Improvement of psoriasis vulgaris after intralesional injections of 15-hydroxyeicosatetraenoic acid (15-HETE)

II Ill II I I

Improvement of psoriasis vulgaris after intralesional injections of 15-hydroxyeicosatetraenoic acid (15-HETE)*

Karsten Fogh, M.D., Helmer S0gaard, M.D., Troels Herlin, M.D., Ph.D., and Knud Kragballe, M.D., Ph.D. Aarhus, Denmark

Psoriatic skin lesions are characterized by elevated levels of 5- and 12-1ipoxygenase products (leukotrienes B4, C4, and D4, and 12-hydroxyeicosatetraenoic acid [12-HETE]), which can stimulate epidermal proliferation and induce skin inflammation. 15-Hydroxyeicosatetraenoic acid (15-HETE) has the potential to inhibit the activity of 5- and 12-1ipoxygenases. The purpose of the present study was to determine the therapeutic effect of intralesional injections of 15-HETE. 15-HETE was formed by oxidation of arachidonic acid by soybean lipoxygenase, purified by reversed-phase high-performance liquid chromatography, and identified by mass spectrometric analysis. Thirteen patients took part in the investigation. Plaques with a diameter of approximately 1 cm were injected with 0.1 ml of 10 tzmol/L 15-HETE, 0.1 ml of 1 ~mol/L 15-HETE, or 0.1 ml of saline weekly. After 3 weeks the effect was evaluated clinically and histologically by an observer uninformed of the treatment given. We found that plaques injected with 10 ~mol/L 15-HETE had cleared completely in four patients and improved considerably in seven. In one patient minimal improvement only was seen and in one patient no change was observed. Injection of 1 p~mol/L 15-HETE was without effect in 11 patients and improvement was observed in two patients. Of the plaques injected with saline, minimal improvement was observed in one patient; otherwise the plaques had not changed. Injection of 0.1 ml of 10 Ixmol/L 15-HEPE (identical to 15-HETE except for five double bonds instead of four) induced only minimal improvement in one of four patients. The results imply that 15-HETE by a dose-dependent and stereospecific mechanism can improve psoriasis. Whether a specific stimulation of 15-1ipoxygenase activity (thus increasing the level of 15-HETE) may be a new approach to the treatment of psoriasis awaits investigation. (J AM ACAD DERMATOL 1988;18:279-85.)

From the Department of Dermatology, Marselisborg Hospital, and the Department of Pathology, Aarhus Komrnunehospital, Univer- sity of Aarhus.

Supported by the Danish Medical Research Foundation (Grant 12- 6262) and the Psoriasis Research Foundation in Denmark.

Accepted for publication July 27, 1987.

Reprint requests to; Dr. Karsten Fogh, Department of Dermatology, Marselisborg Hospital, University ofAarhus, DK-8000 Aarhus C, Denmark.

*Part of this study has been published as a letter to the editor in Lancet 1986;2:509.

Certain 5-1ipoxygenase products of arachidonic acid metabolism (leukotrienes 94, C4, and D4) present in biologically active amounts in psoriatic skin lesions 13 can stimulate epidermal proliferation 4,5 and induce skin inflammation characterized by neutrophil invasion. 6.7 The 12-1ipoxygenase product 12-hydroxyeicosatetraenoic acid (12- HETE), which also is present in psoriatic lesions) '8 possesses similar properties although far less potent? Therefore it has been suggested that

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280 Fogh e t a l Journal of the

American Academy of Dermatology

Table I, Effect of intralesional injections of 15-HETE in psoriatic plaques

0,1 ml of 10.0 txmol/L

15-HETE

0.1 ml [ of 1.0 0.1 ml

ttmol/L of 15-HETE saline

Clearing 4 0 0 Marked improvement 7 1 0 Minimal improvement 1 1 1 No effect 1 11 12

The results among the three groups are significantly different, p < 0.0001, by means of Kruskal-Wallis test. Clearing: Complete flattening of plaques including borders, plaques may be outlined by pigmentation; marked improvement: nearly complete flattening of plaques, but borders of plaque still palpable; minimal improvement: slightly less scale and/or erythema.

chemoattractant lipoxygenase products play a pathogenic role in psoriasis.

15-Hydroxyeicosatetraenoic acid (15-HETE) is another arachidonic acid product formed by the 15-1ipoxygenase pathway. In contrast to leukotriene B 4 and 12-HETE, it has no proinflammatory properties. On the contrary, it can inhibit the activity of 5- and 12-1ipoxygenase enzymes in certain cell types. ~~ In addition, 15-HETE can inhibit leukotriene-B4-induced chemotaxis of neutrophils. ~3 In other words, 15-HETE has the potential to inhibit both the formation and the activity of proinflammatory eicosanoids. Furthermore 15- HETE and its precursor 15-hydroperoxyeicosa- tetraenoic acid (15-HPETE) can inhibit certain functions of T lymphocytes ~4'~s that have been reported as abnormal in psoriasis. 16

To examine the hypothesis that a relative shortage of 15-HETE in psoriatic skin lesions might contribute to the clinical manifestation of psoriasis, we have injected purified 15-HETE into psoriatic skin lesions.

PATIENTS AND METHODS Synthesis of 15-HETE and 15-HEPE

15-HETE and 15-hydroxyeicosapentaenoic acid (15- HEPE) were prepared by oxygenation of arachidonic acid (5,8,11,14-eieosatetraenoic acid, Sigma Chemi- cal Co., St. Louis, MO) and eicosapentaenoic acid (5,8,11,14,17-eicosapentaenoic acid, Sigma), respec- tively, by soybean lipoxygenase (soybean lipoxygenase type IV, Sigma). 1~ The incubation was carried out at

4 ~ C in 100 ml of 0.1 M Tris-HC1 buffer (pH 8.5) containing 1 mM phenol. Six mg lipoxygenase enzyme was added to the buffer followed by the addition of 50 mg fatty acid (previously dissolved in 4 ml of 0.01 M NH4OH). After 35 minutes of incubation, the reaction was terminated by acidification with 1 N HC1 to pH 3.0. Lipids were extracted into ether in a separatory funnel. The pooled extract was washed three times with ice-cold water. Then 50 mg of triphenylphosphine (Sigma) previously dissolved in ether was added (to reduce hydroperoxyeicosanoids to hydroxyeicosa- noids). 10 The solvent was evaporated under a stream of nitrogen gas and the residue dissolved in ethylacetate and hexane (ratio of 5:95 by volume) and further purified on silica SEP-PAK cartridges (Waters Instru- ments, Rochester, MN). The collected solvent was evaporated under a stream of nitrogen gas and further purified by reversed-phase high-performance liquid chromatography by use of a semipreparative, Hypersil 5 txm, Cls column (120 • 8 mm), eluted with methanol and water (ratio of 75:25 by volume) at 2.0 ml/min. Ultraviolet (UV) absorption was monitored at 235 nm. 15-HETE and 15-HEPE were identified by coelution on reversed-phase high-performance liquid chromatography with authentic 15-HETE and 15-HEPE, respec- tively (Cayman Chemical, Ann Arbor, MI), by UV spectrometry, and finally verified by mass spectrometric analysis*). The purified (99%) 15-HETE and 15-HEPE were evaporated under a stream of nitrogen gas and dissolved in absolute ethanot for storage under nitrogen at - 7 0 ~ C. The concentrations were determined by integrated optical density at 235 nm. Just before injection the hydroxyfatty acids were diluted to the desired concentration with sterile saline (0.88% NaC1). The final ethanol concentration was 0.1%.

Patients

Informed consent was given by 13 patients (10 male and 3 female) with stable psoriasis vulgaris. The patients had not received medication 2 weeks before the study. In the same anatomic region, three psoriatic plaques (appearing as chronic lesions) of equal size (approximately 1 cm in diameter) and of similar se- verity, judged by scaling, infiltration, and redness, were chosen. Solutions of 0.1 ml of 10 ~mol/L (300 ng) 15- HETE, 0.1 ml of 1 p~mol/L (30 ng) 15-HETE, or 0.1 ml of saline with 0.1% ethanol (being the vehicle in which 15-HETE was dissolved) were injected into the plaques. The plaques were infiltrated totally by injection

*We thank Mr. E. Larsen and Mr. H. Egsgaard, Risr National Lab, Denmark, for performing the mass spectrometric analysis.

Volume 18 Number 2, Part 1 February 1988

Improvement of psoriasis after 15-HETE 281

Fig. 1. Ctose-up of typical psoriatic plaque before (A) and 3 weeks after (B) weekly injections of 0.1 ml 15-HETE at 10 ~mol/L.

at a single location and the injection volume Was placed high in the dermis. Injections were given three times at weekly intervals. Before each injection the size, erythema, scaling, and infiltration were evaluated by an observer who was not informed of the treatment given. The patients were not told which site received which treatment. After 3 weeks of treatment, punch biopsy specimens were obtained under local anesthesia (lido- caine 1%). The specimens were stained with hematoxylin and eosin and examined by light microscopy. The pathologist also was not informed of the treatment given and histopathology was evaluated for the presence of psoriatic features (parakeratosis, acanthosis, neutrophil

infiltration in the epidermis, and pattern of the dermal papillae).

RESULTS

The results of intralesional injections of 15- HETE and of solvent (saline with 0.1% ethanol) are seen in Table I. Plaques injected with 15-HETE at 10 }~mol/liter cleared completely in four patients and improved considerably in seven patients as judged by marked reduction of the size (50% to 75%) and by minimal induration, erythema, and scaling. Minimal improvement was observed in

282 Fogh et al Journal of the


Fig. 2. Close-up of typical psoriatic plaque before (A) and 3 weeks after (B) weekly injections of 0.1 ml saline (0.88% NaC1) with 0.1% ethanol.

only one patient. The healing process consisted of an initial decrease in erythema. Infiltration and scaling were reduced after 1 week, and after an additional 2 weeks the plaque was no longer rec- ognizable or palpable. In one patient no effect of 15-HETE at 10 tzmol/L was observed. The effect of 15-HETE appeared to be dependent of the dose because plaques injected with 15-HETE at 1 ~mol/L showed improvement in only two patients (Table I). Fig. 1 shows photographs of a typical plaque before (A) and 3 weeks after (t3) injections with 15-HETE at 10 Izmol/L. It is seen that the plaque resolved, leaving residual pigmen-

tation with only a trace of psoriasis at the periph- ery. Fig. 2 shows photographs of a typical plaque before (A) and 3 weeks after (t?) injections with saline with 0.1% ethanol. Scaling is less prominent in (B) because the plaque was cleaned before each injection; otherwise the plaque had not changed.

To test the specificity of the effect of 15- HETE/15-HEPE, the I5-1ipoxygenase product of eicosapentaenoic acid was injected at 10 tzmol/L into four plaques. Three of these plaques did not change clinically; one showed minimal decrease in erythema (data not shown).

Histologic examination of specimens obtained



Fig. 3. Photomicrograph of specimen obtained from healed psoriatic plaque following injections of 15-HETE at 10 p, mol/L weeny for 3 weeks. (Hematoxylin-eosin stain; x 125.)

from sites cleared clinically after 15-HETE injections showed that there was a complete regression of the psoriatic features (Fig. 3). Plaques injected with saline with 0.1% ethanol showed the typical histopathology of psoriasis (parakeratosis, acanthosis, elongation of the dermal papillae, and intraepidermal neutrophils) (Fig. 4). No time course study was done of the microscopic changes.

DISCUSSION

In this study we have demonstrated that intralesional injections of 15-HETE in a dose-dependent and stereospecific way improve psoriasis. The improvement observed clinically was supported by histologic examination, which showed absence of psoriatic features in the healed lesions.

The reason 15-HETE improved psoriasis is un- known. However, on the basis of the effects that 15-HETE has in vitro, several possibilities exist. The effect may be produced by an inhibition of the 5- and/or 12-1ipoxygenase enzymes in the

psoriatic skin. 15-HETE has been shown to inhibit neutrophil 5-1ipoxygenase 1~ and epidermal 12-1ipoxygenase. 12 Products of the 5- and 12- lipoxygenases have been found in biologically active concentrations in psoriatic skin. 1-3"8 Because Ieukotriene B4 and 12-HETE are both proinflammatory 6'7,9 and cause epidermal proliferation, 4'5 an inhibition of their synthesis by 15-HETE may explain why 15-HETE can induce regression of the psoriatic lesions. Furthermore I5-HETE can specifically inhibit leukotriene-B4-induced chemotaxis of neutrophils 13 and thus potentially pre- vent leukotriene B4 from inducing accumulation of neutrophils in the epidermis. Whether this actually happens in vivo is still open to discussion. Wilkinson et al ~7 have reported that some of the inhibitory effect of 15-HETE on the 5-1ipoxygenase is through competition of 15-HETE with arachidonic acid for the substrate binding site on the 5-1ipoxygenase enzyme. This interaction results in the transformation of 15-HETE into 5,15-

284 Fogh et al Journal of the


Fig. 4. Photomicrograph of specimen obtained from psoriatic plaque injected with 0. l ml saline (0.88% NaC1) with 0.1% ethanol weekly for 3 weeks. (Hematoxylin-eosin stain; x 250.)

dihydroxyeicosatetraenoic acid (5,15-di-HETE), which has not been shown to have biologic activity.

The reason 15-HETE injections were not equally effective in all patients may be that there is a great variation in the content o f lipoxygenase products in psoriatic plaques, 3,~'* which suggests a variation in the activity of the different lipoxygenase enzymes. Plaques with weak 5- and 12- lipoxygenase activities would therefore be less sensitive to 15-HETE supplementation. It is important to realize that 15-HETE is detectable in psoriatic lesions in highly variable concentrations. 3 On the basis of the concentrations of 15-HETE required in vitro to inhibit 5-1ipoxygenase activity, the lesional concentrations of 15-HETE in most cases appear to be too low to cause any signifi-

*Duell EA, Fortune JW, Petersen CJ, Ellis CN, Voorhees JJ. Eicos- anoids (LTB~, 12-HETE, PGE2, PGF2,) quantitated simultaneously from keratomed epidermal strips of psoriatic skin. Sixth Interna- tional Conference on Prostaglandirts and Related Compounds [abstract]. Firenze, Italy, 1986.

cant 5-1ipoxygenase inhibition. However, the concentrations required for lipoxygenase inhibition in vitro and in vivo may not be directly comparable in terms of inhibitory activity; whether these bio- chemical changes actually occur in vivo still re- mains to be established.

In our experiments injection of 0.1 ml of 10 ~mol/L (300 ng) of 15-HETE was required to cause a clinical effect. However, we do not know the actual concentration of 15-HETE obtained intralesionally or for how long a certain concentration was maintained. Therefore it is still open to discussion whether intralesionally injected 15- HETE acted by inhibiting epidermal 5- and 12- lipoxygenases.

Another potential mechanism of action is inter- ference with T-lymphocYte function. 15-HETE is the major lipoxygenase product of T lymphocytes TM and is reported to inhibit the proliferation of mitogen-stimulated unfractionated T lymphocytes. 14 This action of 15-HETE and its precursor 15-HPETE on T-lymphocyte function appears to be the net effect of an inhibition of T-helper



cells and a stimulation of T-suppressor cells, is T-lymphocyte function has been reported as abnormal in psoriasis ~* and it has been postulated that psoriatic lesions erupt where epidermal influx of helper T lymphocytes overrides the normal epidermal suppressor mechanism. 16 A potential role of activated T lymphocytes in the pathogenesis of psoriasis has gained support recently from clinical trials with cyclosporine. 19.2o

In summary, the results of the present study show that intralesionally administered 15-HETE improves psoriasis. These results support the idea that 15-HETE, or rather a relative shortage of 15-HETE, plays an important role in psoriasis. In search for new antipsoriatic drugs, the screen- ing should therefore include not only inhibitors of 5-1ipoxygenase but also stimulators of 15- lipoxygenase, thus increasing the level of the en- dogenous 5-1ipoxygenase inhibitor, 15-HETE.

REFERENCES

1. Brain S, Camp R, Dowd P, et ai. The release of leu- kotdene B,-like material in biologically active amounts from the lesional skin of patients with psoriasis. J Invest Dermatol 1984;14:70-3.

2. Brain SD, Camp RDR, Black AK, et al. Leukotrienes (;4 and D4 in psoriatic skin lesions. Prostaglandins 1985;29:611-9.

3. Fogh K, Kill J, Herlin T, et al. Heterogeneous distribution of lipoxygenase products in psoriatic skin lesions. Arch Dermatol Res [in press].

4. Kragballe K, Desjarlais L, Voorhees JJ. Leukotrienes B4, C4 and D4 stimulate DNA synthesis in cultured human epidermal keratinocytes. Br J Dermatol 1985; 113:43-52.

5. Bauer FW, van de Kerkhof PCM, Maassen-De Grood RM. Epidermal hyperproliferation following the induction of microabscesses by leukotriene B4. Br J Dermatol 1986;114:409-12.

6. Camp RDR, Jones RR, Brain S, et al. Production of intraepidermal microabscesses by topical application of leakolriene B~. J Invest Dermatol 1984;82:202-4.

7. Soter NA, Lewis RA, Core), F_J, Austen KF. Local effect

of synthetic leukotrienes (LTC4, LTD4, LTE4 and LTB4) in human skin. J Invest Dermatol 1983;80:115-9.

8. Hammarstrfm S, Hamberg M, Samuelsson B, et o3. Increased concentrations of nonesterified araehidonic acid, 12L-hydroxy-5,8,10,14-eicosatetraenoic acid, prostaglandin F~ and prostaglandin F~ in epidermis of psoriasis. Proc Natl Acad Sci USA 1975;72:5130-4.

9. Dowd PM, Black AK, Woollard PM, et ai. Cutaneous responses to 12-hydroxy-5,8,1,14-eicosatetraenoic acid (12-HETE). J Invest Dermatol 1985;85:537-41.

10. Vanderhoek JY, Bryant RW, Bailey JM. Inhibition of leukotriene biosynthesis by the leukocyte product 15- hydroxy-5,8,11,13-eicosatetraenoic acid. J Biol Chem 1980;255:10064-5.

11. Vanderhoek JY, Bryant RW, Bailey JM. 15-Hydroxy- 5,8,11,13-eicosatetraenoic acid. A potent and selective inhibitor of platelet lipoxygenase. J Biol Chem 1980; 255:5996-8.

12. Kragballe K, Pinnamaneni G, Desjarlais L, et al. Dermis- derived 1.5-hydroxy-eicosatetraenoic acid inhibits epidermal 12-1ipoxygenase activity. J Invest Dermatol 1986; 87:494-8.

13. Kragballe, K, Ternowitz T, Fogh K. 15-Hydroxyeicosa- tetraenoic acid (15-HETE) specifically inhibits LTB,- induced ehemotaxis of PMNs [abstract]. J Invest Der- matol 1987;89:325.

14. Gualde N, Durgaprasadarao A, Goodwia JS. Effect of lipoxygenase metabolites of arachidonic acid on proliferation of human T cells and T cell subsets. J Immunol 1985; 134:1125-9.

15. Gualde N, Rigaud M, Goodwin JS. Induction of suppressor cells from peripheral blood T-cells by 15- hydroperoxy-eicosatetraenoic acid (15-HPETE). J Im- munol 1985;135:3424-9.

16. Baker BS, Swain AF, Griffiths CEM, et al. Epidermal T lymphocytes and dendritic cells in chronic plaque psoriasis: the effect of PUVA treatment. Clin Exp Immunol 1985;61:526-34.

17. Wilkinson D, Hallam C, Hemsley PE, et al. 15-L(S)- 5z,Sz,llz,13E-Eicosatetraenoic acid is a substrate for 5-1ipoxygenase. Biochem Soc Trans 1985;13:I80-2.

18. Goetzl EJ. Selective feed-back inhibition of the 5-1ipoxygenation of arachidonic acid in human T-lymphocytes. Biochem Biophys Res Commun 1981; 101:344-50.

19. Mueller W, Herrmann B. Cyclosporin A for psoriasis. N Engl J Med 1979;301:555.

20. Ellis CN, Gorsulowsky DC, Hamilton TA, et al. Cyclo- sporJne improves psoriasis in a double blind study. JAMA 1986;256:3110-6.

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Improvement of psoriasis vulgaris after intralesional injections of 15-hydroxyeicosatetraenoic acid (15-HETE)