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Polyclonal antibodies to EB1089 (seocalcitol), an analog of 1α,25-dihydroxyvitamin D3☆
Steroids 66 (2001) 539 –548
Polyclonal antibodies to EB1089 (seocalcitol), an analog of 1␣,25-dihydroxyvitamin D3夞 Lars K.A. Blæhra,*, Fredrik Bjo¨rklinga, Ernst Binderupa, Martin J. Calverleya, Peter Kaastrupb a
Department of Medicinal Chemistry, Leo Pharmaceutical Products Ltd., 55 Industriparken, DK-2750 Ballerup, Denmark b Institute of Medical Microbiology and Immunology, University of Copenhagen, DK-2200 Copenhagen, Denmark Received 5 June 2000; received in revised form 5 September 2000; accepted 11 September 2000
Abstract A hapten derivative of EB1089 [1(R),3(S),25-trihydroxy-26,27-dimethyl-9,10-seco-24-homocholesta-5(Z),7(E),10(19),22(E),24(E)-pentaene], a side-chain analog of 1␣,25-dihydroxyvitamin D3, was synthesized for raising antibodies with a high specificity for EB1089. The A-ring moiety of EB1089 was replaced in the hapten by a linker for conjugation to a protein. Three polyclonal antibodies were obtained by immunizing rabbits with a BSA-conjugate of the hapten. The antibodies were characterized for titer, avidity and specificity using an enzyme immunoassay with covalently bound EB1089. The three antibodies had similar binding profiles and were highly selective for EB1089 and its metabolites over the naturally occurring vitamin D metabolites. Cross-reactivities with 25-hydroxyvitamin D3, the most abundant vitamin D metabolite in serum, were in the range 0.01– 0.2% relative to EB1089. © 2001 Elsevier Science Inc. All rights reserved. Keywords: EB1089; 1␣,25-Dihydroxyvitamin D3 analog; Hapten; Synthesis; Antibody production; Enzyme immunoassay
1. Introduction EB1089 (seocalcitol, 1) is a synthetic, low-calcemic analog of the natural hormone 1␣,25-dihydroxyvitamin D3 (1,25(OH)2D3, 2) that has recently been selected for clinical trials as a potential anti-cancer agent [1]. The analog is 50 –200 times stronger than 1,25(OH)2D3 in inhibiting cellular proliferation, but is only 50% as calcemic, and this makes it a promising drug candidate. The evaluation of EB1089 as a potential anti-cancer agent in the clinic has demanded methods for quantifying the drug in biological samples. Because existing assays were unsuitable for this purpose, we decided to raise polyclonal antibodies specific to EB1089 in order to develop an immunoassay. The use of highly specific antibodies could eliminate the problem of cross-reaction with naturally occurring vitamin D metabolites, especially 25-hydroxyvita夞This work was presented as a poster at the 11th Workshop on Vitamin D, Nashville, TN, May 27–June 1, 2000. The synthesis section was taken from the doctoral thesis of Lars Blæhr (Leo Pharmaceutical Products & Royal Danish School of Pharmacy, Copenhagen, 1998). * Corresponding author. Tel.: ⫹45-44-92-38-00; fax: ⫹45-44-94-55-10. E-mail address: [email protected] (L.K.A. Blæhr).
min D3 (25(OH)D3) which is the major circulating metabolite. In addition, the affinity of the antibodies was expected to be sufficiently high for analysis in the picogram scale. In this paper we report the synthesis of an EB1089 hapten (3, Fig. 1) and the characterization of polyclonal antibodies raised in three rabbits against the hapten conjugated to BSA. The hapten was designed in such a way that only key structural features of the compound of interest were included, namely the CD-ring and the side-chain. This was done in order to avoid possible side effects that could result from long-time immunization with large doses of conjugates of EB1089 itself. In addition, the number of synthesis steps was reduced. Despite the missing A-ring in the hapten, the antibodies raised toward the hapten were expected to cross react with EB1089 to a high degree. The antibodies were characterized with respect to titer, avidity, and specificity in an enzyme immunoassay (EIA). 2. Experimental 2.1. Hapten synthesis Tetrahydrofuran (THF) was dried by distillation under argon from sodium benzophenone ketyl immediately before
0039-128X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 9 - 1 2 8 X ( 0 0 ) 0 0 2 2 5 - 7
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L.K.A. Blæhr et al. / Steroids 66 (2001) 539 –548
Fig. 1. Structures of EB1089 (1), 1␣,25-dihydroxyvitamin D3 (2), and the hapten derivative of EB1089 (3).
use. Other solvents (CH2Cl2, Et2O, and pyridine) were dried by storage over molecular sieves 4 Å. All reagent-grade chemicals were obtained from Aldrich Chemical Co. unless otherwise mentioned and were used as received. Watersensitive reactions were performed in oven-dried (130°C) or flame-dried glassware under argon. Thin layer chromatography was carried out on E. Merck precoated silica gel 60 F254 glass plates. Plates were visualized using UV radiation (254 nm) or with 4 N H2SO4 containing 1.5% cerium sulfate and 1.0% molybdic acid followed by heating to 150°C. Flash chromatography was performed according to the procedure of Still et al. [2] using E. Merck silica gel 60, 40 – 63 m. NMR spectra were obtained in CDCl3. 1D NMR spectra were recorded on a Bruker AC-300 at 300 MHz for 1H and 75 MHz for 13C. 2D NMR experiments were performed on a Bruker ARX500 (500 MHz). Chemical shifts are reported in parts per million downfield from internal tetramethylsilane. For compounds containing silicon, CHCl3 (7.25 ppm) was used as reference in 1H-NMR, and CDCl3 (76.81 ppm) was used as reference in 13C-NMR. High-resolution massspectra were recorded on a VG Autospec using electron impact ionization. All spectra and elemental analyses were recorded at the Department of Spectroscopy at Leo Pharmaceutical Products. Melting points were measured on a Bu¨chi 510 melting point apparatus and are uncorrected. 2.2. (22E,24E)-Des-A,B-26,27-dinor-8-[(tertbutyldimethylsilyl)oxy]-25-carbomethoxy-cholesta-22,24diene (5) Aldehyde 4 [3] (2.04 g, 6.29 mmol) and trimethyl 4-phosphonocrotonate (2.67 g, 11.31 mmol, Lancaster Syn-
thesis Gmbh, Mu¨hlheim am Main, Germany) were dissolved in 20 ml dry THF under argon and cooled to ⫺50°C. Lithium-bis(trimethylsilyl)amide (LHMDS, 1 M, 9.44 ml) was added from a syringe in such a way that the temperature stayed below ⫺45°C. After complete addition, the yellow mixture was stirred for 45 min at ⫺40 to ⫺60°C, warmed to ⫺10°C and quenched with water (6 ml). Additional ether (80 ml) and water (30 ml) were added. The aqueous phase was separated and extracted with ether (2 ⫻ 40 ml). The combined organic layers were washed with water (3 ⫻ 20 ml) and brine (2 ⫻ 10 ml), dried over MgSO4, and concentrated. The residue was purified by flash chromatography with EtOAc/pentanes (1:24) to give 2.43 g (95%) of the conjugated ester 5 as a white solid. The solid was recrystallized in CH2Cl2/methanol, mp 58.5– 60.5°C. 1H-NMR: ␦ -0.02 (3H, s), -0.01 (3H, s), 0.88 (9H, s), 0.92 (3H, C18-CH3, s), 1.03 (3H, C21-CH3, d, J ⬃ 6.6 Hz), 1.1–1.8 (11H, series of m), 1.92 (1H, m), 2.20 (1H, m), 3.72 (3H, COOMe, s), 3.98 (1H, H-8, m), 5.77 (1H, H-25, d, J ⬃ 15.3 Hz), 5.96 (1H, H-22, dd, J ⬃ 15.1, 8.3 Hz), 6.08 (1H, H-23, dd, J ⬃ 15.1, 10.4 Hz), 7.24 (1H, H-24, dd, J ⬃ 15.4, 10.3 Hz). 13 C-NMR: ␦ -5.2, -4.8, 14.0, 17.7, 18.0, 19.6, 23.0, 25.8, 27.4, 34.4, 40.0, 40.6, 42.4, 51.4, 52.9, 56.1, 69.3, 118.5, 125.9, 145.9, 151.0, 167.8. Anal. Calcd for C24H42O3Si: C, 70.88; H, 10.41. Found: C, 70.79: H, 10.42. 2.3. (22E, 24E)-Des-A,B-26,27-dinor-25-carbomethoxycholesta-22,24-dien-8-ol (6) The silyl ether 5 (1.92 g, 4.71 mmol) was suspended in 60 ml CH3CN, and hydrofluoric acid (40% aq., 2.5 ml) was added. A white solid precipitated, which disappeared after 45 min. After stirring at rt overnight the reaction mixture was concentrated under reduced pressure, neutralized with saturated NaHCO3, and extracted with ether (4 ⫻ 50 ml). The combined extracts were washed with brine and dried. Filtration and evaporation gave 6 in a quantitative yield as an oil, which crystallized in the freezer, mp 103.5–106.8°C. 1 H-NMR: ␦ 0.97 (3H, C18-CH3, s), 1.05 (3H, C21-CH3, d, J ⬃ 6.6 Hz), 1.1–2.05 (14H, series of m), 2.22 (1H, m), 3.73 (3H, COOMe, s), 4.08 (1H, H-8, m), 5.79 (1H, H-25, d, J ⬃ 15.4 Hz), 5.97 (1H, H-22, dd, J ⬃ 15.2, 8.5), 6.10 (1H, H-23, dd, J ⬃ 15.2, 10.7), 7.24 (1H, H-24, dd, J ⬃ 15.4, 10.6 Hz). 13C-NMR: ␦ 13.8, 17.4, 19.6, 22.5, 27.4, 33.6, 40.0, 40.3, 42.1, 51.4, 52.5, 55.9, 69.2, 118.7, 126.0, 145.8, 150.7, 167.8. Anal. Calcd for C18H28O3: C, 73.93; H, 9.65. Found: C, 73.69; H, 9.65. 2.4. (22E,24E)-Des-A,B-24-homo-26,27-dimethyl-cholesta22,24-dien-8,25-diol (7) [4] Lithium wire (0.76 g, 0.11 mmol, flattened and cut into small pieces) was placed in dry ether (30 ml) in a 3-necked flask equipped with a thermometer, argon inlet and septum. The mixture was cooled to ⫺20°C. Ethyl bromide (5.45 g, 0.05 mol) was measured in a syringe, and approximately
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one tenth was added to the lithium suspension. After 5 min, the suspension turned cloudy, and the remaining ethyl bromide was slowly added while maintaining the temperature between ⫺15 and ⫺20°C. The mixture was then stirred for 50 min at the same temperature. The hydroxy ester 6 (773 mg, 2.64 mmol) was dissolved in dry THF (50 ml) under argon, cooled to ⫺78°C and the previously prepared EtLi solution (7 ml, 1.2 M) was added. After stirring at ⫺78°C for 1 h, the mixture was quenched with water and allowed to warm up to ⫺10°C. After concentration under reduced pressure, the residue was diluted in ethyl acetate (50 ml) and washed with water (40 ml). The aqueous phase was extracted with EtOAc (2 ⫻ 20 ml), and the combined organic layers were washed with water and brine and dried with MgSO4. Filtration and concentration gave 909 mg of a white crystalline residue. Recrystallization from n-hexane gave 669 mg (78%) of 7 as a white solid, mp 106 –108°C (lit. 107–108°C) [4]. 1H-NMR: ␦ 0.86 (6H, C27a,27b-2CH3, t, J ⬃ 7.5 Hz), 0.96 (3H, C18-CH3, s), 1.02 (3H, C21-CH3, d, J ⬃ 6.7 Hz), 1.1–2.2 (19H, series of m), 4.08 (1H, H-8, m), 5.51 (1H, H-22, dd, J ⬃ 14.9, 8.4 Hz), 5.52 (1H, H-24a, d, J ⬃ 15.4 Hz), 5.96 (1H, H-23, dd, J ⬃ 15.0, 10.4 Hz), 6.15 (1H, H-24, dd, J ⬃ 15.3, 10.3 Hz). 13C-NMR: ␦ 7.9, 13.7, 17.3, 19.9, 22.4, 27.5, 32.9, 33.5, 39.5, 40.2, 41.8 (C-13), 52.5, 56.3, 69.2 (C-8), 75.3 (C-25), 127.3, 128.9, 136.1, 140.2. Anal. Calcd for C21H36O2: C, 78.70; H, 11.32. Found: C, 78.33; H, 11.25. HRMS: m/z 320.2727 (calcd. for C21H36O2, 320.2715). 2.5. (22E,24E)-Des-A,B-24-homo-26,27-dimethyl-25hydroxy-cholesta-22,24-dien-8-one (8) [4] Dess-Martin periodinane (0.872 g, 1.05 mmol, Lancaster Synthesis Gmbh, Mu¨hlheim am Main, Germany) and pyridine (0.65 ml) were suspended in dry CH2Cl2 (20 ml) under argon. A solution of alcohol 7 (513 mg, 1.60 mmol) in CH2Cl2 (10 ml) was added. The mixture was stirred at rt for 40 min. After cooling in an ice-bath, the mixture was diluted with ether (60 ml) and poured into an ice-cold 1:1 mixture of saturated NaHCO3 and 1 M Na2S2O3. The aqueous phase was extracted with ether (3 ⫻ 25 ml). The ether layers were successively washed with sat. NaHCO3, CuSO4 aq. (0.15 M), water and brine (each 50 ml), and dried over MgSO4. Filtration and concentration gave 590 mg crude product as an oil, which crystallized in the freezer. Recrystallization from ether-pentanes gave 460 mg (90%) of ketone 8 as a white solid, mp 83.5– 85.0°C. 1H-NMR: ␦ 0.66 (3H, C18CH3, s), 0.86 (6H, C27a,27b-2CH3, t, J ⬃ 7.5 Hz), 1.08 (3H, C21-CH3, d, J ⬃ 6.7 Hz), 1.25–2.35 (17H, series of m), 2.45 (1H, H-14, dd, J ⬃ 10.8, 7.4 Hz), 5.51 (1H, H-22, dd, J ⬃ 15.1, 8.5 Hz), 5.53 (1H, H-24a, d, J ⬃ 15.1 Hz), 5.98 (1H, H-23, dd, J ⬃ 15.0, 10.6 Hz), 6.16 (1H, H-24, dd, J ⬃ 15.2, 10.4 Hz). 13C-NMR: ␦ 7.9, 12.7, 19.0, 20.3, 24.0, 27.6, 33.0, 38.8, 39.6, 40.9, 49.8, 56.4, 61.9, 75.4, 127.9, 128.6, 136.2, 139.2, 211.8. Anal. Calcd for C21H34O2: C, 79.19; H, 10,76.
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Found: C, 78.41; H, 10.55. HRMS: m/z 318.2569 (calcd. for C21H34O2, 318.2559). 2.6. (22E,24E)-Des-A,B-24-homo-26,27-dimethyl-8-[(E)ethoxycarbonyl-methylidene]-cholesta-22,24-dien-25-ol (9) A suspension of NaH (55– 65% suspension in oil, 250 mg) in dry THF (10 ml) was cooled in an ice-bath under argon, and triethyl phosphonoacetate (1.76 g, 7.85 mmol) was slowly added from a syringe. After complete addition, the mixture was stirred at rt for 40 min, then placed in an ice-bath again. After 10 min, a solution of ketone 8 (250 mg, 0.785 mmol) in THF (6 ml) was added in one portion. The mixture was stirred overnight at 28°C. The reaction mixture was cooled in an ice-bath and quenched with water (25 ml), extracted with ether, and dried over Na2SO4. After filtration, the solvent was removed under reduced pressure. Flash chromatography of the crude material using EtOAc/pentanes (1:4) afforded 228 mg (76%) of unsaturated ester 9. 1 H-NMR: ␦ 0.60 (3H, C18-CH3, s), 0.88 (6H, C27a,27b-2CH3, t), 1.06 (3H, C21-CH3, d, J ⬃ 6.6 Hz), 1.2–2.2 (17H, series of m), 1.28 (3H, Et, t), 3.88 (1H, H-9, m), 4.14 (2H, CH2OCO, q), 5.45 (1H, H-7, s), 5.52 (1H, H-24a, d, J ⬃ 15.3 Hz), 5.53 (1H, H-22, dd, J ⬃ 15.0, 8.4 Hz), 5.98 (1H, H-23, dd, J ⬃ 15.0 Hz, 10.4 Hz), 6.14 (1H, H-24, dd, J ⬃ 15.3, 10.3 Hz). HRMS: m/z 388.2988 (calcd. for C25H40O3, 388.2977). 2.7. (22E,24E)-Des-A,B-24-homo-26,27-dimethyl-8-[(E)carboxy-methylidene]-cholesta-22,24-dien-25-ol (10) The unsaturated ester 9 (228 mg, 0.59 mmol) was treated with KOH in methanol (1.5 M, 8 ml) and stirred for 3 days at rt. After cooling in an ice-bath, the pH was carefully adjusted to 5 with diluted HCl, and the solution was quickly extracted with cold ethyl acetate. The extracts were washed with water and brine and dried over MgSO4. After filtration and concentration, the crude product was purified by flash chromatography with EtOAc/pentanes (11:9) yielding 116 mg (55%) of acid 10. 1H-NMR: ␦ 0.61 (3H, C18-CH3, s), 0.86 (6H, C27a,27b-2CH3, t, J ⬃ 7.5 Hz), 1.06 (3H, C21-CH3, d, J ⬃ 6.6 Hz), 1.2–2.3 (18H, series of m), 3.85 (1H, H-9, m), 5.49 (1H, H-8, bs), 5.52 (1H, H-22, d, J ⬃ 14.7, 8.5 Hz), 5.53 (1H, H-24a, d, J ⬃ 15.1 Hz), 5.98 (1H, H-23, dd, J ⬃ 14.7, 10.3 Hz), 6.15 (1H, H-24, dd, J ⬃ 15.1, 10.3 Hz). 2.8. (22E,24E)-Des-A,B-24-homo-26,27-dimethyl-8-[(E)N-(2-(ethoxycarbonyl)-ethyl)-carbamoylmethylidene]cholesta-22,24-dien-25-ol (11) A solution of crude carboxylic acid 10 (70 mg, 0.194 mmol), -alanine ethyl ester hydrochloride (30 mg, 0.194 mmol) and Et3N (22 mg) in dry CH2Cl2 (4 ml) was cooled in an ice-bath and DMAP (2 mg) was added. The solution was stirred for 5 min upon which solid N,N⬘-dicyclohexylcarbodiimide (DCC, 44 mg, 0.213 mmol) was added. Stir-
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ring was continued overnight in the melting ice-bath. Dicyclohexylurea was removed by filtration. The filtrate was concentrated and redissolved in ethyl acetate, washed with saturated NaHCO3, water and brine, dried over Na2SO4, and concentrated. The crude material was purified by flash chromatography with EtOAc/pentanes (7:13) giving 52 mg (58%) of amido-ester 11 as an oil. 1H-NMR: ␦ 0.59 (3H, C18-CH3, s), 0.87 (6H, C26a-CH3, t), 1.05 (3H, C21-CH3, d), 1.26 (3H, COOCH2CH3, t), 1.2–2.2 (17H, series of m), 2.56 (2H, CH2COO, t), 3.55 (2H, CH2NH, dt), 3.82 (1H, H-9, m), 4.13 (2H, COOCH2CH3, q), 5.30 (1H, H-7, s), 5.51 (2H, H-22 and H-24a, m), 5.96 (1H, H-23, dd), 6.05 (1H, N-H, t), 6.16 (1H, H-24, dd). 13C-NMR: ␦ 7.6, 12.1, 13.9, 20.1, 21.9, 23.4, 27.2, 29.1, 32.8, 33.9, 34.2, 39.8, 46.4, 56.2, 60.4, 75.2, 114.4, 127.5, 128.6, 136.3, 139.6, 157.3, 166.7 (CONH), 172.6 (COOEt). HRMS: m/z 459.3360 (calcd. for C28H45NO4, 459.3349). 2.9. (22E,24E)-Des-A,B-24-homo-26,27-dimethyl-8-[(E)N-(2-carboxyethyl)-carbamoylmethylidene]-cholesta22,24-dien-25-ol (3) Aqueous NaOH (2N, 61 l) was added to a solution of amido-ester 11 (50 mg, 0.11 mmol) in dioxane-water 9:1 (1.5 ml), and the resulting mixture was stirred at rt for 3 h. Phosphate buffer (pH 7.4) was added, and the aqueous solution was extracted with ethyl acetate until the extracts no longer contained any carboxylic acid (checked on TLC plate/UV detection). The combined organic layers were washed with brine, dried over Na2SO4 and filtered. The filtrate was concentrated to give 49 mg of 3 as an oil in a quantitative yield, sufficiently pure for analysis and conjugation to BSA. 1H-NMR: ␦ 0.59 (3H, C18-CH3, s), 0.86 (6H, C26a-CH3, t), 1.05 (3H, C21-CH3, d), 1.1–1.8 (17H, series of m), 2.60 (2H, CH2COOH, t), 3.55 (2H, CH2NH, m), 3.78 (1H, H-9␣, m), 5.31 (1H, H-7, s), 5.53 (2H, H-24a, d and H-22, dd), 5.96 (1H, H-23, dd), 6.14 (2H, H-24 and N-H, m). 13C-NMR: ␦ 7.9, 12.3, 20.4, 22.2, 23.7, 27.5, 29.5, 33.0, 33.7, 34.3, 34.7, 40.1, 46.7, 56.5, 56.5, 75.6 (C-25), 114.4, 127.7, 128.9, 136.5, 139.9, 158.2, 167.6 (CONH), 176.4 (COOH). UV (99% ethanol): max 232 nm (⑀ 41 500). HRMS: m/z 413.2951 (M-H2O) (calcd. for C26H39NO3 (MH2O), 413.2930). 2.10. (1R,3S,5Z,7E,22E,24E)-9,10-secocholesta5,7,10(19),22,24-pentaen-1,25-diol-24-homo-26,27dimethyl-3-yl-(2-trimethylsilyl-ethyl)-succinate (12) A mixture of EB1089 [1] (1, 105 mg, 0.222 mmol), 2-(trimethylsilyl)-ethyl hydrogen succinate [5] (58 mg, 0.266 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 47 mg, 0.244 mmol) and DMAP (2 mg) in dry THF was stirred under an argon atmosphere for 2 h at rt. After quenching with water, the solution was diluted with ethyl acetate, washed with water and brine and dried over MgSO4. After filtration and con-
centration, the residue was purified by flash chromatography (5–50% EtOAc/pentanes). Four compounds were isolated of which the third most polar compound (Rf 0.71 in EtOAc/pentanes 1:1) was the desired mono-ester 12. 1HNMR: ␦ 0.01 (9H, s), 0.54 (3H, s), 0.84 (6H, t), 0.95 (1H, m), 1.03 (3H, d), 1.2–2.1 (21H, series of m), 2.38 (1H, dd), 2.57 (5H, m), 2.80 (1H, m), 4.1– 4.2 (2H, m), 4.38 (1H, m), 4.98 (1H, s), 5.21 (1H, m), 5.32 (1H, s), 5.50 (2H, d and dd), 5.97 (2H, m), 6.13 (1H, dd), 6.31 (1H, d). 2.11. (1R,3S,5Z,7E,22E,24E)-9,10-secocholesta5,7,10(19),22,24-pentaen-1,25-diol-24-homo-26,27dimethyl-3-yl-hydrogen succinate (13) Tetrabutylammonium fluoride (TBAF, 50 mg, 0.157 mmol) was added to a solution of trimethylsilylethyl ester 12 (35 mg, 0.052 mmol) in dry THF (1.5 ml) under an argon atmosphere. The resulting mixture was stirred at rt for 3 h, diluted with ethyl acetate and washed with water and brine. After drying and removal of the solvent, the residue was purified by flash chromatography (MeOH/CHCl3) to give 28 mg (97%) of hemisuccinate 13. 1H-NMR: ␦ 0.56 (3H, s), 0.86 (6H, t), 1.05 (3H, d), 1.2–2.2 (20H, series of m), 2.40 (1H, dd), 2.60 (5H, m), 2.82 (1H, m), 4.38 (1H, m), 5.01 (1H, m), 5.24 (1H, m), 5.35 (1H, m), 5.54 (2H, d and dd), 5.96 (1H, dd), 6.02 (1H, d), 6.15 (1H, dd), 6.32 (1H, d). 2.12. Preparation of NHS-EB1089 succinate (for EIA) A solution of EB1089 3-O-hemisuccinate 13 (28 mg, 0.05 mmol) in dry THF (1 ml) was treated with N-hydroxysuccinimide (12 mg, 0.10 mmol), EDC (20 mg, 0.10 mmol), and a catalytic amount of DMAP. The reaction was stirred overnight at 4°C and extracted with ethyl acetate and water. The organic layer was washed with brine, dried, and the solvent was removed. The crude product was passed through a short column of silica gel, affording 14 mg of the NHS-ester. A stock solution in dry DMSO was immediately prepared, which was stored at ⫺18°C. 2.13. BSA conjugate formation The hapten 3 was conjugated to bovine serum albumin (BSA) via an N-succinimidyl ester according to a standard procedure [6]. N-hydroxysuccinimide (16 mg, 0.14 mmol) and EDC (27 mg, 0.14 mmol) were added to a solution of 3 (15 mg, 35 mol) in 95% dioxane aq. (0.2 ml). The mixture was stirred at room temperature for 3 h, diluted with water, and extracted with ethyl acetate. The combined extracts were washed with water, dried, and concentrated under reduced pressure. The crude NHS ester was dissolved in pyridine (0.75 ml), and a solution of BSA (25 mg, crystallized, Sigma) in phosphate buffer (0.05 M, 0.75 ml) was added dropwise. After stirring overnight at 4°C, the suspension was dialyzed against PBS (pH 7.0) and cold water (each for 2 ⫻ 12 h) and freezedried, affording 32 mg of
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conjugate. UV (Tris buffer pH 8.4): max 226. (A232 ⫽ 1.2 at a concentration of 0.05 g/l). 2.14. Antibody production [7] The BSA conjugate (6 mg) was suspended in 1.5 ml of an aqueous solution of NaCl (0.1 M) and NaN3 (15 mM). The suspension was added dropwise to Freund’s incomplete adjuvant (1.5 ml) under vigorous mixing. Three rabbits (Danish Whites), 3– 6 months old, were immunized subcutaneously (s.c.) in the neck region with 100 l of the immunogen-adjuvant emulsion (2 g/l). This procedure was repeated every two weeks for 2 months, then once every month. After 12 weeks following the first immunization, blood samples were collected from an ear vein. Blood samples were subsequently collected every two weeks.
Fig. 2. Reagents, conditions and yields: a) trimethyl 4-phosphonocrotonate, LHMDS, THF, ⫺78°C; 95%; b) 2.5% HF, CH3CN-H2O; 100%; c) EtLi (2 eq.), THF; 78%.
2.15. EIA procedure (standard) [8] CovaLink™ and MicroWell™ polypropylene microtiter plates were purchased from Nunc (Roskilde, Denmark). Swine anti-rabbit horseradish peroxidase and TMB one-step substrate system were purchased from DAKO (Glostrup, Denmark). Metabolites of vitamin D and of EB1089 used for cross-reaction studies were synthesized at Leo Pharmaceutical Products, except for 24R,25-dihydroxyvitamin D3, which was obtained from Solvay Duphar B.V. (Weesp, The Netherlands). The buffers used were phosphate-buffered saline (PBS) (0.15 M NaCl, 8.5 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, pH 7.0), coating buffer (PBS with 40 M chloramine T), washing buffer (PBS with 0.1% Tween 20, pH 7.0), and assay buffer (PBS with 1% casein, pH 7.2). Plates were washed 4 times between incubations. All incubations were run for 1 h at room temperature on shakers unless otherwise stated. OD was measured on a SLT Rainbow Scanner (SLT Labinstruments) at 450 nm. All determinations were performed in duplicate. CovaLink plates were incubated overnight at 4°C with 100 l NHS-EB1089 succinate (0.40 g/ml in coating buffer, diluted from a stock solution in DMSO). After washing, wells were blocked with 200 l assay buffer. Antiserum was diluted in assay buffer and 100 l were applied as primary antibody in each well, and the plates were incubated and washed. The secondary antibody (swine antirabbit horseradish peroxidase) was diluted 1:4000 in assay buffer and 100 l were applied per well. After washing, 100 l of TMB one-step substrate were added per well, and the plates were incubated for 30 min in the dark. The reaction was then stopped with 2N H2SO4 (100 l/well), and the absorbance was measured at 450 nm. 2.15.1. Chaotropic ion elution EIA The standard EIA procedure was followed using an antiserum dilution of 1:1000, except that the following step was inserted between the primary and the secondary anti-
body incubations: A serial dilution of NH4SCN in PBS (100 l/well, concentrations 0 – 0.062– 0.125– 0.250 – 0.50 –1.0 – 2.0 – 4.0 M) was applied, and the plate was incubated for 20 min, followed by washing. 2.15.2. Cross-reaction study Stock solutions of EB1089, EB1089 metabolites and selected vitamin D metabolites in ethanol were prepared, and the exact concentrations were determined from the UV absorbance ( 264 nm, ⑀ 17 500 for vitamin D metabolites and 232 nm, ⑀ 44 000 for EB1089 and related metabolites). Aliquots (500 –10 000 pmol depending on the metabolite) were applied in the first column of a MicroWell polypropylene plate, and the solvent was allowed to evaporate. Acetonitrile (20 l) was then added to each well in the column, followed by assay buffer (180 l). In the remaining wells of the plate, 100 l assay buffer containing 10% acetonitrile was applied. Serial dilutions of each metabolite were then made row-wise by transferring 100 l from column to column with mixing. From the last column, 100 l were discarded. The primary antibody was diluted 1:4000 in assay buffer, and 100 l were added to each well. The plate was incubated at 4°C overnight. The standard EIA procedure was used, except that the preincubated antibody was applied as primary antibody (100 l/well) by transfer from the MicroWell plate.
3. Results The target hapten 3 (Fig. 1) was synthesized from the known aldehyde 4 [3]. Assembly of the side-chain from the aldehyde is outlined in Fig. 2 and is similar to the reported procedure for preparing EB1089 [1]. The aldehyde was converted to the unsaturated ester 5 by Horner-Emmons olefination with trimethyl 4-phosphonocrotonate. Only the
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Fig. 4. Reagents, conditions, and yields: a) (CH3)3Si(CH2)2OOC(CH2)2 COOH, EDC, DMAP, THF; 24% b) TBAF䡠3H2O, THF; 100%.
Fig. 3. Reagents, conditions, and yields: a) Dess-Martin periodinane, CH2Cl2; 90%; b) Triethyl phosphonoacetate, NaH, THF; 76%; c) KOH, MeOH; 55%; d) H2N(CH2)3COOEt䡠HCl, DCC, Et3N, CH2Cl2; 58%; e) NaOH, dioxane/H2O; 100%.
trans-trans product was isolated, which was indicated by large coupling constants of the vinylic hydrogens in 1HNMR. The exclusive formation of the trans alkene geometry is characteristic when using stabilized phosphonate anion reagents. Before completing the side-chain assembly by formation of a terminal allylic alcohol by addition of 2 equivalents ethyl lithium to the ester group, the TBS-group on the 8-hydroxyl was removed. Deprotection of the 8-TBS was not possible with TBAF using standard conditions and required more rigorous conditions using hydrofluoric acid. Due to the inherent lability of an allylic alcohol to acid conditions, the formation of this alcohol was deferred until after the deprotection. It turned out that the addition of ethyl lithium to the ester proceeded smoothly and in high yield even in the presence of the unprotected hydroxyl group. A small amount of ethyl-adduct resulting from 1,4-addition was also formed in this step. Following the side-chain assembly, a spacer was attached at C-8 in the C-ring (Fig. 3). Compound 7 was oxidized to 8 with Dess-Martin periodinane. Horner-Emmons olefination with excess triethyl phosphonoacetate and NaH giving rise to 9 was achieved in moderate yield without protection of the terminal tertiary alcohol. The reaction temperature was critical; the optimal temperature was found to be 25–30°C. Lower temperatures prolonged the reaction time considerably and higher temperatures resulted in the formation of the C-14 epimer as a byproduct. The configuration of the newly formed double bond in 9 was established by ROESY. A NOE was observed between H-7 and H-15, which indicated that the double bond had the E-configuration as drawn. The ester 9 was hydrolyzed with base to the carboxylic acid 10. The reaction mixture was acidified to pH 5.5, and the product was obtained by multiple extractions of the
aqueous phase. Acidifying the mixture below pH 5.5 resulted in allylic rearrangement of the side-chain. Despite the fact that the carboxylic acid was expected to be deprotonated at pH 5.5, it was nevertheless possible to extract the compound from the aqueous phase, probably due to the highly lipophilic character of the compound. Compound 10 was converted to the amido-ester 11 by carbodiimide coupling with -alanine ethyl ester. Selective hydrolysis of the ethyl ester with NaOH in dioxane-water afforded the hapten 3 after extraction, as described above. 3.1. Preparation of N-hydroxysuccinimide esters of 3 and of EB1089-hemisuccinate Immunizations were carried out with a BSA-conjugate of hapten 3. The conjugation procedure involved conversion of the hapten to its N-hydroxysuccinimide (NHS) ester and subsequent reaction with BSA. The NHS-ester of 3 was prepared by treating 3 with N-hydroxysuccinimide and EDC [6]. The hapten/BSA ratio was estimated to be 41 based on the ultraviolet absorbance at 232 nm by subtraction of the contribution of BSA. To characterize the antibodies, we used an ELISA with immobilized EB1089 in CovaLink microtiter plates. We used EB1089 and not hapten 3 in the assay because EB1089 was the actual target of interest. For the purpose of immobilizing EB1089, we prepared EB1089 3-hemisuccinate (13, Fig. 4). Carbodiimide coupling of EB1089 and 2-(trimethylsilyl)-ethyl succinate [5] afforded a separable mixture of 1- and 3-monoesters as well as diester and unreacted EB1089. The 3-ester 12 was treated with TBAF in THF to afford the 3-O-hemisuccinate of EB1089. The NHS-ester was finally prepared as described for the hapten 3. 3.2. Determination of titer Antisera from three rabbits (As-1, As-2 and As-3) were obtained by immunizations with the prepared BSA-conjugate of 3. The first blood samples were obtained after 12 weeks of immunization. Antibody dilution curves were de-
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Fig. 7. Chaotropic ion elution of As-1 at various dilutions. Fig. 5. Antibody dilution curves, weeks 12 and 26. The diagram shows the resulting reaction of antisera with covalently immobilized EB1089 hemisuccinate.
termined by measuring the binding of a series of concentrations of antiserum to covalently bound EB1089 hemisuccinate in Covalink microtiter plates (Fig. 5). Bound antibody was detected with a swine anti-rabbit peroxidase labeled secondary antibody. The titer of all three antisera had increased 3–5 fold during the three-month period, as measured by the lateral displacement of the absorption curves. 3.3. Avidity measurement Measurement of avidity was performed using chaotropic ion elution [9 –11]. A chaotropic ion (thiocyanate) was added in serial dilutions to the bound antibody in microtiter plates. The chaotropic ion decreases hydrophobic interactions, resulting in a dissociation of the complex. The avidity index was defined as the concentration of thiocyanate at which the maximal binding to immobilized EB1089 was reduced to 50%. Fig. 6 shows the development of the avidity index as a function of time after immunization for the three antisera. It
Fig. 6. Avidity index vs. time post immunization of rabbit antisera (error bars indicate the standard deviation).
should be noted that there was some difficulty in reproducing the experiment, especially because the assay seemed to be sensitive to small variations in the thiocyanate elution step. Apparently the avidity increased as a function of time for all antisera. As-1 and As-3 exhibited a similar, modest increase, while antiserum As-2 showed the most marked increase in avidity. In order to ensure that the observed increase in avidity was not merely an effect of the increase in titer, the avidity was measured for a single antibody at various dilutions. Antiserum As-1 (week 20) was diluted 1:1000, 1:2000, and 1:4000 and tested in the thiocyanate elution assay. Fig. 7 shows the result, which clearly indicates that the avidity index was indeed independent of concentration. The data point for 1:4000 dilution at 4 M thiocyanate concentration may be erratic, because the measured absorbance was equal to the background absorbance for the assay. 3.4. Cross-reactivity In order to assess the specificity of the antisera, the cross-reactivities with various vitamin D metabolites were measured. The most common vitamin D metabolites were selected for the cross-reaction study. These were 1,25(OH)2D3, 25-hydroxyvitamin D3, 24R,25-dihydroxyvitamin D3, vitamin D2 and vitamin D3. Four metabolites of EB1089 itself have been identified [12], and cross-reactivities with these compounds were also examined. Structures of the metabolites of EB1089 are shown in Fig. 8. The metabolite EB1446 has been identified as the major metabolite of EB1089. Antisera from week 26 diluted at a concentration of 1:4000 were preincubated with serial dilutions of metabolite overnight, and the resulting reaction to immobilized EB1089 in Covalink plates was determined. The IC50 value was measured as the concentration of metabolite that was necessary to reduce the maximal binding to 50%. The crossreactivity with a metabolite was then calculated as the
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Fig. 8. Identified metabolites of EB1089.
percentage of the IC50 value of EB1089 to that of the metabolite. The results are listed in Table 1. The specificity profiles of the three antibodies are similar. The antibodies all discriminated EB1089 from endogenous metabolites of vitamin D. In particular, the crossreactivity with 25-hydroxyvitamin D3 was 0.01% for the antibody As-3, which is excellent, taking into account the large amounts of this metabolite in serum (up to 1 ng/ml in humans). The cross-reactivity with the active metabolite 1,25(OH)2D3 was generally slightly higher, but this form is present in equimolar amounts in serum compared to the expected serum levels of EB1089. Cross-reactivities with vitamin D2 and vitamin D3 were negligible for the three antibodies. All three antisera showed relatively high cross-reactivities with the four metabolites of EB1089. Thus, additional hydroxylation of the side chain of EB1089 did not affect the binding to the antibodies significantly.
4. Discussion The analysis of EB1089 and other vitamin D related compounds in serum represents a challenge due to the low serum concentrations, which usually are in the picomolar range for biologically active compounds. Furthermore, the Table 1 Cross-reactivities of three antisera to naturally occurring vitamin D metabolites and to metabolites of EB1089. The cross-reactivity is defined as the percentage of the IC50 value of EB1089 to that of the metabolite Compound
EB1089 1,25(OH)2D3 25(OH)D3 24R,25(OH)2D3 Vitamin D2 Vitamin D3 EB1446 EB1436 EB1445 EB1470
Antibody (Week 26) As-1
As-2
As-3
100 0.3 0.09 0.04 ⬍0.01 ⬍0.01 26 16 26 26
100 0.78 0.16 0.07 ⬍0.01 ⬍0.01 35 28 78 70
100 0.1 ⬍0.01 0.07 ⬍0.01 ⬍0.01 40 29 44 27
presence of closely related, endogenous metabolites in serum interfere. The analysis therefore requires a specific and sensitive method. Recently, a mass spectrometric method has been developed for the quantification of EB1089 in serum samples [13]. However, this method requires preliminary extraction and isolation of EB1089 prior to analysis. We are aiming at developing an immunoassay for EB1089 with specific antibodies that will allow direct measurement in serum, and for this purpose we raised polyclonal antibodies to EB1089. For characterizing the antibodies, we used an enzyme immunoassay that involved covalent binding of EB1089 in special microtiter plates, a method that has been recently described for 25-hydroxyvitamin D3 [8]. Antibodies to vitamin D derivatives have conventionally been raised against their BSA-conjugates, in which the derivative has been coupled to BSA through a spacer [14, 15]. However, we have previously experienced that such antigens in some cases are toxic during long-time immunization, probably due to in vivo hydrolysis and liberation of the hapten. Although EB1089 is a low-calcemic analog of 1,25(OH)2D3, the amounts of EB1089 that potentially can be liberated from immunizing doses of a BSA conjugate are large compared to therapeutic doses. We therefore pursued a different strategy. EB1089 possesses a side-chain that is not found in any natural metabolite of vitamin D or in steroids. It was believed that a hapten containing only the CD-ring and side-chain of EB1089 (Fig. 1) would be sufficient to raise antibodies to the analog that discern EB1089 from closely related endogenous vitamin D metabolites. The absence of the A-ring would render the hapten noncalcemic while preserving the unique structural features of EB1089. In the hapten 3, the A-ring of EB1089 was replaced by a carboxylic acid spacer for carrier conjugation. By introducing an exocyclic double bond at C-8, it was expected that the conformation of the CD-ring system should be similar to that of the original compound. The present study showed that antibodies raised against this ‘truncated’ form of EB1089 recognized the analog as a whole, all the more with a good specificity. All antibodies exhibited an increase in titer toward EB1089 during the immunization period. Although commonly encountered in the literature, affinity constants for polyclonal antibodies are somewhat difficult to measure experimentally. Equilibrium mixtures of polyclonal antibody and antigen are complex, and any observed affinity represents an average of all individual antibody-antigen equilibria. It is therefore more correct to use the less specific term avidity for polyclonal sera. We used chaotropic ion elution as a means to determine relative avidities of the produced antisera. Chaotropic ions disrupt the structure of water and decrease the surface tension. This in turn decreases hydrophobic interactions that are expected to dominate in the binding between the antibodies and EB1089, and this results in a dissociation of the complex. It must be noted that the chaotropic elution method is somewhat controversial, because chaotropic ions denature the
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antibodies and affect intermolecular interactions in addition to affecting the binding to ligands. Nevertheless, it was shown that avidity was independent of antibody concentration (Fig. 7), indicating that the measured parameter is an intrinsic quality of the antibodies. Also, it was generally observed that avidity increased in time (Fig. 6), reflecting a change in the distribution of immunoglobulin characterized by a higher proportion of high-affinity antibodies. The cross-reaction study was performed with the naturally occurring metabolites of vitamin D3 and with metabolites of EB1089 (Table 1). After 26 weeks, the antibodies showed low cross-reactivities with the naturally occurring vitamin D metabolites. It is somewhat surprising that there was a difference in the cross-reactivity with 1,25(OH)2D3 and 25(OH)D3 (0.1% and 0.01%, respectively, for As-3). These two metabolites only differ in the A-ring. Yet the antibodies were raised against a hapten with no A-ring and are therefore not expected to recognize this moiety. It therefore appears that the presence of the 1␣ hydroxyl group does make a difference, perhaps due to random hydrogen bonding. It is fortunate that the selectivity was in favor of 1,25(OH)2D3 due to the much lower concentrations of this metabolite in serum compared to 25(OH)D3. The cross-reactivity of the antibody As-3 with 25(OH)D3 was satisfactorily low (0.01%) to avoid interfering binding of this metabolite, which is present in serum in up to 1000 times the expected concentration of EB1089. Also 24R,25(OH)2D3, a metabolite that also occurs in relatively large concentrations in serum, was easily discriminated (0.04 – 0.07%). A much higher cross reactivity was observed with the metabolites of EB1089 (16 –78%). This indicates that the diene is an important epitope, which is recognized by the antibodies. The additional hydroxyl group in the terminal end of the side-chain of the metabolites reduced binding by only one fifth in the best case compared to EB1089. Similar reactivity patterns have previously been reported, where antibodies to vitamin D analogs fail to recognize slight changes in the side chain [14]. This relatively high crossreactivity could cause interference in the determination of serum levels of EB1089, but the concentrations of these metabolites are expected to be low compared to that of EB1089. One might speculate whether a better specificity could have been obtained by immunizing with BSA-conjugates of non-truncated EB1089 instead of 3. We do not believe that this would improve the selectivity. The presence of additional epitopes created by the A-ring would presumably increase the binding affinity of the antibodies to EB1089, but the affinity to EB1089 metabolites would also increase, because the same A-ring is present in these compounds. One might even obtain less selective antibodies because the unique side-chains would provide a relatively smaller contribution to the total binding between the antibody and metabolite. The sensitivity of the employed immunoassay was not
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optimized for measuring serum levels of EB1089. Phase I clinical studies have suggested therapeutic doses up to 20 g/day per patient [1], but the majority of the drug is accumulated in the liver. Circulating levels of the drug are therefore estimated to be in the region of 10 –100 pg/ml serum. The sensitivity of this assay needs to be improved 10 –100 fold in order to detect these levels. There is ample room for improvement in the assay, especially with respect to the secondary antibody, which may be replaced by biotinstreptavidin or enzyme-cascade amplified visualization. Alternatively, a radioimmunoassay may be developed, requiring radioisotope labeled EB1089. In conclusion, we successfully prepared antibodies specific to EB1089 by immunization of rabbits with a truncated form of the molecule. The antibodies showed a good selectivity for EB1089 over endogenous vitamin D metabolites, enough to allow direct measurement in serum of the analog. The further development of an immunoassay with these antibodies could therefore obviate the need for preliminary isolation of EB1089, which is a requirement in existing methods.
Acknowledgments This work was partly financed by the Danish Academy of Technical Sciences. The authors wish to thank Sven Erik Godtfredsen at DAKO A/S for facilitating the production of antisera and Grethe Aagaard and staff at the Department of Spectroscopy at Leo for the recording and interpretation of spectra.
References [1] Hansen CM, Hamberg KJ, Binderup E, Binderup L. Seocalcitol (EB 1089): a vitamin D analog of anti-cancer potential. Background, design, synthesis, pre-clinical and clinical evaluation. Curr Pharm Des 2000;6:881–906. [2] Still WC, Kahn M, Mitra A. Rapid chromatographic technique for preparative separations with moderate resolution. J Org Chem 1978; 43:2923–5. [3] Ferna´ndez B, Pe´rez JAM, Granja JR, Castedo L, Mourin˜o A. Synthesis of hydrindan derivatives related to vitamin D. J Org Chem 1992;57:3173– 8. [4] Posner GH, Lee JK, White MC, Hutchings RH, Dai H, Kachinski JL, Dolan P, Kensler TW. Antiproliferative hybrid analogs of the hormone 1␣,25-dihydroxyvitamin D3: design, synthesis, and preliminary biologic evaluation. J Org Chem 1997;62:3299 –314. [5] Kita Y, Maeda H, Takahashi F, Fukui S. A convenient synthesis of dicarboxylic monoesters using isopropenyl esters: synthesis of oxaunomycin derivatives. Chem Commun 1993;4:410 –2. [6] Kobayashi N, Takama A, Shiomura K, Tabata Y, Takagi K, Shimada K. Production of a group-specific antibody to 1␣,25-dihydroxyvitamin D and its derivatives having the 1␣,3-dihydroxylated A-ring structure. Steroids 1994;59:404 –11. [7] Harboe NMG, Ingild A. Immunization, isolation of immunoglobulins and antibody titre determination. Scand J Immunol 1983;17(Suppl. 10):345–51.
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[8] Lind C, Chen J, Byrjalsen I. Enzyme immunoassay for measuring 25-hydroxyvitamin D3 in serum. Clin Chem 1997;43:943–9. [9] Pullen GR, Fitzgerald MG, Hosking CS. Antibody avidity determination by ELISA using thiocyanate elution. J Immunol Methods 1986;86:83–7. [10] Appleyard G, Wilkie BN, Kennedy BW, Mallard BA. Antibody avidity in Yorkshire pigs of high and low immune response groups. Vet Immunol Immunopathol 1992;31:229 – 40. [11] Ferreira MU, Katzin AM. The assessment of antibody affinity distribution by thiocyanate elution: a simple dose-response approach. J Immunol Methods 1995;187:297–305. [12] Kissmeyer AM, Binderup E, Binderup L, Hansen CM, Andersen NR, Makin HLJ, Schroeder NJ, Shankar VN, Jones G. Metabolism of the vitamin D analog EB1089: identification of in vivo and in vitro liver
metabolites and their biologic activities. Biochem Pharmacol 1997; 53:1087–97. [13] Kissmeyer AM, Sonne K, Binderup E. Determination of the vitamin D analog EB 1089 (seocalcitol) in human and pig serum by liquid chromatography tandem mass spectrometry. J Chromatogr B 2000; 740:117–28. [14] Kobayashi N, Higashi T, Saito K, Murayama T, Douya R, Shimada K. Specificity of polyclonal antibodies raised against a novel 24,25-dihydroxyvitamin D3-BSA conjugant linked through the C-11␣ or C-3 position. J Steroid Biochem Molec Biol 1997;62: 79 – 87. [15] Kobyashi N, Asano T, Kitahori J, Shimada K, Kubodera N, Watanabe H. Production and specificity of anti-22-oxacalcitriol antisera. Chem Pharm Bull 1992;40:1520 –2.
Polyclonal antibodies to EB1089 (seocalcitol), an analog of 1␣,25-dihydroxyvitamin D3夞 Lars K.A. Blæhra,*, Fredrik Bjo¨rklinga, Ernst Binderupa, Martin J. Calverleya, Peter Kaastrupb a
Department of Medicinal Chemistry, Leo Pharmaceutical Products Ltd., 55 Industriparken, DK-2750 Ballerup, Denmark b Institute of Medical Microbiology and Immunology, University of Copenhagen, DK-2200 Copenhagen, Denmark Received 5 June 2000; received in revised form 5 September 2000; accepted 11 September 2000
Abstract A hapten derivative of EB1089 [1(R),3(S),25-trihydroxy-26,27-dimethyl-9,10-seco-24-homocholesta-5(Z),7(E),10(19),22(E),24(E)-pentaene], a side-chain analog of 1␣,25-dihydroxyvitamin D3, was synthesized for raising antibodies with a high specificity for EB1089. The A-ring moiety of EB1089 was replaced in the hapten by a linker for conjugation to a protein. Three polyclonal antibodies were obtained by immunizing rabbits with a BSA-conjugate of the hapten. The antibodies were characterized for titer, avidity and specificity using an enzyme immunoassay with covalently bound EB1089. The three antibodies had similar binding profiles and were highly selective for EB1089 and its metabolites over the naturally occurring vitamin D metabolites. Cross-reactivities with 25-hydroxyvitamin D3, the most abundant vitamin D metabolite in serum, were in the range 0.01– 0.2% relative to EB1089. © 2001 Elsevier Science Inc. All rights reserved. Keywords: EB1089; 1␣,25-Dihydroxyvitamin D3 analog; Hapten; Synthesis; Antibody production; Enzyme immunoassay
1. Introduction EB1089 (seocalcitol, 1) is a synthetic, low-calcemic analog of the natural hormone 1␣,25-dihydroxyvitamin D3 (1,25(OH)2D3, 2) that has recently been selected for clinical trials as a potential anti-cancer agent [1]. The analog is 50 –200 times stronger than 1,25(OH)2D3 in inhibiting cellular proliferation, but is only 50% as calcemic, and this makes it a promising drug candidate. The evaluation of EB1089 as a potential anti-cancer agent in the clinic has demanded methods for quantifying the drug in biological samples. Because existing assays were unsuitable for this purpose, we decided to raise polyclonal antibodies specific to EB1089 in order to develop an immunoassay. The use of highly specific antibodies could eliminate the problem of cross-reaction with naturally occurring vitamin D metabolites, especially 25-hydroxyvita夞This work was presented as a poster at the 11th Workshop on Vitamin D, Nashville, TN, May 27–June 1, 2000. The synthesis section was taken from the doctoral thesis of Lars Blæhr (Leo Pharmaceutical Products & Royal Danish School of Pharmacy, Copenhagen, 1998). * Corresponding author. Tel.: ⫹45-44-92-38-00; fax: ⫹45-44-94-55-10. E-mail address: [email protected] (L.K.A. Blæhr).
min D3 (25(OH)D3) which is the major circulating metabolite. In addition, the affinity of the antibodies was expected to be sufficiently high for analysis in the picogram scale. In this paper we report the synthesis of an EB1089 hapten (3, Fig. 1) and the characterization of polyclonal antibodies raised in three rabbits against the hapten conjugated to BSA. The hapten was designed in such a way that only key structural features of the compound of interest were included, namely the CD-ring and the side-chain. This was done in order to avoid possible side effects that could result from long-time immunization with large doses of conjugates of EB1089 itself. In addition, the number of synthesis steps was reduced. Despite the missing A-ring in the hapten, the antibodies raised toward the hapten were expected to cross react with EB1089 to a high degree. The antibodies were characterized with respect to titer, avidity, and specificity in an enzyme immunoassay (EIA). 2. Experimental 2.1. Hapten synthesis Tetrahydrofuran (THF) was dried by distillation under argon from sodium benzophenone ketyl immediately before
0039-128X/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 9 - 1 2 8 X ( 0 0 ) 0 0 2 2 5 - 7
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Fig. 1. Structures of EB1089 (1), 1␣,25-dihydroxyvitamin D3 (2), and the hapten derivative of EB1089 (3).
use. Other solvents (CH2Cl2, Et2O, and pyridine) were dried by storage over molecular sieves 4 Å. All reagent-grade chemicals were obtained from Aldrich Chemical Co. unless otherwise mentioned and were used as received. Watersensitive reactions were performed in oven-dried (130°C) or flame-dried glassware under argon. Thin layer chromatography was carried out on E. Merck precoated silica gel 60 F254 glass plates. Plates were visualized using UV radiation (254 nm) or with 4 N H2SO4 containing 1.5% cerium sulfate and 1.0% molybdic acid followed by heating to 150°C. Flash chromatography was performed according to the procedure of Still et al. [2] using E. Merck silica gel 60, 40 – 63 m. NMR spectra were obtained in CDCl3. 1D NMR spectra were recorded on a Bruker AC-300 at 300 MHz for 1H and 75 MHz for 13C. 2D NMR experiments were performed on a Bruker ARX500 (500 MHz). Chemical shifts are reported in parts per million downfield from internal tetramethylsilane. For compounds containing silicon, CHCl3 (7.25 ppm) was used as reference in 1H-NMR, and CDCl3 (76.81 ppm) was used as reference in 13C-NMR. High-resolution massspectra were recorded on a VG Autospec using electron impact ionization. All spectra and elemental analyses were recorded at the Department of Spectroscopy at Leo Pharmaceutical Products. Melting points were measured on a Bu¨chi 510 melting point apparatus and are uncorrected. 2.2. (22E,24E)-Des-A,B-26,27-dinor-8-[(tertbutyldimethylsilyl)oxy]-25-carbomethoxy-cholesta-22,24diene (5) Aldehyde 4 [3] (2.04 g, 6.29 mmol) and trimethyl 4-phosphonocrotonate (2.67 g, 11.31 mmol, Lancaster Syn-
thesis Gmbh, Mu¨hlheim am Main, Germany) were dissolved in 20 ml dry THF under argon and cooled to ⫺50°C. Lithium-bis(trimethylsilyl)amide (LHMDS, 1 M, 9.44 ml) was added from a syringe in such a way that the temperature stayed below ⫺45°C. After complete addition, the yellow mixture was stirred for 45 min at ⫺40 to ⫺60°C, warmed to ⫺10°C and quenched with water (6 ml). Additional ether (80 ml) and water (30 ml) were added. The aqueous phase was separated and extracted with ether (2 ⫻ 40 ml). The combined organic layers were washed with water (3 ⫻ 20 ml) and brine (2 ⫻ 10 ml), dried over MgSO4, and concentrated. The residue was purified by flash chromatography with EtOAc/pentanes (1:24) to give 2.43 g (95%) of the conjugated ester 5 as a white solid. The solid was recrystallized in CH2Cl2/methanol, mp 58.5– 60.5°C. 1H-NMR: ␦ -0.02 (3H, s), -0.01 (3H, s), 0.88 (9H, s), 0.92 (3H, C18-CH3, s), 1.03 (3H, C21-CH3, d, J ⬃ 6.6 Hz), 1.1–1.8 (11H, series of m), 1.92 (1H, m), 2.20 (1H, m), 3.72 (3H, COOMe, s), 3.98 (1H, H-8, m), 5.77 (1H, H-25, d, J ⬃ 15.3 Hz), 5.96 (1H, H-22, dd, J ⬃ 15.1, 8.3 Hz), 6.08 (1H, H-23, dd, J ⬃ 15.1, 10.4 Hz), 7.24 (1H, H-24, dd, J ⬃ 15.4, 10.3 Hz). 13 C-NMR: ␦ -5.2, -4.8, 14.0, 17.7, 18.0, 19.6, 23.0, 25.8, 27.4, 34.4, 40.0, 40.6, 42.4, 51.4, 52.9, 56.1, 69.3, 118.5, 125.9, 145.9, 151.0, 167.8. Anal. Calcd for C24H42O3Si: C, 70.88; H, 10.41. Found: C, 70.79: H, 10.42. 2.3. (22E, 24E)-Des-A,B-26,27-dinor-25-carbomethoxycholesta-22,24-dien-8-ol (6) The silyl ether 5 (1.92 g, 4.71 mmol) was suspended in 60 ml CH3CN, and hydrofluoric acid (40% aq., 2.5 ml) was added. A white solid precipitated, which disappeared after 45 min. After stirring at rt overnight the reaction mixture was concentrated under reduced pressure, neutralized with saturated NaHCO3, and extracted with ether (4 ⫻ 50 ml). The combined extracts were washed with brine and dried. Filtration and evaporation gave 6 in a quantitative yield as an oil, which crystallized in the freezer, mp 103.5–106.8°C. 1 H-NMR: ␦ 0.97 (3H, C18-CH3, s), 1.05 (3H, C21-CH3, d, J ⬃ 6.6 Hz), 1.1–2.05 (14H, series of m), 2.22 (1H, m), 3.73 (3H, COOMe, s), 4.08 (1H, H-8, m), 5.79 (1H, H-25, d, J ⬃ 15.4 Hz), 5.97 (1H, H-22, dd, J ⬃ 15.2, 8.5), 6.10 (1H, H-23, dd, J ⬃ 15.2, 10.7), 7.24 (1H, H-24, dd, J ⬃ 15.4, 10.6 Hz). 13C-NMR: ␦ 13.8, 17.4, 19.6, 22.5, 27.4, 33.6, 40.0, 40.3, 42.1, 51.4, 52.5, 55.9, 69.2, 118.7, 126.0, 145.8, 150.7, 167.8. Anal. Calcd for C18H28O3: C, 73.93; H, 9.65. Found: C, 73.69; H, 9.65. 2.4. (22E,24E)-Des-A,B-24-homo-26,27-dimethyl-cholesta22,24-dien-8,25-diol (7) [4] Lithium wire (0.76 g, 0.11 mmol, flattened and cut into small pieces) was placed in dry ether (30 ml) in a 3-necked flask equipped with a thermometer, argon inlet and septum. The mixture was cooled to ⫺20°C. Ethyl bromide (5.45 g, 0.05 mol) was measured in a syringe, and approximately
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one tenth was added to the lithium suspension. After 5 min, the suspension turned cloudy, and the remaining ethyl bromide was slowly added while maintaining the temperature between ⫺15 and ⫺20°C. The mixture was then stirred for 50 min at the same temperature. The hydroxy ester 6 (773 mg, 2.64 mmol) was dissolved in dry THF (50 ml) under argon, cooled to ⫺78°C and the previously prepared EtLi solution (7 ml, 1.2 M) was added. After stirring at ⫺78°C for 1 h, the mixture was quenched with water and allowed to warm up to ⫺10°C. After concentration under reduced pressure, the residue was diluted in ethyl acetate (50 ml) and washed with water (40 ml). The aqueous phase was extracted with EtOAc (2 ⫻ 20 ml), and the combined organic layers were washed with water and brine and dried with MgSO4. Filtration and concentration gave 909 mg of a white crystalline residue. Recrystallization from n-hexane gave 669 mg (78%) of 7 as a white solid, mp 106 –108°C (lit. 107–108°C) [4]. 1H-NMR: ␦ 0.86 (6H, C27a,27b-2CH3, t, J ⬃ 7.5 Hz), 0.96 (3H, C18-CH3, s), 1.02 (3H, C21-CH3, d, J ⬃ 6.7 Hz), 1.1–2.2 (19H, series of m), 4.08 (1H, H-8, m), 5.51 (1H, H-22, dd, J ⬃ 14.9, 8.4 Hz), 5.52 (1H, H-24a, d, J ⬃ 15.4 Hz), 5.96 (1H, H-23, dd, J ⬃ 15.0, 10.4 Hz), 6.15 (1H, H-24, dd, J ⬃ 15.3, 10.3 Hz). 13C-NMR: ␦ 7.9, 13.7, 17.3, 19.9, 22.4, 27.5, 32.9, 33.5, 39.5, 40.2, 41.8 (C-13), 52.5, 56.3, 69.2 (C-8), 75.3 (C-25), 127.3, 128.9, 136.1, 140.2. Anal. Calcd for C21H36O2: C, 78.70; H, 11.32. Found: C, 78.33; H, 11.25. HRMS: m/z 320.2727 (calcd. for C21H36O2, 320.2715). 2.5. (22E,24E)-Des-A,B-24-homo-26,27-dimethyl-25hydroxy-cholesta-22,24-dien-8-one (8) [4] Dess-Martin periodinane (0.872 g, 1.05 mmol, Lancaster Synthesis Gmbh, Mu¨hlheim am Main, Germany) and pyridine (0.65 ml) were suspended in dry CH2Cl2 (20 ml) under argon. A solution of alcohol 7 (513 mg, 1.60 mmol) in CH2Cl2 (10 ml) was added. The mixture was stirred at rt for 40 min. After cooling in an ice-bath, the mixture was diluted with ether (60 ml) and poured into an ice-cold 1:1 mixture of saturated NaHCO3 and 1 M Na2S2O3. The aqueous phase was extracted with ether (3 ⫻ 25 ml). The ether layers were successively washed with sat. NaHCO3, CuSO4 aq. (0.15 M), water and brine (each 50 ml), and dried over MgSO4. Filtration and concentration gave 590 mg crude product as an oil, which crystallized in the freezer. Recrystallization from ether-pentanes gave 460 mg (90%) of ketone 8 as a white solid, mp 83.5– 85.0°C. 1H-NMR: ␦ 0.66 (3H, C18CH3, s), 0.86 (6H, C27a,27b-2CH3, t, J ⬃ 7.5 Hz), 1.08 (3H, C21-CH3, d, J ⬃ 6.7 Hz), 1.25–2.35 (17H, series of m), 2.45 (1H, H-14, dd, J ⬃ 10.8, 7.4 Hz), 5.51 (1H, H-22, dd, J ⬃ 15.1, 8.5 Hz), 5.53 (1H, H-24a, d, J ⬃ 15.1 Hz), 5.98 (1H, H-23, dd, J ⬃ 15.0, 10.6 Hz), 6.16 (1H, H-24, dd, J ⬃ 15.2, 10.4 Hz). 13C-NMR: ␦ 7.9, 12.7, 19.0, 20.3, 24.0, 27.6, 33.0, 38.8, 39.6, 40.9, 49.8, 56.4, 61.9, 75.4, 127.9, 128.6, 136.2, 139.2, 211.8. Anal. Calcd for C21H34O2: C, 79.19; H, 10,76.
541
Found: C, 78.41; H, 10.55. HRMS: m/z 318.2569 (calcd. for C21H34O2, 318.2559). 2.6. (22E,24E)-Des-A,B-24-homo-26,27-dimethyl-8-[(E)ethoxycarbonyl-methylidene]-cholesta-22,24-dien-25-ol (9) A suspension of NaH (55– 65% suspension in oil, 250 mg) in dry THF (10 ml) was cooled in an ice-bath under argon, and triethyl phosphonoacetate (1.76 g, 7.85 mmol) was slowly added from a syringe. After complete addition, the mixture was stirred at rt for 40 min, then placed in an ice-bath again. After 10 min, a solution of ketone 8 (250 mg, 0.785 mmol) in THF (6 ml) was added in one portion. The mixture was stirred overnight at 28°C. The reaction mixture was cooled in an ice-bath and quenched with water (25 ml), extracted with ether, and dried over Na2SO4. After filtration, the solvent was removed under reduced pressure. Flash chromatography of the crude material using EtOAc/pentanes (1:4) afforded 228 mg (76%) of unsaturated ester 9. 1 H-NMR: ␦ 0.60 (3H, C18-CH3, s), 0.88 (6H, C27a,27b-2CH3, t), 1.06 (3H, C21-CH3, d, J ⬃ 6.6 Hz), 1.2–2.2 (17H, series of m), 1.28 (3H, Et, t), 3.88 (1H, H-9, m), 4.14 (2H, CH2OCO, q), 5.45 (1H, H-7, s), 5.52 (1H, H-24a, d, J ⬃ 15.3 Hz), 5.53 (1H, H-22, dd, J ⬃ 15.0, 8.4 Hz), 5.98 (1H, H-23, dd, J ⬃ 15.0 Hz, 10.4 Hz), 6.14 (1H, H-24, dd, J ⬃ 15.3, 10.3 Hz). HRMS: m/z 388.2988 (calcd. for C25H40O3, 388.2977). 2.7. (22E,24E)-Des-A,B-24-homo-26,27-dimethyl-8-[(E)carboxy-methylidene]-cholesta-22,24-dien-25-ol (10) The unsaturated ester 9 (228 mg, 0.59 mmol) was treated with KOH in methanol (1.5 M, 8 ml) and stirred for 3 days at rt. After cooling in an ice-bath, the pH was carefully adjusted to 5 with diluted HCl, and the solution was quickly extracted with cold ethyl acetate. The extracts were washed with water and brine and dried over MgSO4. After filtration and concentration, the crude product was purified by flash chromatography with EtOAc/pentanes (11:9) yielding 116 mg (55%) of acid 10. 1H-NMR: ␦ 0.61 (3H, C18-CH3, s), 0.86 (6H, C27a,27b-2CH3, t, J ⬃ 7.5 Hz), 1.06 (3H, C21-CH3, d, J ⬃ 6.6 Hz), 1.2–2.3 (18H, series of m), 3.85 (1H, H-9, m), 5.49 (1H, H-8, bs), 5.52 (1H, H-22, d, J ⬃ 14.7, 8.5 Hz), 5.53 (1H, H-24a, d, J ⬃ 15.1 Hz), 5.98 (1H, H-23, dd, J ⬃ 14.7, 10.3 Hz), 6.15 (1H, H-24, dd, J ⬃ 15.1, 10.3 Hz). 2.8. (22E,24E)-Des-A,B-24-homo-26,27-dimethyl-8-[(E)N-(2-(ethoxycarbonyl)-ethyl)-carbamoylmethylidene]cholesta-22,24-dien-25-ol (11) A solution of crude carboxylic acid 10 (70 mg, 0.194 mmol), -alanine ethyl ester hydrochloride (30 mg, 0.194 mmol) and Et3N (22 mg) in dry CH2Cl2 (4 ml) was cooled in an ice-bath and DMAP (2 mg) was added. The solution was stirred for 5 min upon which solid N,N⬘-dicyclohexylcarbodiimide (DCC, 44 mg, 0.213 mmol) was added. Stir-
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ring was continued overnight in the melting ice-bath. Dicyclohexylurea was removed by filtration. The filtrate was concentrated and redissolved in ethyl acetate, washed with saturated NaHCO3, water and brine, dried over Na2SO4, and concentrated. The crude material was purified by flash chromatography with EtOAc/pentanes (7:13) giving 52 mg (58%) of amido-ester 11 as an oil. 1H-NMR: ␦ 0.59 (3H, C18-CH3, s), 0.87 (6H, C26a-CH3, t), 1.05 (3H, C21-CH3, d), 1.26 (3H, COOCH2CH3, t), 1.2–2.2 (17H, series of m), 2.56 (2H, CH2COO, t), 3.55 (2H, CH2NH, dt), 3.82 (1H, H-9, m), 4.13 (2H, COOCH2CH3, q), 5.30 (1H, H-7, s), 5.51 (2H, H-22 and H-24a, m), 5.96 (1H, H-23, dd), 6.05 (1H, N-H, t), 6.16 (1H, H-24, dd). 13C-NMR: ␦ 7.6, 12.1, 13.9, 20.1, 21.9, 23.4, 27.2, 29.1, 32.8, 33.9, 34.2, 39.8, 46.4, 56.2, 60.4, 75.2, 114.4, 127.5, 128.6, 136.3, 139.6, 157.3, 166.7 (CONH), 172.6 (COOEt). HRMS: m/z 459.3360 (calcd. for C28H45NO4, 459.3349). 2.9. (22E,24E)-Des-A,B-24-homo-26,27-dimethyl-8-[(E)N-(2-carboxyethyl)-carbamoylmethylidene]-cholesta22,24-dien-25-ol (3) Aqueous NaOH (2N, 61 l) was added to a solution of amido-ester 11 (50 mg, 0.11 mmol) in dioxane-water 9:1 (1.5 ml), and the resulting mixture was stirred at rt for 3 h. Phosphate buffer (pH 7.4) was added, and the aqueous solution was extracted with ethyl acetate until the extracts no longer contained any carboxylic acid (checked on TLC plate/UV detection). The combined organic layers were washed with brine, dried over Na2SO4 and filtered. The filtrate was concentrated to give 49 mg of 3 as an oil in a quantitative yield, sufficiently pure for analysis and conjugation to BSA. 1H-NMR: ␦ 0.59 (3H, C18-CH3, s), 0.86 (6H, C26a-CH3, t), 1.05 (3H, C21-CH3, d), 1.1–1.8 (17H, series of m), 2.60 (2H, CH2COOH, t), 3.55 (2H, CH2NH, m), 3.78 (1H, H-9␣, m), 5.31 (1H, H-7, s), 5.53 (2H, H-24a, d and H-22, dd), 5.96 (1H, H-23, dd), 6.14 (2H, H-24 and N-H, m). 13C-NMR: ␦ 7.9, 12.3, 20.4, 22.2, 23.7, 27.5, 29.5, 33.0, 33.7, 34.3, 34.7, 40.1, 46.7, 56.5, 56.5, 75.6 (C-25), 114.4, 127.7, 128.9, 136.5, 139.9, 158.2, 167.6 (CONH), 176.4 (COOH). UV (99% ethanol): max 232 nm (⑀ 41 500). HRMS: m/z 413.2951 (M-H2O) (calcd. for C26H39NO3 (MH2O), 413.2930). 2.10. (1R,3S,5Z,7E,22E,24E)-9,10-secocholesta5,7,10(19),22,24-pentaen-1,25-diol-24-homo-26,27dimethyl-3-yl-(2-trimethylsilyl-ethyl)-succinate (12) A mixture of EB1089 [1] (1, 105 mg, 0.222 mmol), 2-(trimethylsilyl)-ethyl hydrogen succinate [5] (58 mg, 0.266 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 47 mg, 0.244 mmol) and DMAP (2 mg) in dry THF was stirred under an argon atmosphere for 2 h at rt. After quenching with water, the solution was diluted with ethyl acetate, washed with water and brine and dried over MgSO4. After filtration and con-
centration, the residue was purified by flash chromatography (5–50% EtOAc/pentanes). Four compounds were isolated of which the third most polar compound (Rf 0.71 in EtOAc/pentanes 1:1) was the desired mono-ester 12. 1HNMR: ␦ 0.01 (9H, s), 0.54 (3H, s), 0.84 (6H, t), 0.95 (1H, m), 1.03 (3H, d), 1.2–2.1 (21H, series of m), 2.38 (1H, dd), 2.57 (5H, m), 2.80 (1H, m), 4.1– 4.2 (2H, m), 4.38 (1H, m), 4.98 (1H, s), 5.21 (1H, m), 5.32 (1H, s), 5.50 (2H, d and dd), 5.97 (2H, m), 6.13 (1H, dd), 6.31 (1H, d). 2.11. (1R,3S,5Z,7E,22E,24E)-9,10-secocholesta5,7,10(19),22,24-pentaen-1,25-diol-24-homo-26,27dimethyl-3-yl-hydrogen succinate (13) Tetrabutylammonium fluoride (TBAF, 50 mg, 0.157 mmol) was added to a solution of trimethylsilylethyl ester 12 (35 mg, 0.052 mmol) in dry THF (1.5 ml) under an argon atmosphere. The resulting mixture was stirred at rt for 3 h, diluted with ethyl acetate and washed with water and brine. After drying and removal of the solvent, the residue was purified by flash chromatography (MeOH/CHCl3) to give 28 mg (97%) of hemisuccinate 13. 1H-NMR: ␦ 0.56 (3H, s), 0.86 (6H, t), 1.05 (3H, d), 1.2–2.2 (20H, series of m), 2.40 (1H, dd), 2.60 (5H, m), 2.82 (1H, m), 4.38 (1H, m), 5.01 (1H, m), 5.24 (1H, m), 5.35 (1H, m), 5.54 (2H, d and dd), 5.96 (1H, dd), 6.02 (1H, d), 6.15 (1H, dd), 6.32 (1H, d). 2.12. Preparation of NHS-EB1089 succinate (for EIA) A solution of EB1089 3-O-hemisuccinate 13 (28 mg, 0.05 mmol) in dry THF (1 ml) was treated with N-hydroxysuccinimide (12 mg, 0.10 mmol), EDC (20 mg, 0.10 mmol), and a catalytic amount of DMAP. The reaction was stirred overnight at 4°C and extracted with ethyl acetate and water. The organic layer was washed with brine, dried, and the solvent was removed. The crude product was passed through a short column of silica gel, affording 14 mg of the NHS-ester. A stock solution in dry DMSO was immediately prepared, which was stored at ⫺18°C. 2.13. BSA conjugate formation The hapten 3 was conjugated to bovine serum albumin (BSA) via an N-succinimidyl ester according to a standard procedure [6]. N-hydroxysuccinimide (16 mg, 0.14 mmol) and EDC (27 mg, 0.14 mmol) were added to a solution of 3 (15 mg, 35 mol) in 95% dioxane aq. (0.2 ml). The mixture was stirred at room temperature for 3 h, diluted with water, and extracted with ethyl acetate. The combined extracts were washed with water, dried, and concentrated under reduced pressure. The crude NHS ester was dissolved in pyridine (0.75 ml), and a solution of BSA (25 mg, crystallized, Sigma) in phosphate buffer (0.05 M, 0.75 ml) was added dropwise. After stirring overnight at 4°C, the suspension was dialyzed against PBS (pH 7.0) and cold water (each for 2 ⫻ 12 h) and freezedried, affording 32 mg of
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conjugate. UV (Tris buffer pH 8.4): max 226. (A232 ⫽ 1.2 at a concentration of 0.05 g/l). 2.14. Antibody production [7] The BSA conjugate (6 mg) was suspended in 1.5 ml of an aqueous solution of NaCl (0.1 M) and NaN3 (15 mM). The suspension was added dropwise to Freund’s incomplete adjuvant (1.5 ml) under vigorous mixing. Three rabbits (Danish Whites), 3– 6 months old, were immunized subcutaneously (s.c.) in the neck region with 100 l of the immunogen-adjuvant emulsion (2 g/l). This procedure was repeated every two weeks for 2 months, then once every month. After 12 weeks following the first immunization, blood samples were collected from an ear vein. Blood samples were subsequently collected every two weeks.
Fig. 2. Reagents, conditions and yields: a) trimethyl 4-phosphonocrotonate, LHMDS, THF, ⫺78°C; 95%; b) 2.5% HF, CH3CN-H2O; 100%; c) EtLi (2 eq.), THF; 78%.
2.15. EIA procedure (standard) [8] CovaLink™ and MicroWell™ polypropylene microtiter plates were purchased from Nunc (Roskilde, Denmark). Swine anti-rabbit horseradish peroxidase and TMB one-step substrate system were purchased from DAKO (Glostrup, Denmark). Metabolites of vitamin D and of EB1089 used for cross-reaction studies were synthesized at Leo Pharmaceutical Products, except for 24R,25-dihydroxyvitamin D3, which was obtained from Solvay Duphar B.V. (Weesp, The Netherlands). The buffers used were phosphate-buffered saline (PBS) (0.15 M NaCl, 8.5 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, pH 7.0), coating buffer (PBS with 40 M chloramine T), washing buffer (PBS with 0.1% Tween 20, pH 7.0), and assay buffer (PBS with 1% casein, pH 7.2). Plates were washed 4 times between incubations. All incubations were run for 1 h at room temperature on shakers unless otherwise stated. OD was measured on a SLT Rainbow Scanner (SLT Labinstruments) at 450 nm. All determinations were performed in duplicate. CovaLink plates were incubated overnight at 4°C with 100 l NHS-EB1089 succinate (0.40 g/ml in coating buffer, diluted from a stock solution in DMSO). After washing, wells were blocked with 200 l assay buffer. Antiserum was diluted in assay buffer and 100 l were applied as primary antibody in each well, and the plates were incubated and washed. The secondary antibody (swine antirabbit horseradish peroxidase) was diluted 1:4000 in assay buffer and 100 l were applied per well. After washing, 100 l of TMB one-step substrate were added per well, and the plates were incubated for 30 min in the dark. The reaction was then stopped with 2N H2SO4 (100 l/well), and the absorbance was measured at 450 nm. 2.15.1. Chaotropic ion elution EIA The standard EIA procedure was followed using an antiserum dilution of 1:1000, except that the following step was inserted between the primary and the secondary anti-
body incubations: A serial dilution of NH4SCN in PBS (100 l/well, concentrations 0 – 0.062– 0.125– 0.250 – 0.50 –1.0 – 2.0 – 4.0 M) was applied, and the plate was incubated for 20 min, followed by washing. 2.15.2. Cross-reaction study Stock solutions of EB1089, EB1089 metabolites and selected vitamin D metabolites in ethanol were prepared, and the exact concentrations were determined from the UV absorbance ( 264 nm, ⑀ 17 500 for vitamin D metabolites and 232 nm, ⑀ 44 000 for EB1089 and related metabolites). Aliquots (500 –10 000 pmol depending on the metabolite) were applied in the first column of a MicroWell polypropylene plate, and the solvent was allowed to evaporate. Acetonitrile (20 l) was then added to each well in the column, followed by assay buffer (180 l). In the remaining wells of the plate, 100 l assay buffer containing 10% acetonitrile was applied. Serial dilutions of each metabolite were then made row-wise by transferring 100 l from column to column with mixing. From the last column, 100 l were discarded. The primary antibody was diluted 1:4000 in assay buffer, and 100 l were added to each well. The plate was incubated at 4°C overnight. The standard EIA procedure was used, except that the preincubated antibody was applied as primary antibody (100 l/well) by transfer from the MicroWell plate.
3. Results The target hapten 3 (Fig. 1) was synthesized from the known aldehyde 4 [3]. Assembly of the side-chain from the aldehyde is outlined in Fig. 2 and is similar to the reported procedure for preparing EB1089 [1]. The aldehyde was converted to the unsaturated ester 5 by Horner-Emmons olefination with trimethyl 4-phosphonocrotonate. Only the
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Fig. 4. Reagents, conditions, and yields: a) (CH3)3Si(CH2)2OOC(CH2)2 COOH, EDC, DMAP, THF; 24% b) TBAF䡠3H2O, THF; 100%.
Fig. 3. Reagents, conditions, and yields: a) Dess-Martin periodinane, CH2Cl2; 90%; b) Triethyl phosphonoacetate, NaH, THF; 76%; c) KOH, MeOH; 55%; d) H2N(CH2)3COOEt䡠HCl, DCC, Et3N, CH2Cl2; 58%; e) NaOH, dioxane/H2O; 100%.
trans-trans product was isolated, which was indicated by large coupling constants of the vinylic hydrogens in 1HNMR. The exclusive formation of the trans alkene geometry is characteristic when using stabilized phosphonate anion reagents. Before completing the side-chain assembly by formation of a terminal allylic alcohol by addition of 2 equivalents ethyl lithium to the ester group, the TBS-group on the 8-hydroxyl was removed. Deprotection of the 8-TBS was not possible with TBAF using standard conditions and required more rigorous conditions using hydrofluoric acid. Due to the inherent lability of an allylic alcohol to acid conditions, the formation of this alcohol was deferred until after the deprotection. It turned out that the addition of ethyl lithium to the ester proceeded smoothly and in high yield even in the presence of the unprotected hydroxyl group. A small amount of ethyl-adduct resulting from 1,4-addition was also formed in this step. Following the side-chain assembly, a spacer was attached at C-8 in the C-ring (Fig. 3). Compound 7 was oxidized to 8 with Dess-Martin periodinane. Horner-Emmons olefination with excess triethyl phosphonoacetate and NaH giving rise to 9 was achieved in moderate yield without protection of the terminal tertiary alcohol. The reaction temperature was critical; the optimal temperature was found to be 25–30°C. Lower temperatures prolonged the reaction time considerably and higher temperatures resulted in the formation of the C-14 epimer as a byproduct. The configuration of the newly formed double bond in 9 was established by ROESY. A NOE was observed between H-7 and H-15, which indicated that the double bond had the E-configuration as drawn. The ester 9 was hydrolyzed with base to the carboxylic acid 10. The reaction mixture was acidified to pH 5.5, and the product was obtained by multiple extractions of the
aqueous phase. Acidifying the mixture below pH 5.5 resulted in allylic rearrangement of the side-chain. Despite the fact that the carboxylic acid was expected to be deprotonated at pH 5.5, it was nevertheless possible to extract the compound from the aqueous phase, probably due to the highly lipophilic character of the compound. Compound 10 was converted to the amido-ester 11 by carbodiimide coupling with -alanine ethyl ester. Selective hydrolysis of the ethyl ester with NaOH in dioxane-water afforded the hapten 3 after extraction, as described above. 3.1. Preparation of N-hydroxysuccinimide esters of 3 and of EB1089-hemisuccinate Immunizations were carried out with a BSA-conjugate of hapten 3. The conjugation procedure involved conversion of the hapten to its N-hydroxysuccinimide (NHS) ester and subsequent reaction with BSA. The NHS-ester of 3 was prepared by treating 3 with N-hydroxysuccinimide and EDC [6]. The hapten/BSA ratio was estimated to be 41 based on the ultraviolet absorbance at 232 nm by subtraction of the contribution of BSA. To characterize the antibodies, we used an ELISA with immobilized EB1089 in CovaLink microtiter plates. We used EB1089 and not hapten 3 in the assay because EB1089 was the actual target of interest. For the purpose of immobilizing EB1089, we prepared EB1089 3-hemisuccinate (13, Fig. 4). Carbodiimide coupling of EB1089 and 2-(trimethylsilyl)-ethyl succinate [5] afforded a separable mixture of 1- and 3-monoesters as well as diester and unreacted EB1089. The 3-ester 12 was treated with TBAF in THF to afford the 3-O-hemisuccinate of EB1089. The NHS-ester was finally prepared as described for the hapten 3. 3.2. Determination of titer Antisera from three rabbits (As-1, As-2 and As-3) were obtained by immunizations with the prepared BSA-conjugate of 3. The first blood samples were obtained after 12 weeks of immunization. Antibody dilution curves were de-
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Fig. 7. Chaotropic ion elution of As-1 at various dilutions. Fig. 5. Antibody dilution curves, weeks 12 and 26. The diagram shows the resulting reaction of antisera with covalently immobilized EB1089 hemisuccinate.
termined by measuring the binding of a series of concentrations of antiserum to covalently bound EB1089 hemisuccinate in Covalink microtiter plates (Fig. 5). Bound antibody was detected with a swine anti-rabbit peroxidase labeled secondary antibody. The titer of all three antisera had increased 3–5 fold during the three-month period, as measured by the lateral displacement of the absorption curves. 3.3. Avidity measurement Measurement of avidity was performed using chaotropic ion elution [9 –11]. A chaotropic ion (thiocyanate) was added in serial dilutions to the bound antibody in microtiter plates. The chaotropic ion decreases hydrophobic interactions, resulting in a dissociation of the complex. The avidity index was defined as the concentration of thiocyanate at which the maximal binding to immobilized EB1089 was reduced to 50%. Fig. 6 shows the development of the avidity index as a function of time after immunization for the three antisera. It
Fig. 6. Avidity index vs. time post immunization of rabbit antisera (error bars indicate the standard deviation).
should be noted that there was some difficulty in reproducing the experiment, especially because the assay seemed to be sensitive to small variations in the thiocyanate elution step. Apparently the avidity increased as a function of time for all antisera. As-1 and As-3 exhibited a similar, modest increase, while antiserum As-2 showed the most marked increase in avidity. In order to ensure that the observed increase in avidity was not merely an effect of the increase in titer, the avidity was measured for a single antibody at various dilutions. Antiserum As-1 (week 20) was diluted 1:1000, 1:2000, and 1:4000 and tested in the thiocyanate elution assay. Fig. 7 shows the result, which clearly indicates that the avidity index was indeed independent of concentration. The data point for 1:4000 dilution at 4 M thiocyanate concentration may be erratic, because the measured absorbance was equal to the background absorbance for the assay. 3.4. Cross-reactivity In order to assess the specificity of the antisera, the cross-reactivities with various vitamin D metabolites were measured. The most common vitamin D metabolites were selected for the cross-reaction study. These were 1,25(OH)2D3, 25-hydroxyvitamin D3, 24R,25-dihydroxyvitamin D3, vitamin D2 and vitamin D3. Four metabolites of EB1089 itself have been identified [12], and cross-reactivities with these compounds were also examined. Structures of the metabolites of EB1089 are shown in Fig. 8. The metabolite EB1446 has been identified as the major metabolite of EB1089. Antisera from week 26 diluted at a concentration of 1:4000 were preincubated with serial dilutions of metabolite overnight, and the resulting reaction to immobilized EB1089 in Covalink plates was determined. The IC50 value was measured as the concentration of metabolite that was necessary to reduce the maximal binding to 50%. The crossreactivity with a metabolite was then calculated as the
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Fig. 8. Identified metabolites of EB1089.
percentage of the IC50 value of EB1089 to that of the metabolite. The results are listed in Table 1. The specificity profiles of the three antibodies are similar. The antibodies all discriminated EB1089 from endogenous metabolites of vitamin D. In particular, the crossreactivity with 25-hydroxyvitamin D3 was 0.01% for the antibody As-3, which is excellent, taking into account the large amounts of this metabolite in serum (up to 1 ng/ml in humans). The cross-reactivity with the active metabolite 1,25(OH)2D3 was generally slightly higher, but this form is present in equimolar amounts in serum compared to the expected serum levels of EB1089. Cross-reactivities with vitamin D2 and vitamin D3 were negligible for the three antibodies. All three antisera showed relatively high cross-reactivities with the four metabolites of EB1089. Thus, additional hydroxylation of the side chain of EB1089 did not affect the binding to the antibodies significantly.
4. Discussion The analysis of EB1089 and other vitamin D related compounds in serum represents a challenge due to the low serum concentrations, which usually are in the picomolar range for biologically active compounds. Furthermore, the Table 1 Cross-reactivities of three antisera to naturally occurring vitamin D metabolites and to metabolites of EB1089. The cross-reactivity is defined as the percentage of the IC50 value of EB1089 to that of the metabolite Compound
EB1089 1,25(OH)2D3 25(OH)D3 24R,25(OH)2D3 Vitamin D2 Vitamin D3 EB1446 EB1436 EB1445 EB1470
Antibody (Week 26) As-1
As-2
As-3
100 0.3 0.09 0.04 ⬍0.01 ⬍0.01 26 16 26 26
100 0.78 0.16 0.07 ⬍0.01 ⬍0.01 35 28 78 70
100 0.1 ⬍0.01 0.07 ⬍0.01 ⬍0.01 40 29 44 27
presence of closely related, endogenous metabolites in serum interfere. The analysis therefore requires a specific and sensitive method. Recently, a mass spectrometric method has been developed for the quantification of EB1089 in serum samples [13]. However, this method requires preliminary extraction and isolation of EB1089 prior to analysis. We are aiming at developing an immunoassay for EB1089 with specific antibodies that will allow direct measurement in serum, and for this purpose we raised polyclonal antibodies to EB1089. For characterizing the antibodies, we used an enzyme immunoassay that involved covalent binding of EB1089 in special microtiter plates, a method that has been recently described for 25-hydroxyvitamin D3 [8]. Antibodies to vitamin D derivatives have conventionally been raised against their BSA-conjugates, in which the derivative has been coupled to BSA through a spacer [14, 15]. However, we have previously experienced that such antigens in some cases are toxic during long-time immunization, probably due to in vivo hydrolysis and liberation of the hapten. Although EB1089 is a low-calcemic analog of 1,25(OH)2D3, the amounts of EB1089 that potentially can be liberated from immunizing doses of a BSA conjugate are large compared to therapeutic doses. We therefore pursued a different strategy. EB1089 possesses a side-chain that is not found in any natural metabolite of vitamin D or in steroids. It was believed that a hapten containing only the CD-ring and side-chain of EB1089 (Fig. 1) would be sufficient to raise antibodies to the analog that discern EB1089 from closely related endogenous vitamin D metabolites. The absence of the A-ring would render the hapten noncalcemic while preserving the unique structural features of EB1089. In the hapten 3, the A-ring of EB1089 was replaced by a carboxylic acid spacer for carrier conjugation. By introducing an exocyclic double bond at C-8, it was expected that the conformation of the CD-ring system should be similar to that of the original compound. The present study showed that antibodies raised against this ‘truncated’ form of EB1089 recognized the analog as a whole, all the more with a good specificity. All antibodies exhibited an increase in titer toward EB1089 during the immunization period. Although commonly encountered in the literature, affinity constants for polyclonal antibodies are somewhat difficult to measure experimentally. Equilibrium mixtures of polyclonal antibody and antigen are complex, and any observed affinity represents an average of all individual antibody-antigen equilibria. It is therefore more correct to use the less specific term avidity for polyclonal sera. We used chaotropic ion elution as a means to determine relative avidities of the produced antisera. Chaotropic ions disrupt the structure of water and decrease the surface tension. This in turn decreases hydrophobic interactions that are expected to dominate in the binding between the antibodies and EB1089, and this results in a dissociation of the complex. It must be noted that the chaotropic elution method is somewhat controversial, because chaotropic ions denature the
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antibodies and affect intermolecular interactions in addition to affecting the binding to ligands. Nevertheless, it was shown that avidity was independent of antibody concentration (Fig. 7), indicating that the measured parameter is an intrinsic quality of the antibodies. Also, it was generally observed that avidity increased in time (Fig. 6), reflecting a change in the distribution of immunoglobulin characterized by a higher proportion of high-affinity antibodies. The cross-reaction study was performed with the naturally occurring metabolites of vitamin D3 and with metabolites of EB1089 (Table 1). After 26 weeks, the antibodies showed low cross-reactivities with the naturally occurring vitamin D metabolites. It is somewhat surprising that there was a difference in the cross-reactivity with 1,25(OH)2D3 and 25(OH)D3 (0.1% and 0.01%, respectively, for As-3). These two metabolites only differ in the A-ring. Yet the antibodies were raised against a hapten with no A-ring and are therefore not expected to recognize this moiety. It therefore appears that the presence of the 1␣ hydroxyl group does make a difference, perhaps due to random hydrogen bonding. It is fortunate that the selectivity was in favor of 1,25(OH)2D3 due to the much lower concentrations of this metabolite in serum compared to 25(OH)D3. The cross-reactivity of the antibody As-3 with 25(OH)D3 was satisfactorily low (0.01%) to avoid interfering binding of this metabolite, which is present in serum in up to 1000 times the expected concentration of EB1089. Also 24R,25(OH)2D3, a metabolite that also occurs in relatively large concentrations in serum, was easily discriminated (0.04 – 0.07%). A much higher cross reactivity was observed with the metabolites of EB1089 (16 –78%). This indicates that the diene is an important epitope, which is recognized by the antibodies. The additional hydroxyl group in the terminal end of the side-chain of the metabolites reduced binding by only one fifth in the best case compared to EB1089. Similar reactivity patterns have previously been reported, where antibodies to vitamin D analogs fail to recognize slight changes in the side chain [14]. This relatively high crossreactivity could cause interference in the determination of serum levels of EB1089, but the concentrations of these metabolites are expected to be low compared to that of EB1089. One might speculate whether a better specificity could have been obtained by immunizing with BSA-conjugates of non-truncated EB1089 instead of 3. We do not believe that this would improve the selectivity. The presence of additional epitopes created by the A-ring would presumably increase the binding affinity of the antibodies to EB1089, but the affinity to EB1089 metabolites would also increase, because the same A-ring is present in these compounds. One might even obtain less selective antibodies because the unique side-chains would provide a relatively smaller contribution to the total binding between the antibody and metabolite. The sensitivity of the employed immunoassay was not
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optimized for measuring serum levels of EB1089. Phase I clinical studies have suggested therapeutic doses up to 20 g/day per patient [1], but the majority of the drug is accumulated in the liver. Circulating levels of the drug are therefore estimated to be in the region of 10 –100 pg/ml serum. The sensitivity of this assay needs to be improved 10 –100 fold in order to detect these levels. There is ample room for improvement in the assay, especially with respect to the secondary antibody, which may be replaced by biotinstreptavidin or enzyme-cascade amplified visualization. Alternatively, a radioimmunoassay may be developed, requiring radioisotope labeled EB1089. In conclusion, we successfully prepared antibodies specific to EB1089 by immunization of rabbits with a truncated form of the molecule. The antibodies showed a good selectivity for EB1089 over endogenous vitamin D metabolites, enough to allow direct measurement in serum of the analog. The further development of an immunoassay with these antibodies could therefore obviate the need for preliminary isolation of EB1089, which is a requirement in existing methods.
Acknowledgments This work was partly financed by the Danish Academy of Technical Sciences. The authors wish to thank Sven Erik Godtfredsen at DAKO A/S for facilitating the production of antisera and Grethe Aagaard and staff at the Department of Spectroscopy at Leo for the recording and interpretation of spectra.
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