5-Lipoxygenase and 12-Lipoxygenase: Attractive Targets for the Development of Novel Antipsoriatic Drugs. 5-Lipoxygenase und 12-Lipoxygenase: Attraktive Target-Enzyme für die Entwicklung

Lipoxygenases and Antipsoriatic Drugs

Reviews and Trends

5-Lipoxygenase and 12-Lipoxygenase: Attractive Targets for the Development of Novel Antipsoriatic Drugs 5-Lipoxygenase und 1ZLipoxygenase: Attraktive Target-Enzyme fur die Entwicklung neuer Antipsoriatika

Klaus Miiller

lnstitut fur Pharmazie, Pharmazeutische Chernie I, Universitilt Regensburg. Universitatsstr. 31, D-93040 Regensburg, Germany

Received August 6, 1993

1. Introduction

Psoriasis is a chronic skin disease occuring in about 2% of the population in Western c0untries.l). The phenotype is characterized by silvery scaling erythematous papules or plaques. The usual expression of the disease is psoriasis vulgaris2). Another form is pustular psoriasis, occuring when polymorphonuclear leukocytes (PMNL) collect within the dermis in sufficient quantities to produce pustules3). Although psoriatic lesions may occur on any part of the skin, favoured areas are elbows, knees, and buttocks2).

Etiological factors that precipitate or aggravate the disease are infections, heredity, stress, and certain drugs4). The course of the disease tends to be chronic and punctuated by unpredictable periods of remission and exacerbation2). The most frequent age of onset is before 30 years of age4).

The pathogenesis is at yet unknown, but it has been suggested that both the proliferative and the inflammatory component are important parts of the disease process4). The mitotic cycle of the epidermal cell is increased and the tum- over time of the epidermis is reduced from 28 days to 3-4 days and this rapid transit time of the epidermal cells results in disturbed keratini~ation~). Clinically and histologically psoriasis is characterized by hyperproliferation of the epidermis, incomplete differentiation of the epidermis, and inflammatory infiltration of macrophages and neutrophils mediated by arachidonic acid metabolites6). Factors responsible for these events have not yet been established. The recent recognition of the lipoxygenase products as mediators of inflammation has led to a better understanding of the pathogensis of psoriasis and provides new targets for thera- peutical interventions. For comprehensive reviews on leukotrienes and lipoxygenases, see e.g.

The purpose of this article is to discuss chemical, biological, and clinical aspects of arachidonic acid metabolism in psoriasis and the interference with’ known antipsoriatic agents. Special emphasis is placed on the potential targets and mechanisms of compounds that may limit the biological effects of lipoxygenase products. In particular, the anthrone class of LO inhibitors will be discussed. None of the arachidonic acid metabolites are stored in tissues but are biosynthesized from fatty acids upon chemical, mechanical, or immunological stimuli. Thus, there is considerable interest in the development of antipsoriatic drugs that either con-

3

trol the synthesis of these mediators by inhibiting appropriate enzymes or antagonizing their activity. These agents may find their place in the future treatment of psoriasis.

2. Biosynthetic Pathways of Lipoxygenuse Products

Arachidonic acid [(5,8,11,14)-eicosatetraenoic aicd, AA] metabolism in skin has been described6,’). Particularly the epidermal layer is an active site of its metabolism. Arachi- donic acid is esterified in the 2-position of the glycerol moiety of membrane phospholipids and its release is controlled by the activity of either phospholipase A2 or by the combined action of phospholipase C and a diglyceride lipa- se’O). Activity of these enzymes in human skin has been demonstrated’ ‘9”). Depending on the cell types and tissues free AA is transformed under the influence of different enzymes to a variety of oxygenated metabolites. These biologically active compounds are involved in mediating symptoms associated with various diseases (e.g. asthma, inflammatory disease states) and are collectively called eicosanoids.

The major enzymatic routes of AA metabolism in mammalian cells are the cyclooxygenase (C0)- and the lipoxygenase (LO) pathway (Fig. 1). The former catalyzes the production of prostaglandins (PG), prostacyclin (PG12), and thromboxane (TXA2)13), with PGE2 and PGD2 as the main products in human epidermis’? On the other hand, the LO pathway results in formation of hydroperoxyeicosatetaeno- ic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs) and particularly leukotrienes, which are important for the pathogenesis of psoriasis. The term leukotriene stems from the fact that these mediators were first discovered in leukocytes and contain a conjugated triene str~cture’~). The sub- scripts denote the number of double bonds present.

2.1.5-Lipoxygenuse

5-Lipoxygenase activity (EC 1.13.1 1.34) was first reported in polyrnorphonuclear leukocyte^'^). Contrary to cyclooxygenase 5-LO is restricted to neutrophils, eosino- phils, monocytes, macrophages, mast cells, and keratino- c y t e ~ ’ ~ . ’ ~ ) . These cells are considered to be “inflammatory cells”’ 8).

Arch. Pharm. (Weinheim) 327,3-19 (1994) 0 VCH Verlagsgesellschaft mbH, D-6945 I Weinheirn. 1994 0365-6233/94/0101-003 $5.00 + .25/0

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~~-~ipoxygenase

Miiller

Membrane Phospholipids

Phospholipase A& 1 5-HPETE 12-HPETE

t PGG;,

5HETE LT&

I \

Fig. 1: Biosynthelic pathway of arachidonic acid

The biosynthesis of leukotrienes catalyzed by 5-LO shows several characteristical features: A prerequisite for lipoxygenation by any lipoxygenase is the (lZ,4Z)-pentadiene structure of the fatty acid'"'. Arachidonic acid has three such overlapping units and is highly susceptible for enzymatic transformation by lipoxygenases.

2.1.1.5-Lipoxygenme Products

Arachidonic acid is converted to (5s)-hydroperoxy- (6E,8Z, 1 IZ, 142)-eicosatetraenoic acid (5-HPETE) by an antarafacial process20'. The initial step is the stereospecific abstraction of the pro4 7-H of AA, as evidenced by the use of (7R)-deuterioarachidonic acid and complete retention of the 1abel'O). Insertion of molecular oxygen at C-5 of the AA radical gives 5-HPETE2'), which implies EZ-conjugation of the pentadiene double bonds. The involvement of singlet oxygen in this reaction is extremely unlikely since this would require both hydrogen abstraction and oxygen addition to take place on the same side of the plane of the allylic system22).

The name 5-LO of the enzyme is derived from the posi- tional specificity of the oxygenation process. The position of the carbon bearing the hydroperoxy group is identified relative to the carboxyl group carbon of AA, which is de- signated as l'3).

There is evidence that 5-LO activity catalyzing the subsequent step (LTA4 synthase activity) resides in a single protein. The reaction proceeds from 5-HPETE through a sec- ond stereospecific removal of the pro-R 10-H followed by delocalization of the resulting radical and formation of the epoxide LTA4[(5S)-5E,6E-oiido-7E,9E,1 lZ,14Z-eicosatetraenoic acid]'4). This unstable product25) is enzymatically

hydrolyzed by the zinc-containing LTA4 hydr~lase'~) (E.C. 3.3.2.6) to form LTB4 [(SS,12R)-dihydroxy-6Z,8E, 10E,14Z- tetraenoic acid]26). The mechanisms has been proposed to involve a general base (from a carboxylate) and a Lewis acid (Zn2+), the latter of which may coordinate the nucleo- philic water molecule to facilitate the base catalysis").

In addition, LTA4 is a precursor of the peptide leukotrienes (LTC4, LTD4, LTE4), formerly named slow reacting substances of anaphylaxis (SRS-A), which are important mediators of hypersensitivity reaction^^^.^^). They can be implicated as mediators of certain respiratory, cardiovascu- lar, renal, gastrointestinal, and central nervous system dis- orders3'). Thus LTA4 may undergo two diverse biochemical reactions. It produces LTB4 and may be conjugated with glutathione by glutathione transferase to produce LTC422). Removal of glutamic acid by y-glutamyl transpeptidase gives LTD4. Further removal of a glycine residue by a dipeptidase results in the formation of LTE4I6).

5-HETE is produced by reduction of 5-HPETE or LTA4 by glutathione peroxidase (GPO)3').

Because of the multitude of factors influencing 5-LO and their complex interactions, an enzymological understanding of the enzyme is difficult to achieve.

2.1.2. Properties of 5-Lipoxygenase

The isolation and purification or partial purification of 5- LO from mammalian sources has been described, among others from human leukocyte^^^). The purified enzyme has a molecular weight of 72-76 kDa.

There are a number of issues that complicate the 5-LO assay33). The kinetic properties are complex and often con- fusing. The reaction profile consists of three distinct phas-

Arch. Pharm. (Wciriheini) 327.3-19 (1994)

Lipoxygenases and Antipsoriatic Drugs 5

12 AracMdUlk Add

p o . 0 2

CHPElE

- H a LTA4-Synfhase I OH

L%

SHm

and it has been suggested that FLAP may activate 5-LO by binding and transferring this substrate to the enzyme for the concerted synthesis of LTA443).

2.2.12-Lipoqgenase

The 12-lipoxygenase (EC 1.13.1 1.3 1 ) pathway is the main metabolic pathway of AA in the skin, 12-HETE being the major product in most species4j). The formation of 12- HPETE in platelets was the first report on the lipoxygenation of AA in mammalian ~ e l l s ~ ~ , ~ ~ ) . The initial product of the 12-LO pathway in platelets is I2(S)-HPETE, which is reduced to 12(S)-HETE by a glutathione-dependent peroxi- dase4’).

112-LO, 4

0

OH

Fig. 2: Metabolism of arachidonic acid by 5-lipoxygenase 1 GPO

esI9): 1) an initial lag phase, where the velocity of the reaction, which is initially low, gradually increases and reaches a maximum, 2) the propagation phase, where the enzyme is operating at its optimal velocity, and 3) the inactivation phase, where the velocity gradually decreases to zero. This is due to factors other than substrate depletion or product inhibition. It is an irreversible process, which is mainly caused by lipid hydro peroxide^^^). Another observation suggests that superoxide or hydroxyl radicals may inactivate the enzyme. The stimulation of mast cells or macrophages pretreated with superoxide dismutase or catalase results in the formation of larger amounts of 5-LO products‘@.

The optimal enzyme activity is dependent on a number of cellular factors, among others Ca’+, ATP, and phosphatidyl- choline. Furthermore, small amounts of hydro peroxide^^^) and H202 can activate the en~yrne~~.~’’ . Full activation of 5- LO requires a Ca2+-dependent translocation of the enzyme from the cytosol to a membrane-bound ~ i t e ~ ’ . ~ ~ ) . This translocation is possibly due to a change from a hydrophilic to hydrophobic conf~rmation~~).

Besides 5-L0, another factor required for the cellular biosynthesis of leukotrienes is a 18 kDa membrane protein, which has been isolated from human leukocytes. It was termed “five-lipoxygenase-activating protein” (FLAP) and plays an essential role in the translocation of 5-LO from the cytosol to the membrane41.42’. FLAP specifically binds AA,

0

OH

1 2 4 m

Fig. 3: Metabolism of arachidonic acid by 12-lipoxygenase

The initial step in the production of 12-HPETE is stereospecific removal of the p ro3 10-H. Subsequent insertion of molecular oxygen at C-12 from the opposite side forms the S-configurated enan t i~mer~~) . Contrary, 12-HETE derived from lesional psoriatic scales is stereochemically different from platelet 12(S)-HETE and is in fact its enantiomer 1 ~ ( R ) - H E T E ~ ~ ~ . ~ ~ ) . In contrast, the normal epidermis synthe- sizes predominantly 12(S)-HETE“). The properties of I2(R)-HETE and possible explanations for its formation in psoriatic scales are considered in more detail below (Sec- tion 4.2). 12-LO activity in the skin is localized mainly in the cytosol of the epidermal layeru).

Observations that potent inhibitors of the epidermal 12- LO did not inhibit plurelet derived 12-L052953) suggest the existence of two distinct 12-LO enzymes. With psoriasis as target for therapy, potential 12-LO inhibitors have to be evaluated using epidermal 12-LO.

Arch. Pharni. (Weinheim) 327,3-19 (1994)

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2.3. Active Oxygen Species as Secondary Products of the Arachidonic Acid Metabolism

3. Biological Properties of Lipoxygenase Products

The lipoxygenase products can be classified as newly formed mediators, i.e. none of them is preformed and stored in tissues. They are synthesized directly before their release upon appropriate stimuli and are distinguished by their inflammatory effects.

It is generally accepted that phagocytic cells release oxygen radicals in the environment which result in injury to the i s s ~ e ~ ~ . ~ ~ ) . The presence of components in psoriatic scales that stimulate the generation of oxygen radicals in PMNL has been assessed by chemilumine~cence~~). Nevertheless, the relationship between leukotriene biosynthesis and active oxygen species is not well defined. Since the enzymatic 3*1. LTB4

pathways lead to the formation of polyunsaturated fatty acid hydroperoxides, the catalytic decomposition of these compounds is of biological interest. Given the proper conditions or catalysts, the hydroperoxides can be further transformed by radical routes. Homolytic cleavage of the hydroperoxy group can afford peroxyl radicals and alkoxyl radi- c a l ~ ~ ~ ) . The self reaction of secondary peroxyl radicals involves a cyclic transition state (tetroxide), which decom- poses to give a molecule each of alcohol, ketone, and oxy- ged9). This Russell mechanism involves the elimination of singlet oxygen in order to conserve spin (Fig. 4)60). Peroxyl radicals derived from fatty acid hydroperoxides are possible candidates for such a mechanism because the secondary C-H bond necessary to complete the transition state is present5@. The production of singlet oxygen from soybean lipoxygenase isozymes has been demonstrated by 1268 nm emission characteristics of this species.61).

k H

Fig. 4: Production of singlet oxygen from secondary peroxyl radicals by the Russell mechanism

Soybean lipoxygenase catalyzes the breakdown of polyunsaturated fatty acid hydroperoxides to generate superoxide, peroxyl, and hydroxyl radicals6*). Corey postulated that allenic hydroperoxides may undergo 0-0 bond homolysis to form hydroxyl radicals").

The skin is particularly vulnerable and potentially the target of significant oxidative damage because of its lipid-rich membranes. Thus, epidermal cells and dermal cells can be targets of biological oxidants in skin during normal or altered metabolic activity&). A model of skin inflammation induced by active oxygen species has been reported65). The formation of active oxygen species is an important factor in psoriasis since the activity of superoxide dismutase (SOD) is decreased in this disease66-68). SOD is an enzyme that catalytically accelerates the spontaneous dismutation of superoxide to HzOz and molecular oxygen by a factor of 2 . lo4 69) and provides an important defense mechanism against deleterious effects of oxygen radicals. Consequently, the removal of these species is diminished in psoriasis and superoxide produced from the breakdown of hydroperoxides will deplete the source of local antioxidants and finally exacerbate the inflammatory process.

LTB4 fulfils the criteria for a mediator of inflammation. Of particular interest are the chemokinetic and chemotactic properties, which are common to all HETEs, although it appears that mono-HETEs are relatively less important as chemokinetic agents70). The production of LTB4 is an important factor in the local accumulation of neutrophils in the inflammatory response and LTB4 is regarded as the most potent lipid ~hemoattractant~'). Higher concentrations of LTB4 cause degradation of PMNL72.73), stimulate the release of lysosomal enzymes and the generation of active oxygen species such as s ~ p e r o x i d e ~ ~ ) . These effects of LTB4 on PMNL are not mediated by its 5,lZdihydroxy isomers which can be formed by a non-enzymatic hydrolysis of LT&, documenting the stereospecificity of LTB25). In addition, LTB4 has the ability to activate the human neutrophil 5-LO and is more potent than its A6-trans- 12-epi isomers76). These finding raise the possibility that LTB, may positive feedback on its own synthesis.

The stereospecificity of the stimulation of human PMNL chemotaxis and other functions by LTB4 suggests the presence of distinct receptors. Stereospecific binding sites for [3H]-LTB4 were identified on human PMNL that had properties consistent with their being functionally important receptors for LTB477*78). Studies on human PMNL indicate the presence of two classes of receptors that are specific for LTB4 and distinct from the receptors defined for the peptide leuk~tr ienes~~). Each of the receptor classes appears to be coupled to a different set of PMNL functions. Occupation of the high-affinity receptors for LTB4 evokes calcium mobilization and mediates chemokinesis and chemotaxis. A low affinity class of receptors mediates release of lysoso- ma1 enzymes and activated o ~ y g e n ~ ~ * ~ ~ ) . Further studies suggest the involvement of a guanine nucleotide-binding protein (G-protein) in the regulation of the signal transduc- tion initiated by occupancy of the high-affinity LTB4 recep- t o r ~ ~ ~ ) . Evidence for LTB4 binding sites in a human epidermal cell line and on human keratinocytes has been present- ed81982).

3.2.12-HETE

12(R)-HETE is biologically more potent than the S-enantiomer as a chemokinetic agent for human Ph4NLg3) and in the induction of leukocyte aggregationg4). Because 12(R)- HETE contains a 12(R)-hydroxy-group as does LTB4, it is binding to the LTB4 receptor and competitively displaces [3H]-LTB4 from its receptor on leukocyte membranesE4).

Arch. Pharm. (Weinheim) 327,3-19 (1994)

Lipoxygenases and Antipsoriatic Drugs

antipsoriatic drugs

0

However, the presence of a high-affinity receptor for 12- HETE in a human epidermal cell line revealed higher affinity for 12(S)-HETE than for the R-enantiome?').

f

arachidonic acid ,+@ lipoxygenase products

\

4. Pathogenetic Role of Lipoxygenase Products in Psoriasis

There is considerable evidence that lipoxygenase products are important mediators of the pathophysiology of psoriasis. A variety of data demonstrates that these products are elevated in association with psoriasis and that administration of these mediators can mimic the (Fig. 5).

On the other hand, only a slight elevation of cyclooxygenase products was observedg7). In the following, observations that suggest an involvement of the LO products in the pathogenesis of psoriasis will be discussed.

leukocyte migration, enhanced keratinocyte

proliferation

4.1. Formation of Lipoxygenase Products in the Skin

There are cells present in normal and inflamed skin that produce LO derived products. Besides keratinocytes, tissue macrophages and tissue mast cells representing the constitutive cells, accumulation of neutrophils, monocytes, and erythrocytes having migrated from blood vessels are potential sources of LO products. The mere presence of LTB4 in leukocytes' exudates does not state, whether this mediator is the cause or the effect of the leukocyte infiltrate in the

epidermis. Nevertheless, it has been demonstrated that constitutive cells within the skin contain 5-LO activity, although low. Thus, synthesis of 5-LO products in culturedgg) and freshly isolatedg9) human keratinocytes has been shown. Healthy epidermis has a very active 12-L090).

4.2. Increased Formation of Lipoxygenase Products in Psoriasis

There are several lines of evidence for a disturbed AA metabolism in psoriatic skin. By use of keratome sampling technique Hammarstrom et al. were the first who showed markedly elevated amounts of free AA and 12-HETE in lesional psoriatic sking7). Further studies have been carried out using psoriatic scales and skin chamber f l ~ i d ~ ' . ~ ~ ) . The samples were analyzed by different techniques, i.e. TLC, GC-MS, HPLC in combination with bioassay or radioim- munoassay. Biologically active concentrations of LTB4 and monohydroxyfatty acids have been detected. LTB4, 5- HETE, and 12-HETE have also been identified in psoriatic scales and skin b i o p s i e ~ ~ ~ . ~ ~ ) and in the serum of psoriatic patients97). Moreover, only LTB4 is present in acute guttate psoriatic skin lesions in concentrations able to exert biological effects and may, therefore, be involved in the early inflammatory changes of psoriasis9*). The low level of 12- HETE seems to indicate that this mediator is produced later

1) cells in normal 2) psoriatic lesions and inflamed skin

0 lipoxygenase products

4)

antipsoriatic drugs

0 arachidonic acid ,+@ lipoxygenase

products

Fig. 5: Pathogenetic role of the lipoxygenase products in psoriasis: 1) Constitutive cells and immigrating cells in skin are potential sources of LO-products, 2) elevated levels of LO-products in psoriatic lesions, 3) correlation of the biological effects of LO-products with several pathological features of psoriasis, and 4) antipsoriatic drugs as inhibitors of lipoxygenase enzymes


8 Miiller

during the development of psoriatic lesion, and it appears that 12-HETE may be responsible for maintaining chronic plaque psoriasisg8).

Reasons for the elevated levels of LO products in psoriatic lesions may be among others the raised activities of phospholipase A2999100) and phospholipase C, which is also higher in psoriatic plaques than in uninvolved psoriatic skini2). In addition, an enhancement of 5-LO activity in sol- uble preparations of human psoriatic plaques has been observed"").

As mentioned above, the most prominent feature of psoriasis is the increased formation of 12-HETE. Cultured epidermal cells from involved and uninvolved psoriatic patients exhibit a pronounced defect of the 12(S)-HETE receptor as compared with healthy patientsIo2). This decrease in the number of 12-HETE receptors may explain the markedly elevated 12-HETE concentration resulting from diminished intracellular 12-HETE uptake, but the question concerning the stereochemical characteristic remains unanswered. It has been widely assumed that the product of 12-LO in psoriatic skin is similar to that formed in platelets and, therefore. is 1 2(S)-HETE8'). However, it was clearly distinct from the platelet compound and has been shown by Wollurdso) to be the 12(R)-enantiomer, an important discovery from the biological as well as pharmacological point of view. In virro finding^^',^^.'^) that 12(R)-HETE is more potent than 12(S)-HETE to promote PMNL chemotaxis and induce PMNL aggregation under- line the importance of this fact. In addition, the absol. con- figuration at C-12 is equivalent to that of LTB4, which also contains a 12(R)-hydroxyl group. This is in accordance with the relative binding affinity to the LTB4 receptor site on human neutrophil membranesg4). There are several explanations for the origin of 12(R)-HETE'''): 1) The existence of an epidermal 12(R)-lipoxygenase may be a possible explanation, although it has been shown that epidermal homogenates synthesize 12(S)-HETE as the predominant enan- tiome9'). 2) Auto- and photooxidation of AA by molecular oxygen or active oxygen species would form racemic 12- HETE, whereupon the S-enantiomer would be preferentially esterified into cellular phospholipids. 12(R)-HETE would be left as the product found in psoriatic scale. However, the predominance of a single diastereomeric derivative after derivatization of the C- 12 enantiomers derived from lesion- a1 psoriatic scales argues for minimal contribution of such an explanation and rather suggests a specific biosynthetic process"). 3) An enzyme other than the 12-LO may be involved: Cytochrome P-450 monooxygenases, e .g . , produce predominantly 12(R)-HETE rather than its enantio- mer1O4). Some data tend to exclude the P-450 system as the source of 12(R)-HETE. because carbon monoxide did not inhibit its synthesis in skin homogenates neither did NADPH stimulate its synthesis in human epidermal homo- genates5'). On the other hand, isolated epidermal cells have the capacity to selectively convert AA to a mixture of 12(S) and 12(R)-HETE (the molar ratio averaging 5: 1) by the activity of a novel membrane-bound monooxygenase that is NADPH sensitive and was inhibited by an inhibitor of P-

450 oxygenations, carbon monoxide'05). AS a result of this study the presence of distinct mitochondria1 and microso- ma1 enzyme systems which exhibit 12(S) "stereoprefer- ence" has been suggestedlos'.

4.3 Effects of Lipoxygenase Products in the Skin

Generally accepted in vitro effects of LTB4 and the mono- HETEs are chemotaxis for neutrophils. These effects have been demonstrated in skin using various model systems. Thus, leukocyte migration was observed after abrasions of forearm skin and application of LTB4'06) and after intrader- ma1 injection of LTB4'07~'08). Topical application under occlusion of this mediator showed intraepidermal microab- scesses109), indicating that LTB4 can produce lesions similar to those of pustular psoriasis. On the other hand, topical application of LTB4 to psoriatic patients did not lead to the development of psoriatic lesions' lo) . Consequently, this mediator does not seem not be an initial factor of psoriasis.

12(R,S)-HETE, photochemically produced from AA with singlet oxygen, produced a neutrophil polymorphonuclear and mononuclear infiltrate in the dermis after intradermal infusion'"-"2). The topical administration resulted in an erythematous response'' ').

Along with the inflammatory reaction another key histo- phathological feature of psoriasis is enhanced keratinocyte proliferation. LTB, and 12-HETE are involved in this process. In contrast to its isomers LTB4 is a potent stimulator of keratinocyte proliferation, which has been demonstrated in cultured human keratinocytes by increased incorporation of [3H]thymidine"3). 12-HETE as well is a stimulator of keratinocyte proliferation' 14) . In addition, stimulation of epidermal proliferation in vivo has been described for LTB4 and 12-HETE1I5). Similar hyperproliferative effects have been observed after chronic intracutaneous administration of both compounds''6). Addition of platelet-derived 12- HETE to cultured human keratinocytes stimulated DNA synthesis' ").

4.4. Known Antipsoriatic Drugs as Lipoxygenase Inhibitors

Further support for the involvement of LO products in psoriasis comes from observations that drugs known to be beneficial in this disease inhibit the LO pathway. Glucocor- ticoids are indirect inhibitors of the formation of LO products through the generation of the polypeptide lipomodulin (macrocortin), an inhibitor of phospholipase A2' 18). Gluco- corticoids exhibit an inhibitory effect on eicosanoid synthesis' 19) in psoriasis and their topical application (clobetasol) significantly suppresses LTB4 levels in the treatment of psoriasis'20).

Evidence for a correlation between antipsoriatic activity and LO inhibition of eight known antipsoriatic drugs of diverse structure came from assays using soybean LO'21). This study suggested that a common underlying mechanism of action might be involved and indicated that inhibition of LO is a viable approach to the treatment of psoriasis. Anthralin is an inhibitor of leukotriene production in human

Arch. Pharm. (Weinheirti) 327,3-I9 (1994)


Hretinate

o y 3

0

OH 9 OH CH3

5.1. Methods of Evaluation of Lipoxygenase Inhibitors

Numerous in vitro test systems have been described to study 5-LO activity and to discover inhibitors of this enzyme. A variety of preparations using both intact cells and cell-free systems have been employed including isolated human PMNL38913"132), rat, bovine, and porcine PMNL'33- 13s), peritoneal macrophages from various and cell lines such as rat basophilic leukemic cells (RBL-1)138-

Methods for identification and quantification of 5-LO products are mostly based upon addition of radiolabelled AA, extraction, separation by TLC or HPLC and measurement by liquid scintillation c o ~ n t i n g ' ~ ~ ~ ' ~ ~ - ' ~ ~ ) , LTB4 radio- immunoassays (RIA)13'.143314), or separation by HPLC and UV detection of the eicosanoid.~~~~'~~~'~~~'~~). Furthermore, bioassays and GC-MS are reliable techniques for the determination of LO products. Assay methods for various lipoxygenase products have been surveyed by Young and Gir-

140)

ard 14.5 ).

Anthralln Benoxaproten 5.2. Precautions and Pitfalls

Fig. 6: Antipsoriatic drugs as inhibitors of 5- and 12-LO product formation

neutrophils (ICso = 7 pM)12?) and inhibits the production of 12-HETE in human platelets and epidermal strips of mouse tail skin (ICSo values are 10 p.M and 50 pM, respective- 1 ~ ) ' ~ ~ ' . Some retinoids are also inhibitors of 5- and 12-LO in human and rat n e u t r o p h i l ~ ' ~ ~ ~ ' ~ ~ ) . However, two major metabolites of etretinate, the 4-hydroxyphenyl analog and the 1 3 4 s analog were even more potent inhibitors of 5-LO in rat basophilic leukemic (RBL) cells with ICS0 values of 0.34 pM and 34 pM, respectively, and 12-LO in human platelets (ICso values are 35 pM and 25 pM, respective- 1 ~ ) ' ~ ~ ) . Benoxaprofen, which has been drawn off the mar- ket, inhibits 5-LO product formation'zs~'26) and improves psoriasisiz7). Lonapalene is topically effective in psoria- sis128) and is a potent inhibitor of 5-LO in human leukocytes and RBL cell-free preparations (ICso values are 15 pM and 0.5 pM, re~pectively)'~~).

5 . Inhibition of the Formation and Action of Lipoxygenase Products: Assays and Mechanisms

Of the possible targets for inhibition of leukotriene biosynthesis, it is not surprising that most efforts have been directed to the design and development of 5-LO inhibitors. This enzyme is involved in the initial steps of the metabolic AA cascade and its inhibition would have the widest possible effects. Another approach to regulate the effects of LO products is to block their receptors. Since 5-LO inhibitors block the formation and, therefore, the action of the full spectrum of leukotrienes it is suggested that they may be superior to leukotriene receptor antagonist, which effect the action of only one leukotriene.

Many reports of 5-LO inhibition have appeared in recent years. Most of these reports have used the release of 5-LO products from stimulated cellular preparations. None of these evaluation methods is specific for 5-LO inhibition, but may be responding to other biological effects of the test compounds. Thus, the results obtained from whole cell systems must be interpreted cautiously. This section discus- ses some effects of test compounds other than 5-LO inhibition and shows where the use of intact cell systems would be misleading.

5.2.1. Cytotoxic Effects

While inhibition of the release of 5-LO products from intact cell systems is a widely used method, one must exclude a direct cytotoxic effect of the test compounds on the enzyme source by the measurement of cell viability. This is accomplished by the exclusion test with trypan blue. Trypan stainability after incubation of the cells with the test compound can be estimated by microscopic examination of the cells. Cells that show blue-coloured nuclei or cytoplasm are regarded as dead or defect.

5.2.2. Inhibition of Phospholipase A2

Inhibition of leukotriene or prostaglandin production seen with certain compounds (e.g. corticoids) is not always due to direct inhibition of 5-LO and cyclooxygenase, but has been attributed to the inhibition of endogeneous AA mobilization by phospholipase AZ. If at least one of the pathways of AA metabolism is not inhibited by the test compound, a mechanism involving inhibition of AA release can be ruled

If eicosanoid formation of all pathways is decreased, the amounts of LO product formation determined from endogeneous AA should be compared with those obtained from exogenous AA at different concentrations.

Arch. Pharm. (Weinheim) 327.3-19 (1994)

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5.2.3. Inhibition of LTAl Hydrolase

There are reports of compounds that do not inhibit 5 - HETE but do inhibit the formation of LTB, or show differential inhibition of 5-HETE formation and the latter product. If only LTB, formation is determined to show 5-LO inhibitory activity of the test compound, detection of reduced amounts of this metabolite may not necessarily be related to 5-LO inhibition. While inhibition of the formation of LTB4 without inhibition of 5-HETE would suggest an inhibitory effect on LTA, hydrolase, inhibition of the synthesis of both would indicate blockage of the formation of 5-HPETE and LTA4 (Fig. 2), due to the two catalytic activities of 5-LO. As 5-LO inhibitors display a concomi- tant inhibition of 5-HETE and LTB4 biosynthesis, decreased amounts of both 5-HETE and LTB4 levels have to be demonstrated to exclude selective inhibition of LTA4 hydrolase. LTA4 hydrolase has been discussed as a site for pharmacological intervention and the development of selective inhibitors has been r ep~r t ed*~? '~ ' - ' ~~) .

5.3. Lipoxygenase Inhibitors

The literature of LO inhibition is vast and several reviews on LO inhibitors have appeared18*22~40~152-'54). In this section only some significant examples from each class of inhibitors will be discussed.

Among LO inhibitors described both natural and synthetic compounds have been reported. At least four classes of inhibitors may be distinguished regarding the chemical structures and the mechanisms of inhibition, as far as structure-activity relationships can be obtained at all from the partly controversial data concerning the biological activity. The difficulty in interpreting these data is complicated by the wide variety of evaluation methods and activity criteria. Classes of LO inhibitors include 1) antioxidants or free radical scavengers, 2) iron chelators, 3) substrate or product analogues, and 4) structurally unrelated compounds. In order to categorize LO inhibitors a representation for the LO mechanism of action is desirable.

5.3.1. Enzyme Mechanism 5.2.4. Lack of Cell Penetration

Upon activation 5-LO migrates from the cytosol to the membrane where AA metabolism takes place24). Therefore, the concentration of the test compound in the cell membrane is important for inhibition of the enzyme. Since the use of whole cells would require penetration of the test compound into the cell, the observed inhibition is dependent on the lipophilicity of the inhibitor, i.e. the fraction of the inhibitor that is partitioned into the membrane13'). Determination of the octanol-water partition coefficient (log P) of the test compound may help evaluating penetration problems. To confirm that failure of the test compound to inhibit the enzyme is simply related to the fact that the inhibitor does not penetrate cells, the use of a PMNL homogenate is recommended. This can be achieved by son- ification of the cells, the homogenate is then incubated with exogeneous AA149).

5.2.5. Inhibition of FLAP

Some compounds inhibit 5-LO activity in intact cell systems, but do not inhibit activity in cell-free systems. These compounds have no direct effect on 5-LO activity, but prevent membrane association of 5-LO by binding to 5- lipoxygenase-activating-protein (FLAP)150.151). Thus, it is essential to examine 5-LO activity under cell-free conditions, and it has to be demonstrated that no higher concentrations of the test compound are required to inhibit 5-LO product formation compared to that using intact cells.

On the other hand, FLAP may be a novel target for future drug development (translocation inhibitor^)'^^).

In summary, 5-LO inhibition by a test compound cannot be assumed based on inhibition of the release of LO products from intact cells, unless specificity for the formation of these metabolites or direct 5-LO inhibition in cell-free systems is also shown.

Lipoxygenases contain a non-heme iron per molecule in the enzyme active as high-spin Fe(I1) in the nati- ve state, and high-spin Fe(II1) in the activated ~ t a t e ' ~ ~ . ' ~ ~ ) . Iron is also present in human 5-LO and is essential for enzyme activity'60). It is supposed that iron changes its valence state during the catalytic reaction161*162). Two mechanistic possibilities have been proposed for the lipoxygenation: 1) Fe(II1)-enzyme abstracts a hydrogen atom from the substrate to produce the Fe(I1)-enzyme, a proton, and the substrate radical. This radical reacts with molecular oxygen to form a peroxyl r a d i ~ a l ' ~ ~ . ' ~ ) , which in turn accepts an electron from the Fe(I1)-enzyme and a proton leading to hydroperoxide formation and the regenerated Fe(III)-en~yme'~**'~~) (Fig. 7):

? + H a v - H h f EnzymeFe(l I )

? Enzyme-Fe(lll)

? + H a B Enzyme-Fe(lll) EnzyrnsFe(l1)

Fig. 7: Free radical mechanism of 5-LO reaction

2) On the other hand, Corey has developed an alternative mechanistic working This mechanism involves an organo-iron intermediate, which is formed by

Arch. Pharm. (Weinheim) 327,3-I9 (1994)

Lipoxygenases and Antipsoriatic Drugs 1 1

proton transfer from the substrate to a basic group of the enzyme and electrophilic addition of Fe(II1) to the substrate at the catalytic site. Insertion of oxygen into a o-iron bond yields the LO product hydroperoxide with retention of con- figuration followed by hydrolytic cleavage of Fe(II1)-0 bond (Fig. 8). The observation of fatty acid peroxyl radical^'^^,'^) may dispute this mechanism. At any rate, both mechanisms are consistent with the requirement of an Fe3+ ion for enzyme activity.

I knzyme-Fe(lll) Enzyme

Fig. 8: Organo-iron mediated mechanism of 5-LO reaction according to Corey

5.3.2. Antioxidants and Free Radical Scavengers

It is not surprising that most LO inhibitors are antioxidants or free radical scavengers, since lipoxygenation occurs via a carbon centered radical. Thus, compounds that inhibit the formation of this radical or trap it once formed would be expected to be LO inhibitor^'^^).

These compounds, which encompass both natural and synthetically modified natural products, often act by formation of a lower energy radical thus interfering with the intended pathway of the reactionIS2). Moreover, many LO inhibitors also inhibit lipid p e r o ~ i d a t i o n ' ~ ~ - ' ~ ~ ) acting by scavenging chain-propagating peroxyl free radicals (Lipid- OO.)I7*). Hence it is widely assumed that LO inhibition by antioxidants is due to scavenging of similar radicals of the substrate that are generated within the active site of the enzyme36*163.173,174). Some studies suggest a relationship between LO inhibition and the ability of the inhibitors to reduce Fe3+ at the active site to the catalytically inactive

The largest and most thoroughly studied class of natural LO inhibitors (Fig. 9) are the flavonoids, cirsiliol being the most potent repre~entative'~~), other examples are caffeic acid178), and nordihydroguaiaretic acid179) (NDGA, meso- form). The latter compound has been used extensively as a standard to compare LO inhibitors to. Furthermore, there are structurally diverse compounds such as phenidone and BW755C leading to one-electron oxidation species which play a key role in LO inactivationIs0). Since superoxide dismutase decreases the rate of LO inactivation by these compounds, and moreover, even a direct effect of active oxygen species on LO has been demonstrated181.182), superoxide

Fez+ 175-177)

radical or H202 may be responsible for LO inactivation by these compounds. Further examples for redox inhibitors are antioxidant-based compounds which have been designed using the antioxidant di-tert-butylhydroxytoluene as a tem- plate132,144, 183-185)

NDGA

Fig. 9 Antioxidants as 5-LO inhibitors

5.3.3. Iron Chelators

Corey has synthesized analogs of AA bearing a hydroxamic acid function (Fig. 10) and has demonstrated that these compounds inhibit LO186). Since hydroxamic acids are excellent ligands for Fe(III), inhibition is presumably related to its coordination with a catalytically crucial Fe3+- ion. The full eicosanoid chain is not required for effective inhibition.

H mN'" yc-0

Bufoxsmsc

Fig. 10: Hydroxamic acids as 5-LO inhibitors

Subsequent studies by S ~ m m e r ~ ~ ~ ~ ~ ~ ~ ~ - ~ ~ ~ ) devised a sim- ple hypothesis about the nature of enzyme-inhibitor in which the structures of inhibitors were aligned to a proposed AA conformation when bound to the active site of 5- LO141). In addition, quantitative structure-activity relationships were discussedlg7). The only compound with a hydroxamic acid function used as a drug to date is bufex-


12 Miiller

amac, although with an ICsO of 27 pM markedly less potent than other derivative~'~~). Further improvements of hydroxamic acid derivatives have led to orally active compounds such as hydroxyurea derivative^'^^'^').

However, recently it has been reported that this class of inhibitors also features an antioxidant component, and it has been demonstrated that hydroxamic acid-containing 5-LO inhibitors are able to reduce the active-site-Fe'+ of soybean LO'77). During this process the NOH-moiety is oxidized to the corresponding nitroxide"".

OH

'I' L ETYA

enantiomers have differential effects on 5-LO. Examples are (methoxyalkyl)thiazoles, which represent the first class of inhibitors in which inhibition is mediated by specific, enantioselective interactions with the e n ~ y m e ' ~ ' . ' ~ ~ ) . The (+)-enantiomer of ICI 216800 (Fig. 12) was 50-150 fold more potent in various 5-LO assays than the (-)-enantio- 131e1-I~~). Another report of stereoselective 5-LO inhibition came from a series of indazolinones in which the R-enantiomer of a 1-naphthylethyl derivative (Fig. 12) was the eutomerIw).

0

ICI 218800

Fig. 12: Enantiospecific 5-LO inhibitors 0

6. Lipoxygenase Inhibitors as Future Antipsoriatic Drugs OH

15-HETE-Anslog S,&Methano-LTA, The viability of 5-LO inhibition has been most widely recognized in the treatment of psoriasis, and a variety of

Fig. 11: Substrate and product analogs as 5-LO inhibitors compounds now identified as LO inhibitors are targeted for topical or systemic treatment of this disease2m'.

5.3.4. Substrate and Product Analogs 6.1. Anthrones

This inhibitor class comprises two types of compounds, which have provided valuable information on the mechanism of the lipoxygenation reaction, but have limited potential as drugs because of poor in virro activityIs2).

On the one hand they are based on AA as substrate that either competes for the active site of the enzyme, but is not converted by the enzyme, or the analog is acted on by the LO enzyme to form a reactive species which gives rise to suicide inhibi t i~n '~~) . The other type imitates LO products such as LTA4, 5-HETE and 15-HETE which have been reported to inhibit the enzyme. In this case the LO enzyme is regulated by a feedback An example of a substrate analog is 5,8,11,14-eicosatetraynoic acid45-'94) (ETYA), which irreversibly inactivates LO. Examples of product analogs are 5,6-methan0-LTA~'~~) and the analog of 15-HETE"6' (Fig. 11).

5.3.5. Structurally Unrelated Compounds

Finally, there are many structurally unrelated compounds that have been prepared with the goal of developing LO inhibitors or have been discovered as such. Because of the structural diversity of these compounds and the fact that they do not act upon the LO enzyme by the mechanisms described above, a classification is not possible. Some of these compounds may be representatives of a new class of enantioselective inhibitors, since it has been reported that

Anthralin (dithranol, 1,8-dihydroxy-9( 1 OH)-anthrace- none) is among the most widely used drugs in the treatment of psoriasis. However, patient compliance is limited by its undesirable irritant effects and staining of the skin2"). In order to design new compounds based on anthralin it was desirable to develop a mechanistic working hypothesis for the molecular mode of action of this antipsoriatic drug. The biochemical basis for the mode of action of anthralin and induction of side effects is uncertain, but there is growing evidence that they are related to its redox activity leading to the production of anthralin free radical and active oxygen species2"). These latter species which include singlet oxy-

superoxide a n i ~ n ~ ' ~ ~ ~ ' ~ ) , hydrogen peroxide204), and hydroxyl r a d i ~ a l ~ ' ~ ~ ~ ~ ~ ) , are all formed by anthralin under aerobic conditions. In the following the test systems used to characterize the redox properties of antipsoriatic anthrones and techniques applied to quantify the pro- and antioxidant effects, the implication in LO inhibition, and the application of this information to drug design will be discussed.

An analysis of the structural factors in the drug chromo- phore has helped to elucidate the characteristic parts of antipsoriatic anthrones responsible for formation of active oxygen species2'*). The methylene moiety at C-10 initiates the production of superoxide radical, whereas singlet oxygen sensitizing properties are related to the anion of the hydroxyl group at C-1 associated with the carbonyl group.

Arch. Pharni. (Weitiheini) 327.3-19 (1994)


Two reasonable mechanistic possibilities for LO inhibition by anthralin have to be considered: Firstly, as stated above, compounds possessing redox properties can be expected to inhibit the LO reaction by reducing a radical

reductive inactivation of the enzyme from the ferric to the ferrous state may be important in the regulation of LO activity (Fig. 13).

species. Because anthralin is a one electron donator2"), H +

7 1 U

H

Lipoxygenase 5-HETE kachiinic Acid +- LTB,

H 1 PHETE

e l Fig. 14: LO inactivation by active oxygen species

H

I OH 0' OH

Fig. 13: Anthralin as a one-electron donator for LO in the activated high spin-Fe(II1) state

Secondly. in view of the instability of anthralin it is unlikely that the molecule itself is responsible for the biological properties. Preferentially, activated species arising from anthralin autoxidation are thought to mediate its a ~ t i o n ~ ~ ~ , ~ ~ ~ ) . Since no single mechanism is operative, it may be concluded that all known biological effects of the drug represent a common mechanism caused by "activation" of anthralin and generation of reactive species. There is evidence that enzyme inactivation by active oxygen species is quite possible (Fig. 14). 1) Larger amounts of 5-LO products were observed when mast cells or macrophages were first treated with catalase or superoxide dismutase and then stimulated, suggesting that superoxide and possibly hydroxyl radicals may inactivate the leukotriene path- way16). 2) 12-LO from bovine platelets was markedly inhibited by oxygen radicals and H 2 0 2 at micromolar concentrations's'), whereas 5-LO from bovine PMNL was only mod- erately influenced by active oxygen species. On the other hand, singlet oxygen was completely ineffectiveIs'). In addition, H202 suppressed the production of 12-HETE in rabbit platelets'82). 3) Because LO inhibition by standard inhibitors such as phenidone and BW755C (Fig. 9) only occurs after oxidation of these compounds, it has been suggested that reactive species formed during this process, inter alia superoxide radical and H202, may be responsible for LO inactivation'80).

Syntheses and evaluation of new anthralin analogs were undertaken in which one or both active methylene protons at C- 10 were replaced by suitable substituents which permit control over the release of active oxygen species. The following evaluation methods have been used to characterize the pro- and antioxidant effectszo7): Pro-oxidant activity was determined using the deoxyribose assay, which is a sensitive test for the production of hydroxyl radicals2"). The release of thiobarbituric acid reactive material (expressed as malondialdehyde, MDA) is a measure for hydroxyl radical formation (Fig. 15)212-214). Antioxidant (radical scavenging) activity of the test compounds was studied by inhibition of lipid peroxidation in liposomes stimulated by Fe3+/ascorbic acid (Fig. 15)2'7,21x). In addition, the free radical scavenging activity was determined by kinetic studies on the reduction of the stable free radical, 2,2-diphenyl- I -picrylhydra- zy12'5,2'6) (DPPH, Fig. 16) by the test compounds.

Phenylacyl substitution at C-10 of anthralin substantially reduced redox activity as compared to anthralin in accordance with decreased formation of oxygen radicals. Thus, deoxyribose degradation was diminished by a factor of 15. On the other hand, substitution on the phenyl moiety of the side chain by two or three hydroxyl groups dramatically increased redox activity reflecting the reducing capability of the phenolic h y d r ~ x y l ~ ' ~ ) . Substitution of both C-10 hydrogen atoms of anthralin by phenylalkyl groups abol- ished redox activity as well as biological efficacy.

Generally, the antioxidant approach, especially for orally active compounds, is controversial because of possible side effects'"). However, several topically active 5-LO inhibitors have shown clinical efficacy with no reports on side effec ts40).

Among our new anthralin derivatives are highly potent 5- LO inhibitors that can be considered as redox active (e .g . the pyrogallol derivative in Fig. 17) and non-redox active (e.g. 10-phenylbutyrylanthralin, Fig. 17). The IC,o values for 5-LO inhibition in bovine PMNL are 0.3 pM each2I9). Thus, the presence of the phenolic hydroxyl in the attached phenyl rest is not required for biological activity and two classes of compounds that act by different mechanisms but are both active 5-LO inhibitors were obtained. In particular,

Arch. Pharm. (WeinheinlJ327,3-19 (1994)

14 Miiller

Fig. 15: Tests to characterize redox properties of 5-LO inhibitors

RH - R ' DPPH

Fig. 16: Reduction of the stable free radical DPPH to the corresponding hydrazine by an antioxidant

the non-redox active compounds may be expected to be devoid of skin imitating properties, if active oxygen species such as hydroxyl radicals are responsible for the inflammation of the healthy skin. For these compounds no appre- ciable hydroxyl radical production was measured as compared to anthralin. Because redox inhibitors are oxidized during their action on LO, their stability is limited. Hence it is of importance to develop LO inhibitors acting by a non- redox mechanism.

Structure-activity relationships of the 10-phenylacyl series showed that a terminal aromatic ring is required for activity. The mere presence of an acyl-substituent at C-10 of anthralin does not necessarily lead to enhanced 5-LO inhibition. Furthermore, the potency of the non-redox active inhibitors increased with the length of the acyl chain with three methylene units being the optimum, suggesting a specific enzyme interaction which would not be expected for nonspecific redox inhibitors.

o i s 2 \TI4

0

HO OH

lq3,4,5-Trlhydroxyphenyl> acetylanthralln

0

532 nm

Fig. 17: Redox-active and non-redox active anthrones

6.2. Naphthols, Phenols, and Quinones

Similar to anthralin, 6-chloroisonaphthazarin is an inhibitor of 12-LO in epidermal mouse h~mogenate"~) with an IC50 of 25 pM, and this compound is a potent antipsoriatic agent but suffers from drawbacks such as staining and irri- tating of the skin129). Modification of this compound resulted in the development of lonapalene (Fig. 6), the efficacy of which was established in Phase 111 clinical trials. The activity of lonapalene as a 5-LO inhibitor has been confir- med in human PMNL (IC50 = 15 pM) and RBL cells (IC50 = 0.5 F M ) ' ~ ~ ) . In addition, analysis of skin chamber fluid samples showed a significant reduction in levels of LTB4 from lonapalene treated psoriatic lesions, but 12-HETE levels were not reduced220).

Structure-activity relationships revealed that net lipophilicity played an important role, compounds with log P values greater than 5.5 were less active. Hydrolysis products of



8 OAc

CI a; cl&@ 0

and RBL cells with an ICsO of 0.4 pM and 0.1 pM, respectively. Human platelet 12-LO was inhibited with an ICsO of 5.9 pMI4O). Furthermore, this compound blocks the Ca-ionophore induced increase of LTB4 and epidermal hyperproliferation in the guinea pig ea?25). The observed 5-LO inhibition in the 2,3-dihydrobenzofuranol series was suggested to be dependent on the ability of the inhibitor to reduce a radical species kinetically controlled, and on the fraction of inhibitor that is partitioned into the cell membrane224).

The most prominent compound of the quinones is docebe- none (AA 861), which selectively inhibits 5-LO of guinea- pig peritoneal leukocytes (IC50 = 0.8 pM) without inhibiting 12-LO from bovine platelets226). However, it is interest- ing that it does inhibit the mouse epidermal 12-LO (ICso = 1.9 PM)’~).

6.3. Hydroxamic Acids and Hydro.ylamines

The 9-phenylnonanohydroxamic acid BMY 30094 (Fig. 19), which has been selected for clinical trials in psoriasis, is an inhibitor of human PMNL and shows local anti- inflammatory activity2”).

DUP (154 La1896

0

0

Fig. 18: Naphthols, phenols, and quinones as LO inhibitors

lonapalene (monoesters) are implicated as the putative 5- LO inhibitory species. Introduction of a fused 2,3-alkyli- denedioxy ring (Fig. l8), in place of the 2,3-dimethoxy groups, caused a modest diminution in potency221).

In a similar series of 1,4-naphthohydrquinones, bunapro- last (Fig. 18) and an analog (l-acetoxy-2,3-diethy1-4- methoxynaphthalene) were the most potent representatives in ex vivo LTB4 inhibition in Ca-ionophore stimulated blood from rats222).

2-Benzyl- 1 -naphthol (DuP 654) has been selected for clinical evaluation as a topical antipsoriatic agent on the basis of its pharmacological profile223). This compound is among the most potent 5-LO inhibitors with an ICso of 0.019 pM in the RBL-1 5-LO assay142). Structure-activity relationships showed that the 1 -naphthol ring system was required for activity whereas the identity of the 2-substituent was relatively unimportant, as long as it was lipophilic and linked to the naphthol through a non-oxidized carbon atom142). Using soybean LO as a model, EPR spectroscopy revealed that these compounds exert a significant amount of their potency by reducing the LO active-site iron from the active ferric state to the inactive ferrous state177). Increas- ingly electron-withdrawing 4-substituents at the naphthol ring decreased the oxidation potential and lessened this ability. 2,3-Dihydro-5-benzofuranols have provided potent 5-LO

inhibitor^'^'.^^^). L-65 1896 inhibits 5-LO in human PMNL

OA 208-199

Fig. 19: Hydroxamic acids and hydroxylamines

In a series of hydroxylamines QA 208-199 (Fig. 19) was the most potent 5-LO inhibitor in human PMNL (IC50 = 0.4 pM)228) and significantly inhibited the formation of LTB4 and mono-HETEs from [I4C]-AA incubated with psoriatic skin h o m o g e n a t e ~ ~ ~ ~ ) . The mechanism of inhibition was proposed to involve either interaction of the hydroxylamine group with the radical generating center of the enzyme by H’ donation leading to the reduced enzyme or formation of a partial “one-electron bond” between the electron lone pairs of the 0-atom with the radical; alternatively, iron che- lation was considered228).

6.4. Heterocycles

The phenothiazinone L-651392 (Fig. 20) is a potent inhibitor of purified 5-LO from porcine leukocytes (IC50 = 0.3- 0.5 pM)230), but the inhibition is dependent on the presence of NADH, suggesting the reduced hydroxyphenothiazine to be the active form. Moreover, inhibition of the Ca-ionophore induced epidermal hyperprofileration in guinea-pig skin was reportedzz5).

Arch. Pharm. (Weinheim) 327,3-I9 (1994)

16

CHSO aNV” S 6r 0

L-651392

8

9

10 1 1

12

13 14

15

16

17

R 68151

Fig. 20: Miscellaneous heterocycles as 5-LO inhibitors

18 19

20 21

22 23

24 25

26 A quinoline-containing inhibitor of 5-LO in human

PMNL (IC50 = 0.02 pM), ETH-615 (Fig. 20), is also an inhibitor of interleukin-8 produ~t ion~~.~”) . This compound is in clinical development as a topical agent for the treatment of psoriasis and other skin diseases.

The selective topical 5-LO inhibitor R 68 15 1 (Fig. 20) has been shown to improve psoriasis232’.

Conclusion

27

28 29 3o

31 32

33

The development of new antipsoriatic drugs is still hin- dered by lack of knowledge of the pathogenesis of psoriasis. Moreover, no satisfactory strict animal model that resembles the disease is available. Recent findings suggest a role for LO products, in particular LTB4 and 12-HETE, in the pathogenesis of psoriasis. Research into new treatments is focused on the manipulation of existing drugs to reduce adverse effects and on the development of new compounds, a number of which are under investigation regarding their ability to inhibit the formation of LO products.

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