- Email: [email protected]
Second-generation beta-oxidation resistant 3-oxa-lipoxin A4 analogs
ARTICLE IN PRESS
Prostaglandins, Leukotrienes and Essential Fatty Acids 73 (2005) 245–250 www.elsevier.com/locate/plefa
Second-generation beta-oxidation resistant 3-oxa-lipoxin A4 analogs William J. Guilforda,, John F. Parkinsonb a
Department of Medicinal Chemistry, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA b Department of Immunology, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA
Abstract Lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 are structurally and functionally distinct eicosanoids, with potent antiinflammatory and immunomodulatory actions. Therapeutic use of LXA4 is greatly limited by its rapid metabolism in vivo and chemical instability. First-generation synthetic LXA4 analogs such as methyl (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate (2, ATLa), were designed to minimize metabolism from the o-end of the molecule. Pharmacokinetic analysis of ATLa revealed b-oxidation as a novel route for LXA4 metabolism, prompting the development of second-generation 3-oxa-LXA4 analogs with improved pharmacokinetic disposition. Second-generation 3-oxa-LXA4 analogs such as (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-3-oxa-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoic acid (3), have shown potency and efficacy comparable to ATLa in diverse animal models after topical, intravenous or oral delivery. These include several acute (2–24 h) inflammatory reactions: calcium ionophore-induced skin edema and inflammation (topical), LTB4/PGE2-induced skin inflammation and vascular leak (topical), zymosan A-induced peritonitis (i.v. and oral) and ischemia-reperfusion-induced secondary organ injury (i.v.). Remarkably, 3-oxa-LXA4 analogs have potent once daily oral efficacy in preventing and promoting the resolution of established colitis induced by the hapten trinitrobenzene sulphonic acid (TNBS), an acute/chronic 7–14-day model of Crohn’s disease. The second-generation 3-oxa-LXA4 analogs thus provide new stable pharmacophores with which to explore the emerging role of lipoxins as a new therapeutic principle for regulating inflammation, allergy and immune dysfunction in preclinical and clinical research. r 2005 Elsevier Ltd. All rights reserved.
1. Introduction
2. Design of second-generation LXA4 analogs
Lipoxins constitute the first recognized class of endogenous anti-inflammatory lipid-based autacoids, which function as endogenous ‘‘stop signals’’ that downregulate or counteract the formation and actions of pro-inflammatory mediators [1] and promote the resolution of inflammation [2]. The development of lipoxin A4 (1, LXA4) as a therapeutic agent, however, has been limited by its metabolic and chemical instability. In this review, we describe the design and in vivo evaluation of our second-generation LXA4 analogs.
First- and second-generation LXA4 analogs are listed Fig. 1 [3]. First-generation analogs were designed to minimize rapid inactivation of LXA4 by metabolism via o-oxidation or oxidation at the 15(S)-alcohol by prostaglandin dehydrogenase (PGDH) [4]. This was achieved by replacing the alkyl tail with a fluorinated phenoxy group and by inverting the stereochemistry at C-15 to the R isomer (15-epi). The stereochemical inversion at C-15 was expected to increase metabolic stability and not expected to reduce the bioactivity of the analog due to the discovery that 15-epi- or aspirintriggered LXA4 was equipotent in assays to LXA4, but was a poorer substrate for PGDH. These structural elements were combined in the first-generation LXA4 analogs as seen in 2, methyl (5R, 6R, 7E, 9E, 11Z, 13E, 15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7, 9, 11,
Corresponding author. Tel.: +1 510 669 4065; fax: +1 510 669 4310. E-mail address: [email protected] (W.J. Guilford).
0952-3278/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2005.05.012
ARTICLE IN PRESS W.J. Guilford, J.F. Parkinson / Prostaglandins, Leukotrienes and Essential Fatty Acids 73 (2005) 245–250
246
OH
HO
OH
O OH
3
15 1, lipoxin A4 OH
HO
OH
O
O X
F
O
R
2, ATLa,X = CH2, R =Me 3, ZK-142, X= O, R = H 7, X = O,R= Me HO
F
generation 3-oxa-LXA4 analog 3 was designed, which incorporated the tetraene structure. Despite providing a single stable pharmacophore in vivo, the pharmacokinetic profile of 3 was similar to 2, suggesting tetraene has an intrinsically short half-life in vivo. Similar biological activity with a better pharmacokinetic profile was seen for the corresponding E,E,E-trien-11-yne 3-oxa-LXA4 analog 4.
3. Chemistry O
OH X
O
R
O OH 6, X = CH2, R = H 8, X = CH2, R =Me 4, ZK-994, X = O, R = H Fig. 1. First- and second-generation LXA4 analogs. The figure shows the structure of endogenous lipoxin A4 (1), first-generation analogs (2,6,8), and second-generation 3-oxa-lipoxin analogs (3,4,7).
13-hexadecatetraenoate (15-epi-16-(para-fluoro)-phenoxy-LXA4 (referred to in the literature as ATLa or ATLa2). The potent anti-inflammatory and immunomodulatory properties of 2 were demonstrated in a series of in vivo models. These included ovalbumininduced allergic airway inflammation and hyperreactivity [5], granulocyte and T-cell-dependent skin reactions [6], the acute phase of dextran sodium sulfate (DSS)induced colitis [7], and a lethal T-helper type-1 (Th1)driven adaptive immune response to Toxoplasma gondii infection in 5-lipoxygenase-deficient mice [8]. Taken together, these studies provide evidence that 2 has pronounced anti-inflammatory effects in granulocytedriven acute reactions and that it directly or indirectly modulates T-cell effector functions both in the setting of adaptive Th1- and allergic Th2-dependent immune dysfunction. Although 2 was efficacious in these models it was cleared from the mouse within 15 min after intravenous injection [9]. Moreover, the oral pharmacokinetic disposition of 2 was not described and its potential for systemic uses not fully elaborated. A more complete pharmacokinetic profile for 2 was measured after i.v. and p.o. dosing and showed rapid oral absorption, but with a short plasma half-life [3]. Careful analysis of the plasma samples indicated the presence of a secondary metabolite, which was consistent with a b-oxidation product and with lipid metabolism seen in the prostaglandin [10,11] and leukotriene [12] fields. To eliminate this catabolic pathway [13] and to potentially increase the in vivo half-life, a second-
Since LXA4 was first reported in 1984 [14], the trihydroxytetraene structure has attracted the attention of synthetic chemists, as seen in the number of reported syntheses for 1 [15–18] and 2 [19]. The synthetic challenge with 2 is to prepare a linear chain of 17 carbons, in which all but three carbon atoms are either unsaturated or substituted with oxygen. The challenge is slightly different with the 3-oxa analogs, 3 and 4, in that all of the carbon atoms in the chain are either unsaturated or substituted with oxygen. The synthesis of the 3-oxa-lipoxin analogs is based on the Nicolaou synthesis of LXA4 (1) [20] and reported earlier [3].
4. Efficacy of 3-oxa-LXA4 analogs in inflammation models—i.v. dosing The efficacy of ATLa and the new 3-oxa-LXA4 analogs in animal models of inflammation is summarized in Table 1. Replacement of the cis-11, 12-double bond in 2 for the corresponding trienyne in 6 changes the shape of the lipoxin analog and could alter receptor binding. So the in vivo activity of 2 and 6 were compared in the TNFa-induced inflammatory reaction in the murine dorsal air pouch [22–24]. Both 2 and 6 were equally efficacious, significantly decreasing the total number of leukocytes and neutrophils infiltrating into the air pouch [3]. With the activity of both the tetraene and the trienyne analogs established for the first-generation LXA4 analog series, the 3-oxa analogs were evaluated in lung inflammation after hind-limb ischemia-reperfusion using an i.v. dosing protocol (500 mg/kg prior to ischemia plus 500 mg/kg prior to reperfusion) [25]. The inhibition of lung inflammation was enhanced with novel analogs 3 and 4. While 2 reduced inflammation by only 17%, the novel analogs, 3 and 4, demonstrated 32% and 53% inhibition, respectively. These results indicate that the 3-oxa substitution enhances this lung protective impact of LXA4 analogs [26]. Examination of lung histology indicates that compound 3 reduced leukocyte accumulation, edema, and tissue injury in the lung tissue.
ARTICLE IN PRESS W.J. Guilford, J.F. Parkinson / Prostaglandins, Leukotrienes and Essential Fatty Acids 73 (2005) 245–250
247
Table 1 Summary of 3-oxa-LXA4 analog efficacy in inflammation models Modela
Route of administration
LXA4 or 3-oxa-LXA4 analog and dose
Mouse air-pouch model of inflammation [3] Calcium ionophore-induced acute skin inflammation [3] LTB4/PGE2stimulated acute ear skin inflammation [25] Ischemia-reperfusion-induced lung injury [25] Zymosan A-induced peritonitis [25] Zymosan A-induced peritonitis [25] TNBS-induced colitis [30]
Local Topical Topical Intravenous Intravenous Intragastric Intragastric
2 and 6 efficacious at 10 ng/pouch 2, 3, and 4 efficacious at 300 mg/cm2 2, 3, and 4 efficacious at 20 mg/ear 3, 4 efficacious at 500 mg/kg 2, 7, 8 efficacious at 50 mg/kg 1, 2, 4 efficacious at 5 mg/kg 3 efficacious at 300–1000 mg/kg
a
Data derived from the noted reference.
500 400 300 200 * *
4
3
300
100
300
100
600
300
100
Ionophore
*
*
600
*
0
600
100
vehicle
Elastase Activity (pmol AMC)
Neutrophil Infiltration
2
Topical Dose (µg/cm2)
Fig. 2. Topical ATLa and 3-oxa-LXA4 analogs prevent neutrophil infiltration in calcium ionophore-induced ear inflammation in mice. The figure shows the increase in elastase content as a marker of neutrophil infiltration in mouse ears 24 h after topical application of calcium ionophore. Topical co-application of ATLa (2) or the tetraene (3) or the trienyne (4) 3-oxa-LXA4 analog dose-dependently prevents neutrophil infiltration with ED50 o300 mg/cm2 for all compounds tested. Data are redrawn and reproduced with permission from Guilford et al. [3]. Copyright 2004 Am. Chem. Soc.
5. Efficacy of 3-oxa analogs in inflammation—topical dosing Topical efficacy for native LXA4 and 2 has been demonstrated in several models of skin inflammation [6]. The calcium ionophore model was chosen to compare 3oxa analogs 3 and 4 with 2. Calcium ionophore A-23187 applied topically to the dorsal surface of mouse ears of mouse ears induces acute inflammation with edema and granulocyte infiltration that peaks at approximately 24 h. Anti-inflammatory effects were determined by measuring inhibition of edema formation, granulocyte infiltration, and neutrophil infiltration (Fig. 2). The data clearly show that 3-oxa analogs 3 and 4 inhibited neutrophil infiltration in a dose-dependent manner and at similar doses to 2. Neutrophil infiltration was completely inhibited at the highest dose tested. The
IC50 for 2, 3, and 4 on all three topical efficacy parameters was o300 mg/cm2. The novel 3-oxa analogs can thus substitute for carbon analogs in a topical model of inflammation. The topical efficacy of 2, 3, and 4 was confirmed in blocking dermal inflammation induced by LTB4 plus PGE2, as indicated by marked reductions in neutrophil infiltration and vascular leak [25]. Results on vascular leak are shown in Fig. 3. At 20 mg/cm2, 2 appeared to have the most robust efficacy on both endpoints, being somewhat more effective than 3. The trienyne 4 appeared less effective than either tetraene compound, with a slower and less robust effect on vascular leak. This result suggests the absorption or penetration of 4 into mouse ear skin tissue is slower than for the tetraene analogs. In line with earlier observations [27], neither 2 (20 mg; 1 mg/kg) nor 3 (20 mg; 1 mg/kg)
ARTICLE IN PRESS W.J. Guilford, J.F. Parkinson / Prostaglandins, Leukotrienes and Essential Fatty Acids 73 (2005) 245–250
248
30
80 60 40 20 0 2 3 4 Topical Lipoxin Analog, 20 ug/ear
Fig. 3. ATLa and 3-oxa-LXA4 analogs block LTB4+PGE2-induced vascular leak in mouse ears. The figure shows the % inhibition of LTB4+PGE2-induced Evans blue dye accumulation in mouse ear homogenates as a measure of vascular leak at 7 (black bars) and 20 h (grey bars). LTB4+PGE2 was topically with 20 mg ATLa (2) or the tetraene (3) or the trienyne (4) 3-oxa-LXA4 analogs. Data are redrawn and reprinted by permission from Bannenberg et al. [25]. Copyright 2004 Macmillan Publishers Ltd.
Change of body weight (%of basal)
% Inhibition Vascular Leak
100
20 10
**
**
0 -10 ** -20 -30
* control
10
0.1
0.3
1
TNBS
mg/kg prednisolone, p.o. once daily mg/kg trienyne analog 4,p.o.once daily
gave statistically significant inhibitory effects with LTB4 plus PGE2-initiated vascular leakage in the outer ear when administered by tail vein injection. These results indicate specific local effects of lipoxins in dermal inflammation that cannot be achieved by systemic delivery, and imply direct local actions of lipoxins on skin cells.
Fig. 4. Trienyne 3-oxa-LXA4 analog inhibits weight loss in TNBSinduced colitis in mice. The figure shows the dose-dependent prevention of weight loss in TNBS-induced colitis in mice by the trienyne 3-oxa-LXA4 analog (4) at 0.1, 0.3 and 1 mg/kg in comparison to 10 mg/kg prednisolone. Both agents were administered by oral gavage once daily from day 1 to day 14. Control mice gained some weight by day 14, but TNBS-treated mice receiving vehicle lost 20% of their body weight (P ¼ 0:05 versus control). Animals treated with 10 mg/kg prednisolone and 0.3 or 1 mg/kg 3-oxa-LXA4 analog had significant protection from TNBS-induced weight loss ( Po0:05 versus TNBS plus vehicle). Data are redrawn from Fiorucci et al. [30]. Copyright 2004 National Academy of Sciences, USA.
6. Efficacy of 3-oxa analogs in inflammation—oral dosing To assess the extent to which lipoxins and their analogs can be used as potential systemic anti-inflammatory agents after oral administration, 2, 3, and 4, as well as native LXA4 and LXB4 were all evaluated for oral efficacy in acute zymosan-induced peritonitis in [25]. Systemic anti-inflammatory effects in the peritonitis model were observed with doses as low as 1–10 ng/ mouse (50–500 ng/kg), with increasing effects at higher doses (5–500 mg/kg). Similar degrees of systemic antiinflammatory efficacy of 2, 3 and 4 were observed whether administered intravenously or orally via intragastric administration. The oral efficacy of the tetraene analogs is consistent with their pharmacokinetic profiles [3], but is surprising given the acid-labile nature of the tetraene pharmacophore [28]. These tetraene analogs degrade rapidly in aqueous buffers at gastric pH 1.5 (W. Guilford and J. Bauman, unpublished results). Nevertheless, all these tetraenes are rapidly absorbed after intragastric oral administration, so they must clearly be protected from acid-catalyzed degradation in the gastric environment. Human neutrophils appear to have an LXA4 transporter [29] and it can be speculated that a similar mechanism may be present in the GI tract that facilitates LXA4 and LXA4 analog absorption. The oral bioavail-
ability of lipoxins has implications for understanding the results of other studies, such as the efficacy of 2 in the acute phase of dextran sodium-sulfate-induced colitis in mice [7]. In the colitis study, 2 was administered ad libitum at 10 mg ml1 in the drinking water for an estimated systemic exposure of 50 mg/kg/day (based on typical daily water consumption rates in mice and the oral absorption profile). Based on the potent oral effects of 2 in acute peritonitis, the efficacy of 2 in the colitis model can be assumed to be a sum of immunomodulatory effects after systemic absorption and topical effects in the GI tract due to local actions of 2 that remains in GI transit. The efficacy of the trienyne 3-oxa-LXA4 analog 4 in an animal model of Crohn’s disease was recently established [30]. At daily oral doses of 0.3–1 mg/kg/day analog 4 was as efficacious as 10 mg/kg daily oral prednisolone in preventing (dosing days 1–7) and treating (dosing days 3–14) the severe transmural colon disease observed in this model. Data on weight loss are shown in Fig. 4. Detailed analysis of mucosal markers in immunocompetent Balb/c mice and T- and B-lymphocyte-deficient SCID mice revealed pronounced antiinflammatory and immunomodulatory actions of analog 4. Efficacy was correlated to attenuation of proinflammatory chemokine/cytokine networks and the
ARTICLE IN PRESS W.J. Guilford, J.F. Parkinson / Prostaglandins, Leukotrienes and Essential Fatty Acids 73 (2005) 245–250
prevention of Th1-type immune dysfunction driven by myeloid and lymphoid cellular subsets. 7. Conclusion The second generation, 3-oxa-lipoxin analogs, 3 and 4, which were designed to increase the metabolic and chemical stability of LXA4, are equipotent to 2 in animal models of inflammation. Metabolic stability was improved by eliminating metabolism by b-oxidation and chemical stability by replacing the tetraene unit. Taken together, the results provide new insights on the in vivo catabolism of lipoxins and structure–activity relationships essential for in vivo actions. Second-generation 3oxa-LXA4 analogs provide superior pharmacophores that can be dosed via oral, topical and systemic routes. The 3-oxa-analogs represent state-of-the-art tools with which to explore pre-clinical and clinical concepts for lipoxins in the resolution of inflammation, immunomodulation, allergy, adaptive immunity and autoimmunity.
Acknowledgements The authors wish to acknowledge the support of Drs. H. Daniel Perez and Gary B. Phillips for this work. References [1] C.N. Serhan, in: P. Hedqvist, J.R. Kalden, R. Mu¨ller-Peddinghaus, D.R. Robinson (Eds.), Advances in Rheumatology and Inflammation, Eular, Basel, 1991, pp. 141–153. [2] C.N. Serhan, E. Oliw, Unorthodox routes to prostanoid formation: new twists in cyclooxygenase-initiated pathways, J. Clin. Invest. 107 (2001) 1481–1489. [3] W.J. Guilford, J.G. Bauman, W. Skuballa, et al., Novel 3-oxa lipoxin A4 analogs with enhanced chemical and metabolic stability have anti-inflammatory activity in vivo, J. Med. Chem. 47 (2004) 2157–2165. [4] C.B. Clish, B.D. Levy, N. Chiang, H.H. Tai, C.N. Serhan, Oxidoreductases in lipoxin A4 metabolic inactivation: 15-oxaprostaglandin 13-reductase/leukotriene B4 12-hydroxydehydrogenase is a multifunctional eicosanoid oxidoreductase, J. Biol. Chem. 33 (2000) 25372–25380. [5] B.D. Levy, G.T. De Sanctis, P.R. Devchand, et al., Multi-pronged inhibition of airway hyper-responsiveness and inflammation by lipoxin A4, Nat. Med. 8 (2002) 1018–1023. [6] A.J. Schottelius, C. Giesen, K. Asadullah, et al., An aspirin-triggered lipoxinA4 stable analog displays a unique topical anti-inflammatory profile, J. Immunol. 168 (2002) 7064–7070. [7] A.T. Gewirtz, L.S. Collier-Hyams, A.N. Young, et al., Lipoxin A4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis, J. Immunol. 168 (2002) 5260–5267. [8] J. Aliberti, C.N. Serhan, A. Sher, Parasite-induced lipoxin A4 is an endogenous regulator of IL-12 production and immunopathology in Toxoplasma gondii infection, J. Exp. Med. 196 (2002) 1253–1262.
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[9] C.B. Clish, J.A. O’Brien, K. Gronert, G.L. Stahl, N.A. Petasis, C.N. Serhan, Local and systemic delivery of a stable aspirintriggered lipoxin prevents neutrophil recruitment in vivo, Proc. Natl. Acad. Sci. USA 96 (1999) 8247–8252. [10] C.K. Ellis, M.D. Smigel, J.A. Oates, O. Oelz, B.J. Sweetman, Metabolism of prostaglandin D2 in the monkey, J. Biol. Chem. 254 (1979) 4152–4163. [11] S. Sturzebecher, M. Haberey, B. Muller, et al., Pharmacological profile of a novel carbacyclin derivative with high metabolic stability and oral activity in the rat, Prostaglandins 31 (1986) 95–109. [12] Y. Guindon, D. Delorme, C.K. Lau, R. Zamboni, Total synthesis of LTB4 and analogues, J. Org. Chem. 53 (1988) 267–275. [13] W. Skuballa, E. Schillinger, C. St. Stuerzebecher, H. Vorbrueggen, Synthesis of a new chemically and metabolically stable prostacyclin analogue with high and long-lasting oral activity, J. Med. Chem. 29 (1986) 313–315. [14] C.N. Serhan, M. Hamberg, B. Samuelsson, Trihydroxytetraenes: a novel series of compounds formed from arachidonic acid in human leukocytes, Biochem. Biophys. Res. Commun. 118 (1984) 943–949. [15] J. Adams, B.J. Fitzsimmons, J. Rokach, Synthesis of lipoxins: total synthesis of conjugated trihydroxy eicosatetraenoic acids, Tetrahedron Lett. 25 (1984) 4713–4716. [16] E.J. Corey, W.G. Su, Simple synthesis and assignment of stereochemistry of lipoxin A, Tetrahedron Lett. 26 (1985) 281–284. [17] K.C. Nicolaou, S.E. Webber, A general strategy for the total synthesis of the presumed lipoxin structures, J. Chem. Soc. Chem. Commun. (1985) 297–298. [18] A.R. Rodriguez, B.W. Spur, Total synthesis of aspirintriggered 15-epi-lipoxin A4, Tetrahedron Lett. 42 (2001) 6057–6060. [19] E.D. Phillips, H.-F. Chang, C.R. Holmquist, J.P. McCauley, Synthesis of methyl (5R, 6R, 7E, 9E, 11Z, 13E, 15S)-16-(4fluorophenoxy)-5,6,15-trihydroxy-7, 9, 11, 13-hexadecatetraenoate, an analogue of 15R-Lipoxin A4, Bioorg. Med. Chem. Lett. (2003) 3223–3226. [20] K.C. Nicolaou, C.A. Veale, S.E. Webber, H. Katerinopoulos, Stereocontrolled total synthesis of lipoxins A, J. Am. Chem. Soc. 107 (1985) 7515–7518. [22] T. Takano, C.B. Clish, K. Gronert, N. Petasis, C.N. Serhan, Neutrophil-mediated changes in vascular permeability are inhibited by topical application of aspirin-triggered 15-epi-lipoxin A4 and novel lipoxin B4, J. Clin. Invest. 101 (1998) 819–826. [23] M. Hachicha, M. Pouliot, N.A. Petasis, C.N. Serhan, Lipoxin (LX)A4 and aspirin-triggered 15-epi-LXA4 inhibit tumor necrosis factor alpha-initiated neutrophil responses and trafficking: regulators of a cytokine-chemokine axis, J. Exp. Med. 189 (1999) 1923–1930. [24] C.N. Serhan, T. Takano, C.B. Clish, K. Gronert, N. Petasis, Aspirin-triggered 15-epi-lipoxin A4 and novel lipoxin B4 stable analogues inhibit neutrophil-mediated changes in vascular permeability, Adv. Exp. Med. Biol. 469 (1999) 287–293. [25] G. Bannenberg, R.-L. Moussignac, K. Gronert, et al., Lipoxins and novel 15-epi-lipoxin analogs display potent anti-inflammatory actions after oral administration, Br. J. Pharmacol. 143 (2004) 43–52. [26] N. Chiang, K. Gronert, C.B. Clish, J.A. O’Brien, M.W. Freeman, C.N. Serhan, Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins and aspirin-triggered lipoxins in reperfusion, J. Clin. Invest. 104 (1999) 309–316. [27] T. Takano, S. Fiore, J.F. Maddox, H.R. Brady, N.A. Petasis, C.N. Serhan, Aspirin-triggered 15-epi-lipoxin A4 (LXA4) and LXA4 stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors, J. Exp. Med. 185 (1997) 1693–1704.
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[28] C.N. Serhan, K.C. Nicolaou, S.E. Webber, C.A. Veale, S.E. Dahle´n, T.J. Puustinen, B. Samuelsson, Lipoxin A. Stereochemistry and biosynthesis, J. Biol. Chem. 261 (1986) 16340–16345. [29] L. Simchowitz, S. Fiore, C.N. Serhan, Carrier-mediated transport of lipoxin A4 in human neutrophils, Am. J. Physiol. 267 (1994) C1525–1534.
[30] S. Fiorucci, J.L. Wallace, A. Mencarelli, et al., A b-oxidationresistant lipoxin A4 analog treats hapten-induced colitis by attenuating inflammation and immune dysfunction, Proc. Natl. Acad. Sci. USA 101 (2004) 15736–15741.
Prostaglandins, Leukotrienes and Essential Fatty Acids 73 (2005) 245–250 www.elsevier.com/locate/plefa
Second-generation beta-oxidation resistant 3-oxa-lipoxin A4 analogs William J. Guilforda,, John F. Parkinsonb a
Department of Medicinal Chemistry, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA b Department of Immunology, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA
Abstract Lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 are structurally and functionally distinct eicosanoids, with potent antiinflammatory and immunomodulatory actions. Therapeutic use of LXA4 is greatly limited by its rapid metabolism in vivo and chemical instability. First-generation synthetic LXA4 analogs such as methyl (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate (2, ATLa), were designed to minimize metabolism from the o-end of the molecule. Pharmacokinetic analysis of ATLa revealed b-oxidation as a novel route for LXA4 metabolism, prompting the development of second-generation 3-oxa-LXA4 analogs with improved pharmacokinetic disposition. Second-generation 3-oxa-LXA4 analogs such as (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-3-oxa-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoic acid (3), have shown potency and efficacy comparable to ATLa in diverse animal models after topical, intravenous or oral delivery. These include several acute (2–24 h) inflammatory reactions: calcium ionophore-induced skin edema and inflammation (topical), LTB4/PGE2-induced skin inflammation and vascular leak (topical), zymosan A-induced peritonitis (i.v. and oral) and ischemia-reperfusion-induced secondary organ injury (i.v.). Remarkably, 3-oxa-LXA4 analogs have potent once daily oral efficacy in preventing and promoting the resolution of established colitis induced by the hapten trinitrobenzene sulphonic acid (TNBS), an acute/chronic 7–14-day model of Crohn’s disease. The second-generation 3-oxa-LXA4 analogs thus provide new stable pharmacophores with which to explore the emerging role of lipoxins as a new therapeutic principle for regulating inflammation, allergy and immune dysfunction in preclinical and clinical research. r 2005 Elsevier Ltd. All rights reserved.
1. Introduction
2. Design of second-generation LXA4 analogs
Lipoxins constitute the first recognized class of endogenous anti-inflammatory lipid-based autacoids, which function as endogenous ‘‘stop signals’’ that downregulate or counteract the formation and actions of pro-inflammatory mediators [1] and promote the resolution of inflammation [2]. The development of lipoxin A4 (1, LXA4) as a therapeutic agent, however, has been limited by its metabolic and chemical instability. In this review, we describe the design and in vivo evaluation of our second-generation LXA4 analogs.
First- and second-generation LXA4 analogs are listed Fig. 1 [3]. First-generation analogs were designed to minimize rapid inactivation of LXA4 by metabolism via o-oxidation or oxidation at the 15(S)-alcohol by prostaglandin dehydrogenase (PGDH) [4]. This was achieved by replacing the alkyl tail with a fluorinated phenoxy group and by inverting the stereochemistry at C-15 to the R isomer (15-epi). The stereochemical inversion at C-15 was expected to increase metabolic stability and not expected to reduce the bioactivity of the analog due to the discovery that 15-epi- or aspirintriggered LXA4 was equipotent in assays to LXA4, but was a poorer substrate for PGDH. These structural elements were combined in the first-generation LXA4 analogs as seen in 2, methyl (5R, 6R, 7E, 9E, 11Z, 13E, 15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7, 9, 11,
Corresponding author. Tel.: +1 510 669 4065; fax: +1 510 669 4310. E-mail address: [email protected] (W.J. Guilford).
0952-3278/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2005.05.012
ARTICLE IN PRESS W.J. Guilford, J.F. Parkinson / Prostaglandins, Leukotrienes and Essential Fatty Acids 73 (2005) 245–250
246
OH
HO
OH
O OH
3
15 1, lipoxin A4 OH
HO
OH
O
O X
F
O
R
2, ATLa,X = CH2, R =Me 3, ZK-142, X= O, R = H 7, X = O,R= Me HO
F
generation 3-oxa-LXA4 analog 3 was designed, which incorporated the tetraene structure. Despite providing a single stable pharmacophore in vivo, the pharmacokinetic profile of 3 was similar to 2, suggesting tetraene has an intrinsically short half-life in vivo. Similar biological activity with a better pharmacokinetic profile was seen for the corresponding E,E,E-trien-11-yne 3-oxa-LXA4 analog 4.
3. Chemistry O
OH X
O
R
O OH 6, X = CH2, R = H 8, X = CH2, R =Me 4, ZK-994, X = O, R = H Fig. 1. First- and second-generation LXA4 analogs. The figure shows the structure of endogenous lipoxin A4 (1), first-generation analogs (2,6,8), and second-generation 3-oxa-lipoxin analogs (3,4,7).
13-hexadecatetraenoate (15-epi-16-(para-fluoro)-phenoxy-LXA4 (referred to in the literature as ATLa or ATLa2). The potent anti-inflammatory and immunomodulatory properties of 2 were demonstrated in a series of in vivo models. These included ovalbumininduced allergic airway inflammation and hyperreactivity [5], granulocyte and T-cell-dependent skin reactions [6], the acute phase of dextran sodium sulfate (DSS)induced colitis [7], and a lethal T-helper type-1 (Th1)driven adaptive immune response to Toxoplasma gondii infection in 5-lipoxygenase-deficient mice [8]. Taken together, these studies provide evidence that 2 has pronounced anti-inflammatory effects in granulocytedriven acute reactions and that it directly or indirectly modulates T-cell effector functions both in the setting of adaptive Th1- and allergic Th2-dependent immune dysfunction. Although 2 was efficacious in these models it was cleared from the mouse within 15 min after intravenous injection [9]. Moreover, the oral pharmacokinetic disposition of 2 was not described and its potential for systemic uses not fully elaborated. A more complete pharmacokinetic profile for 2 was measured after i.v. and p.o. dosing and showed rapid oral absorption, but with a short plasma half-life [3]. Careful analysis of the plasma samples indicated the presence of a secondary metabolite, which was consistent with a b-oxidation product and with lipid metabolism seen in the prostaglandin [10,11] and leukotriene [12] fields. To eliminate this catabolic pathway [13] and to potentially increase the in vivo half-life, a second-
Since LXA4 was first reported in 1984 [14], the trihydroxytetraene structure has attracted the attention of synthetic chemists, as seen in the number of reported syntheses for 1 [15–18] and 2 [19]. The synthetic challenge with 2 is to prepare a linear chain of 17 carbons, in which all but three carbon atoms are either unsaturated or substituted with oxygen. The challenge is slightly different with the 3-oxa analogs, 3 and 4, in that all of the carbon atoms in the chain are either unsaturated or substituted with oxygen. The synthesis of the 3-oxa-lipoxin analogs is based on the Nicolaou synthesis of LXA4 (1) [20] and reported earlier [3].
4. Efficacy of 3-oxa-LXA4 analogs in inflammation models—i.v. dosing The efficacy of ATLa and the new 3-oxa-LXA4 analogs in animal models of inflammation is summarized in Table 1. Replacement of the cis-11, 12-double bond in 2 for the corresponding trienyne in 6 changes the shape of the lipoxin analog and could alter receptor binding. So the in vivo activity of 2 and 6 were compared in the TNFa-induced inflammatory reaction in the murine dorsal air pouch [22–24]. Both 2 and 6 were equally efficacious, significantly decreasing the total number of leukocytes and neutrophils infiltrating into the air pouch [3]. With the activity of both the tetraene and the trienyne analogs established for the first-generation LXA4 analog series, the 3-oxa analogs were evaluated in lung inflammation after hind-limb ischemia-reperfusion using an i.v. dosing protocol (500 mg/kg prior to ischemia plus 500 mg/kg prior to reperfusion) [25]. The inhibition of lung inflammation was enhanced with novel analogs 3 and 4. While 2 reduced inflammation by only 17%, the novel analogs, 3 and 4, demonstrated 32% and 53% inhibition, respectively. These results indicate that the 3-oxa substitution enhances this lung protective impact of LXA4 analogs [26]. Examination of lung histology indicates that compound 3 reduced leukocyte accumulation, edema, and tissue injury in the lung tissue.
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Table 1 Summary of 3-oxa-LXA4 analog efficacy in inflammation models Modela
Route of administration
LXA4 or 3-oxa-LXA4 analog and dose
Mouse air-pouch model of inflammation [3] Calcium ionophore-induced acute skin inflammation [3] LTB4/PGE2stimulated acute ear skin inflammation [25] Ischemia-reperfusion-induced lung injury [25] Zymosan A-induced peritonitis [25] Zymosan A-induced peritonitis [25] TNBS-induced colitis [30]
Local Topical Topical Intravenous Intravenous Intragastric Intragastric
2 and 6 efficacious at 10 ng/pouch 2, 3, and 4 efficacious at 300 mg/cm2 2, 3, and 4 efficacious at 20 mg/ear 3, 4 efficacious at 500 mg/kg 2, 7, 8 efficacious at 50 mg/kg 1, 2, 4 efficacious at 5 mg/kg 3 efficacious at 300–1000 mg/kg
a
Data derived from the noted reference.
500 400 300 200 * *
4
3
300
100
300
100
600
300
100
Ionophore
*
*
600
*
0
600
100
vehicle
Elastase Activity (pmol AMC)
Neutrophil Infiltration
2
Topical Dose (µg/cm2)
Fig. 2. Topical ATLa and 3-oxa-LXA4 analogs prevent neutrophil infiltration in calcium ionophore-induced ear inflammation in mice. The figure shows the increase in elastase content as a marker of neutrophil infiltration in mouse ears 24 h after topical application of calcium ionophore. Topical co-application of ATLa (2) or the tetraene (3) or the trienyne (4) 3-oxa-LXA4 analog dose-dependently prevents neutrophil infiltration with ED50 o300 mg/cm2 for all compounds tested. Data are redrawn and reproduced with permission from Guilford et al. [3]. Copyright 2004 Am. Chem. Soc.
5. Efficacy of 3-oxa analogs in inflammation—topical dosing Topical efficacy for native LXA4 and 2 has been demonstrated in several models of skin inflammation [6]. The calcium ionophore model was chosen to compare 3oxa analogs 3 and 4 with 2. Calcium ionophore A-23187 applied topically to the dorsal surface of mouse ears of mouse ears induces acute inflammation with edema and granulocyte infiltration that peaks at approximately 24 h. Anti-inflammatory effects were determined by measuring inhibition of edema formation, granulocyte infiltration, and neutrophil infiltration (Fig. 2). The data clearly show that 3-oxa analogs 3 and 4 inhibited neutrophil infiltration in a dose-dependent manner and at similar doses to 2. Neutrophil infiltration was completely inhibited at the highest dose tested. The
IC50 for 2, 3, and 4 on all three topical efficacy parameters was o300 mg/cm2. The novel 3-oxa analogs can thus substitute for carbon analogs in a topical model of inflammation. The topical efficacy of 2, 3, and 4 was confirmed in blocking dermal inflammation induced by LTB4 plus PGE2, as indicated by marked reductions in neutrophil infiltration and vascular leak [25]. Results on vascular leak are shown in Fig. 3. At 20 mg/cm2, 2 appeared to have the most robust efficacy on both endpoints, being somewhat more effective than 3. The trienyne 4 appeared less effective than either tetraene compound, with a slower and less robust effect on vascular leak. This result suggests the absorption or penetration of 4 into mouse ear skin tissue is slower than for the tetraene analogs. In line with earlier observations [27], neither 2 (20 mg; 1 mg/kg) nor 3 (20 mg; 1 mg/kg)
ARTICLE IN PRESS W.J. Guilford, J.F. Parkinson / Prostaglandins, Leukotrienes and Essential Fatty Acids 73 (2005) 245–250
248
30
80 60 40 20 0 2 3 4 Topical Lipoxin Analog, 20 ug/ear
Fig. 3. ATLa and 3-oxa-LXA4 analogs block LTB4+PGE2-induced vascular leak in mouse ears. The figure shows the % inhibition of LTB4+PGE2-induced Evans blue dye accumulation in mouse ear homogenates as a measure of vascular leak at 7 (black bars) and 20 h (grey bars). LTB4+PGE2 was topically with 20 mg ATLa (2) or the tetraene (3) or the trienyne (4) 3-oxa-LXA4 analogs. Data are redrawn and reprinted by permission from Bannenberg et al. [25]. Copyright 2004 Macmillan Publishers Ltd.
Change of body weight (%of basal)
% Inhibition Vascular Leak
100
20 10
**
**
0 -10 ** -20 -30
* control
10
0.1
0.3
1
TNBS
mg/kg prednisolone, p.o. once daily mg/kg trienyne analog 4,p.o.once daily
gave statistically significant inhibitory effects with LTB4 plus PGE2-initiated vascular leakage in the outer ear when administered by tail vein injection. These results indicate specific local effects of lipoxins in dermal inflammation that cannot be achieved by systemic delivery, and imply direct local actions of lipoxins on skin cells.
Fig. 4. Trienyne 3-oxa-LXA4 analog inhibits weight loss in TNBSinduced colitis in mice. The figure shows the dose-dependent prevention of weight loss in TNBS-induced colitis in mice by the trienyne 3-oxa-LXA4 analog (4) at 0.1, 0.3 and 1 mg/kg in comparison to 10 mg/kg prednisolone. Both agents were administered by oral gavage once daily from day 1 to day 14. Control mice gained some weight by day 14, but TNBS-treated mice receiving vehicle lost 20% of their body weight (P ¼ 0:05 versus control). Animals treated with 10 mg/kg prednisolone and 0.3 or 1 mg/kg 3-oxa-LXA4 analog had significant protection from TNBS-induced weight loss ( Po0:05 versus TNBS plus vehicle). Data are redrawn from Fiorucci et al. [30]. Copyright 2004 National Academy of Sciences, USA.
6. Efficacy of 3-oxa analogs in inflammation—oral dosing To assess the extent to which lipoxins and their analogs can be used as potential systemic anti-inflammatory agents after oral administration, 2, 3, and 4, as well as native LXA4 and LXB4 were all evaluated for oral efficacy in acute zymosan-induced peritonitis in [25]. Systemic anti-inflammatory effects in the peritonitis model were observed with doses as low as 1–10 ng/ mouse (50–500 ng/kg), with increasing effects at higher doses (5–500 mg/kg). Similar degrees of systemic antiinflammatory efficacy of 2, 3 and 4 were observed whether administered intravenously or orally via intragastric administration. The oral efficacy of the tetraene analogs is consistent with their pharmacokinetic profiles [3], but is surprising given the acid-labile nature of the tetraene pharmacophore [28]. These tetraene analogs degrade rapidly in aqueous buffers at gastric pH 1.5 (W. Guilford and J. Bauman, unpublished results). Nevertheless, all these tetraenes are rapidly absorbed after intragastric oral administration, so they must clearly be protected from acid-catalyzed degradation in the gastric environment. Human neutrophils appear to have an LXA4 transporter [29] and it can be speculated that a similar mechanism may be present in the GI tract that facilitates LXA4 and LXA4 analog absorption. The oral bioavail-
ability of lipoxins has implications for understanding the results of other studies, such as the efficacy of 2 in the acute phase of dextran sodium-sulfate-induced colitis in mice [7]. In the colitis study, 2 was administered ad libitum at 10 mg ml1 in the drinking water for an estimated systemic exposure of 50 mg/kg/day (based on typical daily water consumption rates in mice and the oral absorption profile). Based on the potent oral effects of 2 in acute peritonitis, the efficacy of 2 in the colitis model can be assumed to be a sum of immunomodulatory effects after systemic absorption and topical effects in the GI tract due to local actions of 2 that remains in GI transit. The efficacy of the trienyne 3-oxa-LXA4 analog 4 in an animal model of Crohn’s disease was recently established [30]. At daily oral doses of 0.3–1 mg/kg/day analog 4 was as efficacious as 10 mg/kg daily oral prednisolone in preventing (dosing days 1–7) and treating (dosing days 3–14) the severe transmural colon disease observed in this model. Data on weight loss are shown in Fig. 4. Detailed analysis of mucosal markers in immunocompetent Balb/c mice and T- and B-lymphocyte-deficient SCID mice revealed pronounced antiinflammatory and immunomodulatory actions of analog 4. Efficacy was correlated to attenuation of proinflammatory chemokine/cytokine networks and the
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prevention of Th1-type immune dysfunction driven by myeloid and lymphoid cellular subsets. 7. Conclusion The second generation, 3-oxa-lipoxin analogs, 3 and 4, which were designed to increase the metabolic and chemical stability of LXA4, are equipotent to 2 in animal models of inflammation. Metabolic stability was improved by eliminating metabolism by b-oxidation and chemical stability by replacing the tetraene unit. Taken together, the results provide new insights on the in vivo catabolism of lipoxins and structure–activity relationships essential for in vivo actions. Second-generation 3oxa-LXA4 analogs provide superior pharmacophores that can be dosed via oral, topical and systemic routes. The 3-oxa-analogs represent state-of-the-art tools with which to explore pre-clinical and clinical concepts for lipoxins in the resolution of inflammation, immunomodulation, allergy, adaptive immunity and autoimmunity.
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