6',7'-dihydroxybergamottin, a cytochrome P450 inhibitor in grapefruit

The present invention provides a composition and methods for inhibiting cytochrome P450 enzyme activity and in particular, inhibiting the activity of the cytochrome P450 3A sub-family of enzymes, specifically, CYP3A4. The present invention provides 6',7'-dihydroxybergamottin, a furanocoumarin, as the compound primarily responsible for the inhibitory effects of grapefruit juice on cytochrome P450 enzyme activity. The present invention also provides a novel synthesis scheme for 6',7'-dihydroxybergamottin.

FIELD OF THE INVENTION 
The present invention relates generally to a cytochrome P450 inhibitor 
found in grapefruit juice and, in particular, 6',7'-dihydroxybergamottin 
and methods of making and using same. 
BACKGROUND OF THE INVENTION 
Humans are continuously exposed to potential carcinogens in the diet. Such 
compounds may be natural constituents of food (plant alkaloids), 
contaminants (mycotoxins, pesticides) or formed through food preparation 
(heterocyclic amines). Chadwick, R. W. et al., Drug Metab. Rev. 24:425-492 
(1992) and Wakabayashi, K. et al., Cancer Res. 52:2092s-2098s (1992). Over 
90% of carcinogenic substances, however, are not genotoxic in their native 
form but require activation to the ultimate carcinogen. Enzymes of the 
cytochrome P450 family are highly concentrated in the gut wall (in 
particular the CYP3A sub-family of enzymes) and liver and play a 
significant role in this process. Chadwick, R. W. et al., Drug Metab. Rev. 
24:425-492 (1992); Guengerich, F. P., Cancer Res. 48:2946-2954 (1988) and 
Gonzalez, F. J. et al., Drug Metab. Rev. 26:165-183 (1994). Activation of 
numerous procarcinogens including aflatoxin B.sub.1, 2-aminofluorene and 
3-methylcholanthrene by cytochrome P450 enzymes has been demonstrated 
using the Salmonella mutagenicity assay, cell transformation in culture 
and alterations in DNA. Inducers of cytochrome P450 activity enhance 
formation of genotoxic metabolites and increase the rate of tumor 
formation in animal models. Furthermore, variation in cytochrome P450 
activity due to either genetic or environmental reasons has been found to 
correlate with genotoxin formation. Chadwick, R. W. et al., Drug Metab. 
Rev. 24:425-492 (1992). 
Epidemiologic studies have consistently demonstrated that diet has a 
significant influence on the risk of developing cancer. Greenwald, P. et 
al., Adv. Exp. Med. Biol. 369:229-239 (1995). Most striking is the 
observation that diets high in vegetables and fruits appear to lower 
cancer risk. Block, G. et al., Nutr. Cancer 18:1 (1992). The mechanism by 
which foods such as citrus fruits serve as chemopreventive agents is not 
precisely known. Such fruits contain numerous minor dietary components 
including flavonoids, carotenoids and coumarins which when fed in purified 
form to experimental animals appear to reduce the carcinogenic response. 
Wattenberg, L. W., Cancer Res. 52:2085s-2091s (1992). These compounds may 
act as antioxidants, alter the ability to repair damaged DNA, block 
mutagens from reaching target tissues or affect the formation of the 
activated mutagen by enzymes such as cytochrome P450. Ferguson, L. R., 
Mutat. Res. 307:395-410 (1994). It has recently been found that 
chlorophyllin, a potent antimutagen present in many fruits and vegetables, 
acts primarily through non-specific inhibition of cytochrome P450 
activity. Yun, C-H. et al., Carcinogenesis 16:143-740 (1995). It is this 
mechanism which is of particular interest in examining the role of 
naturally-occurring compounds from citrus fruit in cancer prevention since 
research over the past five years has demonstrated that grapefruit juice 
is a powerful inhibitor of cytochrome P450. 
It has also been recently found that grapefruit juice increases 
bioavailability of drugs. Bailey and co-workers were the first to report 
that oral administration of the calcium channel antagonists nifedipine and 
felodipine with grapefruit juice resulted in several-fold increases in 
bioavailability and blood concentrations. Bailey, D. G. et al., Lancet 
337:268-269 (1991). Since these drugs are highly metabolized on first-pass 
through the gut wall or liver, the increased bioavailability was 
attributed to impaired cytochrome P450 activity. Subsequent studies have 
demonstrated impaired metabolism of triazolam, midazolam, terfenadine and 
cyclosporine in the presence of grapefruit juice. Hukkinen, S. K. et al., 
Clin. Pharmacol. Ther. 58:127-131 (1995); Kupferschmidt, H. H. T. et al., 
Clin. Pharmacol. Ther. 58:20-28 (1995); Benton, R., et al., Clin. 
Pharmacol. Ther. 55:146 (1994); Ducharme, M. P. et al., Br. J. Clin. 
Pharmacol. 36:457-459 (1993) and Ducharme M. P. et al., Clin. Pharmacol. 
Ther. 57:485-491 (1995). All of these drugs are metabolized by CYP3A4, the 
most abundant of the cytochrome P450 enzymes accounting for approximately 
30% of the P450 content of the liver and 70% in the gut wall. Shimada, T. 
et al., J. Pharmacol. Exp. Ther. 270:414-423 (1994) and Watkins, P. B. et 
al., J. Clin. Invest. 80:1029 (1987). This enzyme is involved in the 
activation of a number of carcinogens including aflatoxin B.sub.1. 
Guengerich, F. P., Cancer Res. 48:2946-2954 (1988) and Gonzalez, F. J. et 
al., Drug Metab. Rev. 26:165-183 (1994). Grapefruit juice has also been 
reported to inhibit the metabolism of caffeine and coumarin, substrates 
for the CYPIA2 and CYP2A6 enzymes respectively, suggesting that inhibition 
is not limited to a single cytochrome P450 enzyme. Fuhr, U. et al., Br. J. 
Clin. Pharmacol. 35:431-436 (1993) and Merkel, U. et al., Eur. J. Clin. 
Pharmacol. 46:175-177 (1994). Both of these enzymes are also known to 
participate in the formation of mutagenic metabolites. Gonzalez, F. J. et 
al., Drug Metab. Rev. 26:165-183 (1994). 
Grapefruit juice was found to have a much more pronounced effect on the 
bioavailability of oral cyclosporine compared to intravenous (IV) 
cyclosporine. Since cyclosporine is highly extracted by the gut wall and 
poorly extracted by the liver (Hebert, M. F. et al., Clin. Pharmacol. 
Ther. 52:453-457 (1992)), this suggests that grapefruit juice 
predominantly inhibits gut wall cyclosporine metabolism. Ducharme M. P. et 
al., Clin. Pharmacol. Ther. 57:485-491 (1995). The hypothesis that the 
inhibitor in grapefruit juice is primarily active in the gut is supported 
by studies with midazolam. Grapefruit juice had no effect on the systemic 
clearance of intravenous midazolam but increased oral bioavailability by 
50%. Kupferschmidt, H. H. T. et al., Clin. Pharmacol. Ther. 58:20-28 
(1995). In contrast, oral administration of erythromycin impaired the 
metabolism of midazolam after both oral and IV administration. Olkkola, K. 
T. et al., Clin. Pharmacol. Ther. 53:298-305 (1993). Orally administered 
erythromycin is systemically available and inhibits liver P450 activity. 
The fact that erythromycin inhibits IV midazolam while grapefruit juice 
does not suggests that the inhibitor in grapefruit juice is either not 
absorbed into the systemic circulation in significant enough quantities to 
affect liver P450 activity or that its effects on P450 are too short-lived 
to affect hepatic clearance. A transient or short-lived effect of 
grapefruit juice seems unlikely given the findings of Lundahl, J. et al., 
Eur. J. Clin. Pharmacol. 49:61 (1995), who reported impaired first-pass 
metabolism of oral felodipine when grapefruit juice was ingested up to 24 
hours before administration of the drug. The most plausible explanation 
for the lack of effect of grapefruit juice on IV administered substrates 
with a prolonged effect on oral compounds is that the inhibitor is either 
not bioavailable, is absorbed slowly from the gut or binds to gut P450 in 
such a way as to inhibit metabolism for many hours. Inhibition of gut wall 
cytochrome P450 may be a more effective chemoprotective mechanism than 
inhibition of hepatic enzymes since many orally ingested procarcinogens 
will have been activated to their mutagenic form prior to reaching the 
liver. 
A number of published reports have attempted to identify the active P450 
enzyme inhibitor in grapefruit juice, a difficult task given that 
grapefruit forms one of the most complete metabolic grids in a single 
plant tissue, with dozens of polyphenolic compounds presenting different 
chemical classes. Most have focused on the flavonoids since several of 
these compounds are known inhibitors of cytochrome P450 and grapefruit 
juice contains high concentrations of flavonoids such as naringin 
(concentrations up to 500 mg/L) and quercetin. However, naringin is a weak 
inhibitor of oxidative metabolism in vitro (Miniscalco, A. et al., J. 
Pharmacol. Exp. Ther. 261:1195-1199 (1992) and Guengerich, F. P. et al., 
Carcinogenesis 11:2275-2279 (1990)) and the administration of naringin in 
aqueous solution or capsule form to human subjects did not significantly 
affect the disposition of substrates for CYP3A4. Bailey, D. G. et al., 
Clin. Pharmacol. Ther. 53:637-442 (1993) and Bailey, D. G. et al., Clin. 
Pharmacol. Ther. 54:589-594 (1993). A dose of quercetin far in excess of 
the typical amount contained in grapefruit juice also had no effect on 
cytochrome P450 activity. Rashid, J. et al., Br. J. Clin. Pharmacol. 
36:46-463 (1993). A number of in vitro experiments confirm that while 
grapefruit juice exhibits potent inhibition of CYP3A, naringin and its 
aglycone naringenin do not significantly contribute to this effect. 
Edwards, D. J. et al. Life Sciences 59:1025-1030 (1996). 
In summary, inhibition of cytochrome P450 enzyme activity and in 
particular, inhibition of the activity of the CYP3A sub-family of enzymes, 
is clearly of therapeutic importance. The co-administration of 
cyclosporine with a CYP3A enzyme inhibitor such as ketoconazole increases 
bioavailability and allows for the use of much lower oral doses of this 
expensive medication. Keogh, A. et al. N. Engl. J. Med. 333:628-633 
(1995). In addition, as outlined above, these enzymes play a role in the 
activation of procarcinogens to their genotoxic form (Shimada T. et al., 
Proc. Natl. Acad. Sci. USA 86:462-465 (1989)), suggesting that a 
cytochrome P450 enzyme inhibitor ingested chronically could be useful in 
the prevention of cancer. It would thus be desirable to provide the 
compound in grapefruit juice responsible for inhibition of cytochrome P450 
enzyme activity. 
SUMMARY OF THE INVENTION 
The present invention provides a composition and methods for inhibiting 
cytochrome P450 enzyme activity and in particular, inhibiting the activity 
of the CYP3A sub-family of enzymes, specifically, CYP3A4. The present 
invention provides 6',7'-dihydroxybergamottin, a furanocoumarin, as the 
compound primarily responsible for the inhibitory effects of grapefruit 
juice on cytochrome P450 activity. The composition of the present 
invention thus comprises 6',7'-dihydroxybergamottin preferably in an oral 
unit dosage form. 
The present invention further provides methods of using 
6',7'-dihydroxybergamottin generally comprising the treatment of a patient 
(in need of such treatment) with a therapeutically-effective amount of 
6',7'-dihydroxybergamottin. The methods preferably include administration 
of 6',7'-dihydroxybergamottin in a pharmaceutically-acceptable vehicle via 
modes known to those skilled in the art, e.g. oral, intravenous, etc. The 
methods of the present invention may also include administration of 
6',7'-dihydroxybergamottin in combination with at least one drug that is 
metabolized by cytochrome P450 enzymes, to increase the bioavailability of 
the drug(s). The present invention further includes methods of making 
6',7'-dihydroxybergamottin. 
Additional objects, advantages, and features of the present invention will 
become apparent from the following description, taken in conjunction with 
the accompanying drawings and claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention relates to a composition and methods for inhibiting 
cytochrome P450 enzyme activity and in particular, inhibiting CYP3A enzyme 
activity. The composition, 6',7'-dihydroxybergamottin, is a potent 
inhibitor of CYP3A activity and is primarily responsible for the effects 
of grapefruit juice on decreasing cytochrome P450 activity in humans. In 
accordance with the present invention, a therapeutically-effective amount 
of 6',7'-dihydroxybergamottin is formulated and administered to a patient 
to inhibit cytochrome P450 enzyme activity. The methods may also be used 
in combination with administration of any active agent, e.g. a drug such 
as cyclosporine, that is metabolized by cytochrome P450 enzymes, in 
particular CYP3A enzymes, to increase the bioavailability of the active 
agent. The methods of the present invention may also be used to reduce the 
activation of procarcinogens by inhibiting cytochrome P450 enzyme 
activity. The methods of the present invention thus find use in any 
patient (human or other mammal) wherein the inhibition of cytochrome P450 
enzyme activity is desirable. 
It should be appreciated that while this invention preferably contemplates 
oral administration of the composition of the present invention, nothing 
herein should be construed to limit the mode of delivery. Both oral and 
systemic routes of delivery may be appropriate, particularly in 
combination-therapy regimes, i.e. administration of 
6',7'-dihydroxybergamottin in combination with an additional active agent. 
Further, it should also be appreciated that 6',7'-dihydroxybergamottin and 
any additional active agent need not be administered in the same manner, 
i.e., one may be administered orally while another may be administered 
systemically. It therefore follows that while the compositions selected 
for the methods of the present invention are preferably administered 
concomitantly, the administration need not be co-instantaneously. It is 
preferred that they be administered such that their therapeutic effects 
are synchronized or overlap. Based upon ease of treatment, however, in a 
highly preferred embodiment, the selected compositions are administered 
separately, in individual unit dosage form, including tablet and capsule. 
It will be appreciated that the composition of the present invention may 
also be employed in a pharmaceutically-acceptable form such as an ester, 
salt, or as a prodrug. In addition, it will be appreciated that homologues 
and analogues of 6',7'-dihydroxybergamottin which also decrease cytochrome 
P450 enzyme activity, are further contemplated by the present invention. 
The synthesis schemes for 6',7'-dihydroxybergamottin provided herein may 
be readily adapted by those skilled in the art for the production of 
homologues and analogues of 6',7'-dihydroxybergamottin. 
In practicing the methods of the present invention, the amount of 
6',7'-dihydroxybergamottin to be administered (as well as any other active 
agents in the treatment regime) will vary with the patient being treated 
and will be monitored on a patient-by-patient basis by the physician or 
other health-care provider. Generally, a therapeutically-effective amount 
of the compound of the present invention will be applied for a 
therapeutically-effective duration. By "therapeutically-effective amount" 
and "therapeutically-effective duration" is preferably meant an amount or 
duration effective to achieve a selected desired result in accordance with 
the present invention without undue adverse physiological effects or side 
effects; the desired result generally being a clinically observable 
increase in the bioavailability of certain active agents, i.e., drug(s), 
and/or the inhibition of cytochrome P450 activity. 
It should be appreciated that duration of treatment according to the 
methods of the present invention will vary with many factors and will 
primarily depend upon the specific condition of the patient and the 
specific combination of agents employed. It should also be appreciated 
that treatment agents, dosage and duration will be interdependent and can 
be varied together in order to achieve an optimal clinical response. In 
addition, dosage and duration will also depend on the specific combination 
of agents employed. 
The agents utilized in the compositions and methods of the present 
invention can be administered in accordance with the present invention in 
any pharmaceutically-acceptable carrier, preferably one which is both 
non-toxic and suitable for oral delivery. The compounds may be formulated 
for administration by procedures well-established in the pharmaceutical 
arts. 
As set forth herein, 6',7'-dihydroxybergamottin is a potent CYP3A enzyme 
inhibitor in vitro with a concentration of about 1 to 2 .mu.M being 
required to reduce CYP3A activity by about 50%, using enzyme expressed 
from transfected human CYP3A4 cDNA. Concentrations of 
6',7'-dihydroxybergamottin in naturally occurring grapefruit juice are in 
the range of about 20 to 60 .mu.M (10-30 mg/L). In unit dosage form, about 
5 to about 20 mg of 6',7'-dihydroxybergamottin is preferred and about 10 
mg 6',7'-dihydroxybergamottin is most preferred. 
For example, a pharmaceutical preparation in unit dosage form adapted for 
administration to promote bioavailability of a drug of interest may be 
prepared comprising, per unit dosage, an effective non-toxic amount of 
6',7'-dihydroxybergamottin within the range of from about 5 to about 20 
mg. A pharmaceutical preparation may also be adapted to include both a 
therapeutically-effective amount of 6',7'-dihydroxybergamottin in addition 
to a therapeutically-effective amount of a drug of interest. As used 
herein, "drug of interest" or "active agent" is meant to include any drug 
metabolized by cytochrome P450 enzyme, and in particular, the P450 3A 
family of enzymes, preferably CYP3A4, wherein increased bioavailability of 
the drug is desirable. Such drugs include, but are not limited to, 
felodipine, triazolam, midazolam, terfenadine, cyclosporine, quinidine, 
nifedipine, ethinylestradiol, lovastatin, retinoic acid, steroids, taxol 
and verapamil. 
Such unit dosage preparations may be adapted for oral administration as a 
tablet, capsule, liquid, powder, bolus or the like. They may likewise be 
prepared in unit dosage form in an ingestible or injectable form. 
Art-disclosed formulation ingredients such as tableting agents, colorants, 
flavorants, anti-oxidants, emollients, surface-active agents, 
encapsulation agents, and the like may also be employed. 
The Specific Examples below further describe the compositions and methods 
of the present invention. These examples are for illustrative purposes 
only and are not intended in any way to limit the scope of the invention. 
Specific Example 1 describes the isolation, synthesis and activity of 
6',7'-dihydroxybergamottin. Specific Example 2 describes an additional, 
and more efficient, synthesis scheme for 6',7'-dihydroxybergamottin. 
Specific Example 3 shows the inhibitory effect of 
6',7'-dihydroxybergamottin on cytochrome P450 CYP3A4. Specific Example 4 
describes the quantification of 6',7'-dihydroxybergamottin in naturally 
occurring juice, in vivo absorption and disposition of 
6',7'-dihydroxybergamottin, and the effect of 6',7'-dihydroxybergamottin 
on enzyme activity. Specific Example 5 describes pharmaceutical methods of 
use of 6',7'-dihydroxybergamottin. 
SPECIFIC EXAMPLE 1 
Methods 
Chemicals. Bergamottin was obtained from Indofine Chemical Company, Inc. 
(Somerville, N.J.). Testosterone, 6.beta.-hydroxytestosterone and 
11.beta.-hydroxytestosterone were purchased from Steraloids Inc. (Wilton, 
N.H.). Ketoconazole was obtained from Janssen Pharmaceutica (Belgium). All 
other chemicals were supplied by Sigma Chemical Co. (St. Louis, Mo.). 
Isolation of 6',7'-dihydroxybergamottin by TLC. Grapefruit juice prepared 
from frozen concentrate (Old South.TM., Lykes Pasco, Dade City, Fla.) was 
extracted into methylene chloride. Following evaporation of the organic 
phase, the residue was dissolved in a small volume of methylene chloride, 
applied to TLC plates (Silica Gel, 250 .mu.M, Sigma Chemical Co.) and 
eluted with hexane:acetone (6:4). The R.sub.f for 
6',7'-dihydroxybergamottin was 0.35 under these conditions. The compound 
of interest was isolated from the plates and re-chromatographed using a 
methylene chloride:acetone (3:2) solvent system (R.sub.f =0.6). After 
extraction from the silica gel with ethyl acetate, 
6',7'-dihydroxybergamottin was crystallized from a hexane:ethyl acetate 
mixture. Approximately 5 mg of 6',7'-dihydroxybergamottin was obtained 
from each liter of juice. 
Synthesis of 6',7'-dihydroxybergamottin. 6',7'-dihydroxybergamottin was 
synthesized according to procedures described by Dreyer and Huey. Dreyer, 
D. L. et al., Citrus Macroptera. Phytochem. 12:3011-3013 (1973). The yield 
of 6',7'-dihydroxybergamottin from bergamottin was 16%. 
Measurement of CYP3A Activity. CYP3A activity was assessed by measuring the 
formation rate of 6.beta.-hydroxytestosterone from testosterone in 
dexamethasone-induced liver microsomes from male Sprague-Dawley rats 
(Human Biologics Inc., Phoenix, Az.) using incubation conditions identical 
to those described previously. Edwards, D. J. et al., Life Sci. 
59:1025-1030 (1996). Liver microsomes from rats were studied since they 
have been reported to exhibit comparable inhibition to human liver 
microsomes in studies with other naturally occurring inhibitors such as 
the flavonoids. Siess, M.-H. et al., Toxicol. Appl. Pharmacol. 130:73-78 
(1995). In addition, 6.beta.-hydroxytestosterone formation was also 
measured in human lymphoblast microsomes containing CYP3A4 expressed from 
transfected human CYP3A4 cDNA (Gentest Corporation, Woburn, Mass.). 
6.beta.-hydroxytestosterone formation was linear with respect to 
incubation time, microsomal protein and substrate concentration over the 
incubation period. 
HPLC Procedures and instrumentation. HPLC was used to quantitate 
6.beta.-hydroxytestosterone concentrations in metabolic experiments 
(Brian, W. R. et al., Biochem. 29:11280-11292 (1990)) and to measure the 
concentration of 6',7'-dihydroxybergamottin in grapefruit juice and orange 
juice. The internal standard (11.beta.-hydroxytestosterone) was added to 
0.5 ml of the incubation mixture (to measure 6.beta.-hydroxytestosterone) 
or 0.3 ml of citrus juice (to measure 6',7'-dihydroxybergamottin). 
Methylene chloride (4 ml) was added and samples were vortex mixed for 2 
minutes. After centrifugation, the organic phase was evaporated, the 
residue re-constituted in 200 .mu.l of mobile phase (55% methanol in 
water) and injected (100 .mu.l) onto the column (Partisil 5 ODS-3 25 cm 
C.sub.18 column, Whatman Inc., Clifton, N.J.). A mobile phase flow rate of 
1.2 ml/minute was used with detection of analytes at 254 nm (Waters 490E 
multiwavelength UV detector, Waters Corp., Milford, Mass.). Retention 
times for 6.beta.-hydroxytestosterone, 11.beta.-hydroxytestosterone, 
6',7'-dihydroxybergamottin and testosterone were typically 7, 10, 16 and 
20 minutes respectively. Standard curves for 6.beta.-hydroxytestosterone 
were linear over the range of 2.5 to 20 .mu.M. The coefficient of 
variation of the assay at a concentration of 10 .mu.M was 6.5%. 
6',7'-dihydroxybergamottin was measured over the range of 5 to 80 .mu.M in 
juice. 
Metabolic Experiments. Grapefruit juice (1 ml) was extracted into methylene 
chloride and, following evaporation of the organic phase, the residue was 
reconstituted in mobile phase and chromatographed by HPLC. Fractions of 
the HPLC eluent were collected at 3-4 minute intervals following 
injection, evaporated and the effect of the residue on 
6.beta.-hydroxytestosterone formation examined. The concentration of 
6',7'-dihydroxybergamottin required to reduce the formation of 
6.beta.-hydroxytestosterone by 50% (IC.sub.50) was measured by adding 
synthetic 6',7'-dihydroxybergamottin to the incubation mixture in 
concentrations ranging from 10 to 100 .mu.M. For comparison, the IC.sub.50 
was also determined for ketoconazole and cimetidine. To evaluate the 
contribution of 6',7'-dihydroxybergamottin to the inhibition produced by 
grapefruit juice, the compound was added to orange juice at a 
concentration of 30 .mu.M (similar to the concentration measured in 
grapefruit juice). After adjustment to pH 7.4 using sodium hydroxide, the 
juice was added to the incubation mixture and its effects on CYP3A 
activity compared to grapefruit juice, grapefruit juice extracted with 
methylene chloride to remove 6',7'-dihydroxybergamottin, orange juice and 
orange juice spiked with naringin. Edwards, D. J. et al., Life Sci. 
59:1025-1030 (1996). The effect of these solutions on 
6.beta.-hydroxytestosterone formation was compared using analysis of 
variance (SYSTAT for Windows.TM., Version 5.0) with the Tukey test for 
post-hoc analysis (p&lt;0.05 for significance). 
Results 
Extraction of grapefruit juice into methylene chloride resulted in a 
chromatogram with a number of peaks following injection into the HPLC 
(FIG. 1B). However, only the eluent fraction containing the peak at 15.6 
minutes was capable of inhibiting 6.beta.-hydroxytestosterone formation 
(FIG. 1A). TLC was used to isolate the compound producing this peak. A 
carbon NMR spectrum suggested that the compound contained 21 carbon atoms. 
A proton NMR spectrum (General Electric GE300 NMR spectrometer) in 
deuterated chloroform matched that reported by Tatum and Berry (Tatum, J. 
H. et al., Phytochemistry 18:500-502 (1979)) for 
6',7'-dihydroxybergamottin (C.sub.21 H.sub.24 O.sub.6, MW 372), a 
furanocoumarin (psoralen) isolated from grapefruit peel oil (FIG. 2B). 
Dreyer and Huey (Dreyer, D. L. et al., Citrus Macroptera. Phytochem. 
12:3011-3013 (1973)) had previously identified the same compound in Citrus 
macroptera, a fruit native to the south Pacific islands. 
6',7'-dihydroxybergamottin had not been previously identified in 
grapefruit juice. Authentic 6',7'-dihydroxybergamottin was synthesized and 
found to be identical to the compound isolated from grapefruit juice by 
comparing the proton NMR spectrum, HPLC retention time, EI-MS and CI-MS 
data. The CI/MS indicated a parent (M+H) ion at m/z 373 with other 
prominent ions at m/z 355, m/z 203 and m/z 153 (FIG. 2B). The spectrum is 
dominated by the fragment (M+H) ion at 203 which represents 
5-hydroxypsoralen (bergaptol). The peak at m/z 355 is due to the loss of 
water from the side chain of 6',7'-dihydroxybergamottin with the large 
fragment ion at m/z 153 being associated with the remainder of the 
geranyloxy side chain. 
The concentration of 6',7'-dihydroxybergamottin required to inhibit 
6.beta.-hydroxytestosterone formation by 50% was found to be 25 .mu.M in 
rat liver microsomes. The IC.sub.50 was 1.8 .mu.M for ketoconazole and 
more than 100 .mu.M for cimetidine. 6',7'-dihydroxybergamottin was less 
potent than ketoconazole but was considerably more active than cimetidine. 
6',7'-dihydroxybergamottin was much more potent in inhibiting the human 
CYP3A4 enzyme with an IC.sub.50 of 1-2 .mu.M. 
Concentrations of 6',7'-dihydroxybergamottin in re-constituted frozen 
concentrated grapefruit juice (regular strength) were measured by HPLC and 
found to average 30 .mu.M (12 mg/L). Orange juice (freshly squeezed from 
Navel oranges) contained no measurable 6',7'-dihydroxybergamottin. The 
formation of 6.beta.-hydroxytestosterone in the presence of grapefruit 
juice and orange juice treated in order to vary the native concentration 
of 6',7'-dihydroxybergamottin is listed in Table 1. Orange juice was a 
much weaker inhibitor of CYP3A activity than grapefruit juice (p&lt;0.05). 
There was no significant difference between the degree of inhibition 
produced by grapefruit juice and that produced by orange juice spiked with 
either synthetic 6',7'-dihydroxybergamottin or material extracted from 
grapefruit juice. Extraction with methylene chloride removed essentially 
all 6',7'-dihydroxybergamottin from grapefruit juice and reduced 
inhibitory activity to values comparable to control. 
TABLE 1 
______________________________________ 
Effect of Grapefruit Juice, Orange Juice and Orange Juice 
Spiked with 6',7'-dihydroxybergamottin or Naringin on 
6.beta.-hydroxytestosterone Formation in Rat Liver Microsomes 
Solution Tested (n = 5) 
% of Control Activity (Mean .+-. SD) 
______________________________________ 
Grapefruit Juice with 
28.6 .+-. 1.7 
6',7'-dihydroxybergamottin 30 .mu.M 
Orange Juice spiked with synthetic 
31.6 .+-. 0.8 
6',7'-dihydroxybergamottin 30 .mu.M 
Orange Juice spiked with extracted 
34.7 .+-. 1.1 
6',7'-dihydroxybergamottin 30 .mu.M 
Orange Juice spiked with naringin 
60.4 .+-. 2.0* 
450 mg/L 
Orange Juice 62.2 .+-. 3.3* 
Grapefruit Juice following organic 
82.9 .+-. 1.9* 
extraction with methylene chloride 
______________________________________ 
*Significantly different from grapefruit juice or orange juice containing 
6',7dihydroxybergamottin 
SPECIFIC EXAMPLE 2 
Synthesis of 6',7'-dihydroxybergamottin 
An efficient synthesis of 6',7'-dihydroxybergamottin is provided and 
outlined in FIG. 3. The commercially available furanocoumarin bergapten 
(indicated as compound 3 in FIG. 3) was demethylated using a previously 
published procedure (Miniscalco, A. et al., J. Pharmac Exp. Ther. 
261:1195-1199 (1992)), producing the corresponding phenolic derivative 
bergaptol (indicated as compound 4 in FIG. 3). Geranylation of bergaptol 
(4) under phase transfer conditions (geranyl bromide, benzyl 
tributylammonium bromide, NaOH) then afforded bergamottin (indicated as 
compound 1 in FIG. 3) in 98% yield. Subsequent treatment of bergamottin 
(1) with m-chloroperbenzoic acid then resulted in selective epoxidation of 
the terminal double bond of the geranyl sidechain, affording 
bergamottin-6,7-epoxide 5 in 65% yield. Opening of the epoxide (3% 
perchloric acid in dioxane) then produced the desired 
6',7'-dihydroxybergamottin (indicated as compound 2 in FIG. 3) in 70% 
yield. 
SPECIFIC EXAMPLE 3 
CYP3A4 Inhibition By 6',7'-dihydroxybergamottin 
6',7'-dihydroxybergamottin was evaluated as an inhibitor of CYP3A4 (3A4) in 
a purified enzyme preparation, as well as against human liver microsomes 
and human 3A4 expressed in E. coli membrane. In each case, 
6',7'-dihydroxybergamottin proved to be a potent NADPH- and time dependent 
inactivator of 3A4. 
Methods 
Coexpression of P450 3A4 and reductase in E. coli. The plasmid pB216 
containing human P450 3A4 cDNA and human P450 reductase cDNA was 
transformed into JM 109 cells. Ducharme, M. P. et al., Br. J. Clin. 
Pharmac. 36:457-459 (1993). The growth of the transformed E. coli was 
carried out in the modified Terrific Broth and the expression of P450 3A4 
and reductase was induced by addition of 1 mM IPTG. .delta.-Aminolevulinic 
acid (0.5 mM) was added for heme synthesis. The membrane fraction was 
prepared from the bacterial cells by sonication after treatment with 
lysozyme. It was subsequently isolated by differential centrifugation from 
the bacterial cell homogenate. Ducharme, M. P. et al., Br. J. Clin. 
Pharmac. 36:457-459 (1993). 
Inactivation of P450 3A4 in human liver microsomes and E. coli membranes. 
The human liver microsomes or the E. coli membrane fractions containing 1 
nmol of P450 3A4 were incubated with various concentrations of 
6',7'-dihydroxybergamottin in 1 ml of 50 mM Hepes buffer (pH 7.5) 
containing 2 mM GSH, 1000 U catalase, 0.5 mM EDTA, 30 mM MgCl.sub.2 and 
20% glycerol at 37.degree. C. for various time periods. At end of the 
incubation, 100 .mu.l of the incubation mixture was taken for 
determination of testosterone 6.beta.-hydroxylation activity. The other 
aliquot was used for P450 determination by the method of Omura and Sato. 
Ducharme, M. P. et al., Clin. Pharmacol. Ther. 57:485-491 (1995). The 
human liver microsomes was prepared by differential centrifugation. 
Determination of testosterone 6.beta.-hydroxylation. The human liver 
microsomes or the P450 3A4 and reductase containing E. coli membrane 
fractions (100 .mu.mol of P450) were incubated with 200 .mu.M testosterone 
in 1 ml of 50 mM Hepes buffer (pH 7.5). The reactions were initiated by 
the addition of 1 mM NADPH and terminated by 1 ml of ethyl acetate in ice. 
The 6.beta.-hydoxytestosterone product was determined by HPLC on a C18 
column (Microsorb-MV, 5 .mu.m, 4.6.times.15 cm) eluted with a mobile phase 
of 65% of methanol at flow rate of 1 ml/min and the eluate was monitored 
by UV detection at 254 nm. 
Results 
P450 3A4 activity was significantly inhibited by 6',7'-dihydroxybergamottin 
when either human liver microsomes or E. coli membranes containing the 
expressed human P450 3A4 and reductase were used. As shown in FIG. 4, the 
inhibition of 6.beta.-hydroxytestosterone formation activity in human 
liver microsomes was time and concentration dependent as well as requiring 
catalytic turnover of 6',7'-dihydroxybergamottin, which suggested that the 
inhibition resulted from the mechanism based-inactivation of P450 3A4. 
Guengerich, P. et al., Carcinogenesis 11:2276-2279 (1990). The 
inactivation appeared to be due to a reaction at the active site because 
it was not inhibited by the presence of 2 mM GSH in the incubation system. 
Competitive inhibition of the catalytic activity was also observed for the 
samples without preincubation where approximately 30% of testosterone 
6.beta.-hydroxylase activity was inhibited when 6',7'-dihydroxybergamottin 
was incubated with human liver microsomes at a final concentration of 20 
.mu.M, and about 45% of the activity of the expressed P450 3A4 was 
inhibited when it was incubated with 6',7'-dihydroxybergamottin in the 
absence of NADPH (see Table 2). Despite the loss of 70% of the P450 3A4 
catalytic activity by preincubation with 6',7'-dihydroxybergamottin and 
NADPH, human liver microsomes still retained about 90% of the P450 as 
measured by the reduced-CO spectrum (see Table 2). Similar results were 
obtained with the E coli. expressed P450 3A4 where 90% of catalytic 
activity was lost by 90% after preincubation with 
6',7'-dihydroxybergamottin and NADPH for 15 min, but there was not any 
loss of spectrally detectable P450 (see Table 2). Studies on inactivation 
by another furanocumarin, 8-methoxyporalen, have also shown that there was 
no heme fragment, MI complex and heme adduct formation in in vivo studies 
of 8-methoxyporalen treated rats or in in vitro studies of 
8-methoxyporalen incubated with rat liver microsomes. (Kronbach, T. et 
al., Clin. Pharmac. Ther. 43:630-635 (1988); Edwards, D. J. et al., Drug 
Metab. Dispos. 24:1287-1290 (1996). But, 8-methoxyporalen was extensively 
bound to microsomal apoP450, and the binding was only partially diminished 
by cysteine. Another example involved inactivation of P450 1A1 and 1A2 by 
coriandrin, a linear furoisocoumarin. In this case, covalent binding of 
the coriandrin to apoP450 was demonstrated without significant heme. 
Tatum, J. H. et al., Phytochemistry 18:500-502 (1979). Therefore, 
6',7'-dihydroxybergamottin-mediated inactivation of P450 3A4 appears to be 
primarily a result of modification of the apoprotein, as has been observed 
with 2-or 9-ethynylphenanthrene for the mechanism based inactivation of 
P450 2B1 and 2B4. Chatterjee, A. et al., J. Chem. Soc. 2245-2247 (1961); 
Schonberg, A. et al., J. Am. Chem. Soc. 77:2563-2564 (1955). 
TABLE 2 
______________________________________ 
6',7'-dihydroxybergamottin (DHB)-Mediated 
Inactivation Of P450 3A4* 
Testosterone 
6-.beta. hydroxylation 
P450 Content 
(nmol/nmol P450/min) 
(nmol/ml) 
______________________________________ 
Human liver microsomes 
+NADPH/-DHB 0.54 0.94 
-NADPH/+DHB 0.37 0.93 
+NAHPH/+DHB 0.18 0.87 
Coexpressed P450 3A4 with 
reductase in E. coli membrane 
+NADPH/-DHB 8.13 1.20 
-NADPH/+DHB 4.45 1.16 
+NADPH/+DHB 0.45 1.20 
______________________________________ 
*Human liver microsomes or the E. coli. membrane containing P450 3A4 
coexpressed with reductase (1 nmol/ml) were incubated with 400 .mu.M DHB 
for 15 min at 37.degree. C. in 50 mM Hepes buffer (pH 7.5) containing 30 
mM MgCl.sub.2, 2 mM GSH, 0.5 mM EDTA, 1000 u/ml of catalase and 20% 
glycerol. 
SPECIFIC EXAMPLE 4 
I. Quantification of 6',7'-dihydroxybergamottin in grapefruit and other 
fruits 
Using the high performance liquid chromatographic assay set forth in 
Specific Example 1, the concentration of 6',7'-dihydroxybergamottin is 
measured in grapefruit juice (and juice from a number of sources (fresh 
squeezed from different varieties of grapefruit, commercially prepared 
juice, juice from frozen concentrate)) and at various times of the year. 
Other citrus fruits (e.g. sweet oranges, sour oranges (Seville), lemon, 
pummelo), non-citrus fruits and beverages derived from natural products, 
such as tea, are screened for the presence of this compound. 
II. Absorption and disposition of 6',7'-dihydroxybergamottin 
In human subjects, oral administration of grapefruit juice (containing a 
known amount of 6',7'-dihydroxybergamottin) allows for an estimation of 
bioavailability from urinary recovery studies. Oral clearance, absorption 
rate, and elimination half-life are calculated from plasma concentrations. 
A chronically catherized rat model is used to compare the disposition of 
6',7'-dihydroxybergamottin following oral, intraportal and IV 
administration. Raftogianis, R. B. et al., J. Pharmacol. Exp. Ther. 
276:602-608 (1996) and Kimura, R. E. et al., Pediatr.Res. 23:235-240 
(1988). 6',7'-dihydroxybergamottin is incubated with human liver 
microsomes and metabolic products identified using NMR, mass spectroscopy 
and other standard techniques. 
A. Disposition of 6',7'-dihydroxybergamottin in the rat. The absorption 
process is relatively consistent between species (Lin, J. H., Drug Metab. 
Dispos. 23:1008 (1995)) and the pharmacokinetic profile in the rat 
provides valuable information in designing human studies with this 
compound. Drug is administered intravenously, intraportally or into the 
duodenum with sampling from the aorta or portal vein for up to 21 days. 
Raftogianis, R. B. et al., J. Pharmacol. Exp. Ther. 276:602-608 (1996) and 
Kimura, R. E. et al., Pediatr.Res. 23:235-240 (1988). Pharmacokinetic 
parameters such as clearance, volume of distribution and absolute 
bioavailability require IV administration for precise calculation. In 
addition, oral and intraportal dosing can distinguish between the gut and 
liver first-pass metabolism of compounds. This data is particularly 
relevant for 6',7'-dihydroxybergamottin since it inhibits cytochrome P450 
in the intestinal wall. 
Male Sprague Dawley rats (175-200 gm) are studied. This strain is widely 
used to study xenobiotic disposition in rodents. Animals are allowed to 
acclimatize for 2 weeks before surgery is performed. Animals are 
anesthetized with 20 mg/ml ketamine intramuscularly followed by 10 mg/kg 
pentobarbital intraperitoneally. The abdomen and back of the neck are 
shaved, washed with ethanol and painted with 1% Betadine. Using aseptic 
technique, a 5 cm vertical midline incision is made from the subxiphoid 
process to the suprapubic region. Cannulae (hand prepared from silastic 
tubing, JELCO intravenous catheter placement unit and PE 60 tubing) are 
placed into the aorta, inferior vena cavae, portal vein and duodenum. 
Infusion sets are sutured securely to the back of the rat following 
closing of the abdominal cavity. Catheters are flushed twice daily with 
500 u/mL heparin and 4 mg/ml ampicillin. The entire surgical procedure 
takes approximately 20 minutes. Rats are weighed and monitored daily for 
signs of distress and infection. 
6',7'-dihydroxybergamottin is administered into the duodenum, intravenously 
and directly into the portal vein. Since the surgical model remains intact 
for up to 21 days, each animal receives compound by all three routes with 
a 3 day washout period between treatments. Since preliminary studies 
indicate that the typical dose of 6',7'-dihydroxybergamottin in a glass of 
grapefruit juice is roughly 10-15 mg (0.15-0.2 mg/kg), initial experiments 
use a similar dose (0.2 mg/kg) for all routes of administration. The 
compound is much more soluble in ethanol than water. In order to minimize 
the volume to be administered, 6',7'-dihydroxybergamottin is initially 
dissolved in a small volume of ethanol and diluted to the required 
concentration with water (final ethanol content is expected to be less 
than 5%). Animals are free moving but maintained in metabolism cages 
throughout the experiments. Urine is collected after administration of 
6',7'-dihydroxybergamottin to assess urinary recovery of parent compound 
and metabolites. Blood samples for estimation of standard pharmacokinetic 
parameters are collected (0.25 ml) at various times for several hours 
after administration. Finally, samples are obtained from the portal vein 
following duodenal administration of compound. 
Plasma concentrations of 6',7'-dihydroxybergamottin are determined using 
the HPLC assay described herein. Pharmacokinetic parameters such as 
clearance, volume of distribution, and elimination half-life are 
calculated following IV administration using standard non-compartmental 
techniques (area estimation using the computer program LAGRAN). 
Bioavailability is calculated for oral and intraportal administration of 
the drug by comparing the area under the plasma concentration-time curve 
to that observed with IV administration. Intraportal administration allows 
for an assessment of the intrinsic clearance of the compound by the liver. 
A significant difference between oral and intraportal availability 
suggests poor absorption or gut wall metabolism of 
6',7'-dihydroxybergamottin. Samples obtained from the portal vein 
following duodenal administration determine whether drug is reaching the 
portal circulation intact. A comparison is also made of the plasma 
concentration-time profile of 6',7'-dihydroxybergamottin when administered 
orally as grapefruit juice or purified compound in order to determine 
whether bioavailability is dependent upon oral formulation. Studies with 
8-methoxypsoralen have suggested that formulation is an important 
determinant of oral bioavailability. Hertog, M. G. L. et al., Lancet 
342:1007-1011 (1993). Statistical comparisons are made using analysis of 
variance (when the same parameter is being compared between more than two 
groups of animals) or the t-test for comparisons between 2 groups. A value 
of p&lt;0.05 is considered to be statistically significant. 
B. Disposition of 6',7'-dihydroxybergamottin in humans. Since 
6',7'-dihydroxybergamottin is consumed by a significant proportion of the 
population as part of their usual diet, the oral administration of this 
compound to humans in the form of grapefruit juice poses no significant 
ethical or toxicologic risk. 
Grapefruit juice is administered initially to twenty healthy non-obese 
(within 20% of ideal body weight) subjects (male or female) between the 
ages of 18 and 70 years of age. Subjects are asked to refrain from 
grapefruit and any other food identified as having a high content of 
6',7'dihydroxybergamottin for 1 week prior to study in order to ensure 
that baseline concentrations of 6',7'-dihydroxybergamottin are not 
significant. Following an overnight fast, subjects ingest 300 ml of juice 
prepared from frozen concentrate at approximately 8 am. The concentration 
of 6',7'-dihydroxybergamottin is determined prior to administration in 
order to calculate the administered dose. Based on preliminary data, it is 
expected that 300 ml of juice contains between 10 and 15 mg of 
6',7'-dihydroxybergamottin. No food is allowed until 4 hours after 
administration of grapefruit juice at which time a standard meal 
(containing foods previously screened and found to be free of 
6',7'-dihydroxybergamottin) is given. Initial studies focus on the urinary 
recovery of 6',7'-dihydroxybergamottin and major metabolites identified by 
preliminary studies in order to assess the oral bioavailability of the 
compound. Urine is collected prior to and at timed intervals from 0-2, 
2-4, 4-8 and 8-12 hours after juice administration. Concentrations in 
urine are typical several-fold higher than plasma concentrations for most 
xenobiotics allowing for a more sensitive assessment of bioavailability. 
For example, even if only 1% of the administered dose of 
6',7'-dihydroxybergamottin is found in the urine, concentrations with 
typical urine volumes should be in the range of 0.1 mg/L and detectable by 
the HPLC assay method. 
Further studies are conducted with collection of blood after administration 
of grapefruit juice. Blood samples (5 ml) are collected from an 
antecubital vein immediately prior to administration of juice and at 0.5, 
1.0, 2.0, 3.0, 4.0, 6.0, 8.0 and 12.0 hours after ingestion of juice. 
Samples at later times may be obtained if preliminary data suggests that 
the compound has a long half-life. Blood is collected into red top blood 
collection tubes, allowed to clot and the serum harvested and stored 
pending analysis. Subjects are allowed water ad libitum throughout the 
study and standard meals are given at 4 and 10 hours into the study. 
Plasma and urine are assayed for 6',7'-dihydroxybergamottin concentration 
using the HPLC assay described herein. Pharmacokinetic parameters are 
calculated using standard non-compartmental methods. Area under the plasma 
concentration-time curve is estimated using Lagrange polynomial 
interpolation. Oral clearance (CI/F), apparent volume of distribution 
(Vss/F), elimination half-life, time to reach maximum plasma concentration 
(tmax) and maximum plasma concentration (Cmax) is calculated from the 
plasma data. 
III. Effect of 6',7'-dihydroxybergamottin on enzymes involved in the 
oxidation and conjugation of foreign compounds 
Using isolated pure compound, in vitro studies are conducted with human 
liver microsomes to determine the specificity, potency and mechanism of 
inhibitory effects on substrates for the primary cytochrome P450 enzymes 
involved in pro-carcinogen activation (including, but not limited to, 
CYP1A1, CYP1A2, CYP2A6, CYP2D6, CYP2E1 and CYP3A4). Since conjugation may 
also result in bioreactive metabolites, phenol sulfotransferase, 
UDP-glucuronosyltransferase and glutathione S-transferase activity is 
measured under control conditions and following incubation with 
6',7'-dihydroxybergamottin. 
A. Inhibition of cytochrome P450 enzymes by 6',7'-dihydroxybergamottin. 
Studies are conducted using a similar model as described herein, which 
have been useful in evaluating the inhibitory effects of grapefruit juice. 
It has been established that inhibition is concentration-dependent and 
requires no pre-incubation with microsomes suggesting that 
6',7'-dihydroxybergamottin does not have to be converted to a more active 
form in order to inhibit enzyme activity. However, it is possible that 
pre-incubation could enhance the inhibitory effects of the compound. 
Initial mechanistic studies examine the effect of 
6',7'-dihydroxybergamottin in the formation of 6.beta.-hydroxytestosterone 
as a marker of CYP3A activity. Product formation is be compared in the 
presence of the inhibitor over a wide range of concentrations and the data 
plotted according the methods of Dixon and Comish-Bowden. Dixon, M., 
Biochem. J. 55:470-471 (1953) and Cornish-Bowden, A., Biochem. J. 
137:143-144 (1974). Both methods are employed since the Dixon plot allows 
for discrimination between competitive and uncompetitive inhibition while 
the Cornish-Bowden plot is superior in distinguishing between competitive 
and non-competitive inhibition. Comparisons are made between inhibition 
produced with and without pre-incubation of the inhibitor. In addition, 
P450 concentration is determined during incubation of 
6',7'-dihydroxybergamottin with microsomes in order to examine the 
hypothesis that the compound may exhibit mechanism-based inhibition. 
The specificity and potency of 6',7'-dihydroxybergamottin inhibition of 
individual P450 enzymes is studied by measuring the rates of metabolism of 
known substrates for specific enzymes. These include ethoxyresorufin 
deethylase (CYP1A1 and CYP1A2), (Burke, M. D. et al., Biochem. Pharmacol. 
34:3337-3345 (1985)) coumarin 7-hydroxylation (CYP2A6), (Greenlee, W. F. 
et al., J. Pharmacol. Exp. Ther. 205:596-605 (1978)) bufuralol 
1-hydroxylation (CYP2D6), (Brian, W. R. et al., Biochemistry 
29:11280-11292 (1990)) chlorzoxazone 6-hydroxylation (CYP2E1) (Peter, R. 
et al., Chem. Res. Toxicol. 3:566-573 (1990)) and testosterone 
6.beta.-hydroxylation (CYP3A4). Sonderfan, A. J. et al., Arch. Biochem. 
Biophys. 255:27-41 (1987). Individual cDNA-expressed human cytochrome P450 
enzymes are obtained from Gentest Corporation (Woburn, Mass.) and 
incubated with substrate and NADPH-regenerating system. 
6',7'-dihydroxybergamottin is added to the incubation mixture over a wide 
range of concentrations in order to determine the concentration required 
to reduce the rate of the reaction by 50% (IC.sub.50) for each of the 
common cytochrome P450 enzymes. For comparison, IC.sub.50 values are 
determined for known specific inhibitors of each of these enzymes (e.g. 
furafylline (CYP1A2), quinidine (CYP2D6), ketoconazole (CYP3A4)). 
B. Effect of 6',7'-dihydroxybergamottin on substrate conjugation. Galinsky 
and co-workers Raftogianis, R. B. et al., J. Pharmacol. Exp. Ther. 
276:602-608 (1996) have examined the effect of total parenteral nutrition 
on hepatic drug conjugation in vitro and in vivo. Since the effect of 
grapefruit juice or 6',7'-dihydroxybergamottin on drug conjugation has not 
been previously studied, experiments are conducted in vitro in order to 
assess the need for follow-up studies in humans or animals. 
Microsomal or cytosolic fractions are prepared from male Sprague-Dawley 
rats according to established procedures. Raftogianis, R. B. et al., J. 
Pharmacol. Exp. Ther. 276:602-608 (1996). Cytosolic glutathione 
S-transferase activity is assessed under control conditions and with 
varying concentrations of 6',7'-dihydroxybergamottin using 
1-chloro-2,4-dinitrobenzene as substrate. Acetaminophen and p-nitrophenol 
are used as substrates for phenol sulfotransferase in cytosol and for 
assessment of glucuronosyltransferase activity in microsomes. Raftogianis, 
R. B. et al., J. Pharmacol. Exp. Ther. 276:602-608 (1996); Habig, W. H. et 
al., J. Biol. Chem. 249:7130-7139 (1974) and Ritter, J. K. et al., Drug 
Metab. Dispos. 15:335-343 (1987). Sulfotransferase activity towards 
acetaminophen is determined in 0.1M citric acid-0.2M sodium phosphate 
buffer (pH 5.7) containing 5-10 mg/ml cytosolic protein, 500 .mu.M PAPS, 
and 600 .mu.M acetaminophen. The reaction is terminated by addition of 
perchloric acid after a 10 minute incubation at 25 C. Acetaminophen 
sulfate is measured by HPLC. Corcoran, G. B. et al., J. Pharmcol. Exp. 
Ther. 232:857-863 (1985). For determination of the glucuronidation of 
acetaminophen, the incubation mixture consists of 50 mM tris buffer (pH 
7.85) containing 150 mM KCl, 10 mM MgCl.sub.2, 2 mg/mL microsomal protein, 
60 mM acetaminophen and 0.05% Triton X-100. The reaction is initiated 
through addition of 5 mM UDPGA and terminated after 15 minutes at 37 C. by 
addition of perchloric acid. 
SPECIFIC EXAMPLE 5 
Cyclosporine is widely used as an immunosuppressant drug in order to 
prevent rejection following transplantation. Although highly effective, 
there are significant problems with the use of cyclosporine. It is an 
extremely expensive drug with annual treatment costs which can be in the 
range of several thousand dollars per patient. In addition, it is 
difficult to titrate the dosage to provide adequate immunosuppression with 
minimal toxicity in part due to the poor and highly variable 
bioavailability as a result of CYP3A-mediated gut wall metabolism of the 
drug. Grapefruit juice containing 6',7'-dihydroxybergamottin has been 
demonstrated to increase the bioavailability of cyclosporine by about 50%. 
Larger increases would be anticipated with doses of 
6',7'-dihydroxybergamottin larger than can be ingested conveniently in the 
form of grapefruit juice (approximately 10-20 mg). Increased 
bioavailability reduces the dosage requirement of cyclosporine (a dosage 
reduction of at least one-third would be expected with the administration 
of approximately 5-20 mg of 6',7'-dihydroxybergamottin). Moreover, an 
improvement in the extent of drug availability is typically accompanied by 
more consistent, less variable bioavailability. This results in more 
consistency in blood concentrations and makes it easier for the clinician 
to achieve an optimal dosage. 
Typically, cyclosporine is administered chronically at a dosage of 3-10 
mg/kg/day with the dosage declining over time as the risk of 
transplantation rejection decreases. The daily dose is generally divided 
for administration twice daily. Dosage is adjusted based on measured blood 
concentrations for cyclosporine designed to produce concentrations of 100 
to 400 ng/mL (using assays specific for cyclosporine). Patients receiving 
cyclosporine in combination with 6',7'-dihydroxybergamottin would 
initially be started on a regimen of 6',7'-dihydroxybergamottin 10 mg in 
the morning and 10 mg in the evening with each dose of 
6',7'-dihydroxybergamottin to be given concomitantly with a dose of 
cyclosporine. The starting dose of cyclosporine would be two-thirds of the 
usual starting dose in anticipation of a 50% improvement in 
bioavailability. The dosage of 6',7'-dihydroxybergamottin as well as 
cyclosporine would be adjusted based on measured blood concentrations. For 
example, if blood concentrations of cyclosporine are low, the dosage of 
6',7'-dihydroxybergamottin would be increased in order to increase the 
degree of inhibition, bioavailability and blood concentrations. If blood 
concentrations are too high, the dosage of cyclosporine would be further 
lowered. 
Those skilled in the art can now appreciate from the foregoing description 
that the broad teachings of the present invention can be implemented in a 
variety of forms. Therefore, while this invention has been described in 
connection with particular examples thereof, the true scope of the 
invention should not be so limited since other modifications will become 
apparent to the skilled practitioner upon a study of the drawings, 
specification and claims. 
All publications cited herein are expressly incorporated by reference.