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Drug Metabolism and Disposition Fast Forward
First published on November 18, 2005; DOI: 10.1124/dmd.105.007294

0090-9556/06/3402-324-330$20.00
DMD 34:324-330, 2006

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DEVELOPMENT OF NOVEL FURANOCOUMARIN DIMERS AS POTENT AND SELECTIVE INHIBITORS OF CYP3A4

E. Row1, S. A. Brown1, A. V. Stachulski1, and M. S. Lennard

Academic Unit of Clinical Pharmacology, University of Sheffield, Royal Hallamshire Hospital, Sheffield (E.R., M.S.L.); and SAFC Pharma (formerly Ultrafine), Synergy House, Manchester Science Park, Manchester (S.A.B., A.V.S.), United Kingdom

(Received September 9, 2005; accepted November 15, 2005)


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Grapefruit juice has been found to cause an increase in the oral bioavailability of many therapeutic agents. Such interactions are believed to result from the mechanism-based inhibition of CYP3A4 activity in the intestine. Furanocoumarin dimers present in the juice have been found to be extremely potent inhibitors of CYP3A4 activity. The aim of this work was to synthesize and test a series of dimers with a view to defining the relationship between structure and inhibitory activity and establish whether they might make suitable probes of CYP3A4 activity. Eleven furanocoumarin dimers were synthesized and evaluated as inhibitors of CYP3A4 using human liver microsomes, with testosterone as the marker substrate. Four of the most potent dimers were also investigated for their effects on CYP3A4 activity in the human intestine and on five additional hepatic cytochrome P450 isoforms. The dimers showed potent dose-dependent inhibition of CYP3A4 activity in both liver and intestine; IC50 values ranged from 0.021 ± 0.002 to 0.146 ± 0.041 µM (mean ± S.D. n = 3). Of the four dimers evaluated further, all showed time-dependent inhibition of CYP3A4 activity. 88Prop showed moderate inhibition of both CYP2C19 and CYP1A2 with IC50 values of 4.42 ± 0.01 and 1.98 ± 0.34 µM, 88Octa was found to inhibit CYP2C19 (IC50 = 3.16 ± 0.01 µM) and 58Prop to inhibit CYP1A2 (IC50 = 2.39 ± 0.77 µM). Minimal inhibition of CYP2D6 and CYP2C9 was observed (IC50 > 10 µM). In conclusion, all the dimers tested were extremely potent inhibitors of CYP3A4 activity. In particular, dimer 55EE was highly selective toward the enzyme, suggesting that this compound is a suitable probe for determining the contribution of CYP3A4 to drug metabolism.

There has been substantial interest in the inhibition of CYP3A4 activity by the constituents of grapefruit juice. This effect was first recognized by Bailey et al. (1989Go) from a chance finding in a felodipine-ethanol interaction study in which grapefruit juice was used to mask the taste of the alcohol. Elevated plasma felodipine concentrations were observed both in the alcohol and control phases of the study, and in a later study, grapefruit juice was found to triple the mean area under the plasma concentration-time curve through inhibition of CYP3A4 (Bailey et al., 2004Go). Subsequently, a large number of studies have been performed on the effects of grapefruit juice on a range of drugs of therapeutic importance, such as terfenadine (Benton et al., 1996Go; Rau et al., 1997Go), felodipine (Bailey et al., 2000Go; Dresser et al., 2000Go; Goosen et al., 2004Go), nifedipine (Mohri et al., 2000Go), midazolam (Kupferschmidt et al., 1995Go), diazepam (Ozdemir et al., 1998Go), and cyclosporine (Yee et al., 1995Go). The constituents of grapefruit juice that cause inhibition of CYP3A4 have not been conclusively identified. Strong candidates are the furanocoumarins, particularly the bergamottin series (Ohnishi et al., 2000Go; Kakar et al., 2004Go). However, there is no clear evidence that any one compound is primarily responsible, and it is possible that the interaction arises from the accumulative effect of a number of constituents present in the juice. Of the compounds isolated, three dimers, namely, GF-I-1, GF-I-4, and GF-I-6 (Fig. 1), possibly derived from the geranyloxy monomer (6',7'-dihydroxybergamottin), were the most potent inhibitors of CYP3A4 activity (Fukuda et al., 1997Go; Guo et al., 2000aGo; Tassaneeyakul et al., 2000Go). Potencies were found to exceed that of ketoconazole and were shown to be 1 to 2 orders of magnitude higher than those observed for the monomers bergamottin, 6',7'-epoxybergamottin, and 6',7'-dihydroxybergamottin, when using testosterone as the marker substrate (Guo et al., 2000bGo).


Figure 1
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  		FIG. 1. Furanocoumarin dimers isolated from grapefruit juice (Fukuda et al., 2000Go; Guo et al., 2000aGo).

 
Inhibition of CYP3A4 activity by grapefruit juice is believed to be the result of mechanism-based inactivation (Schmiedlin-Ren et al., 1997Go; He et al., 1998Go), indicating that enzyme activity is lost by a time-dependent degradation of the enzyme. It is believed that the interaction of CYP3A4 with furanocoumarins occurs at the unsaturated furan ring. There is initial formation of an epoxide, which then undergoes ring-opening (by hydrolysis or attack from an internal nucleophile) to form a vic-adduct, which is able to covalently bind to the apoprotein (He et al., 1998Go) of the enzyme, thus irreversibly inactivating it. This mechanism may be similar to the scheme proposed for the inactivation of CYP2B1 and CYP2A6 by furanocoumarins (Koenigs and Trager, 1998Go).

The naturally occurring dimers in grapefruit juice that inhibit CYP3A4 are those substituted at the 5-position and are derivatives of bergamottin. Position 8-substituted dimers, based on 8-geranyloxypsoralen, which is a much more potent CYP3A4 inhibitor than bergamottin (Guo et al., 2000bGo), have not been tested. In the present work a series of dimers were synthesized with differing functional groups at either the 5- or the 8-position on the psoralen (furanocoumarin) ring system, as well as some 5/8-mixed dimers. A number of dimers were designed incorporating five different interlinking chains including cis- or trans-but-2-ene, propyl, octyl, and an ethoxyethane polyether linkage (Table 1).


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  		TABLE 1 Structures of the furanocoumarin dimers synthesised, where R denotes either the 5 or the 8-furanocoumarin ring system

 

The incorporation of cis- or trans-but-2-ene generates a linkage between the two furanocoumarin molecules similar to that seen for resvatrol. The latter is a component of red wine, which has also been shown to be a potent inhibitor of CYP3A4 (Chan and Delucchi, 2000Go). Inactivation of CYP3A4 by resvatrol has been suggested to occur via epoxidation or hydroxylation of the ethylene bridge (Chan and Delucchi, 2000Go). It is possible that inhibition of CYP3A4 by the cis- and trans-but-2-ene dimers may also occur in this manner. Alternatively, the inclusion of an alkene substituent may enhance binding by increased Van der Waals interactions. The inclusion of the propyl and octyl derivatives enables the inclusion of more flexible chains into the molecule compared with the but-2-ene derivatives. Incorporation of the polyether linkage enables a direct comparison with the octyl derivative to establish whether enhanced hydrogen bonding along the chain influences CYP3A4 inhibition.

Based on the above considerations, the aim of this work was to determine the relationship between the chemical structure of furanocoumarin dimers (chain length, chain flexibility, hydrogen bonding capability and lipophilicity) and the inhibition of CYP3A4 activity in liver and intestine. The selectivity of these dimers for CYP3A4 was also investigated by determining their effect on the activities of other human cytochromes P450.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemicals. Unless otherwise stated, all chemicals were of analytical grade or of a higher purity and were purchased from Sigma-Aldrich Company Ltd. (Gillingham, Dorset, UK), Fisher Scientific (Loughborough, UK), Roche Diagnostics Ltd. (Lewes, UK), or VWR International Ltd. (Poole, UK), or were donated by SAFC Pharma (formerly Ultrafine, Manchester, UK).

Synthesis and Characterization of Furanocoumarin Dimers. The 5/5- and 8/8-substituted dimers were synthesized under basic conditions in a reaction between the hydroxylated furanocoumarin derivatives and a dihalogenated-interlinking chain (Castellan et al., 1983Go). Preparation of the 5/8-mixed furanocoumarin dimers was carried out in two steps. First, the reaction between the 8-hydroxypsoralen and the dibromoalkane gave the intermediate bromopropyl and bromooctyl derivatives. These compounds were then coupled with bergaptol, affording the propyl and octyl mixed furanocoumarin dimers in reasonable yields.

1H and 13C NMR spectra were recorded on Bruker AC 250 or Bruker AMX1-400 instruments. Chemical shifts ({delta}H, {delta}C) are reported in ppm, and coupling constants (J) are in Hertz (Hz). Chemical shifts were referenced to residual nondeuterated solvent present in the deuterated sample, e.g., CHCl3 in CDCl3. Infrared spectra were determined by direct sample analysis using a Bruker Goldengate ATR Vector 22 Spectrometer (Bruker, Newark, DE) and are reported by wave numbers (cm–1). Electron impact (EI) and chemical ionization (CI) mass spectrometry was carried out on a Micromass Prospec magnetic sector instrument by Sheffield University Mass Spectrometry Department or a Kratos Concept 1S instrument by Manchester University Mass Spectrometry Department. Melting points were determined on a hot stage microscope apparatus, and are quoted uncorrected in degrees Centigrade. Thin-layer chromatography (TLC) was carried out on aluminum-backed Merck Kiesgel plates (Merck, Darmstadt, Germany) with detection by UV (254 nm) fluorescence. Chromatography was carried out using Merck Silica gel 60 (<63 µm) or Fisher Matrex 35 to 70 µm.

General Procedure 1. The dihalide (0.50 mmol) was added dropwise to a stirred suspension of 8-hydroxypsoralen (200 mg, 0.99 mmol), potassium carbonate (273 mg, 1.98 mmol), and potassium iodide (20 mg, 0.12 mmol) in DMF (10 ml) under argon. The reaction was heated to 80°C for 3 h. On completion, as judged by TLC, aqueous citric acid (10% w/v) was added, which resulted in precipitation of the product. Water (5 ml) was added and the reaction was stirred for a further 30 min. The product was recovered by filtration and washed with ether (20 ml).

General Procedure 2. A solution of 8-hydroxypsoralen (200 mg, 0.99 mmol) in DMF (5 ml) was added dropwise to a stirred suspension of the dibromoalkane (3.96 mmol) and potassium carbonate (273 mg, 1.98 mmol) in DMF (5 ml) under argon. The reaction mixture was heated to 80°C for 45 min. On completion, as judged by TLC, the potassium carbonate was neutralized by the addition of aqueous citric acid (10% w/v). The solution was extracted with ethyl acetate (two times, 20 ml) and the combined organic extracts were washed with water, then brine, and then were dried (MgSO4). Removal of the solvent under reduced pressure gave a translucent yellow oil, which was purified by column chromatography, affording the bromo compound as colorless crystals. The intermediate bromopropyl or bromooctyl derivative (0.31 mmol) was added to a stirred suspension of bergaptol (63 mg, 0.31 mmol), potassium carbonate (85 mg, 0.62 mmol), and potassium iodide (12 mg, 0.08 mmol) in DMF (10 ml) under argon. The reaction mixture was heated to 80°C for 6 h. Aqueous citric acid (10% w/v) was added, resulting in the formation of a pale precipitate, which was recovered by filtration.

88cBUT was prepared by general procedure 1, using cis-1,4-dichlorbut-2-ene and xanthotoxol. The desired compound was recrystallized from dichloromethane/hexane (2:1) yielding off-white crystals (186 mg, 0.41 mmol, 83%). m.p. 182–183°C; {upsilon}max cm–1 1709 (C=O), 1586 (C=C); {delta}H (d6-DMSO; 250 MHz) 8.11 (2H, d, J = 9.6 Hz), 8.06 (2H, d, J = 2.1 Hz), 7.65 (2H, s), 7.06 (2H, d, J = 2.1 Hz), 6.40 (2H, d, J = 9.6 Hz), 5.98 (2H, t, J = 4.2 Hz), 5.04 (4H, d, J = 4.2 Hz); {delta}C (d6-DMSO; 63 MHz) 159.7, 147.9, 147.5, 145.3, 143.0, 130.3, 129.5, 125.8, 116.5, 114.6, 114.3, 107.2, 68.6; m/z (CI) 457 (15%, [M + H]+), 203 (75%, [RarOH + H]+). Found (EI) 456.0841; C26H16O8 requires 456.0845.

88tBUT was prepared by general procedure 1, using trans-1,4-dichlorobut-2-ene and xanthotoxol and yielding the desired compound as small cream crystals (212 mg, 0.46 mmol, 94%). m.p. 226–227°C; {upsilon}max cm–1 1712 (C=O), 1584 (C=C); {delta}H (d7-DMF; 250 MHz) 8.27 (2H, d, J = 9.8 Hz), 8.20 (2H, d, J = 2.0 Hz), 7.79 (2H, s), 7.18 (2H, d, J = 2.0 Hz), 6.53 (2H, d, J = 9.8 Hz) 6.33 (2H, bs), 5.16 (4H, d, J = 3.4 Hz); {delta}C (d7-DMF; 100.6 MHz) 165.5, 153.3, 150.9, 148.9, 136.5, 135.2, 131.7, 122.3, 120.0, 119.9, 112.7, 78.3, not found (2 x OCar) possibly obscured by CarOR at 148.9; m/z (CI) 457 (20%, [M + H]+), 203 (100%, [RarOH + H]+). Found (EI) 456.0841; C26H16O8 requires 456.0845.

88Prop was prepared by general procedure 1, using the 1,3-dibromopropane and xanthotoxol, and yielding the desired compound as pale brown crystals (76 mg, 0.17 mmol, 34%). m.p. 183–185°C; {upsilon}max cm–1 1727 (C=O), 1588 (C=C); {delta}H (CDCl3; 250 MHz) 7.75 (2H, d, J = 9.6 Hz), 7.67 (2H, d, J = 2.2 Hz), 7.33 (2H, s), 6.80 (2H, d, J = 2.2 Hz), 6.34 (2H, d, J = 9.6 Hz), 4.89 (4H, t, J = 6.0 Hz), 2.44 (2H, quin, J = 6.0 Hz); {delta}C (d6-DMSO; 63 MHz) 159.6, 147.8, 147.3, 145.2, 142.8, 131.0, 125.8, 116.5, 114.2, 107.1, 70.2, 30.6; m/z (EI) 444 (45%, M+), 202 (90%, [RarOH + H]+). Found (EI) 444.0845; C25H16O8 requires 444.0845.

88Octa was prepared by general procedure 1, using 1,8-dibromooctane and xanthotoxol, and yielding the desired compound as light yellow crystals (89 mg, 0.17 mmol, 70%). m.p. 143–144°C, Lit. (Castellan et al., 1983Go) 145–146°C; {upsilon}max cm–1 1714 (C=O), 1581 (C=C); {delta}H (CDCl3; 250 MHz) 7.78 (2H, d, J = 9.6 Hz), 7.72 (2H, d, J = 2.2 Hz), 7.37 (2H, s), 6.83 (2H, d, J = 2.2 Hz), 6.39 (2H, d, J = 9.6 Hz), 4.51 (4H, t, J = 6.7 Hz), 1.89 (4H, quin, J = 6.7 Hz), 1.62–1.31 (8H, m); {delta}C (CDCl3; 63 MHz) 160.6, 148.2, 146.6, 144.4, 143.4, 132.1, 126.0, 116.5, 114.7, 112.9, 106.7, 70.1, 29.7, 29.2, 25.6; m/z (EI) 514 (10%, M+), 202 (37%, RarOH). Found (EI) 514.1635; C30H26O8 requires 514.1628.

88EE was prepared by general procedure 1, using 1,2-bis-(2-iodoethoxy)ethane and xanthotoxol. The product was recrystallized from ethyl acetate/hexane (1:1) furnishing light yellow crystals of the desired compound (193 mg, 0.37 mmol, 75%). m.p. 120–121°C, Lit. (Castellan et al., 1983Go) 124–125°C; {upsilon}max cm–1 1703 (C=O), 1580 (C=C); {delta}H (CDCl3; 250 MHz) 7.77 (2H, d, J = 9.6 Hz), 7.71 (2H, d, J = 2.2 Hz), 7.37 (2H, s), 6.82 (2H, d, J = 2.2 Hz), 6.37 (2H, d, J = 9.6 Hz), 4.64 (4H, t, J = 4.8 Hz), 3.92 (4H, m), 3.74 (4H, s); {delta}C (CDCl3; 63 MHz) 160.4, 148.1, 146.7, 144.3, 143.3, 131.9, 125.9, 116.4, 114.6, 113.2, 106.7, 73.1, 70.9, 70.4; m/z (EI) 518 (10%, M+), 202 (77%, RarOH). Found (EI) 518.1209; C28H22O10 requires 518.1213.

55cBUT was prepared by general procedure 1, using cis-1,4-dichlorobut-2-ene and bergaptol. The product recrystallized from dichloromethane/hexane (2:1) yielding an off-white solid (181 mg, 0.40 mmol, 80%). m.p. 244–246°C; {upsilon}max cm–1 1714 (C=O), 1626 (C=C); {delta}H (d7-DMF; 250 MHz) 8.33 (2H, d, J = 9.5 Hz), 8.14 (2H, s), 7.51 (2H, s), 7.43 (2H, s), 6.42 (2H, d, J = 9.5 Hz), 6.28 (2H, s), 5.42 (4H, s); {delta}C (d7-DMF; 100 MHz) 165.8, 163.5, 158.3, 154.1, 151.7, 145.1, 134.7, 119.5, 118.2, 112.8, 111.2, 99.3, 74.4; m/z (EI) 456 (10%, M+) 202 (100%, RarOH). Found (EI) 456.0861; C26H16O8 requires 456.0845.

55tBUT was prepared by general procedure 1, using trans-1,4-dichlorobut-2-ene and bergaptol. The desired compound was recovered as small cream crystals by filtration and washing with ether. The insolubility of this compound in most organic solvents led to difficulties with purification. Numerous attempts were made, but a purity of only 77.4% was obtained. Thus, the compound was not evaluated as an inhibitor of CYP3A4. (454 mg, 0.99 mmol, 80%). m.p. 230–232°C; {upsilon}max cm–1 1716 (C=O), 1624 (C=C); {delta}H (d6-DMSO; 250 MHz) 8.17 (2H, d, J = 9.8 Hz), 8.03 (2H, d, J = 2.1 Hz), 7.37 (2H, s), 7.30 (2H, d, J = 2.1 Hz), 6.33 (2H, d, J = 9.8 Hz), 6.21 (2H, s), 5.09 (4H, s), impurities not included; {delta}C (d7-DMF; 63 MHz) 159.8, 157.7, 152.2, 148.2, 145.7, 139.0, 128.6, 113.4, 112.2, 106.5, 105.0, 93.3, 71.8; m/z (liquid chromatography/mass spectrometry, electrospray) 457 (100%, [M + H]+).

55Prop was prepared by general procedure 1, using 1,3-dibromopropane and bergaptol, and yielding off-white crystals (185 mg, 0.42 mmol, 84%). m.p. decomposed at 285°C; {upsilon}max cm–1 1703 (C=O), 1626 (C=C); {delta}H (d6-DMSO; 250 MHz) 8.16 (2H, d, J = 9.8 Hz), 8.03 (2H, d, J = 2.1 Hz), 7.34 (2H, s), 7.31 (2H, s), 6.21 (2H, d, J = 9.8 Hz), 4.76 (4H, t, J = 6.0 Hz), 2.39 (2H, quin, J = 6.0 Hz); {delta}C (d7-DMF; 100.6 MHz) 161.0, 158.9, 153.5, 149.9, 146.6, 140.1, 113.9, 112.9, 106.3, 93.9, 70.2; not found (2 x CarCOR), expected to lie around 107.0 ppm. The signal for CH2 around 30 ppm was possibly obscured by the DMF peak; m/z (EI) 444 (47%, M+), 203 (5%, [RarOH + H]+). Found (EI) 444.0851; C25H16O8 requires 444.0845.

55Octa was prepared by general procedure 1, using 1,8-dibromooctane and bergaptol, and yielding light brown crystals (140 mg, 0.27 mmol, 55%). m.p. 174–176°C; {upsilon}max cm–1 1717 (C=O), 1621 (C=C); {delta}H (CDCl3; 250 MHz) 8.17 (2H, d, J = 9.7 Hz), 7.61 (2H, s), 7.16 (2H, s), 6.96 (2H, s), 6.29 (2H, d, J = 9.7 Hz), 4.47 (4H, t, J = 6.2 Hz), 1.93–1.88 (4H, m), 1.59–1.33 (8H, m); {delta}C (d7-DMF; 100.6 MHz) 165.9, 163.9, 158.4, 155.0, 151.5, 145.2, 118.9, 118.0, 112.0, 111.4, 98.8, 78.6, 40.8, 39.6, 31.3; m/z (EI) 514 (5%, M+), 202 (100%, RarOH). Found (EI) 514.1630; C30H26O8 requires 514.1628.

55EE was prepared by general procedure 1, using 1,2-bis-(2-iodoethoxy)ethane and bergaptol. The product was recrystallized from dichloromethane, affording pale mauve crystals (170 mg, 0.33 mmol, 66%). m.p. 207–209°C; {upsilon}max cm–1 1715 (C=O), 1626 (C=C); {delta}H (CDCl3; 250 MHz) 8.12 (2H, d, J = 9.7 Hz), 7.59 (2H, d, J = 2.3), 7.11 (2H, s), 6.96 (2H, d, J = 2.3 Hz), 6.26 (2H, d, J = 9.7 Hz), 4.55 (4H, t, J = 4.5 Hz), 3.89 (4H, t, J = 4.5 Hz), 3.77 (4H, s); {delta}C (d7-DMF; 100.6 MHz) 160.9, 158.7, 153.2, 149.7, 146.9, 140.3, 114.9, 113.1, 107.8, 105.9, 94.4, 73.6, 71.1, 70.4; m/z (EI) 518 (23%, M+), 203 (100%, [RarOH + H]+). Found (EI) 518.1214; C28H22O10 requires 518.1213.

58Prop was prepared by general procedure 2, using 1,3-dibromopropane and xanthotoxol. The desired compound was recrystallized from dichloromethane/hexane (2:1), yielding the desired compound as off-white crystals (82 mg, 0.18 mmol, 59%). m.p. 222–224°C; {upsilon}max cm–1 1716 (C = O), 1623 (C = C); {delta}H (CDCl3; 250MHz) 8.09 (1H, d, J = 9.8 Hz), 7.75 (1H, d, J = 9.6 Hz), 7.63 (1H, d, J = 2.4 Hz), 7.59 (1H, d, J = 2.2 Hz), 7.34 (1H, s), 7.17 (1H, d, J = 2.4 Hz), 7.11 (1H, s), 6.81 (1H, d, J = 2.2 Hz), 6.36 (1H, d, J = 9.6 Hz), 6.18 (1H, d, J = 9.8 Hz), 4.88 (2H, t, J = 6.0 Hz), 4.79 (2H, t, J = 5.7 Hz), 2.45 (2H, quin, J = 5.9 Hz); {delta}C (d7-DMF; 100.6 MHz) 165.9, 165.5, 163.9, 158.3, 154.7, 153.4, 153.4, 151.6, 150.8 1, 149.0, 145.0 1, 137.0, 131.8, 122.4, 119.9, 118.9, 117.9, 112.8, 112.0, 111.3 1, 98.0, 76.0, 75.0, 36.1; m/z (EI) 444 (100%, M+), 202 (60%, RarOH), Found (EI) 444.0854; C25H16O8 requires 444.0845.

58Octa was prepared by general procedure 2, using 1,8-dibromooctane and xanthotoxol. The desired compound was purified by column chromatography eluting with ethyl acetate/hexane (2:3); removal of solvent under reduced pressure furnished off-white crystals (82 mg, 0.16 mmol, 64%). m.p. 136–137°C; {upsilon}max cm–1 1708 (C=O), 1620 (C=C); {delta}H (CDCl3; 250 MHz) 8.18 (1H, d, J = 9.8 Hz), 7.77 (1H, d, J = 9.6 Hz), 7.70 (1H, d, J = 2.2 Hz), 7.59 (1H, d, J = 2.4 Hz), 7.36 (1H, s), 7.13 (1H, s), 6.98 (1H, d, J = 2.4 Hz), 6.83 (1H, d, J = 2.2 Hz), 6.37 (1H, d, J = 9.6 Hz), 6.27 (1H, d, J = 9.8 Hz), 4.49 (4H, ot, J = 6.5, 6.5 Hz), 1.90 (4H, quin, J = 6.7 Hz), 1.65–1.39 (8H, m); {delta}C (d7-DMF; 100.6 MHz) 165.9, 165.6, 163.9, 158.4, 155.0, 153.6, 153.4, 151.5, 150.9, 149.1, 145.2, 137.2, 131.9, 122.5, 119.9, 119.7, 118.9, 118.0, 112.8, 112.0, 111.4, 98.7, 79.4, 78.6, 31.2, not found ~35.0 (4 x CH2) obscured by DMF peaks; m/z (EI) 514 (100%, M+), 203 (60%, [RarOH + H]+). Found (EI) 514.1621; C30H26O8 requires 514.1628.

Source and Preparation of Tissue Samples. Samples of liver tissue were obtained with written consent from patients undergoing surgery for the removal of a hepatocellular tumor secondary to colon cancer. Macrosopically normal tissue close to the resection line was used. Samples of upper intestine (duodenum and jejunum) were obtained with written consent from patients undergoing total gastrectomy. These studies were approved by the appropriate Hospital Ethics Committee.

Human liver microsomes and intestinal supernatant S9 fraction were prepared as described previously (Crewe et al., 1997Go). Intestinal S9 samples were pooled from five patients because of limited tissue availability.

Incubation Conditions for the Characterization of CYP3A4 Inhibition. The 6ß-hydroxylation of testosterone was used as the index of CYP3A4 activity. Human liver microsomes and intestinal S9 (0.2 mg/ml) were incubated with testosterone (37 µM) and the test compounds (concentration range 0.5–100 µM) at 37°C in the presence of KCl (1.15%), phosphate buffer (0.2 M, pH 7.4), and a NADPH-generating system, for 10 min in a total volume of 1 ml (Crewe et al., 1997Go). Incubations were performed in triplicate. The reaction was terminated by the addition of ethyl acetate (2 ml). 16-{alpha}-Hydroxytestosterone (1.5 µg) or 11-ß-hydroxytestosterone (1 µg) (depending on the retention time of the test compound) was added as the internal standard. Samples were gently mixed for 15 min, before centrifugation at 1500g for 15 min. The upper organic layer was evaporated to dryness under reduced pressure, and the residue was stored at –20°C until analysis.

Characterization of the Inhibition of the Activities of Other Human Cytochromes P450. The O-demethylation of dextromethorphan (10 µM) was used as the index of CYP2D6 activity (Gorski et al., 1994Go), the 7-hydroxylation of (S)-warfarin (4 µM) as the index of CYP2C9 activity, the O-deethylation of 7-ethoxyresorufin (1 µM) as the index of CYP1A2 activity (Hanioka et al., 2000Go), the 4-hydroxylation of (S)-mephenytoin (200 µM) as the index of CYP2C19 activity (Wrighton et al., 1993Go), and the 6-hydroxylation of chlorzoxazone (20 µM) as the index of CYP2E1 activity (Leclercq et al., 1998Go). The concentrations in brackets are the mean Km values for the reactions and were those used in the present work.

Mechanism-Based Inhibition of CYP3A4. To minimize competitive inhibition by the furanocoumarin dimers, incubations were performed at a testosterone substrate concentration of 250 µM. Human liver microsomes (2 mg/ml) were preincubated with furanocoumarin dimers or DMSO in the presence of a NADPH-generating system for various times, ranging from 0 to 180 s at 37°C. An aliquot (100 µl) was then transferred to another incubation vial containing the testosterone and NADPH-generating system (final volume 1 ml) and assayed for remaining CYP3A4 activity. This resulted in a 1 in 10 dilution of protein (final concentration 0.2 mg/ml) and in final inhibitor concentrations of 12.5, 31, 62.5, 125, and 250 nM. The reaction was terminated after 3 min by the addition of ethyl acetate (2 ml). Samples were then treated as described above. Incubations were performed in triplicate.

Analysis of 6ß-Hydroxytestosterone. Sample residues were reconstituted with mobile phase (150 µl), and an aliquot was injected onto the HPLC. A Hypersil C8 BDS column (150 mm x 4.6 mm; 5-µm particle size) was used. The mobile phase was methanol/water (55:45 v/v) delivered at a flow rate of 1 ml/min. Eluents were detected by UV at 254 nm. The lower limit of determination of the assay was 30 pmol ml–1, and the coefficient of variation at 328 pmol ml–1 was less than 5%.

Data Analysis. Values of IC50 were obtained by nonlinear regression using GraFit version 5.0 (Erithacus Software Ltd., Horley, Surrey, UK). The inactivation parameters kinact and KI were calculated according to the method of Silverman (1988Go). The initial rate constant (k min–1) for the inactivation at each inhibitor concentration was estimated from the slope of the log of the remaining CYP3A4 activity versus the preincubation time. Values for the inactivation parameters were determined from the double reciprocal plot of the initial inactivation rate constants versus the respective inhibitor concentrations (1/k versus 1/[I]). These values were used as initial estimates in solving the following equation with GraFit: kobs = kmax x [I]/KI + [I], where [I] is the initial inhibitor concentration, kinact is the maximum rate constant for inactivation when [I] approximates infinity, and KI is the inhibitor concentration that produces half the maximal rate of inactivation.


Figure 2
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  		FIG. 2. Inhibition of CYP3A4 activity microsomes from liver HL7 by four of the most potent dimers, which were chosen for further study. {blacksquare}, 88Prop; {blacktriangleup}, 58Prop; x, 55EE; {diamondsuit}, 88Octa. Mean ± S.D. from triplicate experiments.

 


Figure 3
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  		FIG. 3. Inhibitory effects of furanocoumarin dimers on five different P450 isoforms in liver microsomes from liver HL7. {blacksquare}, 88Prop; {blacktriangleup}, 58Prop; x, 55EE; {diamondsuit}, 88Octa. Results are expressed as the mean of triplicate incubations ± S.D.

 

   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
All the synthetic furanocoumarin dimers were found to inhibit CYP3A4 in a dose-dependent manner (Table 2) with potencies comparable to and, in most cases, exceeding those obtained for their naturally occurring counterparts (Guo et al., 2000bGo). Within this series 88Prop, 88tBUT, 88Octa, and 55EE are found to be the most potent analogs, with IC50 values of less than 0.030 µM, and causing maximum inhibition of more than 90% over the concentration range studied. The least potent analog was the 55Octa derivative with an IC50 value of 0.140 µM. 88Prop showed approximately a 2-fold increase in potency over the 5/5- and 5/8-derivatives. 88Octa was found to be the most potent inhibitor of the octyl derivatives, showing a 4.8- and 7-fold higher potency compared with the 5/8- and 5/5-octyl dimers, and 58Octa was 1.4-fold more inhibitory than its 5/5-substituted counterpart. Within the 8/8-substituted series, the potency of inhibition seemed to increase with lipophilicity (Table 2). However, for the 5/5- and 5/8-substiuted dimers, a decrease in potency was observed as the lipophilicity of the compounds increased.


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  		TABLE 2 IC50 values for inhibition of CYP3A4 activity in human microsomes from a single liver (HL7) by furanocoumarin dimers (mean ± S.D. data from triplicate experiments)

Log P values were calculated using Accord for Excel v0.5.0.

 

Four of the most potent dimers (88Prop, 58Prop, 88Octa, and 55EE) were selected for further study (Figs. 2 and 3). Inhibition of CYP3A4 by these compounds was assessed in human microsomes from additional livers, again using testosterone as the marker substrate (Table 3). Overall, there was about a 3-fold variation in inhibitory potency between the livers. Furanocoumarin dimers were found to inhibit CYP3A4 activity in intestinal supernatant S9 fractions to an extent similar to that seen with liver microsomes (Table 4). The effects of these four furanocoumarin dimers on the activity of five different P450 isoforms (Fig. 4) were investigated to demonstrate whether the compounds were selective for CYP3A4. All were found to be weak inhibitors of CYP2D6 and CYP2C9 activity, giving IC50 values of more than 10 µM (Table 5). No data were obtained for the inhibition of CYP2E1 activity by the dimers, since DMSO and DMF (both 0.2% v/v), used to dissolve the analogs, were found to be potent inhibitors of chlorzoxazone-6-hydroxylayse activity in vitro (producing a more than 90% loss), data that were consistent with previous findings (Hickman et al., 1998Go). Moderate inhibition of CYP2C19 activity was seen for the two 8/8-substituted derivatives, giving IC50 values approximately 2 orders of magnitude higher than those observed for CYP3A4. 88Prop, 58Prop, and 88Octa produced moderate inhibition of CYP1A2 activity. Inclusion of the propyloxy interlinking chain led to a 2.5-fold higher potency when compared with the longer-chain octyl derivative. The 5/5-ethoxyethane compound (55EE) showed minimal inhibition of the five P450 isoforms.


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  		TABLE 3 Variation between human livers in the inhibition of microsomal CYP3A4 activity by four furanocoumarin dimers Mean (±S.D. from triplicate experiments) IC50 values.

 

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  		TABLE 4 Comparison of the inhibition of CYP3A4 activity in human intestine (S9) (pooled from five patients; mean ± S.D. IC50 data from triplicate experiments) and human liver microsomes [HLM; mean ± S.D. IC50 data from five livers (Table 3)] by furanocoumarin dimers

 

Figure 4
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  		FIG. 4. The four furanocoumarin dimers chosen for further study had propyl octyl or ethoxy-ethane linkages substituted at either the 5- or the 8-position of the ring system.

 

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  		TABLE 5 IC50 values (mean ± S.D. from triplicate experiments) obtained for the inhibition of the activities of five human P450s by four furanocoumarin dimers in microsomes from liver HL7

 

These four furanocoumarin dimers were found to inhibit testosterone CYP3A4 activity in a time- as well as concentration-dependent manner. Pseudo first-order kinetics were observed for the inactivation of enzyme activity. The mean (±S.D. from triplicate experiments) KI values obtained for 88Prop, 58Prop, 88Octa, and 55EE were 0.098 ± 0.015, 0.123 ± 0.041, 0.040 ± 0.013, and 0.123 ± 0.007 µM, respectively, and their inactivation rate constant kinact values were 0.088 ± 0.006, 0.058 ± 0.009, 0.036 ± 0.004, and 0.045 ± 0.001 min–1, respectively, in microsomes from liver HL7.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The dimers synthesized showed a dose-dependent inhibitory effect on CYP3A4 activity in human liver microsomes. The potency of these compounds, which were active at low nanomolar concentrations, was found to exceed that of ketoconazole and other known inhibitors of CYP3A4 (Guo et al., 2000bGo). The dimers exhibited comparable potencies with little or no difference arising from the length of the interlinking chain. This lack of variability may be attributed to the lipophilic nature of these chains, which possibly causes them to "fold up" on themselves within the aqueous medium of the active site of CYP3A4. The highly lipophilic nature of the compounds as a whole may give rise to strong interactions within hydrophobic region(s) of the active site. The IC50 values for the inhibition of CYP3A4 activity were of the same order of magnitude for each dimer in microsomes from five different livers.

Dose-dependent inhibition of the formation of 6ß-hydroxytestosterone in human intestinal (S9) supernatant was also observed. Complete abolition of enzyme activity was found at a concentration of 1 µM for all four dimers, indicating a degree of inhibition similar to that observed in liver. This suggests that the susceptibility of enteric and hepatic CYP3A4 to inhibition is similar, a result that is consistent with published data (Lown et al., 1998Go; Obach et al., 2001Go). The furanocoumarin dimers synthesized in the present work were shown 1) to inhibit CYP3A4 activity in human liver microsomes in a time- and concentration-dependent manner, and 2) along with the naturally occurring dimers GF-I-1 and GF-I-4 (Tassaneeyakul et al., 2000Go), to be extremely potent mechanism-based inhibitors. Their potencies (range of KI values = 0.04–0.32 µM) are 1 to 2 orders of magnitude greater than many other known mechanism-based inhibitors [e.g., verapamil = 2.3 µM (Yeo and Yeo, 2001Go), gestodene = 46 µM (Guengerich, 1990Go), mibedrafil = 2.3 µM (Yeo and Yeo, 2001Go), and L-754,394 = 7.5 µM (Lightning et al., 2000Go)].

Studies with other irreversible CYP3A4 inhibitors such as 8-methoxypsoralen, furafylline, and coumarin compounds suggest that the loss of catalytic activity may be attributed to modification of the heme, denaturation of the apoprotein, or covalent binding of a metabolite of the inhibitor to the enzyme active site. The furanocoumarin monomers bergamottin and 6',7'-dihydroxybergamottin are also mechanism-based inhibitors of CYP3A4 with kinact and KI values of 40 and 5.6 µM, and 0.08 and 0.06 min–1, respectively (Tassaneeyakul et al., 2000Go). The kinact values for the naturally occurring furanocoumarin dimers are very similar to those obtained for the synthetic dimers, suggesting the possibility that the mechanism of inactivation is similar. Thus, the dimers may interact with the enzyme at the unsaturated furan moiety in a manner similar to that described for the inactivation of CYP3A4, CYP2B1, and CYP2A6 by furanocoumarin monomers (He et al., 1998Go; Koenigs and Trager, 1998Go). Initially, an epoxide is formed on the unsaturated furan ring. This epoxide then undergoes ring-opening (by hydrolysis or attack from an internal nucleophile) to form a vic-adduct, which is able to covalently bind to the apoprotein of the enzyme, thus irreversibly inactivating it. Further studies are required to confirm this hypothesis with respect to the synthetic dimers.

Our finding suggests that the 8/8-octyl and propyl derivatives are slightly more selective toward CYP3A4 than are their 5/5- and 5/8-substituted counterparts. Weak inhibition of CYP2D6 and CYP2C9 was observed for the four dimers chosen for further study, with IC50 values exceeding 10 µM. 88Octa and 88Prop were found to be moderate inhibitors of CYP2C19 activity but with IC50 values approximately 2 orders of magnitude greater than those observed for inhibition of CYP3A4 activity. Little difference in potency toward CYP2C19 was observed with increasing chain length, suggesting that the binding sites on the enzyme mainly consist of hydrophobic regions. 88Prop, 88Octa, and 58Prop showed moderate inhibition of CYP1A2 activity but with IC50 values exceeding those observed for CYP3A4 by 2 orders of magnitude. An increase in potency was observed with a decrease in chain length. CYP1A2, like CYP3A4, catalyzes the metabolism of neutral/basic, lipophilic, or planar molecules with at least one hydrogen-bonding site, making both the furanocoumarin monomers and dimers candidates for an interaction with this enzyme. In addition to the compounds tested, flavonoids (Zhai et al., 1998Go; Murray et al., 2001Go), methylxanthines (Murray et al., 2001Go), grapefruit juice, 6',7'-dihydroxybergamottin, bergamottin, and the naturally occurring dimers GF-I-1 and GF-I-4 (Tassaneeyakul et al., 2000Go) have been shown to be potent to moderate inhibitors of CYP1A2 activity, using phenacetin as the marker substrate. The increased selectivity of the 55EE analog suggests different orientations for the 5- and 8-substituted ring systems within the binding domains for CYP1A2 and CYP2C19. For CYP1A2, it appears that the 8-substituted furanocoumarins have a greater affinity for the binding site than the 5/5-substituted dimers. Since inhibitory activity was observed with the mixed 5/8-propyl dimer, it is proposed that the dimer is binding at its 8-rather than its 5-substituted end.

Unfortunately, no data were obtained for CYP2E1, since the DMSO (0.2% v/v) used to dissolve the dimers was found to be a potent inhibitor of chlorzoxazone 6-hydroxylase activity (>90%), the probe reaction used. However, it is unlikely that any interaction would occur between these dimers and CYP2E1, since the enzyme is principally involved with the metabolism of small molecules with a molecular weight of <200. Furthermore, since neither naturally occurring furanocoumarin monomers nor dimers are reported to be potent inhibitors of CYP2E1 activity (Tassaneeyakul et al., 2000Go), no significant inhibitory effects would be envisaged with the synthetic furanocoumarin dimers tested in the present study. The selectivity of our synthetic compounds is similar to that observed by Tassaneeyakul et al. (2000Go), in which furanocoumarin dimers isolated from grapefruit juice significantly inhibited CYP1A2, CYP2C9, CYP2C19, and CYP2D6 activities, but with IC50 values being at least 1 order of magnitude higher compared with those for CYP3A4 inhibition.

In addition to CYP3A4, P-glycoprotein has been reported to play a role in the grapefruit juice interaction (Soldner et al., 1999Go; Spahn-Langguth and Langguth, 2001Go; Wang et al., 2001Go). In this context, a small pilot study was performed on four of the furanocoumarin dimers synthesized. Using the P-glycoprotein-overexpressed human leukemia CEMVBL100 cell line, inhibition of its transport function by all four dimers was observed (data not shown). These preliminary findings are consistent with other published data showing that the furanocoumarins GF-I-1, 6',7'-dihydroxybergamottin, bergamottin, bergapten, and bergaptol (10 µM) inhibit the efflux of [3H]VBL in Caco-2 cells, increasing uptake by 601, 357, 138, 244, and 270%, respectively (Ohnishi et al., 2000Go).

In conclusion, the synthetic furanocoumarin dimers 88Prop, 58Prop, 88Octa, and 55EE have been shown to inhibit the activity of CYP3A4 in a highly selective and potent fashion, suggesting that these compounds are suitable probes for determining the contribution of the enzyme to drug metabolism.


   Footnotes
 
We thank the Biotechnology and Biological Sciences Research Council (BBSRC) for financial support.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.007294.

ABBREVIATIONS: P450, cytochrome P450; EI, electron impact; CI, chemical ionization; TLC, thin-layer chromatography; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; 88cBUT, 9,9'-[(2Z)-but-2-ene-1,4-diylbis(oxy)]bis(7H-furo[3,2-g]chromen-7-one; 88tBUT, 9,9'-[(2E)-but-2-ene-1,4-diylbis-(oxy)]bis(7H-furo[3,2-g]chromen-7-one; 88Prop, 9,9'-[propane-1,3-diylbis(oxy)]bis(7H-furo[3,2-g]chromen-7-one; 88Octa, 9,9'-[(octane-1,8-diylbis(oxy)]bis(7H-furo[3,2-g]chromen-7-one; 88EE, 9,9'-[(ethane-1,2-diylbis(oxyethane-2,1-diyloxy)]bis(7H-furo[3,2-g]chromen-7-one; 55cBut, 4,4'-[(2Z)-but-2-ene-1,4-diylbis(oxy)]bis(7H-furo[3,2-g]chromen-7-one; 55tBut, 4,4'-[(2E)-but-2-ene-1,4-diylbis(oxy)bis(7H-furo[3,2-g]chromen-7-one; 55Prop, 4,4'-[(propane 1,3-diylbis(oxy)]bis(7H-furo[3,2-g]chromen-7-one; 55Octa, 4,4'-[octane-1,8-diylbis(oxy)]bis(7H-furo[3,2-g]chromen-7-one; 55EE, 4,4'-[ethane-1,2-diylbis(oxyethane-1,2-diyloxy)]bis(7H-furo[3,2-g]chromen-7-one; 58Prop, 4-({3-[(7-oxo-7H-furo[3,2-g]chromen-9-yl)oxy]propoxy}-7H-furo[3,2-g]chromen-7-one; 58Octa, 4-({8-[(7-oxo-7H-furo[3,2-g]chromen-9-yl)oxy]octyl}oxy)-7H-furo[3,2-g]chromen-7-one.

1 Current affiliations: Avecia Ltd., Huddersfield, West Yorkshire, U.K. (S.A.B.); and The University of Liverpool, Department of Chemistry, Robert Robinson Laboratories, Liverpool, U.K. (E.R., A.V.S.). Back

Address correspondence to: Dr. M. S. Lennard, Academic Unit of Clinical Pharmacology, Pharmacokinetics and Pharmacogenetics Group, University of Sheffield, M Floor, Royal Hallamshire Hospital, Sheffield, S10 2JF, U.K. E-mail: m.s.lennard{at}sheffield.ac.uk


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 Materials and Methods
 Results
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