Biogenic Synthesis, Purification, and ChemicalCharacterization of Anti-inflammatory Resolvins Derivedfrom Docosapentaenoic Acid (DPAn-6)Received for publication, December 1, 2008, and in revised form, March 25, 2009 Published, JBC Papers in Press, March 26, 2009, DOI 10.1074/jbc.M809014200

Bindi Dangi1, Marcus Obeng, Julie M. Nauroth, Mah Teymourlouei2, Micah Needham, Krishna Raman,and Linda M. Arterburn3

From Martek Biosciences Corporation, Columbia, Maryland 21045

Enzymatically oxygenated derivatives of the �-3 fatty acidscis-4,7,10,13,16,19-docosahexaenoic acid (DHA) and cis-5,8,11,14,17-eicosapentaenoic acid, known as resolvins, havepotent inflammation resolution activity (Serhan, C. N., Clish,C. B., Brannon, J., Colgan, S. P., Chiang, N., and Gronert, K.(2000) J. Exp.Med. 192, 1197–1204;Hong, S., Gronert, K., Devc-hand, P. R., Moussignac, R., and Serhan, C. N. (2003) J. Biol.Chem. 278, 14677–14687). Our objective was to determinewhether similar derivatives are enzymatically synthesized fromother C-22 fatty acids and whether these molecules possessinflammation resolution properties. The reaction of DHA,DPAn-3, and DPAn-6 with 5-, 12-, and 15-lipoxygenases pro-duced oxylipins, whichwere identified and characterized by liq-uid chromatography coupled with tandem mass-spectrometry.DPAn-6 and DPAn-3 proved to be good substrates for 15-li-poxygenase. 15-Lipoxygenase proved to be the most efficientenzyme of the three tested for conversion of long chain polyun-saturated fatty acids to corresponding oxylipins. Since DPAn-6is a major component of Martek DHA-STM oil, we focused ourattention on reaction products obtained from the DPAn-6 and15-lipoxygenase reaction. (17S)-hydroxy-DPAn-6 and (10,17S)-dihydroxy-DPAn-6 were the main products of this reaction.These compounds were purified by preparatory high perform-ance liquid chromatography techniques and further character-ized by NMR, UV spectrophotometry, and tandem mass spec-trometry. We tested both compounds in two animal models ofacute inflammation anddemonstrated that both compounds arepotent anti-inflammatory agents that are active on local intra-venous as well as oral administration. These oxygenatedDPAn-6 compounds can thus be categorized as a new class ofDPAn-6-derived resolvins.

Enzymatically formed oxygenation products of C-20 andC-22 long chain polyunsaturated fatty acids (LC-PUFAs),4 have

important biological roles in inflammation, allergies, and bloodclotting and are thus believed to have therapeutic potential inseveral chronic immune diseases (1–10) Several biologicallyimportant products of cis-5,8,11,14-eicosatetraenoic acid/arachidonic acid (ARA), cis-5,8,11,14,17-eicosapentaenoic acid(EPA), and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA)have been described (4, 11, 12). Proinflammatory oxylipins,such as leukotrienes and some prostaglandins, are derived fromARA, an �-6 fatty acid. Interestingly, the same fatty acid alsoserves as a precursor to anti-inflammatory or proresolutionmolecules like lipoxins (13, 14). Stable analogues of lipoxins arebeing developed as drugs for asthma and other inflammatoryairway diseases (15, 16). Oxylipins derived from �-3 fatty acids,such as DHA and EPA, known as resolvins, are primarilyanti-inflammatory in nature (17). EPA acts as a precursor tothe E-series resolvins that have shown potential in the treat-ment of colitis, arthritis, and periodontitis (18–20). Theresolvins of the D-series derived from DHA are useful asneuroprotective agents. 10,17-Dihydroxy-4,7,11,13,15,19-docosahexaenoic acid (10,17-HDHA) or neuroprotectin D1is a resolvin that is formed endogenously in the human brainand eye and is believed to exert its protective effect againstcell injury-induced oxidative stress (21–23).The main enzymes responsible for the production of these

oxygenated LC-PUFA products are primarily lipoxygenasesand, in addition, cyclo-oxygenases and cytochromes P450.These enzymes produce oxylipins via transcellular activity,often involving multiple cell types (24). This activity mainlyresults in mono-, di-, and tri-hydroxylation products of fattyacids that have varying potencies, depending on the exact struc-ture of the compound. Lipoxygenases are non-heme, iron-con-taining dioxygenases that catalyze the regioselective and enan-tioselective oxidation of polyunsaturated fatty acids containingone or more cis,cis-1,4-pentadienoic moieties to give the corre-sponding hydroperoxy derivatives (25, 26).We thus consideredthat, in addition to DHA and EPA, other C-22 PUFAs contain-ing such methylene interrupted double bonds may also be sub-strates for lipoxygenases and that resulting products may have1 To whom correspondence should be addressed: Martek Biosciences, 6480

Dobbin Rd., Columbia, MD 21045. Tel.: 443-542-2148; Fax: 410-740-2985;E-mail: bdangi@martek.com.

2 Present address: U.S. Pharmacopeia, 12601 Twinbrook Pkwy., Rockville, MD20852.

3 Present address: Alba Therapeutics Corp., 800 W. Baltimore Street, Balti-more, MD 21201.

4 The abbreviations used are: LC-PUFA, long chain polyunsaturated fatty acid;ARA, Na-MES, 2-(N-morpholino)ethanesulfonic acid sodium salt; ARA, cis-5,8,11,14-eicosatetraenoic acid/arachidonic acid; EPA, cis-5,8,11,14,17-ei-cosapentaenoic acid; DHA, cis-4,7,10,13,16,19-docosahexaenoic acid;

10,17-HDHA, 10,17-dihydroxy-4,7,11,13,15,19-docosahexaenoic acid;(17S)-HDHA, (17S)-hydroxy-4,7,10,13,15,19-docosahexaenoic acid; TNF,tumor necrosis factor; HPLC, high performance liquid chromatography;17-HDPAn-6, 17-hydroxy-4,7,10,13,15-docosapentaenoic acid; 10,17-HD-PAn-6, 10,17-dihydroxy-4,7,11,13,15-docosapentaenoic acid; LC, liquidchromatography; MS, mass spectrometry; MS/MS, tandem mass spec-trometry; VCD, vibrational circular dichroism; PBS, phosphate-bufferedsaline; ANOVA, analysis of variance.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 22, pp. 14744 –14759, May 29, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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anti-inflammatory activity similar to DHA-derived resolvins.DPAn-6 (cis-4,7,10,13,16-docosapentaenoic acid) is present inalgal oils, and recent studies have demonstrated that this fattyacid has anti-inflammatory activities in vitro and, in conjunc-tion with DHA, also has anti-inflammatory activity in vivo.5Also, it has been suggested that a combination of DHA andDPAn-6 could be a beneficial natural therapy in neuroinflam-matory conditions like Alzheimer disease. Specifically, in a3�Tg-AD mouse model of Alzheimer disease, DPAn-6 wasshown to reduce levels of early stage phospho-Tau epitopes,which in turn correlated with a reduction in phosphorylatedc-Jun N-terminal kinase, a putative Tau kinase (27). Althoughthe precise mechanism of action of DPAn-6 in these inflamma-torymilieus is not known, it suggests a possible role for oxylipinproducts of DPAn-6 in resolution of inflammation. Also,another LC-PUFA, DPAn-3 (cis-7,10,13,16,19-docosapentae-noic acid) usually present along with DHA and EPA in marineoils is known to be a potent inhibitor of platelet aggregation(28–30). In addition, this LC-PUFA has a potent inhibitoryeffect on angiogenesis through the suppression of VEGFR-2(vascular endothelial-cell growth factor receptor 2) expression.Angiogenesis is known to contribute to tumor growth, inflam-mation, and microangiopathy, again pointing to the possibilitythat anti-inflammatory activity of DPAn-3 might be mediatedthrough resolvin-like products as in the case of DHA and EPA(31).The purpose of this research was to determine whether oxy-

lipins are formed from the C-22 LC-PUFAs, DPAn-6 andDPAn-3, by lipoxygenase activity; to compare them to productsformed from DHA; to chemically characterize products; topurify key oxylipin products from theDPAn-6/15-lipoxygenasereaction; and to test whether these compounds have resolvin-like anti-inflammatory activity. This research also sets the stagefor preparation and isolation of a wide range of other C-22oxylipins that could be evaluated as potential anti-inflamma-tory compounds.

EXPERIMENTAL PROCEDURES

Materials—5-lipoxygenase from potato, 12-lipoxygenasefrom porcine leukocytes, and (17S)-hydroxy-4,7,10,13,15,19-docosahexaenoic acid ((17S)-HDHA) were from CaymanChemicals (Ann Arbor, MI). Tumor necrosis factor-� (TNF�)was from Peprotech, Inc. (Rocky Hill, NJ). All fatty acids werefrom Nu-Chek Prep (Elysian, MN). Filtration units were fromNalgene (Rochester, NY). Acrodisc syringe filters were fromPall Life Sciences (Ann Arbor, MI). All Luna high performanceliquid chromatography (HPLC) columns were from Phenome-nex (Torrance CA), Chiralpak-IA was from Chiral Technolo-gies (West Chester, PA), Chirobiotic-T was fromAstec (Whip-pany, NJ), and all HPLC solvents were from Fisher. All otherreagents andmaterials were from Sigma, whereas animals werefrom Harlan (Indianapolis, IN).

Reaction of DHA, DPAn-3, and DPAn-6 with 15-Lipoxygen-ase for Comparison of Efficiency of Conversion—Soybean 15-li-poxygenase (Type 1-B) at a final concentration of 524 units/mlwas mixed into 100 �M solutions of DHA, DPAn-6, or DPAn-3fatty acids in 0.05 M sodium borate buffer, pH 9.0, and the reac-tion mixtures were incubated at 0 °C. The appearance of themonohydroxy-conjugated diene derivatives of the fatty acidswas monitored through absorbance at 237 nm. Conjugateddiene products were quantified by UV spectrophotometryusing an extinction coefficient of 28,000/M�cm (32).Enzyme Kinetics Measurements of 15-Lipoxygenase with

DHA, DPAn-3, and DPAn-6—Type 1-B soybean lipoxygenase(39.8 units/ml) was used to dioxygenate substrates (DPAn-6,DPAn-3, and DHA at 5–100 �M) at 22 °C in oxygen-saturated0.05 M sodium borate buffer (pH 9.0) with 4.52 � 10�3% (v/v)Tween 20. Final oxygen concentrations exceeded 750 mM,measured using a Clark type oxygen electrode. The probe wascalibrated by bubbling air and directly measuring the concen-tration with immobilized fluorescent ruthenium (OxySense200T; OxySense, Dallas, TX). Approximately 38 s after theenzyme addition, the hydroperoxide product formation wasmonitored viaUV absorbance at 236 nm (as described above) at15-s intervals for 600 s (DU800 spectrophotometer; BeckmanCoulter, Fullerton, CA). The read average time was 0.2 s. Allexperiments were repeated in triplicate. Product formationcurves were smoothed with a negative exponential transformsampling all of the data with a power of 10 calculating intervalsevery second. Product formation curves showed an initial lagperiod followed by a maximum in rate (rmax). Although this isnot the classical V0max, previous authors have shown that itsatisfies the steady-state assumption for application of theMichaelis-Menten kinetic model (33, 34). This model, r �Vmax[S]/(Km � [S]), was used to elucidate the kinetic parame-ters. SigmaPlot 11.0 with the Enzyme Kinetic Module 1.3 (Sys-tat Software, Inc., San Jose, CA) was used to perform the leastsquares fit of the aforementioned model.Reaction of DHA, DPAn-3, and DPAn-6 with 5-, 12-, and

15-Lipoxygenase for Identification of Reaction Products—Forthe 5-lipoxygenase reaction, 200 �l of 10 units/�l potato 5-li-poxygenase was added to a 10-ml reaction mixture containing100�M fatty acid in 0.05 MNa-MES buffer, pH 6.3, 100�MSDS,and 0.02% decaethylene glycol monododecyl ether. For the12-lipoxygenase reaction, 100 �l of 0.75 units/�l porcine leu-kocyte-derived 12-lipoxygenase was added to a 10-ml solutioncontaining 100 �M fatty acid in 0.1 M Tris-HCl, pH 7.5, 5 mMEDTA, and 0.03% Tween 20. For the 15-lipoxygenase reaction,100 �l of 131 kilounits/ml soybean 15-lipoxygenase (Type 1-B)was added to a 10-ml reaction mixture containing 100 �M fattyacids in 0.05 M sodium borate buffer, pH 9.0. Control reactionswere run for all three enzymes under identical conditions with-out adding any enzyme. All reaction mixtures were stirred for30 min at 4 °C. Reaction products were reduced with sodiumborohydride and acidified with glacial acetic before extractionon solid phase DSC-18 cartridges. Final elutions were doneusing anhydrous ethanol and reaction products analyzed byLC-MS/MS (liquid chromatography coupled with tandemmass spectrometry). The reaction of 15-lipoxygenase withDPAn-6 described above was scaled-up for the production of

5 J. M. Nauroth, Y. C. Liu, M. Van Elswyk, R. Bell, E. Bailey-Hall, G. Chung, andL. M., Arterburn, manuscript in preparation. Preliminary work pertaining tothis publication has been presented at the 94th Annual Meeting of theAmerican Association of Immunologists, 2007 (abstract available in Journalof Immunology, 2007, 178, 101.5).

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17-hydroxy-4,7,10,13,15-docosapentaenoic acid (17-HDPAn-6) and 10,17-dihydroxy-4,7,11,13,15-docosapentaenoic acid(10,17-HDPAn-6) from 1 g of DPAn-6.Purification of 17-HDPAn-6 and 10,17-HDPAn-6—All puri-

fications were carried out at a flow rate of 47 ml/min at roomtemperature on a Luna C18 (2), 10 �m, 100 Å, 50 � 250-mmcolumn, set up on an Agilent 1200 preparatory HPLC unit,equipped with a diode array detector. The purification methodinvolved elution with a mixture of solvents A and B. Solvent Aconsisted of 70% water, 30% methanol, and 0.2% ammoniumacetate, and solvent B consisted of 100% acetonitrile. Themethod used for elution of compoundswas 48%B at 0min, 48%B at 25 min, 90% B at 35 min, 90% B at 50 min, 48% B at 52 min,and 48% B at 65 min. Chromatograms were monitored simul-taneously at 236 and 270 nm, and fractions corresponding topeaks of interest were collected and pooled from several runs.Purity of fractions was monitored by UV spectrophotometeryaswell as by LC-MS. Fractionswere concentrated using a rotaryevaporator, and compounds were extracted with ether. Etherextracts were concentrated and treatedwith anhydrous sodiumsulfate to remove any traces of water. This solution was thenfiltered usingAcrodisc syringe filters (Bulk Acrodisc CR 13mmwith 0.2 �M polytetrafluoroethylene membrane, HPLC-certi-fied). Purity was ascertained by LC-MS and LC-MS/MS.UV Spectrophotometry—UV spectrophotometry was carried

out on either an Agilent 8453 (Agilent Technologies, SantaClara, CA) or a Cary 4000 UV-visible spectrophotometer (Var-ian Inc., Palo Alto, CA) or a DU800 spectrophotometer (Beck-man Coulter). Extinction coefficients of 17-HDPAn-6 and10,17-diHDPAn-6 were determined in ethanol.LC-MS and LC-MS/MS—All routine reaction and purity

analysis was done on a Hewlett Packard model 1100 liquidchromatography instrument interfaced with an electrosprayionization mass elective detector. Tandem MS/MS analysis ofthe products of the reactions of fatty acids and different lipoxy-genases was performed on anAgilent 1100 Series HPLC instru-ment (San Paulo, CA) interfaced with an Esquire 3000 ion trapmass spectrometer equipped with an electrospray ionizationsource (Bruker Daltonics, Billerica, MA) or on an Agilent 1200Series HPLC instrument interfaced with an Agilent LC/MSDTrap XCT Plus (San Paulo, CA). Mass spectrometers wereoperated in negative ion detection mode. Nitrogen was used asnebulizing and drying gas with nebulizer pressure at 20 p.s.i.and drying gas flow rate of 7 liters/min. The interface temper-ature was maintained at 330 °C. The chromatographic separa-tionwas carried out on a LUNAC18(2) column (250� 4.6mm,5 �m, 100 Å) using the same mobile phase described for puri-fication of the compounds under similar conditions at a flowrate of 0.4 ml/min.NMR Experiments—All NMR experiments were conducted

byNMRServices (Rochester,NY). 25–50mgof 17-HDPAn-6 or10,17-HDPAn-6 was dissolved in CDCl3 and used for NMRdata acquisition. All NMR data were acquired at 298 K on aBruker model Avance spectrometer operating at a 500-MHzproton NMR frequency. One-dimensional 1H, one-dimen-sional 13C (1H-decoupled), two-dimensional 1H-1H (corre-lation spectroscopy), two-dimensional 1H-1H DQFCOSY(double-quantum-filtered correlation spectroscopy), two-

dimensional 1H-13C-HSQC (heteronuclear single quantumcoherence), and two-dimensional 1H-13C-HMBC (heter-ononuclear multiple bond-correlation) NMR data wereacquired for 17 HDPAn-6. One-dimensional 1H, one-dimen-sional 13C (1H-decoupled), and two-dimensional 1H-1HCOSY were acquired for 10,17-HDPAn-6. All data were pro-cessed using Bruker software, and NMR spectra predictionswere done using ChemDraw Ultra 10.0 and ACDlabssoftware.Chirality—17-HDHA was prepared the same way as 17-

HDPAn-6 except that DHA was used as a substrate. Either17-HDHA or 17-HDPAn-6 was converted to 17-oxo-deriva-tives by dissolving 15 mg of each compound in anhydrousdichloromethane and then oxidizing the compounds at 0 °Cwith 15 mg of Dess-Martin’s reagent. The 17-oxo-derivativeswere then reduced using sodium borohydride to give the cor-responding racemicmixtures. The racemicmixtures were thenseparated on aChiralpak-IA columnusingmethanol/water (80:20) as themobile phase at a flow rate of 0.6ml/min at 25 °C. Thepeaks were assigned as R or S by using (17S)-HDHA as a re-ference standard. The same protocol was followed for 17-HDPAn-6, and the order of elution of the two isomers wasassumed to be the same as for 17-HDHA isomers. The Chi-robiotic-T column was tested with supplier-recommendedmobile phases. Vibrational circular dichroism (VCD) studieswere carried out by Biotools Inc. (Syracuse, NY) for17-HDPAn-6.Log DpH 7.4 and Solubility—LogDpH 7.4 and solubility studies

were conducted by Cerep (Seattle, WA). All predictions of var-ious physicochemical parameters were made using ACDLabssoftware. LogPwas calculated based on the formula, logP� logD � (pH � pKa) (35) Aqueous solubility (�M) was determinedby comparing the peak area of the principal peak in a calibrationstandard (200�M) containing organic solvent (methanol/water,60/40, v/v) with the peak area of the corresponding peak in abuffer sample. Reference compounds used for comparing solu-bility were diethylstilbestrol, haloperidol, ketoconazole, meto-prolol tartrate, phenytoin, rifampicin, simvastatin, and tamox-ifen. Log DpH 7.4 was determined by the shake flask methodusing octanol and PBS, pH 7.4 (35). The total amount of com-pound was determined as the peak area of the principal peak ina calibration standard (100 �M) containing organic solvent(methanol/water, 60/40, v/v). The amount of compound in thebuffer was determined as the combined, volume-corrected, andweighted areas of the corresponding peaks in the aqueousphases of three organic aqueous samples of different composi-tion. An automated weighting system was used to ensure thepreferred use of raw data from those samples with well quanti-fiable peak signals. The amount of the compound in the organicphase was calculated by subtraction. Subsequently, log D wascalculated as log10 of the amount of compound in the organicphase divided by the amount of compound in the aqueousphase.Chemical Stability Studies—17-HDPAn-6 and 10,17-HDPAn-

6 were incubated at a concentration of 10 �M in both PBS andethanol at 4 °C for 14 days and at 22 °C for 24 h and at 37 °C for2 h, and peak heights were monitored by LC-MS/MS. For the14-day stability studies, sampleswere analyzed at 0, 2, 4, 7, 9, 11,

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and 14 days. Samples were analyzed at 0 and 24 h for the 22 °Cstudy and at 0 and 2 h for the 37 °C study.Animal Studies—All animal studies were performed in

accordance with the Guide for the Care and Use of LaboratoryAnimals as adopted and promulgated by theNational Institutesof Health and/or the Final Rules of the Animal Welfare ActRegulation (9CFR).Mouse Air Pouch Study—To generate the dorsal air

pouches, 6–8-week-old female C57Bl/6N mice were anes-thetized, and 6 ml of sterile air was injected subcutaneouslyin the back (36). Pouches were reinflated with 3 ml of sterileair 3 days later. Six days after initial pouch formation, 100 ngof the DPAn-6 oxylipin test compounds or (17S)-HDHAwere administered to 10 animals per group in 1ml of PBS, pH7.4 (�0.005 mg/kg) by intrapouch injection. Within 5 min ofcompound administration, 100 ng of TNF� in 0.1 ml of PBSwas injected into the pouches to initiate an inflammatoryresponse. Indomethacin was administered intraperitoneallyat 2 mg/kg 30 min prior to the TNF� challenge as a positivecontrol. Four h after the TNF� challenge, pouch exudateswere harvested, and leukocytes in the exudates werecounted. A sampling of exudate cells from each animal wascentrifuged onto microscope slides, and the cells were air-dried and Geimsa-stained to quantify granulocyte andmononuclear leukocytes.Rat Hind Paw Edema Studies—Hind paw edema studies

were conducted on male Sprague-Dawley rats (170–200 g,6–8 animals/group) using cross-linked � carrageenan as theinflammatory stimulus (37). Researchers were masked to thetreatment groups in all hind paw studies. The first experi-ment involved administering DPAn-6-derived resolvinseither intravenously (5 �g/animal, �0.025 mg/kg) in the tailvein in 0.1 ml of PBS 2 min prior to carrageenan challenge orper os (by oral gavage) in up to 4 ml of PBS (25 �g/animal,�0.125 mg/kg) 30 min prior to carrageenan challenge. Indo-methacin (5 mg/kg administered intraperitoneally 30 minprior to challenge) and biogenically synthesized (17S)-

HDHA (5 �g/animal administered intravenously 2 min priorto challenge) served as positive and DHA resolvin controls,respectively, in this experiment. Rats were injected with 0.1ml of 10 mg/ml carrageenan solution (prepared in water atleast 2 weeks in advance of the experiment) in the subplantar

FIGURE 1. Comparison of DHA, DPAn-3, and DPAn-6 as substrates forsoybean lipoxygenase. Fatty acids (DHA (E), DPAn-3 (�), and DPAn-6(Œ)) at a concentration of 100 �M were incubated with enzyme at 0 °C, asdescribed under “Experimental Procedures.” Absorbance at 237 nm wasused to estimate the amount of product formed, and theoretical 100%conversion was calculated based on an extinction coefficient of 28,000/M�cm in all three cases. The percentage conversions were verified byLC/MS. The graph shows results from replicate measurements. The errorbars represent ranges.

FIGURE 2. Enzyme kinetics measurements and curve fits for DPAn-6,DPAn-3, and DHA. The reaction was carried out at 22 °C with 39.8 units/ml ofthe enzyme, 4.52 � 10�3 % (v/v) Tween 20 in oxygen-saturated 0.05 M sodiumborate buffer (pH 9.0), and products were quantified by measuring absorb-ance at 236 nm. Shown are actual (circles) and predicted (trace) hydroperox-ide formation rates as a function of substrate concentration for DPAn-3 (A),DPAn-6 (B), and DHA (C). Actual results are averages of triplicates, presentedwith S.E. bars.

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region of the right hind paw. Paw volumes were measured bywater volume displacement at 0, 2, 4, 6, and 24 h post-carra-geenan challenge.

The second experiment involved evaluating oral dose-re-sponse behavior of the two DPAn-6 resolvins. All compounds,including the controls, were administered by oral gavage (4ml/animal) 30 min prior to the carrageenan challenge. The twoDPAn-6 oxylipins were diluted in PBS, pH 7.4, and dosed at 2.5,25, and 250 �g/animal (�0.0125, 0.125, and 1.25 mg/kg bodyweight), and the biogenically synthesized resolvin control17-HDHA was given at a single dose of 25 �g/animal (0.125mg/kg). Indomethacin was administered at 6 mg/animal (30mg/kg). Carrageenan was administered as in the first rat hindpaw experiment. Paw edema volumewasmeasured 2, 4, and 6 hafter the carrageenan challenge. Paw edema volume was calcu-

TABLE 1Kinetic parameters of PUFA substrates with 15-lipoxygenaseSubstrates (5–100 �M) were incubated with 39.8 units/ml of 15-lipoxygenase,4.52 � 10�3% (v/v) Tween 20 in oxygen-saturated 0.05 M sodium borate buffer (pH9.0), and products were quantified using absorbance at 236 nm. Curve fits werecreated using the Michaelis-Menten model.

Parameter DPAn-3 DPAn-6 DHAVmax (�M/min) 672.9 � 31.8 487.1 � 13.9 481.9 � 21.4Km (�M) 36.2 � 4.1 14.3 � 1.5 20.4 � 2.8Vmax/Km (min�1) 18.6 � 2.3 34.1 � 3.7 23.6 � 3.4R2 0.93 0.90 0.85

TABLE 2Products formed upon incubation of DHA, DPAn-3, and DPAn-6 with soybean 15-lipoxygenaseDHA, DPAn-3, and DPAn-6 were incubated with soybean 15-lipoxygenase in 0.05 M sodium borate buffer, pH 9.0, at a concentration of 100 �M, at 4 °C for 30 min, andproducts were reduced to the corresponding hydroxy compounds using sodium borohydride. Reactions were analyzed and product structures were elucidated based onLC-MS/MS analysis of diagnostic fragments. Diagnostic fragments that helped in ascertaining positions of hydroxylation are shown in parentheses.

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TABLE 3Products formed upon incubation of DHA, DPAn-3, and DPAn-6 with porcine 12-lipoxygenaseDHA, DPAn-3, and DPAn-6 were incubated with 12-lipoxygenase obtained from porcine leukocytes in 0.1 M Tris-HCl, pH 7.5, 5 mM EDTA, and 0.03% Tween 20, at aconcentration of 100 �M, at 4 °C for 30 min, and products were reduced to the corresponding hydroxy compounds using sodium borohydride. Reactions were analyzed byLC-MS/MS, and product structures were elucidated based on analysis of diagnostic fragments. Diagnostic fragments that helped in ascertaining positions of hydroxylationare shown in parentheses.

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lated by subtracting right paw volume just prior to carrageenaninjection from the post-carrageenan-treated volume of thesame paw.Statistical Analyses—Statistical analyses for animal studies

were conducted in GraphPad Prism version 4 (San Diego, CA)using Student’s t tests or ANOVA with Dunnett’s post test to

compare treatment groups with control. Data are presented asmeans � S.D.

RESULTS

Substrate Comparison with 15-Lipoxygenase—Fig. 1 showspercentage conversion of DHA, DPAn-3, and DPAn-6 upon

TABLE 4Products formed upon incubation of DHA, DPAn-3, and DPAn-6 with potato 5-lipoxygenaseDHA, DPAn-3, and DPAn-6 were incubated with potato 5-lipoxygenase in 0.05 M Na-MES buffer, pH 6.3, 0.02% C12E10 at a concentration of 100 �M, at 4 °C for 30 min,and products reduced to the corresponding hydroxy compounds using sodium borohydride. Reactions were analyzed by LC-MS/MS, and product structures wereelucidated based on analysis of diagnostic fragments. Diagnostic fragments that helped in ascertaining positions of hydroxylation are shown in parentheses.

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treatment with soybean lipoxygenase at 0 °C. As can be seen,under these conditions, 100% of DPAn-6 was efficiently con-verted to its conjugated diene derivative, whereas about 85% ofDPAn-3 and 50% of DHA were converted to their respectiveconjugated diene derivatives by 15-lipoxygenase. These resultswere also corroborated by LC/MS peak analysis, which showedthat under these conditions, a single product with a conjugateddiene was formed, and this corresponded to the 17-hydroper-oxy derivative in all three cases. In addition, we also assessedenzyme kinetics parameters of the three substrates with 15-li-poxygenase at 22 °C. Fig. 2 shows the enzyme kinetics data withcurve fits, and Table 1 shows the kinetic parameters. Over thesubstrate concentrations analyzed, the kinetic behavior wasMichaelis-Menten. AlthoughDPAn-3 exhibited the highest valueforVmax, the efficiencywithwhich the enzyme convertedDPAn-6to products was higher than that for DHA and DPAn-3.Products Formed upon Reaction of Fatty Acids with 5-, 12-,

and 15-Lipoxygenase—Tables 2–4 give a summary of productsformed when DHA, DPAn-3, and DPAn-6 are reacted with thevarious lipoxygenases. In all cases, the enzymes producedhydroperoxy compounds, which were then reduced nonenzy-matically by sodiumborohydride to the corresponding hydroxycompounds. The hydroperoxy derivatives (data not shown)were detected in nonreduced reactions by LC-MS/MS tech-niques. Final reduced products were identified based on parention masses and diagnostic fragments. MS spectra of all mono-hydroxy compounds showed [M-H], [M-H]-H2O, [M-H]-CO2,and [M-H]-CO2,-H2O, which were 345, 327, 301, and 283 Da,respectively, for monohydroxy-DPAn-6 and -DPAn-3 deriva-tives and were 343, 325, 299, and 281 Da for monohydroxy-DHA derivatives. MS spectra of all dihydroxy compoundsshowed [M-H], [M-H]-H2O, [M-H]-CO2, [M-H]-CO2,-H2O,[M-H]-2H2O, and [M-H]-CO2,-2H2O, which were 361, 343,317, 325, and 281 Da, respectively, for dihydroxy-DPAn-3 andDPAn-6 derivatives and were 359, 341, 315, 323, and 279 fordihydroxy-DHA derivatives. All diagnostic fragments that help

in ascertaining positions of oxygen-ation are outlined in Tables 2–4.Of all of the enzymes tested, 15-li-

poxygenasewas themost efficient inconverting fatty acids to oxylipins,whereas 5-lipoxygenase and 12-li-poxygenase converted less than 10%of the fatty acids to oxylipin-likecompounds. Overall, many of theproducts characterized upon usingDPAn-6 or DPAn-3 as substrateswere similar to those obtained fromDHA, as reported earlier (1, 3, 5).As expected, the 15-lipoxygenaseproductswere primarily oxygenatedat the C-17-position, ultimatelyproducing 17-HDHA, 17-HDPAn-6, and 17-HDPAn-3 from therespective fatty acids. These com-pounds had a conjugated dienestructure, with an absorbance max-imum between 234 and 238 nm. In

addition, 10,17-dihydroxy and 7,17-dihydroxy derivatives wereproduced from all three fatty acids. The 10,17-dihydroxy deriv-atives had an absorbance maximum at 270 nm with character-istic shoulders at 260 and 280 nm (�2 nm), which are charac-teristic of a conjugated triene structure. The 7,17-dihydroxyderivatives had an absorbance maximum at �242 � 2 nm,characteristic of two conjugated dienes interrupted by a meth-ylene group. The main product (�90%) formed upon reacting12-lipoxygenase with fatty acids was the 14-hydroxy derivative(hydroxylation at the �-9-position) in all three cases. In addi-tion, several other mono as well as dihydroxy derivatives weredetected (see Table 3). When 5-lipoxygenase was used, themain product (�90%) formed was due to oxygenation at the�-13-position, resulting in the 10-hydroxy derivative. Otherproducts were also seen (see Table 4). The 10,20-dihydroxyderivative was formed only from DHA and DPAn-3 and notfromDPAn-6, sinceDPAn-6 lacks a double bond at C-19. Noneof the controls for any of the enzymatic reactions showed anyappreciable levels of oxylipins, suggesting that there was noappreciable amount of nonenzymatic autoxidation under theconditions tested.Gram Scale Synthesis and Purification of 15-Lipoxygenase

DPAn-6 Resolvins—Fig. 3 shows the LC profiles of productsobtained when 1 g of DPAn-6 was reacted with 15-lipoxygen-ase. The main products that were formed were 17-HDPAn-6(�85%), 10,17-HDPAn-6 (�4%), and 7,17-HDPAn-6 (�2%),similar to what is described above for the small scale reaction.In addition, several other dihydroxy compounds (retentiontime 12–21 min), trihydroxy derivatives (retention time 5–10min), and oxo and epoxide compounds (retention time 33–36min) were produced. These were detected, based on character-istic fragmentation patterns as well as UV behavior. For exam-ple, the 17-oxo-DPAn-6 showed a UV peak at 280 nm, with a[M-H] of 343 and a diagnostic fragment at 245 along with allother expected fragments. Similarly, the 16,17-epoxide ofDPAn-6 exhibited no UV absorbance between 220 and 350 nm

FIGURE 3. LC profile of the gram scale reaction of DPAn-6 with soybean 15-lipoxygenase. DPAn-6 wasincubated with soybean lipoxygenase in 0.05 M sodium borate buffer at 4 °C for 30 min. Products were reducedwith sodium borohydride, the reaction was acidified, and then products were extracted using DSC-18 material.Products were analyzed by LC/MS as described under “Experimental Procedures.” A, total ion chromatogram;B, UV absorbance at 236 nm; C, UV absorbance at 270 nm.

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and showed the expected [M-H], [M-H]-H2O, [M-H]-CO2, and[M-H]-CO2,-H2O fragments along with 233, 245, 274, and 261nm, which are characteristic of an epoxide at the 16,17-posi-tion. The total fraction accounted for by all these other com-pounds was less than 5% of the main products formed. Purifi-cation of 17-HDPAn-6 and 10,17-diHDPAn-6 resulted in

�98% pure compounds (Fig. 4) thatwere further characterized and thenused for other studies.Structural Characterization of

17-HDPAn-6 and 10,17-diHDAPn-6—As described previously, 17-HDPAn-6 showed a UV spectrumwith an absorbance maximum at236 nm. The extinction coefficientin ethanol at room temperature wascalculated to be 30,000/M�cm. AllMS/MS fragments were consistentwith the 17-HDPAn-6 structure, asshown in Fig. 4. Fig. 5 shows theone-dimensional 1H, one-dimen-sional 13C, and two-dimensional1H,1H COSY spectra of 17-HDPAn-6. The number of peaks,integration, and chemical shifts inboth the one-dimensional 1H andthe one-dimensional 13C spectrawere consistent with the molecularformula C22H34O3 and were con-sistent with spectra predicted byChemdraw and ACDlabs software.The two-dimensional 1H,1H COSYunambiguously showed that hy-droxylation was at the 17-position.The protons belonging to C(17)-H-OH centered at 4.12 ppm pro-duced cross-peaks to C(16)-H andC(18)-H but not with protonsbelonging to C(13) or C(14). A two-dimensional 1H,1H DQFCOSY, atwo-dimensional 1H,13C-HSQC,and a two-dimensional 1H,13CHMBC confirmed all nonoverlap-ping 1H and 13C assignments anddouble bond configurations. Thedouble bond configurations wereconfirmed based on several lines ofevidence as outlined below. Butov-ich et al. (38, 39) have described sig-nature chemical shifts obtainedfrom NMR spectra of several com-pounds that are indicative of atrans-trans configuration. Specifi-cally, a conjugated (EE)-diene frag-ment would have produced a quar-tet with a chemical shift � of 6.15 to6.24 ppm, and this signature quartetis clearly absent in all spectra of the

17-HDPAn-6 produced in this study (see Fig. 5) Coupling con-stant information also suggests that the structure is 17-hy-droxydocosa-(4Z,7Z,10Z,13Z,15E)-pentaenoic acid. This isbased on the fact that the value of J13,14 is 11Hz, indicative of thecis-configuration around this vinyl group, whereas, J15,16 is �15Hz, again clearly indicating that the configuration around these

FIGURE 4. LC/MS/MS of purified 17-HDPAn-6 and 10,17-HDPAn-6. The reaction mixture represented by Fig.2 was purified by preparatory HPLC techniques using conditions described under “Experimental Procedures.”Fractions of interest were pooled from several runs and further concentrated and analyzed for purity usingLC/MS/MS techniques. A, total ion chromatogram of 17-HDPAn-6 with LC conditions, as described under“Experimental Procedures.” The inset shows the UV spectrum of the main peak; B, mass spectra of the mainpeak shown in A with main fragments denoted by their origin. The inset shows 17-HDPAn-6 with the expecteddiagnostic fragments. C, total ion chromatogram of 10,17-HDPAn-6 with an inset showing the UV spectrum ofthe main peak. D, mass spectra of the main peak shown in C with main fragments denoted by their origin. Theinset shows 10,17-HDPAn-6 with the expected diagnostic fragments.

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double bonds is trans. In addition, acomparison of chemical shifts of17-HDPAn-6 with 17-HDHA pro-duced by Butovich et al. (38) areremarkably similar, indicating similardouble bond configurations.10,17-HDPAn-6 produced and

purified to 98% homogeneity had aUV spectrum with a �max at 270nm and shoulders at 260 and 280nm (�1 nm), indicative of a conju-gated triene in the molecule (Fig.4). The extinction coefficient wasestimated to be 38,000/M�cm. Theparent ion mass with m/z of 361along with fragments (Fig. 4) con-firms hydroxylation at C-10 andC-17 of DPAn-6 and is consistentwith the molecular formulaC22H34O4. NMR data for thiscompound are shown in Fig. 6.The 1D-13C spectrum clearlyshows the presence of 22 carbons,with two of the carbons having achemical shift � of 71.62 and 71.02ppm, indicative of hydroxylationsat these two carbons. Again, as inthe case of 17-HDPAn-6, no peakswith chemical shift � at 6.12–6.24ppm were seen that would haveindicated the presence of either(11E,13E,15Z)-, (11Z,13E,15E)-,or (11E,13E,15E)-isomers. The 1Hand 13C chemical shifts and cou-pling constants were similar tothose of potato lipoxygenase-derived as well soybean lipoxy-genase-derived 10,17-dihydroxy-docosahexa-(4Z,7Z,11E,13Z,15E,19Z)-enoic acid for C(1)-C(18) andH(1)-H(18) (38, 39). Coupling con-stants of 15 Hz for trans-, and 10Hz for cis-double bond configura-tions were observed around thetriene structure, strongly suggest-ing that the compound was 10,17-dihydroxydocosa-(4Z,7Z,11E,13Z,15E)-pentaenoic acid. AlthoughNMR data strongly suggest doublebond configurations, as describedabove, synthetic standards withknown double bond configurationswill be needed for final confirmation.Chirality—VCDtechniques,which

are used for ascertaining absoluteconfigurations at chiral centers,were not successful in determina-tion of chirality in the case of

FIGURE 5. NMR data for 17-HDPAn-6. A, 1D-1H spectrum showing 1H assignments. The C(17)-H-OH and theC(1)-COOH protons are not seen due to exchange broadening; thus, the main peaks can be integrated to 32protons. Any extra peaks are solvent-related. B, a quantitative one-dimensional 13C spectrum showing 13Cassignments. Peaks can be integrated to 22 carbon atoms. Any extra peaks are solvent-related. A tetramethyl-silane (TMS) peak was used for referencing. C, two-dimensional 1H-1H COSY showing correlations betweencross-peaks belonging to adjacent carbon atoms.

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17-HDPAn-6. Therefore, chiral chromatography was used forassigning chirality. Order of elution of R- and S-isomers onchiral columns is the same for similar hydroxylated LC-PUFAcompounds when the same chromatographic conditions areused (40, 41). In this case, the 17-HDHA racemate mixture and(17S)-HDHA was used to ascertain the order of elution. TheChirobiotic-T columnwas not successful in separating isomers,

and final separations were success-ful using the Chiralpak-IA column.As seen in Fig. 7, it is clear that theR-isomer of 17-HDHAelutes beforethe S-isomer. A similar separationfor 17-HDPAn-6 shows that thisenzymatically prepared compoundcomprises 95% S-isomer and 5%R-isomer. Hence, 15-lipoxygenaseis primarily pro-S-selective in thecase of DHA as well as DPAn-6 atthe C-17-position.Physicochemical Properties and

Stability—pKa values of both com-pounds were predicted by ACDlabssoftware to be 4.58. Log DpH 7.4 val-ues determined using PBS and octa-nol partitioning were consistentwith those predicted usingACDlabssoftware (Fig. 8A) and were 3.5 for17-HDPAn-6 and 2.7 for 10,17-diHDPAn-6. The correspondingcalculated log P values were 6.32and 5.09, respectively. Experimen-tally, solubility of both compoundsin PBS, pH 7.4, was found to bemore than 200 �M. Solubilities ofreference compounds, as deter-mined in the same assay, were 5.6�M for diethylstilbestrol, 46.3 �Mfor haloperidol, 109.2 �M for keto-conazole, 227.3 �M for metoprololtartrate, 81 �M for phenytoin, 197.8�M for rifampicin, 19.9 �M for sim-vastatin, and 0.6 �M for tamoxifen.Predictions usingACDlabs software(Fig. 8B) suggest that 17-HDPAn-6has a solubility of 1.9 mM and thatthe more polar 10,17-diHDPAn-6has a solubility that is about 6-foldhigher at 12.2 mM in PBS, pH 7.4.The stability of compounds wastested in PBS and ethanol, asdescribed under “Experimental Pro-cedures.” At 4 °C, there was notmore than 4% degradation after 14days. Also, both compounds werecompletely stable (0% degradation)for 24 h at room temperature andfor 2 h at 37 °C.Anti-inflammatory Activity of

DPAn-6 Resolvins in the Air Pouch Model—The murine dorsalair pouch model, mimicking acute inflammation of the syno-vium, was used to assess anti-inflammatory activity of DPAn-6resolvins. Resolvins were administered locally by intrapouchinjection, andwithin 5min, TNF�was injected directly into thepouch to induce the inflammatory response. Exudates wereharvested 4 h later, and infiltrating leukocytes were enumer-

FIGURE 6. NMR data for 10,17-HDPAn-6. A, one-dimensional 1H spectrum showing 1H assignments. TheC(10)-H-OH, C(17)-H-OH, and the C(1)-COOH protons are not seen due to exchange broadening; thus, the mainpeaks can be integrated to 31 protons. Any extra peaks are solvent-related. B, a quantitative 1D-13C spectrumshowing 13C assignments. Peaks can be integrated to 22 carbon atoms. Any extra peaks are solvent-related. Atetramethylsilane (TMS) peak was used for referencing. C, two-dimensional 1H-1H COSY showing correlationsbetween cross-peaks belonging to adjacent carbon atoms.

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ated. Fig. 9 shows the total number of leukocytes in the pouchexudates. As expected, TNF� stimulated an�4-fold increase inleukocyte migration into the pouch. Cell migration wasreduced to the level of the negative control (no TNF� stimula-tion) by both 17-HDPAn-6 and 10,17-HDPAn-6, whereas theresolvin control (17S)-HDHA tended to reduce migration, butthe reduction was not statistically significant. The major effectof the DPAn-6-derived resolvin compounds was to reduce theproportion of granulocytes and increase the proportion ofmac-rophages (Table 5) in the exudates, suggesting a switch fromactive immune response (granulocyte-dominated) to resolu-tion (macrophage-dominated) of the inflammatory response.The two DPAn-6 resolvin compounds administered locally at adose of �0.005 mg/kg (on a whole animal basis) were as effica-cious as indomethacin administered intraperitoneally at a doseof 2 mg/kg in this study.Rat Hind Paw Edema Assay—The two DPAn-6 resolvins

were tested in a rat carrageenan-induced hind paw edemamodel to confirm their anti-inflammatory activity in a secondanimal species and in a second in vivo model of acute inflam-mation. This also served to explore efficacy following systemicrather than local administration. The intravenous doses in thisstudy were 50-fold higher than in themurine air pouch study toaccount for the larger species size and to account for systemicrather than local administration. Oral doses were an additional5-fold higher than intravenous doses to help compensate forpossibly lower bioavailability. Results, shown in Fig. 10, indicatethat both 17-HDPAn-6 and 10,17-H DPAn-6 reduced edemavolume following intravenous administration and resulted in�20–25% reduction in paw edema at the 2, 4, and 6 h timepoints, demonstrating activity of these compounds for at least6 h after intravenous administration. Potency was similar to the

known resolvin 17-HDHA at 4 and6 h, although the 17-HDHAappeared more potent at the 2 htime point. There was no effect ofany compound, including indo-methacin, at the 24 h time point.The 10,17-HDPAn-6 was activeorally, resulting in similar reduc-tions in edema as with the intrave-nous dosing of this compound. The17-HDPAn-6 did not show activityat this dose following oral adminis-tration in this study.A second hind paw edema study

was conducted to explore the dose-response relationship of orallyadministered DPAn-6 resolvins.Data from this study are summa-rized in Fig. 11.The responses in this dose-re-

sponse assay reproduced the resultsfrom the first hind paw assay in thatthe 25-�g dose of 10,17-HDPAn-6produced a significant�20% reduc-tion in paw edema, whereas thesame dose of the 17-HDPAn-6 had

no effect. The response to the resolvin 17-HDHA at 25 �gresembled that of 17-HDPAn-6 with no reduction in edemavolume, whereas the indomethacin reduced swelling by about30%. Although there was no effect of 17-HDPAn-6 at the mid-dle (25-�g) dose, the low (2.5-�g) and high (250-�g) dosesreduced edema at the 4 h time point (Fig. 11C). The high dose of17-HDPAn-6 was most effective and consistently reducededema at all time points (Fig 11A). On the other hand, the mid-dle dose of 10,17-HDPAn-6 was most effective at reducingedema at all time points (Fig. 11B). The low and high doses ofthis compound reduced edema at the 2 h but not later timepoints. Both DPAn-6 resolvin compounds resulted in non-monotonic dose responses (Fig. 11C).

DISCUSSION

Lipoxygenases catalyze the dioxygenation of LC-PUFAs con-taining cis,cis-1,4-pentadienemoieties (26). The primary site ofoxidation in arachidonic acid is commonly used in namingthese enzymes. For example, 15-lipoxygenase catalyzes the pro-duction of 15-hydroperoxyarachidonic acid from ARA (42). Inthe current study, 15-lipoxygenase, 12-lipoxygenase, and 5-li-poxygenase acted on DHA, DPAn-6, and DPAn-3 with posi-tional preferences for dioxygenation. 15-Lipoxygenase, primar-ily oxygenated theC22 substrates at theC-17 or�-6 position, aswould be expected from previous studies with these enzymes(3, 4, 43). In addition, 10,17- and 7,17-dihydroxyoxylipins werealso obtained from DPAn-6 and DPAn-3. These compoundsare analogous in structure to the potent anti-inflammatory17-hydroxy series docosanoids described by Hong et al. (3).Other lipoxygenases like 12-lipoxygenase primarily oxygenatedDHA, DPAn-3, and DPAn-6 at the C-14/�-9-position and 5-li-poxygenase at the C-10-position, consistent with what was

FIGURE 7. Chirality determination of C17 in 17-HDPAn-6. 17-HDHA and 17-HDPAn-6 prepared enzymaticallyusing 15-lipoxygenase were oxidized to the corresponding oxo-compounds and then reduced with sodium boro-hydride to the corresponding racemic mixtures. Separations were performed on Chiralpak-IA using methanol/water (80:20) as mobile phase at a flow rate of 0.6 ml/min at 25 °C. A, racemic 17-HDHA prepared by oxidation-reduction of enzymatically prepared 17-HDHA; B, (17S)-HDHA from Cayman Chemicals; C, 17-HDHA preparedenzymatically using 15-lipoxygenase; D, racemic 17-HDPAn-6 prepared by oxidation-reduction of enzymaticallyprepared 17-HDPAn-6; E, enzymatically prepared 17-HDPAn-6 showing the major isomer assigned as S.

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expected (4, 44–46). Like DHA, other LC-PUFAs, such asDPAn-6 and DPAn-3, and lipoxygenases are found in varyingamounts in blood and several tissues, suggesting that these or

otherDPA-derived resolvins could be produced physiologicallyand act as bioactivemediators (47–52). However, actual biolog-ical production of these DPAn-6-derived resolvins remains tobe investigated. Comparison of substrate reactions with 15-li-poxygenase at 0 °C showed that DPAn-6 was completely con-verted to products, whereas DHA and DPAn-3 were not, thusindicating that at such low temperatures, DPAn-6 reacts with15-lipoxygenase in a facile manner. When enzyme kineticsbehavior was probed at higher temperatures (22 °C) in the pres-ence of a surfactant, DPAn-3 exhibited the highest Vmax. How-ever, DPAn-6 proved to be the substrate that was convertedmost efficiently to products. The 15-lipoxygenase-PUFA reac-tion is a complex one, involving several phenomena, such assubstrate inhibition, product inhibition, product activation,substrate deactivation from decoupling of the radical substrateprior to oxygen insertion, low oxygen concentration, andenzyme thermodynamic inactivation. Additionally, the sub-strates exhibit tendencies toward micelle formation on which15-lipoxygenase does not act effectively (33). The criticalmicelle concentration for linoleic acid at pH9.0 and 20 °C is 150�M (53), and the critical micelle concentration for DHA at pH8.8 and 25 °C is 60–90 �M (54). Although some of the factorsmentioned above were taken into consideration while deter-mining kinetic constants, it was not feasible to evaluate all ofthem as is true for almost all studies evaluating the 15-lipoxy-genase-PUFA reaction. Therefore, the experiments reportedhere are useful for comparing the efficiencywithwhich soybean15-lipoxygenase converts DHA, DPAn-3, andDPAn-6 to prod-ucts, but comparison with other studies remains difficult, dueto the large variability of conditions reported in the literature.Both DHA and DPAn-3 are �-3 fatty acids, and the differentkinetic behavior observed forDPAn-6 couldbedue to the fact thatit is an�-6 fatty acid. This could affect the affinity of the substrateto the enzyme at the reactive site as well as at the site responsiblefor substrate inhibition. In the absence of precise structural infor-mation regarding details of binding interactions between the sub-strate and theenzyme, it isdifficult topinpoint thepreciseoriginofthe differences observed in the kinetic activities.One of themain reasons for focusing our attention onDPAn-

6-derived oxylipins stems from thefact that it is a constituent of ourMartek-DHA-STM oil (27). Studieshave shown that in a carrageenan-induced rat hind paw edemamodel,this Martek-DHA-STM oil per-formed better at reducing paw vol-ume than the Martek-DHA-TTM

oil.5 The major difference betweenthe two oils is that in addition toDHA, the DHA-STM oil containsDPAn-6. We thus reasoned thatDPAn-6 contributes to the anti-in-flammatory activity of this oil andpossibly does this via resolvin-likemediators. Since most resolvins areoxylipins produced by action oflipoxygenases on LC-PUFAs, weexamined if DPAn-6 was a substrate

FIGURE 8. Predicted log D and solubility profile of 17-HDPAn-6 and 10,17-diHDPAn-6. Log D and solubility were calculated for 17-HDPAn-6 and 10,17-HDPAn-6 using ACDLabs software. A, a plot of predicted log D at various pHvalues for 17-HDPAn-6 (E) and 10,17-HDPAn-6 (Œ). B, predicted log solubilityat various pH values for 17-HDPAn-6 (E) and 10,17-HDPAn-6 (Œ).

FIGURE 9. Effects of 17-HDPAn-6 and 10,17-HDPAn-6 on leukocyte migration in a murine air pouchmodel of acute inflammation. The figure shows means (n � 10 animals/group) � S.D. of the number ofleukocytes found in air pouch exudates 4 h after stimulation with TNF�. Test compounds (100 ng) wereadministered intrapouch immediately before injection of the TNF� into the pouch, and indomethacin wasadministered intraperitoneally (2 mg/kg) 30 min prior to the TNF� injection. Groups were compared with theTNF� control group by ANOVA with Dunnett’s post test. *, p � 0.01 compared with TNF� control.

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for lipoxygenases and characterized the products of thesereactions. The major products of the most efficient reaction,17-HDPAn-6 and 10,17-HDPAn-6, were extensively charac-terized and assessed for their “oral drugability” characteris-tics and further examined for their anti-inflammatory poten-tial. Chemical characterization of these resolvins involveddetermination of double bond configurations and chirality atasymmetric carbon atoms, since these are important chem-ical features that affect bioactivity. For example, in the neu-

roprotectin D1 family, the major isomer produced by humanleukocytes, characterized as (10R,17S)-dihydroxy-(4Z,7Z,11E,13E,15Z,19Z)-hexaenoic acid, was found to be morepotent than (10S,17S)-dihydroxy-(4Z,7Z,11E,13Z,15E,19Z)-hexaenoic acid, in reducing neutrophil migration in a murinezymosan-induced peritonitis model (1). It should be pointedout that 18O2 incorporation experiments have shown that theformer is generated via an epoxide intermediate, whereas thelatter is produced primarily via a double lipoxygenation reac-tion. Although another isomer, (10S,17S)-dihydroxy-(4Z,7Z,11E,13E,15Z,19Z)-hexaenoic acid, showed substantial activityin the same study, it was not found to be biologically relevant. Inour study, configurations of double bonds in 17-HDPAn-6 and10,17-HDPAn-6 as elucidated by high resolution NMR werethe same as analogous DHA products described by Butovich etal. (38, 39). In particular, 10,17-HDPAn-6 has double bondgeometry that is different from the main neuroprotectin D1

FIGURE 10. Kinetics of effect of intravenously and orally administeredDPAn-6 resolvins on carrageenan-induced hind paw edema in rats. Ani-mals were treated with 5 �g of resolvins intravenously 2 min prior to carrag-eenan challenge (A) and 25 �g of resolvins orally 30 min prior to carrageenanchallenge (B). Vehicle (E) was administered intravenously 2 min prior to car-rageenan challenge, and indomethacin (�) was dosed at 5 mg/kg intraperi-toneally 30 min prior to carrageenan challenge in both A and B. Resolvinsshown are 17-HDPAn-6 (f), 10,17-HDPAn-6 (F), and 17-HDHA (Œ) in both Aand B. Means (n � 8 animals/group) � S.D. of edema volume measured 2, 4, 6,or 24 h post-carrageenan challenge are shown. **, significantly different fromvehicle control by ANOVA with Dunnett’s post test, p � 0.01.

FIGURE 11. Dose effects of 17-HDPAn-6 and 10,17-HDPAn-6 on carrag-eenan-induced rat paw edema. All compounds were administered by oralgavage 30 min before carrageenan challenge, and paw edema was measured2, 4, or 6 h after carrageenan challenge. The figure shows mean paw edemavolume (n � 6 animals/group) � S.D. after administration of various doses of17-HDPAn-6 (A) or 10,17-HDPAn-6 (B). Groups represented are vehicle (E), 2.5�g (F), 25 �g (f), and 250 �g (Œ). Dose effects of compounds at the 4 h timepoint are shown in C. Resolvin treatment groups were compared with vehiclecontrol. *, p � 0.05; **, p � 0.01 by ANOVA with Dunnett’s post test.

TABLE 5Proportion of granulocytes and macrophages in exudates harvestedfrom murine air pouchesAnimals were injected with 100 ng of resolvins followed immediately by 100 ng ofTNF� intrapouch. Exudates were harvested 4 h later, and granulocytes and macro-phages were quantified after Diff-Quik staining on slides. Means � S.D.

Group Granulocytes Macrophages% %

TNF� control 81.8 � 5.4 18.2 � 5.4No TNF� control 61.2 � 19.4a 38.8 � 22.3a17-HDHA 76.3 � 19.5 23.7 � 19.517-HDPAn-6 67.4 � 4.1a 32.6 � 4.1a10,17-HDPAn-6 66.8 � 15b 33.2 � 15.0bIndomethacin 70.9 � 16.1 29.1 � 16.1

a p � 0.01, significantly different than TNF� control by Student’s t test.b p � 0.05, significantly different than TNF� control by Student’s t test.

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isomer found in human neutrophils and is in effect consistentwith a double lipoxygenation product, the major isomer inmurine exudates and a minor isomer in human leukocytes, asdescribed by Serhan et al. (1, 39).

Soybean lipoxygenase is known to primarily produce LC-PUFAhydroperoxides with an S-configuration at the chiral�-6carbon. This has been seen with several substrates, such asdocosahexaenoic acid, arachidonic acid, and linoleic acid, whenintact soybean lipoxygenase (Type 1-B) was used (38, 55). Ser-han et al. have demonstrated that (17S)-HDHA is the mainproduct of the reaction of DHAwith soybean lipoxygenase andthat in order to obtain (17R)-hydroxylated compounds ofDHA,one needs to use acetylated COX-2 (3, 4). Our chiral chroma-tography data clearly show that DPAn-6 is converted by soy-bean lipoxygenase to (17S)-HDPAn-6. Commonly used tech-niques, such as VCD, that are used to determine configurationsat chiral centers did not work for 17-HDPAn-6. This is primar-ily because this class of molecules is highly flexible, resulting inmultiple conformations, thus complicating the assignment ofVCD/infrared spectra to a single conformer. Formation of10,17-HDPAn-6 as described before seems to occur through adouble lipoxygenation mechanism, and we thus believe thatthis dihydroxy compound possesses the S-configuration atC-17 and C-10, a situation analogous to what is seen for 10,17-HDHA described by Butovich et al. (38, 39).Oral drugability of these two DPAn-6 derived resolvins can

be evaluated using the commonly used Lipinski’s rule of five(56–58). Based on this rule, a good orally administered drugcandidate should have the following features: molecular weightless than 500, fewer than five hydrogen bonddonors, fewer than10 hydrogen bond acceptors, and a partition coefficient, log Pless than 5 (58). The two compounds, 17-HDPAn-6 and 10,17-HDPAn-6, satisfy all of the criteria except that their log P valuesare 6.32 and 5.09, respectively. Despite this apparently highhydrophobicity, the solubility of these compounds was betterthan that of a large number of other commonly used drugs, suchas simvastatin, tamoxifen, haloperidol, and phenytoin. Never-theless, the log P values of both DPAn-6-derived resolvinscould be easily changed with suitable derivitization.17-HDPAn-6 and 10,17-HDPAn-6 used in efficacy testing

were produced biogenically using soybean 15-lipoxygenase,and each was highly purified (�98% purity), ensuring that theresponses seen in the animal studies were indeed due to thesespecific DPAn-6 resolvins. In the air pouch model of acute in-flammation, both 17-HDPAn-6 and 10,17-HDPAn-6 werehighly efficacious at reducing leukocyte migration intoinflamedpouches. The 100-ngDPAn-6 resolvin dose, chosen tomatch doses used by Serhan and colleagues (3, 4) with DHA-resolvins in the same model, reduced leukocyte migration by70–80% andhad effects similar to that reported for (17S)-seriesDHA-derived resolvins, which reduced neutrophil migrationinto air pouches. In our experiment, the 17-HDPAn-6 resolvinswere more efficacious than the same dose of 17S-HDHA,although different stereoisomers of the resolvins may have dif-ferent potencies (1). Importantly, both DPAn-6 resolvinsreduced leukocyte migration to the base-line levels seen in ani-mals not receiving TNF�, and both compounds were as effica-cious as the indomethacin administered intraperitoneally but at

a 400 times higher dose (on awhole animal basis). Interestingly,both compounds proportionately reduced granulocyte andincreased macrophage numbers. This is consistent with a pro-resolution mechanism described by Gilroy et al. (59), whereinneutrophil migration is reduced and macrophages move in tophagocytose the apoptotic neutrophils. Further research isrequired to understand the specific mechanism by which theDPAn-6 resolvins mediate their response, but in the air pouchmodel of acute inflammation, they behave similarly to thestructurally analogous DHA-derived resolvins.The hind paw edema model is a widely used model of acute

inflammation and has been recently used to test the analgesiceffects of lipoxins (60). In our studies, paw inflammation wasinduced by injecting cross-linked carrageenan. Despite thisstrong stimulus, both 17-HDPAn-6 and 10,17-HDPAn-6 sig-nificantly reduced paw edema by 20–25% when administeredsystemically using a 5-�g intravenous dose (�0.025 mg/kg).This response was similar to the response to biogenically syn-thesized (17S)-HDHA, also administered intravenously. Bothcompounds also showed efficacy at early time points (2 h post-carrageenan) when administered orally at the lowest dose (2.5�g) tested. However, both compounds exhibited nonmono-tonic dose-response behavior with the oral administrationroute. The 17-HDPAn-6, for example, was not active at themiddle 25-�g dose (similar to its DHA analog 17-HDHA) buthad activity at the low and high doses. The most effective dosesacross all time points, resulting in about 25% reduction inedema, were 250 �g for 17-HDPAn-6 and 25 �g for 10,17-HDPAn-6. These observations suggest that either 10,17-HD-PAn-6 ismore potent than 17-HDPAn-6, that bioactivemetab-olites of 10,17-HDPAn-6 contribute to its response, or thatearly exposure to 10,17-HDPAn-6 may result in extendedeffects by, for example, altering gene expression patterns.Although we do not know the reason for the nonmonotonicdose-response behavior observed here, possible explanationsinclude dose-dependent metabolism in vivo, interaction withmultiple receptor systems, ormixed agonist/antagonist activityprofiles (61, 62). Based on strong similarities to the DHA-de-rived resolvins, in both their synthesis by lipoxygenases tostructurally analogous compounds and their comparable activ-ity at similar doses in acute inflammationmodels, these DPAn-6-derived oxylipins appear to function like resolvins, which actin a proresolution fashion to down-regulate inflammation andcan thus be categorized as DPAn-6-derived resolvins. Furtherstudies will be required to determine whether persistent, inap-propriate disease-associated inflammation, such as that foundin cardiovascular disease or autoimmune diseases, can bemod-ified by providing either DPAn-6 fatty acid precursors or bydirect administration of these DPAn-6-derived resolvins oranalogs thereof. Although a large number of studies need to bedone to assess the bioefficacy and therapeutic profile of both17-HDPAn-6 and 10,17-HDPAn-6, these could potentially rep-resent a new class of therapeutic resolvins.

Acknowledgments—We thank Dr. Sandip Sur (NMRServices) forexpert technical assistance and Dr. Dutt Vinjamoori, Dr. Paul Zim-mer, and Marcia Morton for helpful discussions.

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Resolvins from DPAn-6

MAY 29, 2009 • VOLUME 284 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 14759

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Krishna Raman and Linda M. ArterburnBindi Dangi, Marcus Obeng, Julie M. Nauroth, Mah Teymourlouei, Micah Needham,

Anti-inflammatory Resolvins Derived from Docosapentaenoic Acid (DPAn-6)Biogenic Synthesis, Purification, and Chemical Characterization of

doi: 10.1074/jbc.M809014200 originally published online March 26, 20092009, 284:14744-14759.J. Biol. Chem. 

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