Pharmacokinetics and Metabolism of Selective Oxoeicosanoid (OXE) Receptor Antagonists and Their Effects on 5-Oxo-6,8,11,14-eicosatetraenoic Acid (5-Oxo-ETE)-Induced Granulocyte Activation in Monkeys

Article abstract of DOI:10.1021/acs.jmedchem.6b00895

The potent eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a 5-lipoxygenase product that acts via the selective OXE receptor, which is present in many species, but not rodents. We previously reported that the indole 230 is

Full text of DOI:10.1021/acs.jmedchem.6b00895

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Article
Pharmacokinetics and metabolism of selective oxoeicosanoid
OXE) receptor antagonists and their effects on 5-oxo-6,8,11,14-
(
eicosatetraenoic acid (5-oxo-ETE)-induced granulocyte activation in monkeys
Chantal Cossette, Shishir Chourey, Qiuji Ye, Chintam Nagendra Reddy, Vivek Gore, Sylvie
Gravel, Irina Slobodchikova, Dajana Vuckovic, Joshua Rokach, and William S. Powell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00895 • Publication Date (Web): 21 Oct 2016
Downloaded from http://pubs.acs.org on October 26, 2016
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Pharmacokinetics and metabolism of selective oxoeicosanoid (OXE) receptor antagonists
and their effects on 5ꢀoxoꢀ6,8,11,14ꢀeicosatetraenoic acid (5ꢀoxoꢀETE)ꢀinduced granulocyte
activation in monkeys
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†
ǁ
‡ǁ
‡
‡
‡┴
Chantal Cossette , Shishir Chourey , Qiuji Ye , Chintam Nagendra Reddy , Vivek Gore ,
†
§
§
‡
Sylvie Gravel , Irina Slobodchikova , Dajana Vuckovic , Joshua Rokach , and William S.
†
Powell *
†
MeakinsꢀChristie Laboratories, Centre for Translational Biology, McGill University Health
Centre, 1001 Decarie Blvd, Montreal, QC H4A 3J1, Canada
‡
Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, 150
West University Boulevard, Melbourne, FL 32901ꢀ6982, USA
§
Department of Chemistry and Biochemistry and PERFORM Centre, Concordia University,
7
141 Sherbrooke St. W., Montréal, QC H4B 1R6, Canada
ǁ
These authors contributed equally to this work
┴
Present address for VG: Navinta LLC, 1499 Lower Ferry Rd, Ewing, New Jersey 08618ꢀ1414,
USA
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ABSTRACT
The potent eosinophil chemoattractant 5ꢀoxoꢀ6,8,11,14ꢀeicosatetraenoic acid (5ꢀoxoꢀETE) is a 5ꢀ
lipoxygenase product that acts via the selective OXE receptor, which is present in many species,
but not rodents. We previously reported that the indole 230 is a potent human OXE receptor
antagonist. The objective of the present study was to determine whether the monkey would be a
suitable animal model to investigate its pharmaceutical potential. We found that monkey
leukocytes synthesize and respond to 5ꢀoxoꢀETE, and that 230 is a potent antagonist of the OXE
receptor in monkey eosinophils. Pharmacokinetic studies revealed that 230 appears rapidly in the
blood following oral administration. Using chemically synthesized standards we identified the
major microsomal and plasma metabolites of 230 as products of ω2ꢀhydroxylation of the alkyl
side chain. These studies demonstrate that the monkey is a promising animal model to
investigate the drug potential of OXE receptor antagonists.
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INTRODUCTION
ꢀOxoꢀ6,8,11,14ꢀeicosatetraenoic acid (5ꢀoxoꢀETE) is formed by oxidation of the 5ꢀlipoxygenase
product 5Sꢀhydroxyꢀ6,8,11,14ꢀeicosatetraenoic acid (5SꢀHETE) by 5ꢀhydroxyeicosanoid
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+
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dehydrogenase (5ꢀHEDH) in the presence of NADP (Fig. 1). Among lipid mediators, it is the
most powerful chemoattractant for human eosinophils, acting through the selective OXE
2
receptor, which is highly expressed on eosinophils, and to a lesser extent on neutrophils.
Another eicosanoid derived from arachidonic acid (AA) with similar properties is prostaglandin
D (PGD ), which also promotes eosinophil migration through its interaction with the DP
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receptor, but does not elicit as strong a response as 5ꢀoxoꢀETE. Leukotriene B (LTB ) is a
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potent chemoattractant for guinea pig eosinophils, but has only weak effects on the migration of
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human and monkey eosinophils, its major target in humans being neutrophils.
Figure 1. Synthesis of granulocyte
chemoattractants from AA. AA is
converted by the 5ꢀLO pathway to
LTB and 5ꢀoxoꢀETE and by the
4
cyclooxygenase (COX) pathway to
PGD , each of which acts via its own
2
selective receptor to induce
chemotaxis (Chtx) and actin
polymerization of eosinophils (Eo)
and/or neutrophils (Ne). Effects
mediated by the OXE receptor can be
blocked by the selective antagonist
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30. Abbreviations: PGDS, PGD
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synthase; LTAH, LTA hydrolase.
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Because of its potent effects on eosinophils, 5ꢀoxoꢀETE could play an important
pathophysiological role in asthma and other eosinophilic diseases and for this reason we set out
to develop selective OXE receptor antagonists. We recently reported two potent and selective
OXE antagonists with IC values of about 30 nM in inhibiting 5ꢀoxoꢀETEꢀinduced calcium
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mobilization and actin polymerization in human eosinophils and neutrophils in vitro. Each of
these compounds has a single asymmetric carbon with the antagonist activity residing almost
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exclusively in the Sꢀenantiomer, which have IC values between 5 and 10 nM. Testing of
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these OXE receptor antagonists in animal models will be required to further explore the
possibility that they may be useful in treating human diseases. Although orthologs of the OXE
receptor are present in many species, including zebrafish, where 5ꢀoxoꢀETE plays an important
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role in regulating leukocyte recruitment in response to injury, there is no ortholog of this
receptor in rodents. Alternative models are therefore required to test the effects of OXE
antagonists. To this end we recently investigated the effects of 5ꢀoxoꢀETE on feline leukocytes
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and found it to be a highly potent granulocyte chemoattractant in this species. However, the
antagonists that we developed against the human OXE receptor were not very effective in cats,
having IC values of ~5ꢀ10 µM, well over 100 times higher than those observed with human
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cells. Presumably differences in the sequences of the feline and human OXE receptors (76%
identity) were too great to permit effective inhibition by the antagonists.
Given the above problems we decided to focus on the monkey, which has an OXE receptor
ortholog that is 96% identical to the human receptor and therefore would be much more likely to
respond to our antagonists than the feline receptor. Our initial objective was to determine
whether monkey leukocytes can synthesize and respond to 5ꢀoxoꢀETE. Once this was
accomplished we wished to determine whether 5ꢀoxoꢀETEꢀinduced granulocyte activation could
be blocked by an OXE receptor antagonist and, if so, to investigate its metabolism and
pharmacokinetics to determine the suitability of the cynomolgus monkey for further in vivo
testing. As preliminary experiments suggested that 230 (Fig. 1) is more resistant to metabolism
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by rat liver microsomes than the other OXE receptor antagonist that we characterized we
decided to initially focus on this compound for further studies in the monkey.
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RESULTS
Monkey leukocytes can synthesize 5ꢀoxoꢀETE
Monkey leukocytes were incubated with 5SꢀHETE in the presence of phorbol myristate acetate
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(
PMA), which triggers the respiratory burst in phagocytic cells, thus raising the intracellular
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levels of the 5ꢀHEDH cofactor NADP . Analysis of the products by RPꢀHPLC revealed that
substantial amounts of 5ꢀoxoꢀETE (t , 8.3 min) were synthesized under these conditions (Fig.
R
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A), whereas in the absence of PMA only small amounts were formed (Fig. 2A, inset). Large
amounts of 5ꢀoxoꢀETE (t , 37.3 min) were also formed by monkey leukocytes incubated with
R
AA in the presence of A23187 and PMA. 5SꢀHETE, 12SꢀHETE, LTB and the cyclooxygenase
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product 12ꢀHHT (12ꢀhydroxyꢀ5,8,10ꢀheptadecatrienoic acid) were also detected (Fig. 2B). The
identity of 5ꢀoxoꢀETE was confirmed by its UV spectrum (λmax, 280 nm; inset to Fig. 2B).
Figure 2. Biosynthesis of 5ꢀoxoꢀETE by monkey
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leukocytes. A: Monkey leukocytes (2 x 10 cells in 1 ml)
were incubated with 5SꢀHETE (4 µM) for 10 min in the
presence of PMA (100 nM) and the products analyzed by
precolumn extraction/RPꢀHPLC as described in the
Experimental Section. HETEs were detected at 235 nm,
whereas 5ꢀoxoꢀETE (5oETE) was detected at 280 nm.
The inset shows a chromatogram of the products from an
identical incubation in the absence of PMA. The scale of
the Xꢀaxis is the same for both chromatograms. B:
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Monkey leukocytes (2 x 10 cells in 1 ml) were
incubated with AA (30 µM) in the presence of A23187 (5
µM) and PMA (100 nM) for 60 min and the products
analyzed by RPꢀHPLC. The inset shows the UV
spectrum of the peak labeled 5oETE with a retention time
of 37.3 min. The results are representative of independent
experiments using cells from 4 different monkeys.
5
ꢀOxoꢀETE elicits actin polymerization and chemotaxis in monkey leukocytes
To determine whether 5ꢀoxoꢀETE can activate monkey granulocytes, leukocytes were pretreated
with APCꢀlabeled antiꢀCD49d, and then exposed to agonists for 20 s. They were then treated
with Nꢀ(7ꢀnitrobenzꢀ2ꢀoxaꢀ1,3ꢀdiazolꢀ4ꢀyl)phallacidin (NBDꢀphallacidin) to stain polymerized
actin and analyzed by flow cytometry. Eosinophils were identified on the basis of high
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expression of CD49d and high side scatter, whereas neutrophils exhibited lower CD49d
expression and side scatter (Fig. 3A). We compared the effects of 5ꢀoxoꢀETE on actin
polymerization to those of LTB and PGD . 5ꢀOxoꢀETE (EC , 1.5 ± 0.4 nM) and LTB (EC ,
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.7 ± 0.3 nM) had similar potencies in activating eosinophils, whereas PGD (EC , 5.0 ± 1.1
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nM) was somewhat less potent (p < 0.05; Fig. 3B). The maximal response to 5ꢀoxoꢀETE was a
little higher than that to LTB (p < 0.05). Both 5ꢀoxoꢀETE (EC , 0.9 ± 0.3 nM) and LTB (EC ,
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0.06 ± 0.03 nM) were very active in stimulating actin polymerization in neutrophils, with LTB4
being the more potent (p < 0.05; Fig. 3C). In contrast to its effects on eosinophils, PGD had no
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measurable effect on actin polymerization in monkey neutrophils.
Figure 3. Effects of eicosanoids and 230 on actin polymerization in monkey granulocytes. The effects of
eicosanoids on actin polymerization in eosinophils and neutrophils were evaluated by flow cytometry in APCꢀantiꢀ
CD49dꢀlabeled leukocytes treated with NBDꢀphallacidin as described in Experimental Procedures. A: Dotplot
showing the identification of populations of eosinophils (high side scatter and antiꢀCD49d labeling) and neutrophils
(
lower side scatter and antiꢀCD49d labeling). Concentrationꢀresponse curves for the effects of 5ꢀoxoꢀETE (●), LTB
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(○) and PGD (▲) on actin polymerization in eosinophils (B) and neutrophils (C) were determined as described
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above. D: Leukocytes were preincubated for 5 min with different concentrations of 230 prior to the addition of 5ꢀ
oxoꢀETE (10 nM) and evaluation of actin polymerization as described above. Data for human granulocytes (○) taken
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from a previous study are shown for comparison.
We also examined the effects of 5ꢀoxoꢀETE, LTB , and PGD on leukocyte migration. Because
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of the limited amount of monkey blood available for these experiments we did not attempt to
fractionate the leukocytes, which had been prepared by dextran sedimentation of red blood cells.
These preparations contained about 39 ± 7 % neutrophils and 1.1 ± 0.2 % eosinophils. As shown
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in Fig. 4A, 5ꢀoxoꢀETE and LTB are potent chemoattractants for monkey leukocytes, whereas
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PGD was virtually inactive, consistent with the predominance of neutrophils over eosinophils in
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these unfractionated leukocytes preparations. Although LTB (EC , 2.5 ± 0.8 nM) was more
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potent than 5ꢀoxoꢀETE (EC , 35 ± 4 nM) (p < 0.005), the maximal response to 5ꢀoxoꢀETE was
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over 3 times greater than that to LTB (p < 0.05).
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Figure 4. Effects of eicosanoids and 230 on monkey
leukocyte migration. A: The effects of 5ꢀoxoꢀETE (●),
LTB (○), and PGD (▲) on leukocyte migration were
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evaluated using microchemotaxis chambers. Vehicle or
different concentrations of eicosanoids were added to the
bottom chambers, whereas unfractionated leukocytes
were added to the top chambers. B: The effects of 230 on
macaque leukocyte chemotaxis (●) were evaluated as
described above. 230 was added to both the top and
bottom chambers. The values are means ± SE (n = 4).
Data for human granulocytes (○) taken from a previous
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study are shown for comparison.
The OXE receptor antagonist 230 is a potent inhibitor of 5ꢀoxoꢀETEꢀinduced granulocyte
activation
We found that 230 is a potent inhibitor of 5ꢀoxoꢀETEꢀinduced actin polymerization in monkey
eosinophils, with an IC of 37 ± 11 nM (Fig. 3D). The concentrationꢀresponse curve for
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monkeys is virtually identical to that which we previously reported for human eosinophils (IC ,
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33 ± 10 nM), which is also shown in Fig. 3D for comparison. Similarly, as shown in Fig. 4B, the
effect of 230 on 5ꢀoxoꢀETEꢀinduced leukocyte migration in monkeys (IC 422 ± 181 nM) is
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very similar to that for humans (454 ± 192 nM). The IC values are higher for the chemotaxis
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assay because a higher concentration of 5ꢀoxoꢀETE (100 vs 10 nM) was employed compared to
the assay for actin polymerization.
Metabolism of 230 by monkey liver microsomes
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To determine the susceptibility of 230 to biological inactivation we investigated its metabolism
by monkey liver microsomes. Because the presence of the 3ꢀmethyl group in the acyl side chain
of 230 should block βꢀoxidation we initially anticipated that it would primarily be metabolized
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by cytochrome P450ꢀcatalyzed ωꢀoxidation to ω1ꢀhydroxy and ωꢀcarboxy metabolites (In this
paper the positions of substituents on the hexyl side chain are defined starting from the terminal
methyl group, which is designated as ω1 and the adjacent methylene group as ω2, etc). To
facilitate the identification of these compounds they were chemically synthesized from the
precursor 5ꢀClꢀindoleꢀ2ꢀcarboxylic acid as shown in Scheme 1.
a
Scheme 1: Synthesis of ωꢀCO Hꢀ230 and ω1ꢀOHꢀ230
2
a
Reagents and conditions: (a) NaH, CH I, DMF, 0 °C ꢀ rt, 18 h, quant; (b) LiAlH , THF, 0 °C ꢀ rt, 12 h, 86%; (c) MnO ,
3
4
2
CH Cl , rt, 16 h, 84%; (d) 5, tBuOK, THF, –78 °C ꢀ rt, 3 h, 70%; (e) 10% Pd/C, EtOH, H , rt, 91%; (f) CH N , Et O, rt, 2
2
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2
2
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min, quant; (g) Me AlCl, CH Cl , 10, CH NO , 0 °C ꢀ rt, 3 h; (h) LiOH, THF, H O, rt, 36 h, 50% over two steps g and h;
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2
3
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(
i) LiAlH , THF, 0 °C – rt, 14 h, 82%; (j) Me AlCl, CH Cl , 13, CH NO , 0 °C – rt, 6 h, 43%.
4 2 2 2 3 2
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Figure 5. Metabolism of 230 by monkey liver microsomes. Monkey liver microsomes were incubated with 230
(
100 µM) for 4 h in the presence of NADPH (2 mM) as described in the Experimental Section. A: The products
were analyzed by precolumn extraction/RPꢀHPLC using a Novapak C18 column (4 µm particle size; 3.9 x 150
mm) with a linear gradient between 50 and 85% MeOH in H O containing 0.02% HOAc over 30 min at a flow
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rate of 1 ml/min and a temperature of 30 °C. B: The products were analyzed by precolumn extraction/RPꢀHPLC
using a Kinetex C18 column (2.6 µm particle size; 4.6 x 100 mm) with a linear gradient between 30 and 65%
MeCN in H O containing 0.02% HOAc over 35 min at a flow rate of 1 ml/min and a temperature of 30 °C. C:
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UV spectra of the major metabolites of 230 obtained using a diode array UV detector. All spectra were from the
MeOH/H O gradient except for ωꢀhydroxyꢀ230 (d) which was from the MeCN/H O gradient. The scale of the
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Yꢀaxis was adjusted for each of the spectra to facilitate comparison. DꢀF: Collisionꢀinduced dissociation of the
ꢀ
[
(
MꢀH] ions of (D) metabolite c (m/z 392.1612) in panel B, (E) metabolite a (m/z 390.1478) and (F) metabolite b
m/z 406.1402). * Ions that are formed as a result of a McLafferty rearrangement.
The products formed after incubation of 230 (50 µM) with monkey liver microsomes (0.8 mg
protein/mL) in the presence of NADPH (2 mM) for 4 h were analyzed by RPꢀHPLC using a
water/methanol gradient (Fig. 5A). Two major products with retention times of 13.95 min (a)
and 15.35 min (c) were detected. Metabolite c cochromatographed with authentic ω1ꢀhydroxyꢀ
230 (data not shown), whereas metabolite a had a retention time somewhat lower than that of
authentic ωꢀcarboxyꢀ230, which cochromatographed with the minor product (b) of intermediate
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polarity (t , 14.65 min). When we repeated the analysis using a water/acetonitrile gradient the
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order of elution of the two major metabolites was reversed (Fig. 5B). Under these conditions, c
(
tR, 13.88 min) no longer cochromatographed with ω1ꢀhydroxyꢀ230, but instead
cochromatographed with the minor product b. With the acetonitrile gradient the minor product d
tR, 14.18 min) cochromatographed with authentic ω1ꢀhydroxyꢀ230 (not shown), whereas the
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(
major product a (t , 15.35 min) had a considerably longer retention time.
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a
Scheme 2. Synthesis of ω2ꢀOHꢀ230 and ω2ꢀoxoꢀ230
a
+ ꢀ
Reagents and conditions: (a) Et N I , BF ꢀEt O, CH Cl , rt, 48%; (b) DHP, PPTS, THF, rt, 90%; (c) PPh ,
4
3
2
2
2
3
CH CN, 85 °C, 47 h; (d) KHMDS, –78 °C ꢀ 0 °C, 3 h, aq. NH Cl solution, 58%; (e) 10% Pd/C, EtOH, H , rt, 3 h,
3
4
2
quant; (f) Me AlCl, 13, CH NO , rt, 3 h, 82%; (g) DMP, CH Cl , rt, 9 h, 64%.
2
3
2
2
2
The reversal in the order of elution of a and c in a water/methanol vs a water/acetonitrile gradient
is characteristic of a pair of compounds that differ from one another by an oxo vs a hydroxyl
9
group. We therefore postulated that 230 is metabolized by ωꢀ1 oxidation (i.e. oxidation of the
ω2 methylene group) to give ω2ꢀhydroxyꢀ230 and ω2ꢀoxoꢀ230. To aid in the identification of
these products the authentic compounds were prepared by chemical synthesis as shown in
Scheme 2. Metabolites a and c cochromatographed with authentic ω2ꢀoxoꢀ230 and ω2ꢀhydroxyꢀ
230, respectively, in both methanol/water and acetonitrile/water gradients (data not shown). The
UV spectra of compounds a ꢀ d (Fig. 5C) were all virtually identical to that of 230, consistent
with an unaltered indole chromophore and modification of one of the side chains. Further
support for the above structural assignments was obtained by LC/MS/MS analysis. Metabolites c
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ꢀ
and d had [MꢀH] ions at m/z 392.1612 and 392.1611, respectively, compared to the theoretical
2
value m/z 392.1634 for a monohydroxy metabolite of 230 (mass accuracy, 5.6 ppm). The MS
ꢀ
fragmentation pattern of the [MꢀH] ion for c (Fig. 5D) is identical to that of authentic ω2ꢀ
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hydroxyꢀ230 (not shown), with major ions at m/z 374 (loss of H O), 348 (loss of CO ), 332 (loss
2
2
of CH C(OH) due to a McLafferty rearrangement), 306 (loss of CH =CHꢀCH ꢀCO H due to a
2
2
2
2
2
different McLafferty rearrangement), and 264 (loss of the acyl side chain).
ꢀ
Metabolite a had an [MꢀH] ion at m/z 390.1457, compared to the theoretical value of 390.1478
2
expected for ω2ꢀoxoꢀ230 (mass accuracy, 5.4 ppm). Furthermore, the MS fragmentation pattern
ꢀ
of the [MꢀH] ion from a (Fig. 5E) is identical to that of authentic ω2ꢀoxoꢀ230 (not shown) and
was similar to that of ω2ꢀhydroxyꢀ230 except that the m/z values of the major ions were 2 mass
units lower: 372 (loss of H O), 346 (loss of CO ), 330 (loss of CH C(OH) due to a McLafferty
2
2
2
2
rearrangement), 304 (loss of CH =CHꢀCH ꢀCO H due to a McLafferty rearrangement), and 262
2
2
2
(loss of the acyl side chain).
The mass spectrum of d was very similar to that of c and identical to that of authentic ω1ꢀ
hydroxyꢀ230 (data not shown). There was also another metabolite with a t (14.65 min) between
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those of d and a (Fig. 5B) with an almost identical mass spectrum (not shown), indicating that it
is a regioisomer of these compounds with the hydroxyl group at a different position in the hexyl
side chain.
ꢀ
Finally, the m/z value for the [MꢀH] ion of metabolite b was 406.1402, compared to the
2
theoretical value of 406.1427 for ωꢀcarboxyꢀ230 (mass accuracy, 6.2 ppm). The MS
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fragmentation pattern of this metabolite (Fig. 5F) is identical to that of authentic ωꢀcarboxyꢀ230
(not shown) with prominent ions with m/z values of 388 (loss of H O), 370 (loss of HCl), 344
2
(
loss of H O and CO ), 326, 320 (loss of CH =CHꢀCH ꢀCO H due to a McLafferty
2 2 2 2 2
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rearrangement), and 260.
a
Scheme 3: Synthesis of 197 and 225
a
Reagents and conditions: (a) 21, LiHMDS, –78 °C ꢀ 0 °C, 3 h, 90%; (b) 10% Pd/C, EtOH, H , rt, 3 h, 91%; (c)
2
KOH, DMSO, CH I, rt, 3 h, 95%; (d) Me AlCl, CH Cl , 13, rt, 3 h, 64%.
3
2
2 2
Effects of a branchedꢀchain alkyl group on antagonist potency
We attempted to reduce the metabolism of our OXE antagonist by replacing the hexyl group
with a branchedꢀchain alkyl group, which we hoped would be more resistant to ωꢀoxidation. To
this end we synthesized compounds 197 and 225, in which the hexyl group of 230 is replaced by
a 5ꢀmethylhexyl or a 4ꢀmethylhexyl group, respectively (Scheme 3). Both compounds were
prepared from the precursor 5ꢀchloroindoleꢀ2ꢀcarbaldehyde as shown in Scheme 3. The effects
of these compounds on 5ꢀoxoꢀETEꢀinduced calcium mobilization in human granulocytes are
shown in Fig. 6. The concentrationꢀresponse curve for 197 (IC , 20 ± 6 nM) is nearly identical
5
0
to that for 230 (IC , 16 ± 4 nM), whereas 225 (IC , 88 ± 26 nM) is over 4 times less potent.
5
0
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Figure 6. Effects on antagonist potency of adding a
methyl group to the hexyl side chain of 230. Calcium
mobilization was measured in human neutrophils
stimulated with 10 nM 5ꢀoxoꢀETE following the addition
of either vehicle, 230 (●), 197 (∆), or 225 (▲). All
compounds were racemic mixtures.
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Metabolism of 197 by monkey liver microsomes
Because 197 is the more potent of the above two branched chain compounds we further
investigated its metabolism. 197 (50 µM) was incubated with monkey liver microsomes in the
presence of NADPH for 4 h and the products were analyzed by RPꢀHPLC using a
water/acetonitrile gradient. Under these conditions 197 was almost completely converted to two
major metabolites, x (t , 13.69 min) and y (t , 23.69 min), along with several minor metabolites
R
R
(Fig. 7A). Analysis of the same sample using a water/methanol gradient gave a very similar
distribution of peaks (data not shown), with no apparent change in the order of elution. As with
the major microsomal metabolites of 230, both x and y had UV spectra virtually identical to that
of 197, suggesting that the indole chromophore had not been altered (Fig. 7B).
ꢀ
Analysis by LCꢀMS revealed that the [MꢀH] ion of metabolite y has an m/z value of 406.1769,
within 3.9 ppm of the theoretical value of 406.1785 for a monohydroxy metabolite of 197. The
2
MS fragmentation pattern of y (Fig. 7C) was dominated by fragmentation of the acyl side chain
and exhibited major ions at m/z 388 (loss of H O), 362 (loss of CO ), 346 (loss of CH C(OH)
2
2
2
2
due to a McLafferty rearrangement), 320 (base peak; loss of CH =CHꢀCH ꢀCO H due to a
2
2
2
2
McLafferty rearrangement), and 278 (loss of the acyl side chain). Since the UV and MS spectra
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of this compound rule out alterations involving the indole and acyl side chain, respectively, it can
be concluded that the additional hydroxyl group in metabolite y is located on the alkyl side chain.
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Figure 7. Metabolism of 197 by monkey liver microsomes. Monkey liver microsomes were incubated with 197
(
100 µM) for 4 h in the presence of NADPH (2 mM) as described in the Experimental Section. A: The products
were analyzed by precolumn extraction/HPLC using a Kinetex C18 column (5 µm particle size; 4.6 x 250 mm)
with a linear gradient between 30 and 65% MeCN in H O containing 0.02% HOAc over 35 min at a flow rate of
2
1 ml/min and a temperature of 35 °C. B: UV spectra of the major metabolites of 197 obtained using a diode array
UV detector. The scale of the Yꢀaxis was adjusted for each of the spectra to facilitate comparison. CꢀE: Collisionꢀ
ꢀ
induced dissociation of the [MꢀH] ions of (C) metabolite y (m/z 406.1769), (D) metabolite x (m/z 422.1721), and
(E) metabolite z (m/z 420.1578). * Ions that are formed as a result of a McLafferty rearrangement.
ꢀ
The second major metabolite (x; t , 13.69 min) has an [MꢀH] ion with an m/z of 422.1721,
R
which is within 3.1 ppm of the theoretical value of 422.1734 expected for a dihydroxy
2
metabolite. The MS fragmentation pattern of this compound (Fig. 7D) has fragment ions with
m/z values of 404 (loss of H O), 378 (loss of CO ), 362 (loss of CH C(OH) due to a McLafferty
2
2
2
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rearrangement), 336 (base peak; loss of CH =CHꢀCH ꢀCO H due to a McLafferty
2
2
2
rearrangement), and 294 (loss of the acyl side chain). The UV spectrum of x, combined with its
mass spectrum containing a prominent McLafferty rearrangementꢀderived ion emanating from
loss of the major part of the acyl side chain, suggest that both hydroxyl groups are situated on the
alkyl side chain.
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a
Scheme 4: Synthesis of ω1ꢀOHꢀ197
a
Reagents and conditions: (a) NaH, DMF, 0 °C ꢀ rt, 2 h, 70%; (b) HBr, Ac O, reflux, 16 h, 72%; (c) PPh ,
2
3
CH CN, reflux, 18 h, 70%; (d) tBuOK,
–
10 °C ꢀ 0 °C, 3 h, 57%; (e) 10% Pd/C, H , EtOH, rt, 98%; (f) LiAlH ,
3
2
4
THF,
70%.
–20 °C ꢀ rt, 4 h, 67%; (g) Me AlCl, CH Cl , 10, CH NO , 0 °C ꢀ rt, 3 h, 70%; (h) LiOH, THF, H O, rt, 48 h,
2 2 2 3 2 2
a
Scheme 5: Synthesis of ω2ꢀOHꢀ197
a
Reagents and conditions: (a) 34, tBuOK, THF, –78 °C ꢀ rt, 3 h; (b) KOH, DMSO, CH I, rt, 3 h, 85% over steps
3
a and b; (c) 10% Pd/C, benzene, H , rt, 3 h, 98%; (d) CH MgBr, THF, 0 °C – rt, 30 min, 85%; (e) Me AlCl,
2
3
2
CH Cl , 13, rt, 3 h, 54%.
2
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Since it was not possible to determine the precise positions of the hydroxyl groups in x and y
from the above mass spectral data we prepared several potential candidates by total chemical
synthesis. The first of these, ω1ꢀOHꢀ197, was prepared as shown in Scheme 4. However, this
compound did not cochromatograph with the major metabolite y, but rather with a minor product
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with a t of 25.92 min (Fig. 7A), the identity of which was confirmed to be ω1ꢀOHꢀ197 based on
R
2
ꢀ
the identity of the MS fragmentation pattern of its [MꢀH] ion with that of the authentic standard
(not shown). We then prepared ω2ꢀOHꢀ197 as shown in Scheme 5. This compound
2
cochromatographed with y and had an MS fragmentation pattern identical to that shown in Fig.
7C for the microsomal metabolite, confirming the identity of metabolite y as ω2ꢀOHꢀ197.
a
Scheme 6: Synthesis of ω1,ω1ꢀdiOHꢀ197
a
Reagents and conditions: (a) 38, tBuOK, CH Cl , THF, −78 °C ꢀ rt, 5 h; (b) Pd/C, EtOAc, 6 h, rt, 73% over two
2
2
steps; (c) LiAlH , THF, 0 °C ꢀ rt, 30 min, 83%; (d) CBr , PPh , CH Cl , rt, 1 h, 94%; (e) 43, NaH, 0 °C ꢀ rt, 14 h,
4
4
3
2
2
7
2
4%; (f) LiAlH , THF, 0 °C ꢀ rt, 4 h, 81%; (g) Me AlCl, CH Cl , 10, 0 °C ꢀ rt, 45 min; (h) LiOH, THF, H O, rt,
2 h, 59% over two steps g and h.
4
2
2
2
2
In an attempt to identify the dihydroxy metabolite of 197 we prepared ω1,ω1ꢀdiOHꢀ197, by total
chemical synthesis as shown in Scheme 6. However, this compound had a t (13.46 min),
R
slightly lower than that of metabolite x (t , 13.65 min), consistent with x being an isomeric
R
dihydroxy metabolite, possibly ω1,ω2ꢀdiOHꢀ197 or ω2,ω3ꢀdiOHꢀ197.
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ꢀ
The mass spectrum of another minor product (z; t 24.56) exhibited an [MꢀH] ion at m/z
R
2
4
20.1578, indicative of an ωꢀcarboxy metabolite (i.e. ωꢀCO Hꢀ197). The MS fragmentation
2
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pattern derived from this ion (Fig. 7E) gave fragment ions at m/z 402 (base peak; loss of H O),
2
384 (loss of 2 molecules of H O), 360 (loss of CH C(OH) due to a McLafferty rearrangement),
2
2
2
3
58 (loss of H O and CO ), 334 (loss of –CH =CHꢀCH ꢀCO H due to a McLafferty
2 2 2 2 2
rearrangement), 292 (loss of acyl side chain), and 274 (loss of acyl side chain + H O).
2
Figure 8. In vivo metabolism of 230 in rats. 230 was administered by oral gavage to rats as described in Materials and
Methods. The rats were sacrificed after different times and plasma samples were analyzed by RPꢀHPLC following solidꢀ
phase extraction using 197 as an internal standard as described in the Experimental Section. A: Chromatogram of an
extract of plasma obtained 4 h after administration of 230 (30 mg/kg). B: UV spectra of the peak labeled “230” in panel A
and authentic 230. C: Plasma levels of 230 after oral administration of a dose of 30 mg/kg. Each point represents a single
rat (blood was taken only once at the time the animal was sacrificed).
Pharmacokinetics of 230 in rats
We conducted a pilot study in rats to determine whether 230 would appear in the blood after oral
administration. 230 (30 mg/kg) was administered by oral gavage and the animals were sacrificed
after different time points. Only a single blood sample was taken by cardiac puncture from each
rat at the end of the experiment. Plasma samples were analyzed by RPꢀHPLC following solidꢀ
phase extraction, using 197 as an internal standard. Considerable amounts of 230 were detected
in plasma after 4 h, with only relatively minor peaks containing potential metabolites being
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detected (Fig. 8A). The identity of 230 was confirmed by its retention time and UV spectrum
(Fig. 8B). 230 rapidly appeared in the blood and was detected at a level of about 1.5 ꢀM after 30
min (Fig. 8C). The plasma concentration rose to about 2.2 ꢀM between 1 and 4 h and then
declined to about 0.7 ꢀM at 6 h, which was the longest time investigated. No adverse effects of
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230 on the rats were observed prior to sacrifice at the end of the experiment.
Figure 9. Pharmacokinetics of 230 and 197 in monkeys.
A: 230 (●; n = 4) and 197 (○; n = 2) were administered
by oral gavage to cynomolgous monkeys as described in
Materials and Methods. Plasma was prepared from blood
samples taken immediately before and 0.5, 1, 2, 4, 8, 18,
and 24 h after administration of antagonist. After solidꢀ
phase extraction the samples were analyzed by
precolumn extraction/RPꢀHPLC using 197 as an internal
standard in the case of 230 and 230 as an internal
standard for analysis of plasma levels of 197. B: The
peaks for 230 (●) and 197 (○) obtained after RPꢀHPLC of
plasma samples were collected and subjected to chiralꢀ
HPLC to determine the proportions of Rꢀ and Sꢀ
enantiomers.
Pharmacokinetics of 230 and 197 in monkeys
We next performed more extensive experiments in cynomolgus monkeys to investigate the
pharmacokinetics and metabolism of 230. The levels of 230 were analyzed as described above in
plasma samples obtained over a period of 24 h following administration by oral gavage. As in
rats, 230 appeared very rapidly in plasma, but the levels were much higher in monkeys, reaching
maximal concentrations of about 100 ꢀM by 30 min and subsequently declining to 2.7 ꢀM by 8
h and to about 0.6 ꢀM at 24 h (Fig, 9A). We also examined the pharmacokinetic profile of 197
and found it to be similar to that of 230, except that the plasma concentrations were somewhat
lower (Fig. 9A). A maximum concentration of 197 of about 30 ꢀM was reached by 30 min,
which declined to about 2 ꢀM by 8 h and 0.3 ꢀM at 24 h. As both 230 and 197 are racemates, we
collected fractions containing the unmetabolized compounds after RPꢀHPLC and determined the
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relative amounts of R and S enantiomers by chiral HPLC. In both cases there was a marked
predominance of the R enantiomers at time points up to 8 h, after which time the ratio of R:S
declined to approach the starting ratio of about 1:1 by 18 h (Fig. 9B).
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Figure 10. In vivo metabolism of 230 in monkeys. 230 was administered by oral gavage to a cynomolgus monkey as
described in the Experimental Section and plasma was prepared from a blood sample taken after 4 h. After solidꢀphase
extraction the sample was analyzed either by RPꢀHPLC using a UV detector or by LCꢀMS. A: RPꢀHPLC of an aliquot of a
plasma extract on a Kinetex C18 column (5 µm particle size; 4.6 x 250 mm) using a gradient between 35 and 65% MeCN
in water containing 0.02% HOAc over 60 min with a flow rate of 1 ml/min and a temperature of 35 °C. B: RPꢀHPLC of an
aliquot of a plasma extract on a Novapak C18 column (4 µm particle size; 150 x 3.9 mm) using a gradient between 50 and
8
0% MeOH in water containing 0.02% HOAc over 40 min with a flow rate of 1 ml/min and a temperature of 30 °C. C:
UV spectra of 230 and metabolites f and g. The scale of the Yꢀaxis was adjusted for each of the spectra to facilitate
ꢀ
3
comparison. D: Collisionꢀinduced dissociation of the [MꢀH] ion (m/z 378) of metabolite e. E: MS spectrum showing
daughter ions derived from the fragment ion of e at m/z 306 shown in panel D. F: Collisionꢀinduced dissociation of the
ꢀ 3
MꢀH] ion (m/z 392) for metabolite e. G: MS spectrum showing daughter ions resulting from collisionꢀinduced
[
dissociation of the ion at m/z 292 in the mass spectrum of f shown in panel F. H: Collisionꢀinduced dissociation of the [Mꢀ
ꢀ
H] ion (m/z 292) of the authentic standard 51 lacking the alkyl side chain. * Ions that are formed as a result of a
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McLafferty rearrangement.
In vivo metabolism of 230 in monkeys
The in vivo metabolism of 230 was investigated using RPꢀHPLC with UV detection as well as by
LCꢀMS. The HPLC profile of UVꢀabsorbing peaks in a plasma sample taken 4 h after
administration of 230 using a water/acetonitrile gradient as the mobile phase is shown in Fig.
10A. As was he case with monkey liver microsomes, the major polar metabolite was identified
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as ω2ꢀOHꢀ230 (t , 20.13 min) by cochromatography with the authentic standard. Smaller
R
amounts of ωꢀcarboxyꢀ230 (t , 19.70 min) and ω2ꢀoxoꢀ230 (t , 22.79 min) were also identified
R
R
by cochromatography with authentic standards. When a water/methanol gradient was used (Fig.
0B) the elution order changed, with ω2ꢀoxoꢀ230 having the shortest t (18.0 min) of the above
1
R
three products, followed by ωꢀcarboxyꢀ230 (t , 18.9 min) and ω2ꢀOHꢀ230 (t , 20.1 min). The
R
R
identity of each of the above three plasma metabolites was confirmed by LCꢀMS analysis, with
2
the exact masses of the MꢀH ions and the MS spectra being identical to those of the authentic
standards (data not shown). We also detected a small amount of a more polar product (e; tR,
14.16 min in Fig. 10A), which cochromatographed with authentic ωꢀcarboxyꢀdinorꢀ230,
ꢀ
synthesized as shown in Scheme 7. Metabolite e has an [MꢀH] ion at m/z 378.1113, within 1.3
2
ppm of the theoretical value expected for ωꢀcarboxyꢀdinorꢀ230 (378.1108), and its MS
fragmentation pattern (Fig. 10D) matched that of the authentic compound, with major ions at m/z
3
60 (loss of H O), 316 (loss of H O and CO ), 306 (loss of CH =CHꢀCO H) and 250 (loss of
2 2 2 2 2
a
Scheme 7: Synthesis of dinorꢀωꢀCO Hꢀ230
2
a
Reagents and conditions: (a) KOH, DMSO, CH I, 0 °Cꢀrt, 10 h, 78%; (b) Me AlCl, CH Cl , 10, 0 °C ꢀ rt, 30
3
2
2
2
min, 41%; (c) LiOH, THF, H O.
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3
acyl side chain). The identity of the ion at m/z 306 was confirmed by its MS spectrum (Fig.
0E), which includes ions at m/z 288 (loss of H O), 262 (loss of CO ), 246 (loss of CH C(OH)
2
1
2
2
2
from the acyl side chain due to a McLafferty rearrangement), 220 (loss of CH =CHꢀCH ꢀCO H
2
2
2
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from the acyl side chain due to another McLafferty rearrangement), and 178 (loss of acyl side
chain).
In addition to the above three ω1 and ω2 oxidation products, a pair of less polar metabolites that
were not present after incubation of 230 with microsomes were observed in plasma extracts (Fig.
1
0A). The UV spectra of metabolites f (t , 37.76 min with water/acetonitrile) and g (t , 38.60
R
R
min with water/acetonitrile) were virtually identical to one another and similar to that of
unmetabolized 230 except that the absorbance maximum above 300 nm underwent a
bathochromic shift from about 303 nm (230) to about 311 nm (f and g) (Fig. 10C). The
absorbance maximum at 251 nm also underwent a slight bathochromic shift of about 1 nm,
whereas the maximum at 226 nm underwent a small hypsochromic shift of about 2 nm.
Metabolites f and g were not resolved with a water methanol gradient and had a t of 30.2 min
R
(Fig. 10B). Analysis by LCꢀMS revealed that metabolites f (m/z 392.1666) and g (m/z 392.1669)
ꢀ
have identical [MꢀH] ions, within 8 ppm of the theoretical value of 392.1634 expected for a
2
monohydroxy metabolite of 230. However, the MS spectra of f and g, which were virtually
identical to one another, were quite different from those of ωꢀOHꢀ230 and ω2ꢀOHꢀ230, and were
dominated by a single major ion at m/z 292, corresponding to loss of the hydroxyalkyl side
3
chain, along with a much less intense ion at m/z 374 (loss of H O) (Fig. 10F). The MS
2
2
fragmentation pattern of the base peak (m/z 292) in the MS spectrum of f revealed a pattern
similar to that of the ω1ꢀ and ω2ꢀhydroxy metabolites, dominated by fragmentation of the
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carboxylic acid side chain, with major ions at m/z 274 (loss of H O), 248 (loss of CO ), and 206
2
2
(
loss of CH =CHꢀCH ꢀCO H due to a McLafferty rearrangement) (Fig. 10G). This spectrum is
2 2 2
2
virtually identical to the MS fragmentation pattern (Fig. 10H) of the authentic standard 51
lacking the hexyl side chain, synthesized as shown in Scheme 8. The facile loss of the
hydroxyalkyl side chain as well as the perturbation of the UV spectrum dominated by the indole
moiety suggest that the hydroxyl groups of f and g are close to the indole, perhaps in the ω6
position of the hexyl side chain (Fig. 10F). Since this would add a second chiral center to the
molecule, f and g could be diastereomers, which could be separated by RPꢀHPLC. However,
confirmation of these structural assignments would require the availability of the authentic
standards.
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a
Scheme 8: Synthesis of 51
a
Reagents and conditions: (a) NaH, CH I, DMF, 0 °C ꢀ rt, 18 h, 94%; (b) Me AlCl, CH Cl , 13, 0 °C ꢀ rt, 4 h,
3
2
2 2
8
1%.
Antagonist potencies of synthetic 230 metabolites
To determine the effects of oxidative metabolism of 230 on antagonist activity we examined the
effects of synthetic metabolites on 5ꢀoxoꢀETEꢀinduced calcium mobilization in human
neutrophils (Fig. 11). Oxidation of 230 dramatically reduced antagonist potency. Of the
compounds tested, ω1ꢀhydroxyꢀ230 was the most potent, with an IC value of 270 ± 20 nM, 14
5
0
times higher than that of 230 (19 ± 5 nM in this series of experiments). The ω2ꢀoxidized
metabolites ω2ꢀhydroxyꢀ230 and ω2ꢀoxoꢀ230 were less potent, with IC values of 1.4 ± 0.3 and
5
0
1.6 ± 0.4 µM, respectively. ωꢀCarboxyꢀ230 was even less potent, with an IC of 25 ± 13 µM.
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Interestingly, dinorꢀωꢀcarboxyꢀ230 had an IC (1.3 ± 0.3 µM) similar to the above ω2ꢀoxidized
5
0
Figure 11. Effects of 230 metabolites on 5ꢀoxoꢀETEꢀ
induced calcium mobilization in human neutrophils.
Calcium mobilization was measured in neutrophils
stimulated with 10 nM 5ꢀoxoꢀETE 2 min after the addition
of either 230 or one of its synthetic metabolites. All
compounds are racemic mixtures. Symbols: 230 (ꢀ), ω1ꢀ
hydroxyꢀ230 (▲), ω2ꢀhydroxyꢀ230 (●), ω2ꢀoxoꢀ230 (○),
ωꢀcarboxyꢀ230 (ꢁ), and dinorꢀωꢀcarboxyꢀ230 (∇). Values
are means ± SE of data from between 4 and 5 independent
experiments.
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metabolites.
DISCUSSION
5ꢀOxoꢀETE is a potent activator of human eosinophils and may be an important mediator in
1
eosinophilic diseases such as asthma. Our previous in vitro studies on 5ꢀoxoꢀETE antagonists
led to the identification of the indole 230 as a potent and selective antagonist of the human OXE
receptor. Because there is no ortholog of this receptor in rodents we could not investigate its in
vivo effects in rat or mouse models of asthma. We recently examined the possibility of using a
feline model of asthma for this purpose but found that although 5ꢀoxoꢀETE is a highly potent
chemoattractant for eosinophils in this species, the OXE receptor antagonists we developed
against the human OXE receptor were only weak antagonists of the feline receptor. We therefore
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decided to evaluate the cynomolgus monkey as a potential animal model to investigate the
effects of our OXE receptor antagonists.
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We found that monkey leukocytes synthesize substantial amounts of 5ꢀoxoꢀETE directly from 5ꢀ
1
0
HETE as well as from the 5ꢀHETE precursor AA and, as in the case with human granulocytes,
this process in enhanced by the NADPH oxidase activator PMA. 12ꢀHETE and 12ꢀHHT were
also detected in incubations with AA, presumably because our leukocyte preparations contained
1
1
platelets bound to monocytes. The results of these experiments indicate that the synthesis of 5ꢀ
oxoꢀETE is regulated similarly in monkey and human leukocytes.
Monkey granulocytes are also very responsive to 5ꢀoxoꢀETE. Using flow cytometry and antiꢀ
CD49dꢀlabeled leukocytes we were able to identify distinct populations of eosinophils and
neutrophils. 5ꢀOxoꢀETE and LTB strongly stimulate actin polymerization in both cell types,
4
1
2
with LTB being more potent in the case of neutrophils, as is the case in humans. In contrast,
4
PGD stimulated actin polymerization only in eosinophils, consistent with the lack of the DP2
2
receptor in human neutrophils and validating the identity of these two cell populations in our
flow cytometry assay.
Both 5ꢀoxoꢀETE and LTB are potent chemoattractants for monkey leukocytes. Although LTB
4
4
is more potent than 5ꢀoxoꢀETE in inducing leukocyte migration, the maximal response to 5ꢀoxoꢀ
ETE is substantially greater. This was not due to high eosinophil numbers in our leukocyte
preparations, which consisted of about 39% neutrophils and only 1% eosinophils. The low
eosinophil numbers in these preparations is consistent with the negligible chemotactic response
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of these cells to PGD . The very high maximal response of monkey leukocytes to 5ꢀoxoꢀETE is
2
rather surprizing in view of similar efficacy of 5ꢀoxoꢀETE and LTB in inducing human
4
1
3
neutrophil migration. It is not clear whether this is because of a difference between species or
to the fact that we used unfractionated leukocytes in these experiments rather than purified
neutrophils.
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Once we established that monkey leukocytes are capable of synthesizing and responding to 5ꢀ
oxoꢀETE we wanted to determine whether the antagonist potency of 230 in this species is similar
to that in humans. The potencies of 230 in blocking the stimulatory effects of 5ꢀoxoꢀETE on both
actin polymerization and leukocyte migration in monkeys were found to be virtually identical to
5
those that we previously reported for humans.
Figure 12. Metabolism of 230 and 197 in monkeys. Major pathways are shown in red.
To investigate the metabolic stability of 230 and to identify its major cytochrome P450ꢀderived
metabolites we examined its metabolism by monkey liver microsomes in the presence of
NADPH. In contrast to prior experiments with rat liver microsomes, which revealed only modest
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metabolism of 230, we found that 230 is almost completely converted by monkey liver
microsomes to two polar metabolites, which were identified as ω2ꢀhydroxyꢀ230 and ω2ꢀoxoꢀ230
(
Fig. 12) by comparing their chromatographic properties using two mobile phases with different
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selectivities and their UV and mass spectra with those of authentic standards prepared by total
chemical synthesis. We originally anticipated that substantial amounts of ω1ꢀoxidation products
14, 15
16
would also be formed, since mediumꢀ and longꢀ chain fatty acids,
prostaglandins, and
1
7
leukotriene B4 are all converted to significant amounts of both ω1ꢀ and ω2ꢀ oxidation products
by hepatic cytochrome P450 enzymes. However, we detected only small amounts of ω1ꢀ
hydroxyꢀ230, ωꢀcarboxyꢀ230, and ωꢀcarboxyꢀdinorꢀ230.
We attempted to reduce the degree of ωꢀoxidation by adding a branched methyl group at the end
of the hexyl side chain, which we hoped would diminish access to the active site of the enzyme
due to steric hindrance. We prepared two such compounds with methyl groups in either the ω2ꢀ
position (197) or the ω3ꢀposition (225) and examined the metabolism of the more potent of
these, 197, which had an IC virtually identical to that of 230. However, 197 was metabolized to
5
0
an extent similar to 230 by monkey liver microsomes, principally to ω2ꢀhydroxyꢀ197, with only
a small amount of ω1ꢀoxidation to ω1ꢀhydroxyꢀ197 and ωꢀcarboxyꢀ197 (Fig. 12). Other
substances containing branched chain alkyl groups are also metabolized by ωꢀoxidation. For
example, ω1ꢀoxidation has previously been reported for alkylꢀcontaining compounds with a
1
8
19
20
terminal isopropyl group, such as phylloquinone, tocopherol, and ibuprofen, which are
converted by cytochrome P450 isozymes to ω1ꢀhydroxy and ωꢀcarboxy metabolites. Ibuprofen is
2
0
also metabolized by ω2ꢀhydroxylation and both ω2ꢀ and ω3ꢀ hydroxylation have been reported
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for tocopherols. We speculate that the dihydroxy metabolite of 197 identified by mass
spectrometry in the present study is formed by a combination of ω1ꢀ and ω2ꢀ hydroxylation.
Our next goal was to determine whether significant levels of 230 would appear in blood
following oral administration. A pilot experiment in rats revealed that administration of 230 (30
mg/kg) by oral gavage results in plasma levels of over 2 ꢀM, over 50 times higher than its IC50
in inhibiting 5ꢀoxoꢀETEꢀinduced calcium mobilization in human neutrophils. Consistent with its
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5
modest metabolism by rat liver microsomes, no major metabolites were observed in rat plasma.
We then conducted more extensive experiments with identical doses of 230 in cynomolgus
monkeys that revealed much higher plasma concentrations (~100 ꢀM) that peaked after about 1
hour and then declined to about 1 ꢀM by 24h. The higher blood levels of 230 in monkeys was
probably due to a higher rate of absorption from the GI tract rather than to a lower rate of
metabolism, as substantial levels of plasma metabolites were observed in this species. 197 also
appeared rapidly in the blood, but at lower concentrations than 230. Interestingly, the plasma
levels of the active Sꢀenantiomers of both 230 and 197 were lower than those of the inactive Rꢀ
enantiomers. In the case of 230, there was a delay in the selective depletion of the Sꢀenantiomer,
as the ratio of S to R enantiomers was about 1:1 for the first hour, and then declined substantially
between 2 and 8 h. This suggests that this difference between the two enantiomers was due to
selective metabolism of Sꢀ230 rather than to selective uptake of the Rꢀenantiomer from the GI
tract. On the other hand, the ratio of the S:R enantiomers of 197 was markedly diminished at the
earliest time point (30 min), raising the possibility that there might be enantiomeric selectivity at
the levels of both absorption and metabolism for this compound. These results suggest that it
would be preferable to use pure Sꢀenantiomers for future studies, which will be facilitated by a
2
2
novel synthetic procedure that we reported very recently.
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The profile of plasma metabolites of 230 was more extensive than that which we observed with
monkey liver microsomes. As with microsomes, the ω2ꢀoxidation products ω2ꢀhydroxyꢀ230 and
ω2ꢀoxoꢀ230 were major in vivo metabolites, along with much smaller amounts of ω1ꢀoxidation
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products (mainly ωꢀcarboxyꢀ230). However, two major metabolites of intermediate polarity were
also detected in plasma. These products, which could be separated from one another by HPLC
using an acetonitrile gradient, but not with a methanol gradient, have identical UV and mass
ꢀ
spectra, suggesting that they could be stereoisomers. Although the [MꢀH] ions for these isomers
2
indicated the presence of a hydroxyl group, their MS spectra differed markedly from those of
the ω1ꢀOH and ω2ꢀOH metabolites discussed above, which are virtually identical to one another.
2
The very intense ion at m/z 292 in these MS spectra would be consistent with a hydroxyl group
in the ω6 position, on the carbon adjacent to the indole, although this would need to be
confirmed by chemical synthesis. This would be similar to benzylic hydroxylation, a wellꢀknown
2
3, 24
cytochrome P450ꢀcatalyzed reaction.
Furthermore, a human cytochrome P450 isoform
(CYP4Z1) has been reported to hydroxylate both laurate and palmitate in the ω6ꢀposition as well
25
as the ω5, ω4, and ω3 positions. Similarly, AA can be hydroxylated in the kidney to give 16ꢀ
2
6
(i.e. ω5), 17ꢀ, 18ꢀ, 19ꢀ, and 20ꢀ hydroxy metabolites. We do not know the site of this putative
ω6ꢀhydroxylation reaction of 230 in the monkey, but it is likely to be catalyzed by a cytochrome
P450 isozyme at a location other than the liver, such as the kidney or the intestines, as we did not
observe these products in incubations with liver microsomes.
The pharmacokinetic profile of 230 in monkeys was quite different from what we initially
observed in our pilot experiment in rats. In rats, 230 reached a maximal concentration of 2.2 µM
by 1 h and remained at that level up to 4 h, before declining to 0.7 µM by 6 h. In monkeys the
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concentration of 230 peaked at over 100 µM after only 30 min and then declined to less than
10% of that level by 4 h. However, in spite of the relatively rapid decline in 230 levels in
monkeys, its plasma concentration after 24 h (2.7 µM) was higher than the maximum level
reached in rats. As we did not detect high levels of 230 metabolites in rats, it is possible that
these differences could be due to different rates of absorption from the gut in the two species.
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