Arch Dermatol Res (1992) 284 [Suppl]: S18-$21

9 Springer-Verlag 1992

Role of prostanoids in the inflammatory reaction and their therapeutic potential in the skin R. Ekerdt and B. Miiller Research Laboratories of Schering AG Berlin/Bergkamen, P.O. Box 650311, W-1000 Berlin 65, Federal Republic of Germany

Summary. Vasodilatory prostaglandins (PG), contributing to the inflammatory reaction, have gained considerable attention. It is becoming apparent that PG have pharmacological effects traceable to biological activities distinct from smooth muscle relaxation. The data from pharmacological experiments presented here indicate the diverse action ofvasodilatory PG analogues in the skin of laboratory animals. Nocloprost, a stable PGE 2 analogue, induced erythema in intact skin of rats when applied topically and inhibited in the same dose range an irritant-induced inflammatory reaction in the ears of mice. Iloprost, a stable PGI 2 analogue, showed proinflammatory activity after local application by enhancing the leukotriene B 4 induced cell infiltration in the skin of mice. The attenuation of the spreading of ear necrosis in mice, on the other hand, indicates an anti-ischemic therapeutic potential of iloprost. Research in the past has elucidated the influence of PG on the vascular component of inflammation, but the role of PG on the cellular component of inflammation is less clear. The diverse effects of PG in skin indicate the need for a better understanding of their local actions. Key words: Iloprost - Nocloprost - Inflammation Animal studies - T h e r a p e u t i c p o t e n t i a l

a c a s c a d e o f m e d i a t o r s . P G were c o n s i d e r e d as b e l o n g i n g to this c a t e g o r y . It is b e c o m i n g i n c r e a s i n g l y a p p a r e n t , h o w e v e r , t h a t P G h a v e p h a r m a c o l o g i c a l effects t r a c e a b l e to b i o l o g i c a l activities distinct f r o m v a s c u l a r s m o o t h m u s c l e r e l a x a t i o n . S o m e r e c e n t findings r e p o r t e d here c o n t r i b u t e to a b e t t e r d e s c r i p t i o n o f the local a c t i o n s o f P G in the skin.

Materials and methods Nocloprost, a stable PGE2 analogue, currently under clinical evaluation for gastric ulcer therapy [5], and iloprost, a stable PGI a analogue, currently under clinical evaluation for therapy of arterial occlusive disease [4], were studied. Ethanol/isopropylmyristate (95/5, v/v) was used as solvent in all studies.

Blood JTow studies The cutaneous circulation was quantitated by means of the noninvasive laser Doppler method. Measurements were taken 4 h after topical application of iloprost and nocloprost to the abdominal skin of anesthetized (urethane) hairless rats (female Wistar rats; 20 gl/10 cmZ). Cutaneous blood flow is expressed as percentage change relative to data from solvent treated skin. Studies on Ca 2 + ionophore A23187 or leukotriene B 4 induced i n f l a m m a t o r y reaction

V a s o d i l a t o r y p r o s t a g l a n d i n s (PG), c o n t r i b u t i n g to the inflammatory reaction, have gained considerable attention. R e s e a r c h o n the role o f P G in skin i n f l a m m a t i o n has b e e n i n s p i r e d to s o m e e x t e n t b y the g e n e r a l l y a c c e p t e d mode of action of nonsteroidal anti-inflammatory drugs ( i n h i b i t i o n o f c y c l o - o x y g e n a s e ) , a n d m o s t efforts h a v e t h e r e f o r e b e e n a i m e d at c h a r a c t e r i z i n g the p r o i n f l a m m a t o r y a c t i o n o f P G . E x p e r i m e n t a l evidence o f their p r o i n f l a m m a t o r y a c t i v i t y a n d the f i n d i n g o f their u b i q u i t o u s l y raised c o n c e n t r a t i o n in d i s e a s e d skin was r e g a r d e d as significant in a d d i t i o n to their v a s o d i l a t o r y action. T h e s e o b s e r v a t i o n s fitted c o n v i n c i n g l y the c o n c e p t i o n o f the i n f l a m m a t o r y r e a c t i o n i n i t i a t e d a n d m a i n t a i n e d b y Correspondence to. R. Ekerdt

The inflammatory reaction was induced by either ionophore A23187 (0.1%, w/v) or leukotriene B e (LTB4) (0.003%, w/v). Ionophore or LTB 4 and either nocloprost or iloprost were coapplied topically to the dorsal aspect of both ears of mice (female NMRI mice; 10 gl/ear). After 24 h ear weight, as a measure of edema formation, and the enzymatic activity of myeloperoxidase [2], as a measure of cellular infiltrate, were determined. Both ear weight and extinction values are expressed as percentage change relative to data obtained from non-PG-treated ears (ionophore studies). To test for statistical significance of the action of PG on blood flow or on the inflammatory reaction, a 95% confidence interval was calculated in terms of the variance of observations within the entire experiment. Studies on ischemic necrosis f o r m a t i o n Ischemia was induced by an incision at the base of the ears of mice (male NMRI mice nu-nu) so that two of the three vascular trunks

S19 supplying the ear were permanently interrupted. Once daily, 100 ng/20 gI iloprost was applied to the medial surface of the right ear. Fluorescein isothiocyanate dextran was injected intravenously to visualize the blood vessels. The vascularized ear area was determined morphometrically before, on day 3, and on day 6 after injury. The necrotic, avascularized area is expressed as percentage change relative to the preincision ear area. Statistical comparison was done using Wilcoxon's rank-sum test (~ = 0.05).

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In the present study, synthetic PG analogues rather than natural PG were used. For the topical route of application chemically modified analogues are of advantage due to their increased stability. Figure 1 shows the effect of iloprost and nocloprost after topical application on the cutaneous blood flow in the skin of hairless rats. Both PG analogues augmented local blood flow in a dosedependent manner. Iloprost enhanced blood flow by a factor of 2 even at the lowest dose tested (70 ng/cm2). To reach similar effects with nocloprost, it was necessary to apply an approximately 100-fold higher dose. The reason for this difference in potency of iloprost and nocloprost is unknown at present. Differences in the penetration behavior, metabolism, or tissue response to the respective prostaglandin may be the cause. These data reiterate the long-known vasodilatory activity of PGE 2 and PGI 2 in skin, exemplified by stable analogues after local application. The hyperemic effects were, as indicated, measured after 4 h. This long-lasting effect of the PG tested suggests a reservoir function of the stratum corneum. The influence of iloprost on the LTB4-induced cell infiltration in the skin of mice is demonstrated in Fig. 2. LTB4, the chemotactically active leukotriene, induced a mainly neutrophilic cell infiltration (data not shown). The time course

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Fig, 2. Influence of topical iloprost on LTB4-induced cell infiltration: time course. Myeloperoxidase activity as a measure of cell infiltration was determined in skin homogenates of mice. lloprost ( l ) and LTB~ (A; both 300 ng/cm z) were applied alone or together to) on the outer aspect of the ears of mice. Data are expressed as mean (_-L-SD; ten animals per group) of the optical densities of the myeloperoxidase activity determinations

of LTB~-induced cell infiltration (topically applied, 300 ng/cm2), as detected by increased myeloperoxidase activity, is characterized by a slow onset. Four hours after application, cell infiltration was still marginal. Topically applied iloprost (300 ng/cm 2) by itself had no effect, whereas the LTB4-induced signal was augmented and the time course accelerated by the coapplied iloprost. These data emphasize the proinflammatory potential of vasodilatory PG, which, as shown here, modulate the response of an inflammatory mediator of cell infiltration. Figure 3 depicts the influence of topically applied nocloprost on the ionophore A23187-induced inflammai

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Fig. 1. Influence of topical iloprost and nocloprost on skin blood flow: dose dependency. Blood flow was quantitated by laser Doppler measurement in the skin of anesthetised hairless rats 4 h after local application of iloprost (o) or nocloprost (A). Blood flow data are expressed as percentage increase in comparison to the laser Doppler measurements of solvent treated group (+__ confidence interval at the 95% level; six animals per group). Dosages are expressed as micrograms per square centimeter of skin

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Fig. 3. Influence of topical nocloprost on the ionophore A23187induced inflammatory reaction: dose dependency. Myeloperoxidase activity (A) as a measure of cell infiltration and weight (o) as a measure of edema were determined in the ears of mice. Nocloprost at the indicated dosages and ionophore were applied together on the outer aspect of the ears of mice. Parameters of the inflammatory reaction were determined 24 h later. Myeloperoxidase activity and weight data of the treated groups are expressed as percentage inhibition (_+ confidence interval; ten animals per group) in comparison to the control groups

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Fig. 4. Influence of topical iloprost on ischemic necrosis formation. Ears which developed necrotic skin areas due to severed pineal blood supply were treated with iloprost once daily for 6 days, and the vascularized area was evaluated by intravital microscopy. A, C, control group at day 3 and day 6; B, D, iloprost-treated group at day 3 and day 6. Data are expressed as boxplots with medians (25-75 percentiles; 9 to 11 animals per group). Squares, outliers; asteriks, significant difference from control e = 0.05; Wilcoxon's rank-sum test) tory reaction in the skin of mice. Myeloperoxidase activity as a measure of cell infiltration and weight as a measure of edema are reduced by nocloprost. Concentrationdependent inhibition was found for 0.3 and 3 gg/cm 2 locally applied nocloprost. Higher doses did not further inhibit the inflammatory reaction; maximal inhibition was determined to be approximately 70%. The data indicate that the vasodilatory activity of a P G E analogue does not exclude anti-inflammatory effects at a similar dose range. The mechanism of the ionophoreinduced inflammatory reaction is unclear. It is conceivable, however, that activation of cells, either those of skin (e.g., mast cells) or from the blood circulation (e.g., neutrophils), take part in this process. The mechanism of the anti-inflammatory activity of nocloprost is unknown, but it may be an activity related to the potential of P G to inhibit cell activation by increasing the concentration of intracellular cAMP. The influence of topical treatment with iloprost on spreading of the ischemic necrosis area in the ears of mice is presented in Fig. 4. Treatment for 3 days with iloprost had no significant influence, but application for 6 days limited the final extension of the ischemic necrosis. The median/25-75 percentile values of necrotic skin area in percentage of total perfused ear area were: for the control group, 28.8%/18.7-33.5%; for the iloprost-treated group: 10.9%/9.3-17.1%. Limitation of ischemic necrosis was accompanied by visible hyperemia. Statements of the anti-ischemic mechanism of iloprost are clearly speculative, but the prevention of the late rather than the early progression of necrosis suggests an influence ofiloprost on revascularization or nutritive skin perfusion.

of having various qualities depending on the experimental and pharmacological conditions. Nocloprost induced erythema in intact skin when applied topically and inhibited in the same dose range an irritant-induced inflammatory reaction. Similar effects were reported for P G in the hamster cheek pouch [7]. Inhibition of acute, mast cell dependent inflammation by topically applied P G in vasodilator concentration was found in this experimental system. The mechanism of the anti-inflammatory activity of nocloprost may be related to the potential of P G to inhibit cell activation by increasing the concentration ofintracellular cAMP. It seems evident, however, that vasodilation and anti-inflammatory activity are two distinct activities of the P G E analogue. An anti-inflammatory activity of P G E or derivatives after systemic administration in animal models of chronic inflammation has been reported by several groups, but systemic activities of P G lie outside the scope of this study. (For a recent review see [3].) Iloprost, the stable PGI2 analogue, showed proinflammatory activity after local application by enhancing the LTBe-induced cell infiltration. Proinflammatory activities of PG, augmenting the effects of mediators such as LTB 4 (influencing cell infiltration) or histamine (influencing edema formation) are well known (e.g., see [6]). These effects are satisfactorily explained by the enhanced tissue blood flow and perfusion. The attenuation of the spreading of ear necrosis indicates, on the other hand, an anti-ischemic therapeutic potential of iloprost. The mechanism of this protective and antiischemic action of iloprost in skin is, again, unclear; however, it cannot be attributed solely to an increase in the affected local blood supply or to the attenuation of activated platelets, also known to be affected by iloprost. Inhibition of leukocyte activation has been proposed as a mechanism for the inhibitory effects of local iloprost on the postischemic permeability increase in the hamster cheek pouch [1]. Whether similar effects also contribute to the protective and anti-ischemic action of iloprost in skin remains speculative. Research in the past has elucidated the influence of P G on the vascular component of inflammation, but the role of P G on the cellular component of inflammation is less clear. The diverse effects of P G in skin indicate the need for a better understanding of the local actions of PG. With the advent of stable P G mimetics it is possible to improve and broaden the comprehension of the mode of action of P G in skin. It seems evident that one can underestimate the potential of P G in skin by regarding them as mere proinflammatory mediators.

Acknowledgements. We thank M. Schmidtke, H. D. Luhm and D. Opitz for the skillful carrying out of the experiments.

References Discussion

The data presented here indicate the diverse action of vasodilatory P G analogues in the skin. P G are capable

1. Erlansson M, Bergqvist D, Persson NH, Svensjo E (1991) Modification of postischemic increase of leucocyte adhesion and vascular permeability in the hamster by iloprost. Prostaglandins 41:157-168

$21 2. Goka AK, Farthing MJ (1987) The use of 3,3',5,5'-tetramethylbenzidine as a peroxidase substrate in microplate enzyme-linked immunosorbent assay. Immunoassay 8:29-41 3. Goodwin JS (1991) Are prostaglandins proinflammatory, antiinflammatory, both or neither? J Rheumatol 18:26-29 4. Gryglewski RJ, Stock G (eds) (1987) Prostacylin and its stable analogue iloprost. Springer, Berlin Heidelberg New York 5. Konturek SJ, Brzotowski T, Drozdowicz D, Krzyzek E, Garlicki J, Majka A, Dembinski A, Stachura J, Amon I (1991) No-

cloprost, a unique prostaglandin E 2 analog with local gastroprotective and ulcer-healing activity. Eur J Pharmacol 195: 347 357 6. Movat HZ (1985) The inflammatory reaction. Elsevier, Amsterdam 7. Raud J (1990) Vasodilation and inhibition of mediator release represent two distinct mechanisms for prostaglandin modulation of acute mast cell-dependent inflammation. Br J Pharmacol 99: 449-454