Proc. Natl. Acad. Sci. USA Vol. 81, pp. 1317-1321, March 1984 Biochemistry

9-Deoxy-A9,A12-13,14-dihydroprostaglandin D2, a metabolite of prostaglandin D2 formed in human plasma (dehydration product of prostaglandin D2/serum albumin/cell growth inhibition) YOSHIHARU KIKAWA*, SHUH NARUMIYA*, MASANORI FUKUSHIMAt, HIROHISA WAKATSUKAt, AND OSAMU HAYAISHI§ *Department of Medical Chemistry, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606, Japan; tDepartment of Internal Medicine and Laboratory of Chemotherapy, Aichi Cancer Center, Chikusa-ku, Nagoya 464, Japan; tResearch Institute, Ono Pharmaceutical Co., Shimamoto, Mishima, Osaka 618, Japan; and §Osaka Medical College, Daigaku-cho, Takatsuki, Osaka 569, Japan Contributed by Osamu Hayaishi, November 8, 1983

ABSTRACT Incubation of prostaglandin D2 (PGD2) with from Sigma. Dimethylisopropylsilyl (Me2iPrSi) imidazole human plasma yielded a product that has been identified as 9- and methoxyamine hydrochloride were from Tokyo Kasei deoxy-9,10-didehydro-12,13-cdidehydro-13,14-dihydro-PGD2 (Tokyo). Sep-pak silica and Sep-pak C18 cartridges were (9-deoxy-_9,9'2-13,14-dihydro-PGD2). The identification was from Waters Associates. Precoated silica gel plates [G60- based on mass spectrometry, UV spectrometry, mobilities and (F254)] with concentration zones and silica gel 60 for column retention time on TLC and HPLC, and NMR. The conversion chromatography were from Merck. Sephadex LH-20 was a of PGD2 to this product was dependent on the incubation time product of Pharmacia. Solvents used in the extraction of and the amount of plasma added to a reaction mixture and was PGD2 metabolites for identification were distilled before use. abolished by prior boiling. The conversion rate ofPGD2 to this All other chemicals were of reagent grade. metabolite was 0.03 nmol/min per mg of protein of whole plas- Pregaration of 9-Deoxy-A9,Al2-13,14-dihydro-PGD2. 9-De- ma at pH 8.0 at 37°C. Similar conversion was also found by oxy-A -GD2 was synthesized as described (12), and conver- incubating PGD2 with human serum albumin added at the sion of 9-deoxy-A9-PGD2 to 9-deoxy-A9,A&12-13,14-dihydro- concentration found in plasma. These results suggest that the PGD2 was carried out as described by Bundy et al. (13) for conversion of PGD2 to this product is catalyzed by the enzy- the conversion of PGD2 to A12-13,14-dihydro-PGD2. 9- matic action of a plasma protein, probably serum albumin. Deoxy-A9-PGD2 (48 mg) was dissolved in 3 ml of tetrahydro- The biological activities of this compound were examined in furan and the solution was stirred at 4°C. 1,5-Diazabicy- several systems. It showed negligible activity in inhibition of clo(4.3.0)non-5-ene (17.8 mg) in 0.18 ml of tetrahydrofuran human platelet aggregation and relaxation of rabbit stomach was added to the solution and the mixture was stirred at strip. On the other hand, it exhibited a three times stronger room temperature overnight. The mixture was diluted with inhibitory activity (IC50, 1.8 ,M) than PGD2 (IC50, 5 ,uM) on 30 ml of water, and the product was separated out by extrac- the growth of L-1210 cultured cells. tion with 50 ml of ethyl acetate. The ethyl acetate extract was washed successively with 1.2 M hydrochloric acid, Prostaglandin (PG) D2 is formed in a variety of tissues and three times with excess water, and finally with a saturated cells and modulates their functions under various physiologi- NaCl solution. After being dried over anhydrous MgSO4 and cal and pathological conditions. For example, it is produced concentrated in vacuo, the crude product was purified in sili- during platelet aggregation and works as a negative feedback ca gel column chromatography (30 g of silica gel 60) with a modulator of the aggregation process (1-3). It is produced by solvent of ethyl acetate/n-hexane/methyl alcohol (50:50:2) and modifies the anaphy- to afford 9 mg of 9-deoxy-A9,Al2-13,14-dihydro-PGD2 as a mast cells during IgE stimulation colorless oil; Rf, 0.36 (silica gel TLC with ethyl acetate/ben- laxis process (4, 5). It is also produced in the central nervous zene/acetic acid, 50:50:2); IR, 2930, 1700, 1640, 1580, 1232, system of mammals and is involved in brain functions such dd 6.56 as hypothermia, sleep, and luteinizing hormone secretion (6- and 1028 cm-1; NMR (C2HCl3), 6 7.5 (1H, C9 H), 10). A potential antineoplastic effect of PGD2 has been re- (1H, t, C13 H), 6.35 (1H, dd, C10 H), 5.48 (2H, m, C5,C6 H), ported (11) and it has been found that 9-deoxy-A9-PGD2, a 3.88 (1H, m, C15 H), and 3.44 (1H, m, C8 H); mass spectrum dehydration product ofPGD2, has about three times stronger (direct inlet), ions at m/z 334 (M), 316, 245, and 236; UV L-1210 leukemia cul- (EtOH) Xmax 244 nm (e 6100). growth inhibitory effect than PGD2 on Preparation of Human Plasma. Blood, 50 ml, was obtained tured cells (12). The present study was undertaken to ex- ml plore the possible formation of this PGD2 dehydration prod- from a healthy volunteer into a plastic tube containing 5 here the conversion in human of 3.8% sodium citrate, and the mixture was centrifuged at uct in mammals. We report 1000 x g for 15 min. Plasma was then dialyzed against two plasma of PGD2 to a compound that has been identified as 9- at 7.4 and describe some prop- changes of 2 liters each of 50 mM Tris HCl buffer pH deoxy-A9,A12-13,14-dihydro-PGD2 or pH 8.0. erties of this metabolite.$ Enzyme Assay. Enzymatic activity was assayed as follows. MATERIALS AND METHODS The standard mixture contained 150 uM [3H]PGD2 (4000 cpm/nmol), dialyzed plasma, and 50 mM Tris HCl buffer Materials. [5,6,8,9,12,14,15-3H]PGD2 (100 Ci/mmol; 1 Ci (pH 7.4 or pH 8.0) in a final reaction volume of 0.4 ml. Reac- = 37 GBq) was purchased from New England Nuclear. tions were carried out by incubating the mixture at 37°C with PGB2, PGD2, and 9-deoxy-A9-PGD2 were synthetic products constant shaking and terminated by acidification with 1 M from Ono Pharmaceutical (Osaka, Japan). Human serum albumin (fatty acid free) was from Miles. ADP was obtained Abbreviations: PG, prostaglandin; Me2iPrSi, dimethylisopropyl- silyl; GC/MS, gas chromatography/mass spectrometry. The publication costs of this article were defrayed in part by page charge part of this work was presented at the Fifty-Sixth Annual Meeting payment. This article must therefore be hereby marked "advertisement" of the Japanese Biochemical Society, Sept. 29-Oct. 2, 1983, Fuku- in accordance with 18 U.S.C. §1734 solely to indicate this fact. oka, Japan.

1317 Downloaded by guest on October 4, 2021 1318 Biochemistry: Kikawa et at Proc. NatL Acad Sci. USA 81 (1984)

HCl to pH 3.0. The acidified mixture was directly applied on Tokyo). Compounds were dissolved in ethanol at a concen- a Sep-pak C18 cartridge, and products were eluted with 10 ml tration of 1 mg/ml and diluted with 50 mM TrisHCl (pH 8.0) of ethyl acetate as described (14). After evaporation in vac- before application. Platelet aggregometry was carried out us- uo, the residues were dissolved in 50 1.d of ethyl ether and ing an aggregometer Hematracer 1 model PAT 2M (Niko applied 2 cm wide on a silica gel thin layer. Authentic PGB2, Bioscience, Tokyo) as described (17). Inhibition of L-1210 PGD2, 9-deoxy-A9-PGD2, and 9-deoxy-A9,A'2-13,14-dihy- cell growth was examined as described (11). dro-PGD2 were applied as markers at least 2 cm away from Miscellaneous. UV absorption spectra were recorded on a the samples. TLC was carried out with a solvent of ben- Shimadzu UV300 spectrophotometer. NMR was recorded in zene/ethyl acetate/acetic acid (50:50:2). After development, C2HCl3 with a Varian NMR spectrometer XL-200. Protein radioactive zones on the TLC plates were located by a ra- concentration was determined according to the method of diochromatogram scanner Packard model 7201, and markers Lowry et al. (18) with bovine serum albumin as standard. were visualized by exposure to iodine vapor. Silica gel zones corresponding to PGD2, 9-deoxy-A9,A12-13,14-dihydro- RESULTS PGD2, and 9-deoxy-A9-PGD2 were scraped, respectively, Formation and Identification of 9-Deoxy-&9,A1213914dihy- and the rest of the silica gel was scraped altogether. Radioac- dro-PGD2. When PGD2 was incubated with dialyzed human tivity in the silica gel was measured with a Packard liquid plasma, it was converted to two products (Fig. 1). The major scintillation spectrometer (model 460C) in a toluene scintilla- product (compound 2) accounted for more than 80% of the tor. Since 3H at position 12 was lost during conversion of total amount of products, while the minor product (com- PGD2 to 9-deoxy-A9,A12-13,14-dihydro-PGD2, quantification pound 1) accounted for only 10%. Boiling of plasma marked- of the latter compound was carried out on the basis of the ly decreased the formation of compound 2 but did not affect specific activity corrected for the loss of this tritium. the formation of compound 1 significantly. These results Isolation of 9-Deoxy-A,9 A2-13,14-dihydro-PGD2. The reac- suggest that compound 2 is formed by the enzymatic action tion product, 9-deoxy-A ,A 2-13,14-dihydro-PGD2, was iso- of human plasma while compound 1 is a nonenzymatically lated on a large scale. The reaction mixture was 50 mM degraded product. To identify the structure of compound 2, Tris HCl, pH 8.0/1 mM [3H]PGD2 (140 cpm/nmol) and a 45- we isolated this product on a large scale and examined it by 70% ethanol fraction of plasma dialyzed at pH 8.0 (800 mg of UV spectrometry, GC/MS, and NMR. The UV absorption protein) in a total vol of 16 ml. The reaction was carried out spectrum of compound 2 is shown in Fig. 2; the compound at for terminated acidification with 370C 180 min and by 1 M has absorption maxima at 244 nm with an e (EtOH) of 6100 HCl to pH 3.0. The acidified reaction mixture was divided and a small shoulder around 300 nm, suggesting an enone into 16 tubes and partially purified by the use of Sep-pak C18 type structure. The mass spectrum of the methyl ester meth- cartridges. After evaporation in vacuo, the residues of ethyl oxime-Me2iPrSi ether derivative of compound 2 is shown in acetate extract from the Sep-pak cartridges were dissolved Fig. 3; ions are found at m/z 477 (M), 462 (M - 15, loss of in 100 Al of ethyl ether and applied 15 cm wide on a silica gel *CH3), 446 (M 31, loss of -OCH3), 434 [M 43, loss of thin layer with a concentration zone. Chromatography was *CH(CH3)2], 406 (M - 71, loss of -C5H11), 359 (M - 118, loss carried out with a solvent of benzene/ethyl acetate/acetic of -HOMe2iPrSi), and 201 (M - 276, base peak, Me2iPrSi- acid (50:50:2). The major product with an Rf value of 0.36 O+=CH-C5H11). The molecular ion at m/z 477 suggest- was extracted from the silica gel with ethyl acetate. After evaporation to dryness, the residues were dissolved in 100 ,l of acetonitrile and the product was further purified by re- versed-phase HPLC. HPLC was carried out using a system I II from Waters Associates (a 6000A solvent-delivering systems I with a model U6K injector) and a semi-preparative Waters + Plasma Bondapak C18 column (7.8 mm x 30 cm). Elution was ac- complished under isocratic conditions with a solvent of0.017 B0 l M KH2PO4/CH3CN/isopropanol (30:17:5) at 1 ml/min. Fractions were collected every 2 ml, and the radioactivity in E an aliquot of each fraction was measured to establish the u

chromatographic profile. 9-Deoxy-A9,A'&2-13,14-dihydro- ._ PGD2 typically eluted at 18 min. ._ Gas Chromatography/Mass Spectrometry (GC/MS) of the Product. Derivatizations were carried out essentially as re- Boiled ._ ported (15) except that methylation with diazomethane was 0 200'0 done at -20°C to avoid the formation of by-products. a GC/MS was carried out using a Hitachi model M80A instru- ment. Data were analyzed with a Hitachi model M-003 data analyzer computer system. The column used was a cross- linked OV-1 fused silica capillary column (25 m x 0.31 mm i.d., Hewlett-Packard). Helium was used as a carrier gas niIA with a flow rate of 10 ml/min. The column temperature was 0 5 10 15 maintained isothermal at 240°C, and samples were applied Distance (cm) via a Van den Berg type solventless injector. Mass spec- trometry was done at an ionization potential of 20 eV and an FIG. 1. TLC of reaction products. Reaction mixtures containing ionization current of 120 uA. 50 jM [3H]PGD2 (200,000 cpm/nmol) in 100 of plasma that had Bioassays. Relaxation of a rabbit transverse stomach strip been dialyzed at pH 8.0 were incubated at 370C for 2 hr. Aliquots were acid was examined as follows. The strip was prepared as de- analyzed with a solvent of benzene/ethyl acetate/acetic (50:50:2). (Upper) Dialyzed human plasma (7 mg of protein) at pH scribed (16). The tissue was suspended vertically and super- 8.0. (Lower) Dialyzed human plasma was boiled (100'C) for 5 min fused with a Krebs solution a antago- containing mixture of prior to incubation. Markers and products were localized by expo- nists at 5 ml/min at 370C (12). Relaxation of the tissue was sure to iodine vapor. D2, PGD2; B2, PGB2; 1, compound 1; 2, com- recorded via an isometric transducer T7-8 (Toyo Boldwin, pound 2. Incubation of PGD2 with undialyzed plasma yielded essen- Tokyo) coupled to an amplifier type 7236 (San-ei Instrument, tially same results. Downloaded by guest on October 4, 2021 Biochemistry: Kikawa et aL Proc. Natd Acad Sci USA 81 (1984) 1319 (m, C15 H), and 3.44 (m, C8 H), which were identical with those of the authentic compound. On TLC, compound 2 showed exactly the same mobilities as authentic 9-deoxy- A9,A12-13,14-dihydro-PGD2 with three solvent systems. Sol- vent systems used in TLC analyses were A [ethyl acetate/ benzene/acetic acid (50:50:2)], B [benzene/dioxane/acetic 0 acid (66:33:1)], and C [chloroform/tetrahydrofuran/acetic .0 acid (10:2:1)]. The Rf values were 0.36, 0.45, and 0.83 with solvents A, B, and C, respectively. On HPLC, compound 2 showed a single peak that had a retention time identical with 01 authentic 9-deoxy-A9,A'2-13,14-dihydro-PGD2. On the basis of these analyses, we have identified compound 2 as 9-de- oxy-A19,A12-13,14-dihydro-PGD2. Compound 1 was not iso- lated on as large a scale as compound 2 and, therefore, the 200 250 300 350 detailed structural analyses were not carried out for this Wavelength (nm) compound. However, it showed the same mobilities as au- thentic 9-deoxy-A9-PGD2 on TLC with the three solvent sys- FIG. 2. Comparison of UV spectra ofcompound 2 (upper curve) 0.81 solvents and authentic 9-deoxy-A9,A'2-13,14-dihydro-PGD2 (lower curve) in tems; the Rf values were 0.30, 0.43, and with ethanol. Concentrations were 0.05 and 0.1 mM for the authentic A, B, and C, respectively. compound and compound 2, respectively. As shown in Fig. 4A, the formation of 9-deoxy-A9,4a2- 13,14-dihydro-PGD2 from PGD2 was proportional to the vol- ed that compound 2 had lost one hydroxyl group from the ume of dialyzed plasma added to the reaction mixture and precursor molecule, PGD2, because it is 118 mass units (= was completely abolished by prior boiling. It was also pro- HOMe2iPrSi) less than that of the methyl ester methoxime portional to the time of incubation up to 120 min (Fig. 4B). Me2iPrSi ether of the latter, m/z 595. Compound 2, howev- The reaction rate at pH 8.0 was about 1.5 times that at pH er, showed the base peak at m/z 201, which was also the 7.4. These results suggested the enzymatic nature of this re- base peak of PGD2, suggesting that the 15-hydroxyl group action. On the contrary, the formation of 9-deoxy-A9-PGD2 was retained in the molecule. These results suggested that was not proportional to either plasma volume in the reaction compound 2 had lost a hydroxyl group at position 9 of PGD2 volume or incubation time (data not shown). Fitzpatrick and and undergone a double bond shift to form an enone struc- Wynalda (19) have reported that, in the presence of serum ture. On the basis of these analyses, we synthesized 9-de- albumin, PGD2 undergoes time-dependent degradation to an oxy-A9,A12-13,14-dihydro-PGD2 and compared its properties unidentified compound. We examined the possibility that se- with those of compound 2. As shown in Figs. 2 and 3, the rum albumin catalyzed the above reaction by adding equiva- UV and mass spectra of the two compounds were identical. lent amounts of human serum albumin instead of dialyzed Identification was further confirmed by NMR and mobilities plasma. As shown in Fig. 4A, serum albumin catalyzed the on TLC and HPLC. Although NMR of purified compound 2 conversion of PGD2 to 9-deoxy-A9,412-13,14-dihydro-PGD2 revealed the presence of contaminating materials in the ali- as efficiently as whole plasma, suggesting that serum albu- phatic regions, it also showed signals at 8 (ppm) 7.57 (dd, C9 min in dialyzed plasma worked as a catalyst in the reaction. H), 6.56 (t, C13 H), 6.35 (dd, C10 H), 5.48 (m, C5,C6 H), 3.88 A B 100 C .vO I N c _i O I ' -E Y c >0 0 I- 50 0 0O o o L .z I >% ._1 M ._

0 0 6 12 5 30 60 c 0 Plasma Protein (mg) Time (min)

U 100 0 4 8 Serum Albumin(mg) 0 FIG. 4. Effects of amounts of dialyzed human plasma and human serum albumin on rate of conversion of PGD2 to 9-deoxy-A9,A'2- 13,14-dihydro-PGD2 (A) and time course of conversion of PGD2 to 50 9-deoxy-A9,'A2-13,14-dihydro-PGD2 (B). (A) Various amounts ofhu- man plasma that had been dialyzed at pH 7.4 (O) or pH 8.0 (e) were added to the reaction mixture (total vol, 0.4 ml) and incubation was carried out at 37°C for 60 min. In control experiments, dialyzed plas- ma at pH 7.4 (O) and pH 8.0 (-) was boiled (100°C) for 5 min before addition to the reaction mixture. Human serum albumin was dis- o ILl solved in 50 mM Tris-HCl at pH 7.4 (A) or at pH 8.0 (A) and then 100 added to the reaction mixture at concentrations equal to its plasma m/z concentration. (B) PGD2 (60 nmol) was incubated with dialyzed hu- man plasma (5.6 mg of protein) at pH 8.0 (-) or pH 7.4 (o) in a total FIG. 3. Mass spectra of the methyl ester methoxime Me2iPrSi vol of 0.4 ml for the indicated times. In control experiments, boiled ether derivatives of compound 2 (A) and authentic 9-deoxy-A94,12- dialyzed plasma was used in incubations at pH 8.0 (n) and pH 7.4 13,14-dihydro-PGD2 (B). (o). Downloaded by guest on October 4, 2021 1320 Biochemistry: Kikawa et aL Proc. NatL Acad Sd USA 81 (1984) Biological Properties of 9-Deoxy-A9,A92-13,14-dihydro- smooth muscle relaxing activity (Fig. SB). PGD2 relaxed the PGD2. We examined the biological activity of 9-deoxy- rabbit transverse stomach strip in a dose-dependent manner A9,A"2-13,14-dihydro-PGD2 in several systems by using the between 5 and 200 ng with an ED50 value of 30 ng. 9-Deoxy- synthetic compounds and compared them with those of A&9,A12-13,14-dihydro-PGD2, on the other hand, caused only PGD2. The antiaggregatory activity of PGD2 is compared 20% relaxation at 1000 ng. Thus, in both inhibition of platelet with that of 9-deoxy-A9,A4 -13,14-dihydro-PGD2 in Fig. 5A. aggregation and relaxation of stomach strip, 9-deoxy-A9,A12- PGD2 inhibited platelet aggregation induced by 10 ,uM ADP 13,14-dihydro-PGD2 showed negligible activity. On the con- in a dose-dependent manner; the IC50 value for PGD2 was 40 trary, this compound elicited much stronger inhibitory activ- nM and at 200 nM PGD2 platelet aggregation was completely ity on growth of L-1210 cultured cells than did the precursor, inhibited. On the contrary, 9-deoxy-A9,A'2-13,14-dihydro- PGD2 (Fig. 5C). 9-Deoxy-A9,A'2-13,14-dihydro-PGD2 inhib- PGD2 (200 nM) inhibited platelet aggregation by only 10% ited cell growth in a dose-dependent manner from 0.5 uM and, even at higher concentrations, the extent of inhibition with an IC50 value of 1.8 gM. Under the same conditions, never exceeded 10%. Similar results were also obtained on a the IC50 value of PGD2 was 5 ,tM.

c 10 DISCUSSION A A strong growth inhibitory activity has been found for 9- 0) deoxy-A9-PGD2, a dehydration product of PGD2, and the .2. possible formation of this compound in biological systems 0 has been suggested (12). This study has revealed that 9- a'0 61 deoxy-A9,A12-13,14-dihydro-PGD2 instead of 9-deoxy-A9- PGD2 is enzymatically formed from PGD2 in human plasma. Q- 4 Dehydration of PGE to PGA has been reported by several 9-Deoxy-3?,&?-13,14-dihydro-PGD2 investigators (20, 21). Recently, Fitzpatrick and Wynalda

0g o O- O -O-- (19) reported that serum albumin catalyzes such dehydration _2 P0' and that similar dehydration also occurs with PGD2 but they did not identify the structure of the PGD2 product. When we 0o1 1.0 10 iC 10 added, instead of dialyzed plasma, human serum albumin to 0 Concentrationf(/4M ) the incubation mixture at concentrations found in plasma, we found that serum albumin catalyzes the conversion of 100 B PGD2 to 9-deoxy-A9,A12-13,14-dihydro-PGD2 with efficiency ;- equal to that of dialyzed plasma (Fig. 4). These results sug- _.. 80 0 gest that the formation of 9-deoxy-A9,A12-13,14-dihydro- c PGD2 in dialyzed plasma is catalyzed by serum albumin it- a PGD2 * ._; 60 self. Because 9-deoxy-A&9-PGD2 was also present in the reac- x C% tion mixture as a plausible nonenzymatic product, one 40 conceivable reaction sequence is that first PGD2 is dehydrat- '- 9-Deoxy-i9,J2-t:14-dihydro-PGD2 ed to and then the latter under- c 9-deoxy-A9-PGD2 compound o&20 . goes the shift of the 13,14 double bond to the 12,13 position. 4, Un , 0 In our preliminary work, we found that when 9-deoxy-A9- ff|. ,--e I [3H]PGD2 is added to dialyzed plasma, it is quickly convert- 0 1 10 100 1,000 1QDOO ed to 9-deoxy-A9,A12-13,14-[3H]dihydro-PGD2. However, Dose ( ng ) isotope trapping experiments using [ H]PGD2 and unlabeled -100 9-deoxy-A -PGD2 failed to trap the radioactivity, because -6 excess amounts of 9-deoxy-A9-PGD2 inhibit the conversion L- of [3H]PGD2 (unpublished observation). Thus, an intermedi- ate role of 9-deoxy-A9-PGD2 remains unsettled. The name of 0 PGJ2 has been proposed for 9-deoxy-A9-PGD2 (12) and, in W- that context, 9-deoxy-A9,A'12-13,14-dihydro-PGD2 should be Z 50 named A12-13,14-dihydro-PGJ2. 0 The natural occurrence of 9-deoxy-A9,A12-13,14-dihydro- PGD2 as well as 9-deoxy-A9-PGD2 has not yet been reported. L) In 1979, Ellis et al. (22) reported detailed analyses of urinary metabolites of PGD2 in monkey. According to their results, PGD2 is either metabolized on its side chain with its ring 1 2 5 10 structure intact or first converted to PGF2a and then further Concentration ( uM ) metabolized. Their findings have led to discovery of the two FIG. 5. Dose-response curves of effects of 9-deoxy-A&9,A2- PGD2-catabolizing enzymes-that is, NADP-linked PGD2 13,14-dihydro-PGD2 and PGD2 on the inhibition of human platelet dehydrogenase (17, 23) and PGD2 11-keto reductase (24-26). aggregation (A), on the relaxation of isolated rabbit transverse stom- Ellis et al. did not refer to any compound with a PGJ2-type ach strip (B), and on the growth of L-1210 leukemia cells (C). (A) ring structure. However, they left about 50% of the urinary Human platelet-rich plasma (250 ;A) was incubated for 1 min at 370C products, containing most of the nonpolar metabolites, un- and then for 1 min with various concentrations of 9-deoxy-A9,A12- analyzed (22). Thus a possibility remains that 9-deoxy- 13,14-dihydro-PGD2 and PGD2, after which aggregation was initiat- A9,A12-13,14-dihydro-PGD2 occurs as a natural metabolite of ed by addition of 10 ;LM ADP. (B) The tissue was suspended verti- PGD2. cally and superfused with a Krebs solution and various doses of compounds were applied directly to the tissue. (C) L-1210 leukemia We have found that 9-deoxy-A9,Al2-13,14-dihydro-PGD2 cells were cultured in the presence of various concentrations of 9- shows a unique spectrum of biological activities. It lacks an- deoxy-A9,Al12-13,14-dihydro-PGD2 and PGD2. After 4 days, the tiaggregatory activity for human blood platelet and also is number of cells that excluded trypan blue was counted. In this ex- inactive for relaxation of some smooth muscle, in both of periment, 100%1o represents a cell number of 281 x 104 cells per ml. which PGD2 is quite active. On the contrary, as 9-deoxy-A9- O, 9-Deoxy-A9,A12-13,14-dihydro-PGD2; *, PGD2. PGD2 (12), it showed three times stronger growth inhibitory Downloaded by guest on October 4, 2021 Biochemistry: Kikawa et aL Proc. NatL Acad Sci USA 81 (1984) 1321

activity on L-1210 cultured cells than PGD2 itself. Because 8. Ueno, R., Narumiya, S., Ogorochi, T., Nakayama, T., Ishika- PGD2 and 9-deoxy-A9-PGD2 are converted to 9-deoxy- wa, Y. & Hayaishi, 0. (1982) Proc. Natl. Acad. Sci. USA 79, A9,A12-13,14-dihydro-PGD2 in the presence of plasma, most 6093-6097. 9. Ueno, R., Ishikawa, Y., Nakayama, T. & Hayaishi, 0. (1982) growth inhibitory activity of the former compounds would Biochem. Biophys. Res. Commun. 109, 576-582. be exerted by their conversion to the latter. Thus, it is likely 10. Kinoshita, F., Nakai, Y., Katakami, H., Imura, H., Shimizu, that 9-deoxy-A9,A12-13,14-dihydro-PGD2 is the ultimate T. & Hayaishi, 0. (1982) Endocrinology 110, 2207-2209. compound that exerts antineoplastic action of PGD2 and, be- 11. Fukushima, M., Kato, T., Ueda, R., Ota, K., Narumiya, S. & cause of the lack of other PGD2 activities, it might serve as a Hayaishi, 0. (1982) Biochem. Biophys. Res. Commun. 105, prototype for further development of antineoplastic PG ana- 956-964. logues. 12. Fukushima, M., Kato, T., Ota, K., Arai, Y., Narumiya, S. & Hayaishi, 0. (1982) Biochem. Biophys. Res. Commun. 109, 626-633. Note Added in Proof. Since completion of this manuscript, the catab- 13. Bundy, G. L., Morton, D. R., Peterson, D. C., Nishizawa, olism of PGD2 by serum albumin and identification ofreaction prod- E. E. & Miller, W. L. (1983) J. Med. Chem. 26, 790-799. ucts have also been reported by Fitzpatrick and Wynalda (27). 14. Powell, W. S. (1980) Prostaglandins 20, 947-957. 15. Miyazaki, H., Ishibashi, M., Yamashita, K., Nishikawa, Y. & Katori, M. (1981) Biomed. Mass Spectrom. 8, 521-526. We are grateful to Drs. T. Miyamoto and Y. Arai of Ono Central 16. Whittle, B. J. R., Mugridge, K. G. & Moncada, S. (1979) Eur. Research Institute for their kind assistance and discussion and to J. Pharmarol. 53, 167-172. M. his encouragement to Prof. Sudo of Fukui Medical College for 17. Watanabe, T., Shimizu, T., Narumiya, S. & Hayaishi, 0. Y.K. This work was supported in part by a grant-in-aid for scientific (1982) Arch. Biochem. Biophys. 216, 372-379. research from the Ministry of Education, Science And Culture of N. A. L. & Japan and by grants from the Japanese Foundation of Metabolism 18. Lowry, Q. H., Rosebrough, J., Farr, Randall, and Diseases and the Japan Brain Foundation. R. J. (195i) J. Biol. Chem. 193, 265-275. 19. Fitzpatrick, F. A. & Wynalda, M. A. (1981) Biochemistry 20, 6129-6134. 1. Oelz, O., Oelz, R., Knapp, H. R., Sweetman, B. J. & Oates, 20. McDonald-Gibson, W. J., McDonald-Gibson, R. G. & J. A. (1977) Prostaglandins 13, 225-234. Greaves, M. W. (1972) Prostaglandins 2, 251-263. 2. Whittle, B. J. R., Moncada, S. & Vane, J. R. (1978) Prosta- 21. Polet, H. & Levine, L. (1974) J. Biol. Chem. 250, 351-357. glandins 16, 373-388. 22. Ellis, C. K., Smigel, M. D., Oates, J. A., Oelz, 0. & Sweet- 3. Watanabe, T.; Narumiya, S., Shimizu, T. & Hayaishi, 0. man, 13. J. (1979) J. Biol. Chem. 254, 4152-4163. (1982) J. Biol. Chem. 257, 14847-14853. 23. Watanabe, K., Shimizu, T., Iguchi, S., Wakatsuka, H., Haya- 4. Steinhoff, M. M., Lee, L. H. & Jakschik, B. A. (1980) Bio- shi, M. & Hayaishi, 0. (1980) J. Biol. Chem. 255, 1779-1782. chim. Biophys. Acta 618, 28-34. 24. Wong, P. Y. K. (1981) Biochim. Biophys. Acta 659, 169-178. 5. Lewis, R. A. & Austen, K. F. (1981) Nature (London) 293, 25. Reingold, D. F., Kawasaki, A. & Needleman, P. (1981) Bio- 103-108. chim. Biophys. Acta 659, 179-188. 6. Abdel-Halim, M. S., Hamberg, M., Sjoquist, B. & Anggard, 26. Watanabe, K., Shimizu, T. & Hayaishi, 0. (1981) Biochem. E. (1977) Prostaglandins 14, 633-643. Int. 2, 603-610. 7. Narumiya, S., Ogorochi, T., Nakao, K. & Hayaishi, 0. (1982) 27. Fitzpatrick, F. A. & Wynalda, M. A. (1983) J. Biol. Chem. Life Sci. 31, 2093-2103. 258, 11713-11718. Downloaded by guest on October 4, 2021