate. One hundred microlitres of the upper butanol phase were mixed with 1 mL Microscint and c.p.m. values were measured with a scintillation counter from Canberra Packard (Schwadorf, Austria) using the settings recommended by the manufacturer for tritium measurements. The potassium salt of Etomoxir was added to the samples. For preactivation of Etomoxir, 2 lL of 100 · stocks (0, 0Æ1, 1Æ0 and 10 mmol L)1 dissolved in dimethylsulphoxide) were incubated at 30 °C for 30 min with 48 lL of activation mix (200 mmol L)1 Tris–HCl pH 7Æ4, 100 mmol L)1 KCl, 12 mmol L)1 adenosine triphosphate, 0Æ1 mmol L)1 CoA, 0Æ6% fatty acid-free BSA, 13 mmol L)1 MgCl2 and 0Æ7 mmol L)1 glutathione) and 50 lL of skin samples containing a fixed amount of total protein. Then 100 lL of reaction mix were added to start the CPT-1 reaction and samples were incubated for 5 min at 30 °C. Samples were then treated as described above. Transplantation of human skin The method used is a modification of that reported by Elliott et al.15 Basically, BNX mice (Harlan, Zeist, the Netherlands) were anaesthetized and a small area (± 5 mm diameter) of the epidermis and dermis was removed down to the pannus carnosum. The nonlesional psoriatic skin of nine donors was placed on to the transplant bed of 27 mice and was held in place using Op-SiteÒ (Smith & Nephew, Hull, U.K.). The transplant was then protected by gauze held in place with 3MÒ surgical foam tape (3M, Leiden, the Netherlands). After 3 weeks the 3M tape was removed.15,16

Materials and methods
Cell culture Primary human skin fibroblasts (normal human dermal fibroblasts from PromoCell, Heidelberg, Germany) were cultivated in Dulbecco’s modified Eagle’s medium (Gibco, Karlsruhe, Germany) with 10% fetal calf serum (Gibco). Cells were seeded in T25 flasks (1Æ7 · 105 cells per flask) and incubated with 0, 0Æ1, 1Æ0 and 10 lmol L)1 of Etomoxir ethylester for 3 days (approximately 72 h) at 37 °C and 5% CO2. For CPT-1 analysis cells were harvested in sucrose buffer [250 mmol L)1 sucrose, 10 mmol L)1 Tris–HCl pH 7Æ4, 0Æ1 mmol L)1 EGTA pH 7Æ4, 1 mmol L)1 phenylmethylsulphonyl fluoride (PMSF) and 5 lg mL)1 leupeptin] and pellets were resuspended in buffer A (150 mmol L)1 KCl, 10 mmol L)1 Tris–HCl pH 7Æ4, 1 mmol L)1 PMSF and 5 lg mL)1 leupeptin). Cells were broken up by repeated freeze–thaw cycles using liquid nitrogen and a 30 °C water bath. Protein concentrations were measured and CPT-1 activity was analysed as described below. Preparation of skin homogenates Fresh skin of hairless SKH1 mice (Charles River Laboratories, Sulzfeld, Germany) was treated for 60 min with 17 mg of a formulation (wool wax, vaseline and cetylstearylalcohol) containing 0%, 1% or 30% Etomoxir. After treatment, formulation which had not penetrated the skin was removed, and 1-cm2 skin samples were further analysed. Untreated skin of a normal shaved mouse was used, and psoriatic and healthy human skin was derived from volunteers. Small pieces of frozen skin (diameter 5–10 mm) were homogenized with a hand mixer (IKA Ultra Turrax T8) and with a Dounce homogenizer in approximately 1 mL cold sucrose buffer. Homogenates were centrifuged at 21 000 g for 20 min at 4 °C. Pellets were

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Etomoxir treatment of psoriasis, F. Caspary et al. 939

Induction of psoriasis lesions in transplanted human skin The method used to induce psoriasis lesions is a modification of that described by Wrone-Smith and Nickoloff.17 The peripheral blood mononuclear cells (PBMC) of the nine donors were isolated using a Ficoll gradient and stored at ) 140 °C until needed. The patients’ PBMC were thawed, cultured and activated for 48 h in Iscove’s modified Dulbecco’s medium (BioWhittaker, Walkersville, MD, U.S.A.; lot no. 2MB0103) with 40 U mL)1 recombinant human IL-2 (Cetus, Emeryville, CA, U.S.A.; 120399) and 1 lg mL)1 Staphylococcus enterotoxin B (Toxin Technology, Sarasota, FL, U.S.A.; lot no. 51497B). After cultivation for 48 h cells were harvested for dermal injection. Before injection of the cells, mice with usable transplants were randomized into four treatments groups each consisting of five mice. The biopsies developed a psoriasis-like lesion which was then topically treated with 30 mg cream once daily for 21 days. One of the four groups was treated with placebo (emulsion of wool wax, vaseline and cetylstearylalcohol), the second with betamethasone (30 mg topically once daily; DiproleneÒ; Schering-Plough BV; batch no. 11173) and the third and fourth groups with 1% and 30% Etomoxir emulsified in wool wax, vaseline and cetylstearylalcohol. Histology Histological staining was performed on cryopreserved tissues. Diagonal cross sections (8 lm) were cut, including all skin layers. Haematoxylin and eosin staining (epidermal thickness) Sections were stained with haematoxylin and eosin and evaluated at a microscopic magnification of · 200. Two sections taken round the middle of the biopsy were evaluated. Thickness measurements were made over the total section using a eyepiece graticule. The mean thickness of the epidermal ridges and of the total epidermis (mean of ridges and inter-ridges) was expressed in micrometres. Ki-67 staining (keratinocyte proliferation) Sections were stained with mouse antihuman Ki-67 monoclonal antibody and evaluated at a microscopic magnification of · 400. Two representative sections were evaluated. The total number of positive cells in the entire section was counted and indicated per unit length. One unit length is defined as a microscopic view at a magnification of · 400. Per section at least four unit lengths were counted and the mean number of Ki-67+ cells per unit length was calculated. Ulex staining (keratinocyte differentiation) Sections were stained with biotinylated Ulex europaeus agglutinin 1 and evaluated at a microscopic magnification of · 200. Two

representative sections were evaluated. The thickness of both the Ulex-positive cell layer (distribution of Ulex) and of the Ulex-negative layer of the total section was measured using an eyepiece graticule. From this the mean ratio of Ulex-positive differentiation was calculated (indicated as percentage of epidermis positive for Ulex). Statistics The program SPSS version 11.5 for Windows (SPSS, Chicago, IL, U.S.A.) was used for the statistical evaluation. In this program the ANOVA method followed by post hoc least significant difference analysis was performed.

Results
Carnitine palmitoyltransferase-1 is expressed and active in skin cells Literature data suggest that fatty acid oxidation is increased in psoriatic lesions because levels of E-FABP are increased.11,12 To support this hypothesis further, CPT-1 expression and activity were analysed. These studies confirmed that in the normal skin of psoriatic donors the CPT-1 mRNA level is about 1Æ6-fold increased compared with the mRNA levels of a healthy donor (data not shown). Furthermore, CPT-1 enzyme activity was approximately 60% higher in lesions compared with normal skin of the same donor (Fig. 1), suggesting that an individual at risk is predisposed to switch to fatty acid oxidation and diseased skin is actually switching to fatty acids as a main source of energy to fuel the excessive cell growth. Both observations lead to the conclusion that rates of fatty acid oxidation might be increased in psoriatic epidermis and that this is possibly due to the increased energy demand of the hyperproliferating cells in psoriasis lesions. Accordingly, inhibition of fatty acid oxidation in the skin might result in the blockade of an important energy source required for cell proliferation and lead to a deceleration of cell growth.

250 CPT1 activity [%] 200 150 100 50 0 psoriasis healthy

Fig 1. Carnitine palmitoyltransferase-1 (CPT-1) activity was higher in lesional skin than in healthy skin of the same psoriatic donor. Lesional and healthy skin biopsies from one patient with psoriasis were analysed for their CPT-1 activity. Results are shown as the mean ± SD of two experiments.

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940 Etomoxir treatment of psoriasis, F. Caspary et al. Table 1 Treatment of different total skin extracts with the salt of Etomoxir prior to measurement of carnitine palmitoyltransferase-1 (CPT-1) activity

CPT-1 activity in percentage of untreated extract Etomoxir concentration (lmol L)1) 0 1 10 100 Healthy mouse skin 100 74Æ3 48Æ2 7Æ6 ± ± ± ± 0Æ7 3Æ1 5Æ2 22Æ6 Healthy human skin 100 95Æ0 64Æ0 32Æ3 ± ± ± ± 7Æ4 1Æ8 6Æ3 7Æ0 Psoriatic human skin 100 50Æ3 30Æ1 11Æ5 ± ± ± ± 7Æ7 1Æ0 2Æ2 1Æ2

Skin samples were derived from a normal shaved mouse, and human healthy and psoriatic skin was derived from a skin cancer patient. CPT-1 activity obtained without Etomoxir (0 lmol L)1 value) was set at 100%. Results are shown as the mean ± SD of two independent experiments.

Etomoxir is a potent and irreversible inhibitor of CPT-1 and could therefore be used to reduce fatty acid oxidation in

A 120
100 CPT1 activity [%] 80 60 40 20 0 0 0.1 Etomoxir [µM] 1 10

psoriatic skin cells. To evaluate whether CPT-1 activity in skin is sensitive to Etomoxir, different skin homogenates were prepared. Homogenates were incubated with the potassium salt of Etomoxir and CPT-1 activity was measured (Table 1). These experiments revealed that CPT-1 activity in mouse skin, healthy human skin and psoriatic human skin is sensitive to Etomoxir in a dose-dependent manner. Thus, this small molecule might be used to study whether inhibition of fatty acid oxidation in psoriatic skin is beneficial for the treatment of psoriasis. Etomoxir is able to enter cells and is converted into its active form, Etomoxir-coenzyme A Etomoxir can inhibit CPT-1 activity only when it is linked to CoA.18 Therefore, the ethylester of Etomoxir (used for the ointment formulation) has to be converted into its active form, Etomoxir-CoA, in order to inhibit CPT-1 activity. Etomoxir-CoA inhibits CPT-1 activity by direct binding to the enzyme, which is located in the outer mitochondrial membrane.13,19,20 This means that Etomoxir has to penetrate the skin and pass through the cell membrane, then transform to Etomoxir-CoA in order to inhibit the activity of CPT-1. To study the efficacy of these steps, Etomoxir was incubated with primary human skin fibroblasts and CPT-1 activity was measured. Viability of these cells was not affected by Etomoxir (data not shown). Figure 2A shows that Etomoxir can inhibit CPT-1 activity in these human skin cells in a concentrationdependent manner. This means that Etomoxir can pass through the cell membrane and is converted into its active form. In order to test whether Etomoxir can also penetrate intact skin, fresh skin of mice was treated for 60 min with a placebo formulation or the same formulation containing 1% or 30% of the ethylester of Etomoxir. The skin samples were then homogenized and CPT-1 activity was measured. As a control, a solution containing 30% Etomoxir ester was added to the placebo-treated homogenate and further tested in the assay. There was no difference between placebo and placebo spiked with the 30% Etomoxir, indicating that the effects seen were not due to the assay procedure (data not shown). Figure 2B shows that after 1 h, CPT-1 activity is substantially

B

120 100

CPT1 activity [%]

80 60 40 20 0 placebo Etomoxir Etomoxir

Fig 2. (A) In vitro inhibition of carnitine palmitoyltransferase-1 (CPT1) activity by the ester of Etomoxir. CPT-1 activity in primary human skin fibroblasts (normal human dermal fibroblasts) was sensitive to Etomoxir. Cells were cultivated with different amounts of Etomoxir prior to analysis of CPT-1 activity. CPT-1 activity without Etomoxir (0 lmol L)1 value) was set at 100%. Results are shown as the mean ± SD of three independent experiments. (B) Etomoxir in an ointment formulation was able to inhibit CPT-1 activity in the skin of mice. The skin of one SKH1 mouse was treated with placebo or with Etomoxir for 60 min. CPT-1 activity was measured after treatment. One hundred and fifty micrograms of total protein were used in each sample. Error bars show SD and are derived from triplicates of CPT-1 activity. The experiment was independently repeated on six different mice with slightly different conditions. Similar results were obtained.

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Etomoxir treatment of psoriasis, F. Caspary et al. 941

Cross sections

Human transplanted psoriatic skin Murine skin Tissue-tec Slide Unit-length

Human epidermis

Cross section biopsy

Fig 3. Procedure of the histological stainings which were performed on cryopreserved tissues. Diagonal cross-sections (8 lm) were cut.

Human dermis

Murine basement

inhibited by a formulation containing 1% Etomoxir ester. With the 30% Etomoxir formulation this effect is stronger, showing that inhibition is dependent on the concentration of Etomoxir. These results show that the ester of Etomoxir can penetrate the mouse skin, is converted into its active form and inhibits CPT-1. Etomoxir is therefore active in the systems analysed and may be used to reduce fatty acid oxidation in psoriatic skin.

Treatment of psoriatic human skin in a xenogeneic mouse transplant model To study whether inhibition of fatty acid oxidation by Etomoxir is beneficial for the treatment of psoriasis, the effects of Etomoxir on human transplanted psoriatic skin were evaluated using the BNX psoriasis transplant model.17 This model is so far the most predictive animal model for psoriasis.21,22 Mice

A
placebo 1 % Etomoxir

30 % Etomoxir

betamethasone

Fig 4. Etomoxir significantly reduces the epidermal thickness of transplanted human psoriasis skin. (A) Placebo-, Etomoxir- (1% and 30%) and betamethasone-treated human skin transplants on BNX mice. Located at the top layer are the stratum corneum and epidermis of the transplanted human biopsy and at the bottom the murine tissue, forming a base for the transplanted human skin (haematoxylin and eosin; original magnification · 200). (B) Mean ± SD thickness of the epidermal ridges and of the total epidermis. *P < 0Æ001 compared with placebo; #P ¼ 0Æ005 (30% Etomoxir) and P ¼ 0Æ017 (betamethasone) compared with placebo.

B

250 epidermal ridges epidermal ridges 200 total epidermal total epidermal

Thickness [µm]

150 # # 50

100

0 placebo 1% Etomoxir 30% Etomoxir betamethasone

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942 Etomoxir treatment of psoriasis, F. Caspary et al.

A
placebo 1 % Etomoxir

30 % Etomoxir

betamethasone

B
Ki-67 positive cells/unit-length

80 70 60 50 40 30 20 10 0 placebo 1% Etomoxir 30% Etomoxir betamethasone

Fig 5. Effect of treatment with Etomoxir on the number of (Ki-67+) proliferating cells of transplanted human psoriasis skin. (A) Results of the histological staining with mu-aHU monoclonal antibody (original magnification · 400). (B) Quantification of (A). *P < 0Æ001 and P ¼ 0Æ001 (1% Etomoxir) compared with placebo. Results are shown as mean ± SD.

A
placebo 1 % Etomoxir betamethasone

B
% epidermis ULEX positive

80

60

40

20

0 placebo betamethasone

Fig 6. Effect of treatment with Etomoxir on the differentiation rate (distribution of Ulex) of transplanted human psoriasis skin. (A) Results of the histological staining with biotinylated agglutinin antibody (original magnification · 200). (B) Quantification of (A). *P < 0Æ001 compared with placebo. Results are shown as mean ± SD.

were treated topically with either placebo, 1% Etomoxir formulation, 30% Etomoxir formulation or betamethasone for 3 weeks, beginning on the day that the transplants were injec-

ted with the donor’s activated T cells. After 3 weeks of treatment histological skin staining was performed on the transplanted-treated human skin (see Fig. 3) to determine epi-

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Etomoxir treatment of psoriasis, F. Caspary et al. 943

dermal thickness, keratinocyte proliferation (number of Ki-67+ cells per unit length) and differentiation (percentage of Ulex-positive cells in the epidermis). Treatment with placebo resulted in a mean ± SD epidermal thickness of 138 ± 24 lm (Fig. 4), a proliferation rate (Ki67) of 55 ± 37 positive cells per unit length (Fig. 5) and a percentage of Ulex-positive cells in the epidermis of 55 ± 19% (Fig. 6). Treatment with the positive control betamethasone showed a significant reduction (P ¼ 0Æ017) of the mean epidermal thickness from 138 ± 24 lm to 81 ± 9 lm (Fig. 4). The number of Ki-67+ cells was significantly decreased (P < 0Æ001) from 55 ± 37 to 10 ± 6 positive cells per unit length (Fig. 5). Finally, treatment with betamethasone showed a significant decrease (P < 0Æ001) in the percentage of Ulexpositive cells in the epidermis from 55 ± 19% to 24 ± 7% (Fig. 6) when compared with the placebo group. The epidermis of the mice treated with 1% Etomoxir and mice treated with 30% Etomoxir showed a significant reduction (P < 0Æ001) in epidermal thickness from 138 ± 24 lm to 88 ± 10 lm and 69 ± 4 lm, respectively, when compared with the placebo group (Fig. 4). The reductions in epidermal thickness were comparable with those observed when transplants were treated with betamethasone (Fig. 4). Furthermore, treatment with 1% and 30% Etomoxir showed a significant decrease (P < 0Æ001) in number of Ki-67+ cells from 55 ± 37 to 15 ± 18 and 9 ± 7 cells per unit length, respectively, when compared with the placebo group (Fig. 5). Finally, treatment with 1% Etomoxir resulted in a significantly lower percentage of Ulex-positive cells (P < 0Æ001) in the epidermis from 55 ± 19% to 17 ± 11% when compared with the placebo group (differentiation rate; Fig. 6). The reduction in values was similar to the effects of betamethasone as positive control. Comparing 1% Etomoxir treatment with 30% Etomoxir, the higher concentration of Etomoxir resulted in a more pronounced reduction of epidermal thickness (Fig. 4) and of the number of Ki-67+ cells (Fig. 5), showing that these effects are concentration dependent. In summary, these experiments demonstrate that Etomoxir is as efficacious as betamethasone concerning the antipsoriatic effect on human psoriatic lesions. However, the mechanism of action of Etomoxir (blockade of fatty acid metabolism) is different from that of corticosteroids (blockade of immune activation).

Discussion
Etomoxir is able to inhibit CPT-1 activity in the skin, a crucial rate-controlling enzyme of the fatty acid oxidation pathway. We have shown that the inhibition of this metabolic pathway is beneficial for the treatment of psoriasis, as the thickness, cell proliferation and cell differentiation rate of the psoriatic skin lesions were reduced in mice carrying human psoriatic skin. Therapies for psoriasis and other autoimmune diseases in general are mainly focused on the suppression of the immune system. Psoriasis has been shown to be induced by the activation of the immune system due to infection with bacteria and ⁄or viruses.2–6 Blocking the immune response is effective in

treating psoriasis, as can be seen in the murine transplant experiment with betamethasone treatment and also by data from the literature.14 However, this treatment has only shortterm effects.23 Most treatment regimens used currently consist of corticocosteroids, vitamin D analogues and ultraviolet radiation treatment. Treatments focused on blocking cell proliferation, e.g. methotrexate, mitomycin C and coal tar, have also been used in the past, but these therapies provoked severe sideeffects. Fish oil or highly unsaturated long chain fatty acids have also been used to treat psoriasis,24–27 but these treatments have low efficacy because they reduce only some of the symptoms that play a role in the autoimmune disease. The data presented here show that at least two mechanisms play a role in the development and progression of the disease. One mechanism is the induction of the inflammation due to infection by pathogens and subsequent activation of the immune system, and the other is a systemic metabolic mechanism in which the target tissue changes its metabolism. These two mechanisms explain the beneficial effects of immune blockers but also of fish oil diets on psoriasis. A vegetarian diet, consisting predominantly of glucose and unsaturated fatty acids, also has a beneficial effect on psoriasis and fits in the model we propose.28 Etomoxir blocks fatty acid oxidation, thereby blocking the hyperproliferation and autostimulation of cells under attack by the immune system. The vicious cycle between skin cells producing inflammatory signals and cells of the immune system is thus broken. The direct effect is that the protective fatty acid layer that prevents successive pathogenic infections is restored and the immune response is slowed down dramatically. The human skin transplanted animal experiments show that a fatty acid metabolism blocker such as Etomoxir is at least as efficacious as blocking the inflammatory response with a corticosteroid. The efficacy can be seen in the reduction of the number of proliferating Ki-67+ cells, as well as the reduced skin thickness. The psoriasis model with skin transplanted on immune-deficient BNX mice has the advantage that apart from the injected activated human PBMC the immune response is not replenished by freshly activated PBMC that could continuously attack the transplanted skin. Once the immune response is shut down, the transplant can no longer be stimulated by freshly activated PBMC. However, the transplanted skin is still growing. This suggests that the fatty acid metabolism is still upregulated as untreated skin becomes more and more psoriatic, even though the PBMC from the lesions have disappeared within 1 week after injection (G. Elliott, unpublished data). This supports our hypothesis that after the skin has been activated and the metabolism has changed, fatty acid oxidation intermediates continue to stimulate psoriatic skin cells, thereby worsening the lesion, as can be seen in the placebo group. This effect can also be seen in psoriatic patients who are treated with standard therapies such as corticosteroid creams. In these patients the lesions disappear during the therapy; however, as soon as the therapy is discontinued the lesions recur at exactly the same place where they were previously (our unpublished observations). This indicates that there is a local change in the skin cells, that makes the inflammation visible when not treated.

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944 Etomoxir treatment of psoriasis, F. Caspary et al.

In cell culture experiments with primary human skin fibroblasts as little as 1 lmol L)1 of Etomoxir resulted in a maximal inhibition of CPT-1 activity, whereas, as expected, in the fresh mouse skin experiments more Etomoxir was required to inhibit CPT-1 activity. The differences in concentration needed to block CPT-1 in the different assays can be explained by the fact that only a part of the Etomoxir which is dissolved in the cream is able to penetrate the skin. Furthermore, the skin was incubated with Etomoxir for only 1 h whereas in cell culture systems Etomoxir had a longer time to act on CPT-1. The data gained from the experiments described here suggest that by treatment of both the inflammation and the fatty acid metabolism, the immune cells and the skin cells show reduced activity, thus leading to an extended disease-free period. This would be an improvement, because at the moment with standard therapies recurrence is imminent within a couple of weeks after discontinuation of therapy.

Acknowledgments
¨ We thank P. Jager and B. Koehler for critical reading of the manuscript and B. Koehler for helping us with formatting the manuscript.

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Ó 2005 British Association of Dermatologists • British Journal of Dermatology 2005 153, pp937–944


				
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