Garlic Flavonoids and Organosulfur Compounds: Impact on the Hepatic Pharmacokinetics of Saquinavir and Darunavir

Drug Metab. Pharmacokinet. 25 (6): 521–530 (2010).

Regular Article Garlic Flavonoids and Organosulfur Compounds: Impact on the Hepatic Pharmacokinetics of Saquinavir and Darunavir Katja BERGINC1,*, Irina MILISAV2 and Albin KRISTL1 2Institute

1Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: The therapeutic efficacy of saquinavir and darunavir is affected by the presence of xenobiotics (such as garlic compounds) capable of modifying transporter-enzyme interplay. To ascertain the mechanism of interactions between antiretroviral drugs and garlic supplements and to identify the garlic constituents responsible, the hepatic pharmacokinetics of two antiretroviral drugs was investigated in the presence of garlic phytochemicals and aged garlic extract. For this purpose, rat liver slices and isolated rat hepatocytes were used. Aged garlic extract significantly inhibited saquinavir efflux from rat hepatocytes, while the efflux of darunavir significantly increased. Phytochemicals inducing distribution changes of saquinavir and darunavir were most probably flavonoids and lipophilic organosulfur compounds, respectively. All tested phytochemicals (except S-allyl L-cysteine) and aged garlic extract inhibited CYP3A4 metabolism of both drugs and modulated hepatic distribution of the corresponding saquinavir and darunavir metabolites. The competition between saquinavir and garlic constituent(s) for the same binding site on the efflux transporter and the positive cooperative effect between darunavir and garlic phytochemical(s), which bind to separate binding places on transporter, are the most probable mechanisms explaining the plasma profile changes, which could occur in vivo during concomitant consumption of antiretrovirals and garlic supplements. Keywords: aged garlic extract; darunavir; rat liver slices; isolated rat hepatocytes; saquinavir

to either therapeutic failure or unwanted side effects and toxicity. Depending on the different processing methods utilized for the production of garlic supplements, final garlic products can be classified as preferentially lipophilic (garlic oils and macerates, powders obtained at temperatures below 609C) or hydrophilic (different waterethanol extracts).6) Therefore, the final effect of the applied garlic supplement on the pharmacokinetics of administered drugs will depend on the phytochemical mixture contained in the garlic supplement. Crude garlic bulbs contain mainly organosulfur compounds (gglutamylcystein peptides, alliin and its degradation products formed upon disruption of cell membranes). Some of the lipophilic constituents have been shown to interact with cytochromes (CYPs) and P-glycoprotein (Pgp) and to exert a cholesterol-lowering effect.7) However, according to recent studies, garlic flavonoids (nobiletin, tangeretin, rutin)8) and steroids9) could also significantly contribute to pharmacological activity and thus cause pharmacokinetic modulation of transporter-en-

Introduction Among the most popular food supplements sold worldwide in 2008, those prepared from garlic, echinacea and ginseng caused the majority of unwanted side effects.1) The alleged preventive and curative properties of herbal medicines encourage polypharmacy patients (38.2 billion patients in the USA in 2002) to consume these products2) despite pharmacokinetic and/or pharmacodynamic interactions witnessed in numerous case reports.3,4) Cardiac and diabetic patients and the HIV-infected population often combine the prescribed therapy with garlic preparations to improve plasma lipid and glucose levels and to exploit other health-beneficial properties of garlic (hypotensive, antioxidative, anticancer, immunomodulatory, hepatoprotective, antimicrobial, neuroprotective).5) Unfortunately, garlic phytochemicals have the capacity to modulate the transporter-enzyme interplay in the liver and intestine and thus profoundly modify drug plasma profiles, which can lead

Received; June 15, 2010, Accepted; July 20, 2010, J-STAGE Advance Published Date; October 1, 2010 *To whom correspondence should be addressed: Katja BERGINC, Faculty of Pharmacy, University of Ljubljana, As¾ ker ¾ceva 7, 1000 Ljubljana, Slovenia. Tel. ＋386-1-476-95-00, Fax. ＋386-1-425-80-31, E-mail: katja.berginc＠ffa.uni-lj.si

521

522

Katja BERGINC, et al.

zyme interplay. Namely, nobiletin, tangeretin and rutin have been shown to modulate Pgp and multidrug resistance-associated protein 2 (MRP-2)-mediated transport,8) whereas the inhibition of CYP3A4 has been noted in the case of tangeretin.10) The biochemical abilities, relative non-toxicity and abundance of flavonoids in our diet make these phytochemicals attractive to clinicians and the pharmaceutical industry as potential Pgp inhibitors, whose presence could increase the absorption of drugs that are substrates for efflux transporters.11) Previous in vitro studies indicated that in the presence of aged garlic extract (AGE), the activities of intestinal solute carrier transporters (SLC) and ATP-binding cassette (ABC) transporters in rat intestine, Caco-2 cells and HepG2 cells significantly changed and CYP3A4 inhibition was observed.12,13) Concomitant long-term consumption of saquinavir and garlic supplements caused a significant decrease of the extent of saquinavir absorption in one clinical study, indicating a profound impact of garlic constituents on the transcriptional, translational and/or posttranslational regulation of transporter and/or enzyme expression and thus on the therapeutic efficacy of saquinavir.14) Because the hepatic distribution and metabolism of HIV-protease inhibitors (HIV-PIs) in noncancerous in vitro hepatic models has not yet been evaluated to explain the mechanism of interactions between HIV-PIs and garlic phytochemicals, we decided to address this question and to identify garlic phytochemicals, responsible for the above-mentioned pharmacokinetic changes. For this purpose, we first evaluated the effect of garlic phytochemicals (flavonoids, organosulfur compounds) on Pgp and CYP3A4 activities. According to these results, each component could be classified as a Pgp/CYP3A4 inhibitor, a Pgp/CYP3A4 activator or a compound with no effect. Then, the effects of AGE on the hepatic pharmacokinetics of two HIV-PIs (saquinavir—Saq, darunavir—Dar) were monitored in rat liver slices and isolated rat hepatocytes. With the obtained data, we were able to identify garlic phytochemicals that were most probably responsible for the observed interactions in the presence of AGE and to explain the interaction mechanisms responsible for the pharmacokinetic effects of Saq and Dar.

Materials and Methods Materials: Salts for the incubation salines, nicotinamide adenine dinucleotide phosphate (NADPH), rhodamine 123 (Rho123), flavonoids (tangeretin, nobiletin, rutin, naringin, naringenin, bergamottin, quercetin), diallyl disulfide (DADS), allyl methyl sulfide (AMS), S-allyl L-cysteine (SAC) and all the necessary ingredients for cell cultivation were purchased from Sigma Aldrich Chemie (Deisenhofen, Germany). Saq and ritonavir (Rito) were purchased from Sequoia Research Products (Reading, UK). All chemicals used in this study were of the highest

grade available. Kyolic liquid aged garlic extract was produced by Wakanuga of America Co., Ltd. (Mission Viejo, CA, USA), lot number 5H04A. The AGE used in this study was standardized to 1.27 g/l S-allyl-L-cysteine content, the compound normally used for the standardization of AGE. A more thorough description of AGE composition is given in Berginc et al.12) Dar was extracted from Prezista tablets (300 mg) by ethanol extraction, evaporation and reconstitution in DMSO. The purity of extracted Dar was confirmed by HPLC analysis at 240 nm; the Dar peak surface represented 97% of the total chromatogram surface area. Methods: The influence of garlic phytochemicals on Pgp To evaluate the influence of garlic constituents on Pgp activity, the apparent permeability (Papp) of Pgp substrate Rho12312) was determined in the presence of individual garlic phytochemicals and compared to the reference values (Papp determined in the presence of garlic phytochemical was divided by reference Papp). For this purpose, rat ileum was chosen as an appropriate in vitro model because Pgp expression in this segment of the small intestine is the highest.15) Furthermore, the samples obtained during permeability experiments were free of protein debris and did not need additional preparation steps prior to fluorescence measurements. These facts justified the use of intestinal rather than hepatic tissue to evaluate the impact of garlic constituents on Pgp activity. When tested garlic phytochemicals modulated Pgp activity in the intestine, we assumed that they would have the same effect in the liver. The experiments conducted with rat intestinal tissue conform to the Law for the Protection of Animals (Republic of Slovenia) and were registered at the Veterinary Administration of Republic of Slovenia. Rat small intestine was obtained from male Wistar rats (250–320 g) fasted 18 hours before the experiments. After the rats were euthanized and laparotomized, the intestine was rinsed with ice-cold 10 mM glucose Ringer solution. Ileum located 20 cm proximally from the ileo-cecal junction was used. The intestinal tissue was cut into 3-cmlong segments, excluding visible Peyer's patches. Intestinal segments were opened along the mesenteric border, stretched onto inserts with an exposed tissue area of 1 cm2 and placed between two compartments of EasyMount side-by-side diffusion chambers (Physiologic Instruments, San Diego, USA). A total of 2.5 mL of bathing solution (Ringer buffer) on each side of the rat ileum was maintained at 379C and continuously oxygenated and circulated by bubbling with carbogen (95% O2, 5% CO2). Glucose (625 mM) and mannitol (625 mM) were always added to the serosal (S) and mucosal (M) sides, respectively, to give 10 mM final concentrations. After 25 min of preincubation, serosal solutions were replaced by donor solution containing 20

Garlic Phytochemicals and HIV-PI Hepatic Pharmacokinetics

mM Rho123 and 10 mM glucose. Rho123 transport was monitored in the serosal-to-mucosal (S-M) direction. To test the influence of flavonoids on Pgp-mediated Rho123 efflux, 20 mM of individual flavonoid was added to mucosal side of rat ileum. Samples (250 mL) were withdrawn from the acceptor (M) side every 20 min up to 80 min and replaced by the fresh Ringer buffer containing all necessary ingredients at appropriate concentrations. The Rho123 recovery determined at the end of the experiments was 85–90%, indicating that garlic phytochemicals did not influence the nonspecific binding of Rho123 to intestinal tissue but rather the Pgp activity. Fluorescence of samples was measured and the apparent permeability coefficient for Rho123 was determined. The diffusion chambers were also equipped with two pairs of Ag/AgCl electrodes for measuring the transepithelial potential difference and the short-circuit current with a multi-channel voltage-current clamp (model VCC MC6, Physiologic Instruments), enabling the evaluation of tissue integrity and viability during the experiments as described in detail in Berginc et al.12) The influence of garlic phytochemicals on CYP3A4mediated Saq metabolism CYP3A4 experiments were performed in 0.2 M Tris buffer (pH 7.4). Human hepatic microsomes were diluted with reaction buffer to give 10 mg/ml final protein concentration. All microsome suspensions were preincubated for 3 min at 379 C with NADPH (1.2 mM) and the reactions were started by the addition of dimethyl sulfoxide (DMSO)-concentrated Saq solution to yield 1 mM drug concentration. The final DMSO concentration in the reaction mixture was 5% in all tested samples (reference samples and samples with tested garlic phytochemicals). The incubation reactions (100 mL) were terminated after 5 min by the addition of 25 mL of ice-cold acetonitrile containing 100 mg/L of haloperidol as internal standard. Afterwards, the samples were vigorously vortexed for a short time, placed on ice for 15 min and centrifuged for 10 min at 15,000×g. The supernatants were transferred to HPLC vials and analyzed by LC/MS/MS. When the impact of garlic constituents was monitored, they were added to Tris incubation medium to give a final concentration of 1 mM of garlic phytochemical. pH was adjusted to 7.4. The amount of metabolites formed in the presence of garlic phytochemicals was compared to the amounts obtained in the reference experiment. The influence of garlic constituents on Saq metabolism and distribution in rat liver slices To evaluate the influence of individual garlic phytochemicals on Saq's hepatic pharmacokinetics, rat liver slices were incubated in a medium containing Saq and individual garlic constituents. Samples (100 mL) were withdrawn at 30 and 60 min from the incubation medium and at the end of the experiment (at 60 min) liver slices were homogenized and extracts were prepared.

523

The LC/MS/MS analysis of the liver extract samples and of the withdrawn samples from the incubation medium at different time points was performed. The transport rate (the rate of metabolite concentration increase in the incubation medium) of two major Saq metabolites (SaqM1 and Saq-M3)16) was determined. In the liver extracts, concentrations of Saq and its metabolites were measured. Comparing concentrations of Saq and its metabolites in the extracts and the metabolite transport rates with the corresponding reference values (i.e., concentrations of Saq and its metabolites or metabolite transport rates determined without garlic phytochemicals), we were able to determine the impact of garlic phytochemicals on the activity of efflux transporters (i.e., transporters that efflux drugs and their metabolites from hepatocytes into bile) and of CYP3A4. After decapitation of the rats, rat liver was preserved in ice-cold transportation medium, consisting of Williams medium E supplemented with L-glutamine (0.29 mg/mL), insulin (0.13 I.U/mL), gentamicin (50 mg/mL) and ampicillin (100 mg/mL). Rat liver slices (250 mm thick) for the experiments were prepared and incubated in 1.5 mL of this incubation medium. Saq and garlic phytochemicals were added to give a final concentration of 20 mM of each substance. Samples of incubation medium were prepared for LC/MS/MS analysis in the same way as described for CYP3A4 microsomes. The liver extracts were prepared in the following manner: liver slices were weighed (wet tissue weight was used to normalize LC/MS/MS measurements) and homogenized and the homogenate was precipitated with acetonitrile (1:1). The supernatant was analyzed by LC/MS/MS. The influence of AGE on HIV-PI metabolism and distribution in rat liver slices and primary rat hepatocytes Saq and Dar were tested at 20 mM, whereas AGE or Rito were added to give a final 1 v/v% or 50 mM concentration, respectively. Because Saq hepatic distribution and metabolism were faster than those of Dar (preliminary data, not shown), Dar samples were withdrawn at 45 and 90 min, whereas Saq samples were withdrown at 30 and 60 min to avoid reaching steady state. Experimental protocols afterwards were identical to that described above (Methods, previous section). Rat primary hepatocytes were isolated from adult male Wistar rats (180–280 g) by reverse two-step collagenase perfusion as described by Milisav et al.17) The plated cells were incubated in William's medium E with insulin (0.1 IU/ml) and 1 mM hydrocortisone hemisuccinate, penicillin and streptomycin (50 IU/ml each) in a humidified atmosphere of 95% air and 5% CO2 at 379C over-night. Then, hepatocytes were incubated in the defined incubation medium (see section 3 of Methods) with 1 v/v% AGE or 50 mM Rito at 379 C and continuously shaken. Samples of incubation medium at designed time points for

524

Katja BERGINC, et al.

Saq and Dar were withdrawn, and finally, cell lysates were prepared. Further sample preparation was identical to that described for the rat liver slices. LC/MS/MS analysis of Saq and Dar samples LC/MS/MS analysis of incubation samples was performed on a Varian 1200L triple-quadrupole LC-MS (Palo Alto, CA, USA). The mass spectrometer was coupled to ProStar 210 binary pumps, a Varian 420 autosampler and Varian 510 column oven. For chromatographic separation, a Phenomenex Kinetex 50×2.0 mm C-18 column with 2.6-mm particles was used for Dar, and a Phenomenex Gemini 150×2.0 mm C-18 column with 3-mm particles for Saq. The injection volume was 10 mL and the column temperature was 509C. The mobile phase consisted of water and acetonitrile containing 0.1% formic acid. The elution of Dar and its metabolites was performed using a linear gradient from 20% to 65% of acetonitrile in 4 min with a flow rate of 0.4 mL/min, whereas that of Saq was performed using a linear gradient from 20% to 36% acetonitrile over 12 min with a flow rate of 0.5 mL/min. For the purpose of this study, two Dar metabolites [Dar-M1—Dar carbamate hydrolysis (tr 3.3 min), Dar-M4—Dar hydroxylation (tr 5.2 min)] (17) and two Saq metabolites [SaqM1—Saq oxidation at (1,1-dimethylethyl)amino group (tr 8.1 min), Saq-M3—Saq oxidation at benzyl group (tr 5.9 min)]16) were monitored. Data analysis The apparent permeability coefficient ( Papp) of Rho123 was calculated according to Eq. (1): dc V Papp＝ (1) dt c0A where dc/dt represents changes in Rho123 concentration in the acceptor compartment per unit time under steadystate conditions, V is the volume of the acceptor compartment, A is the exposed surface area (1 cm2) and c0 is the initial donor concentration of Rho123. Afterwards the ratios (R) were determined according to Eq. (2) by dividing Rho123 Papp values measured in the presence of garlic Phy phytochemical ( P app ) by the corresponding reference Papp Ref ( Papp ) values: Phy P app R＝ Ref (2) Papp Results in Tables and Figures are presented as means± SD of at least 3 measurements. Data were evaluated statistically using SPSS 16.0 for Windows. Where appropriate, the F-test for testing the equality of variances and, afterwards, the 2-tailed student t-test (a＝0.05) were used. Otherwise, one-way ANOVA followed by the Bonferroni post-hoc test were applied.

Table 1. The influence of selected garlic flavonoids on Pgpmediated Rho123 efflux through the rat ileum Flavonoid

R

p

Tangeretin

0.52±0.12

0.02

Nobiletin

0.66±0.08

0.04

Rutin

0.51±0.14

0.04

Quercetin

0.39±0.07

0.00

Bergamottin

0.49±0.03

0.00

Naringenin

0.41±0.08

0.04

Naringin

1.04±0.03

0.35

Garlic

Grapefruit

The results are presented as means±SD of 3 measurements. The values R represent the ratio between S-M Papp Rho123 values determined in the presence of 20 mM flavonoids and S-M Papp Rho123 values determined without flavonoids (reference values). All flavonoids except naringin significantly inhibited Pgp (pº0.05).

Results Garlic constituents modify Pgp activity: Regarding the literature data, 10 mM flavonoid concentrations are the lowest needed for Pgp inhibition.8) Since the amount of flavonoids consumed in a regular diet in Western Europe is 20–200 mg/day (30–50 mM) or even higher,8,18) we applied 20 mM concentrations on the mucosal side of the rat ileum (Table 1). Grapefruit flavonoids naringin, naringenin (naringin metabolite formed after the removal of sugar moiety) and bergamottin were included in the set of tested flavonoids as positive controls to compare their Pgp inhibition to that observed for garlic flavonoids. According to Table 1, all tested flavonoids, except naringin, significantly inhibited Pgp-mediated Rho123 efflux, although it is known from the literature that all four grapefruit flavonoids are potent Pgp inhibitors.19) Naringin and rutin are glycosylated flavonoids whose sugar moieties are removed inside the gastrointestinal lumen to give an aglycone structure that interacts with membrane transporters.8) However, in the case of naringin, the transformation of this glycoside to aglycone is probably very slow; therefore, the aglycone probably could not exert its effect on Pgp in the timeframe of permeability experiments, or the concentration of naringin or its metabolite naringenin, formed on the mucosal side of intestine, were too low. The removal of sugars from rutin, a quercetin analog, was obviously fast enough to inhibit Pgp in the timeframe of the experiments. Garlic constituents inhibit CYP3A4 activity: To evaluate the effect of selected garlic phytochemicals on CYP3A4 HIV-PI metabolism, Saq was chosen as a model HIV-PI drug because of clinical studies14) reporting the importance of transporter-enzyme interplay in limiting Saq bioavailability. The concentrations of garlic

525

Garlic Phytochemicals and HIV-PI Hepatic Pharmacokinetics

phytochemicals after absorption into the portal blood are mostly unknown, although there are some data in the literature reporting that flavonoid hepatic concentrations after application of garlic supplements are in the Table 2. The influence of garlic phytochemicals on CYP3A4 Saq metabolism in human hepatic microsomes Garlic phytochemical

% of Saq remaining

% Saq-M1

% Saq-M3

Reference

39.9±5.4

100

100

Tangeretin

43.8±2.1S 47.0±1.1S

89.0±2.0S 83.7±4.1S

85.3±6.1S 85.2±4.6S

51.5±2.7S 61.8±3.2S

81.7±1.4S 76.7±0.3S

83.0±3.7S 82.1±4.1S

70.9±1.0S 49.3±2.9S

77.0±1.1S 88.5±4.9S

84.4±3.8S 91.2±5.4S

39.9±4.2 47.8±1.5S

90.0±5.7S 82.1±4.0S

90.6±0.5S 84.3±1.2S

57.0±0.6S 42.4±1.9

79.7±1.9S 100.1±1.1

81.9±4.0S 94.1±4.5

Nobiletin Rutin Naringin Naringenin Bergamottin Quercetin DADS AMS SAC

The results are represented as means±SD of 4 measurements. At the end of the reaction we determined the percentage (%) of the remaining concentration of non-metabolized Saq and the percentage (%) of formed metabolites (Saq-M1 and Saq-M3) and compared these values to the reference values. S Significantly higher/lower % of remaining Saq/formed metabolites (Saq-M1 and Saq-M3) in the presence of flavonoids compared to the reference values.

micromolar range.18) Therefore, we decided to test all garlic constituents at 1 mM. The formation of two Saq metabolites (Saq-M1, Saq-M3) and the remaining concentration of Saq were measured in human hepatic microsome samples and compared to the reference values (Table 2). All tested flavonoids and organosulfur compounds, except SAC, significantly inhibited CYP3A4 metabolism (significantly higher percentage of remaining Saq and significantly lower percentage of formed metabolites). The impact of garlic constituents on Saq metabolism and distribution in rat liver slices: The influence of garlic phytochemicals on the complex process of transporter-enzyme interplay was evaluated for Saq in rat liver slices. The transport rates of Saq metabolites (Saq-M1 and Saq-M3) from rat liver slices into the incubation medium and the concentrations of Saq, Saq-M1 and Saq-M3 in the liver extracts at the end of the experiments were determined and divided by the corresponding reference values (obtained in the absence of garlic phytochemicals). We decided to classify tested substances into 4 groups based on the observed changes of Saq-M1 and Saq-M3 transport rates (Table 3). In group A, SAC did not influence the transport rate of any metabolite. Tangeretin, nobiletin and rutin in group B significantly lowered the transport rate for Saq-M1 but

Table 3. Classification into 4 groups (A-D) of investigated garlic phytochemicals according to their influence on Saq metabolite distribution Group

Tested phytochemicals

Saq-M1 transport rate

Saq-M3 transport rate No change

A

SAC

No change

B

Tangeretin, nobiletin, rutin

Significantly decreased

No change

C

Naringin, AMS, DADS

Significantly decreased

Significantly increased

D

Naringenin, bergamottin, quercetin

Significantly decreased

Significantly decreased

Table 4. The influence of garlic phytochemicals on Saq metabolism and distribution in rat liver slices Relatiue rate of appearance

Relatiue concentration in extracts

Substance Saq-M3 Tangeretin Nobiletin Rutin Quercetin DADS AMS SAC Naringin Naringenin Bergamottin

Saq-M1

Saq

Saq-M3

Saq-M1

1.12±0.17 0.93±0.16

S

0.45±0.15 0.47±0.18S

S

1.42±0.20 1.21±0.09S

S

0.56±0.16 1.43±0.27S

0.55±0.17S 1.50±0.10S

0.92±0.10 0.61±0.13S

0.86±0.03S 0.56±0.15S

1.19±0.04S 1.26±0.04S

1.58±0.12S 1.16±0.08S

1.62±0.19S 1.27±0.04S

3.11±0.45S 4.81±0.59S

0.81±0.08S 0.52±0.19S

0.62±0.10S 0.62±0.17S

0.52±0.13S 0.54±0.09S

0.61±0.16S 0.65±0.08S

1.14±0.21 1.53±0.17S

1.17±0.24 0.37±0.09S

0.91±0.11 1.29±0.04S

0.95±0.07 1.39±0.16S

1.02±0.07 1.79±0.21S

0.50±0.19S 0.63±0.10S

0.45±0.12S 0.51±0.11S

0.85±0.06S 1.41±0.18S

1.18±0.04S 1.32±0.12S

1.41±0.25S 1.36±0.09S

The results are presented as means±SD of 4 measurements. Values are ratios between rate of metabolite appearance in the incubation medium/concentration of Saq, Saq-M1 and Saq-M3 in the extracts determined in the presence of garlic constituents and reference values (without addition of garlic constituents). S Significantly lower/higher rates of metabolite appearance in the incubation medium or concentrations in the extract compared to the reference values.

526

Katja BERGINC, et al.

had no influence on the transport rate of Saq-M3, whereas naringin, AMS and DADS (group C) significantly decreased the transport rate of Saq-M1 and caused a significant increase of the Saq-M3 transport rate. In group D (quercetin, bergamottin, naringenin), the transport rates of both metabolites significantly decreased (Table 4). One can also observe that in the presence of the tested garlic constituents the concentrations of Saq, Saq-M1 and Saq-M3 in the liver extracts changed. Although all phytochemicals except SAC inhibited CYP3A4 metabolism (Table 2), their influences on Pgp activities were different: tangeretin, nobiletin, rutin, naringenin, bergamottin and quercetin (Table 1) inhibited Pgp, whereas organosulfur compounds DADS, AMS and SAC increased Pgp transport.12) Furthermore, phytochemicals that inhibited both CYP3A4 and Pgp most probably did not inhibit them to the same degree; CYP3A4 inhibition could be more or less profound than that of Pgp. SAC (no CYP3A4 influence and increased Pgp activity) did not affect any intracellular concentrations; therefore, we assumed that there would be no intrahepatic in vivo pharmacokinetic alteration in its presence. For nobiletin and rutin (group B), naringin (group C) and naringenin, bergamottin and quercetin (group D), we observed that intracellular concentrations of Saq and its metabolites significantly increased. These phytochemicals inhibited CYP3A4 (Table 2) and Pgp (Table 1) (except naringin, but this glycoside could be metabolized into naringenin in hepatocytes much more efficiently than measured with enterocytes), leading to higher Saq intrahepatic concentrations. However, since the concentrations of Saq-M1 and Saq-M3 also significantly increased, we assumed that Pgp inhibition was more profound than CYP3A4 inhibi-

tion since final metabolite concentrations in the liver extracts were higher. In the presence of tangeretin (group B), intracellular Saq concentration significantly increased due to Pgp inhibition, but that of metabolites significantly decreased, indicating that in this case CYP3A4 inhibition was more important than Pgp inhibition. Finally, the addition of DADS and AMS (group C) to the rat liver slices induced a significant decrease of intrahepatic Saq and metabolites concentrations. Although both substances inhibited CYP3A4 (Table 2) they also activated Pgp transport.12) Lower intrahepatic concentrations were thus most probably due to efficient Pgp extrusion of Saq and metabolites and CYP3A4 inhibition. The impact of 1 v/v% AGE on metabolism and distribution of HIV-PIs in rat liver slices and in isolated primary rat hepatocytes: To ascertain the effect of AGE (and therefore a mixture of different garlic phytochemicals) on the hepatic distribution and metabolism of Saq and Dar, further experiments with garlic supplement (AGE) and Rito (a known CYP3A4, Pgp and MRP-2 inhibitor20)) were performed. We used two different rat in vitro hepatic models due to certain (dis)advantages associated with these models. The enzymatic activity after isolation of rat hepatocytes quickly deteriorates during cultivation, so these cells should be used immediately after isolation. They also lose their typical liver architecture, which can affect enzymatic and transporter activities. However, with this model one can evaluate the contribution of parenchymal cells (hepatocytes) to the overall liver pharmacokinetics, whereas, with rat liver slices, a relatively low-cost, high-throughput model, one could avoid the loss of architecture, and, if used immediately after decapitation, also the loss of timedependent liver-specific functions. Furthermore, phar-

Fig. 1. Hepatic metabolism and distribution of Saq and Dar in rat liver slices and isolated rat hepatocytes The concentration of HIV-PIs (Saq, Dar) and their metabolites (Saq-M1, Saq-M3, Dar-M1, Dar-M4) in the extract of rat liver slices (Figs. 1A, 1C) and in the lysates of isolated rat hepatocytes (Figs. 1B, 1D) presented as means±SD of 4 measurements.

527

Garlic Phytochemicals and HIV-PI Hepatic Pharmacokinetics

Table 5. The rate of Saq and Dar metabolite transport from rat liver slices or isolated rat hepatocytes into the incubation medium The rate of metabolite appearance in the incubation medium Liver slices Saq

Dar

Saq-M1 (*10－ 14)

Saq-M3 (*10－14)

Dar-M1 (*10－12)

Dar-M4 (*10－ 16)

HIV-PI

16.8±0.9

HIV-PI＋AGE HIV-PI＋Rito

11.6±0.2S 9.7±0.9S

20.7±2.0 16.3±0.9S

17.6±1.4 3.5±0.7S

22.8±1.6 15.9±1.5S

10.5±0.2S

4.3±0.7S

2.6±0.8S

Saq-M1 (*10－ 13)

Saq-M3 (*10－13)

Dar-M1 (*10－12)

Dar-M4 (*10－ 12)

HIV-PI

9.6±0.1

HIV-PI＋AGE HIV-PI＋Rito

1.0±0.0S

207.5±12.1 15.8±0.4S

1.0±0.2 1.2±0.1

6.4±0.4 0.4±0.0S

NI

NI

NI

NI

(mol/min*mg)

Primary rat hepatocytes

Saq

Dar

The results are presented as means±SD of 4 measurements. S Significantly (pº0.05) lower rates of metabolite appearance in the incubation in the presence of AGE (HIV-PI＋AGE) or Rito (HIV-PI＋Rito) compared to reference values (HIV-PI). NI; metabolites were not identified in the incubation medium.

macokinetic changes observed with rat liver slices would simulate in vivo situation better, since the participation of nonparenchymal liver cells could be evaluated.21) The concentrations of HIV-PIs and their metabolites at the end of the experiments in the extracts of rat liver slices and in the lysates of isolated rat hepatocytes are shown in Figure 1. In the presence of 1 v/v% AGE, the intracellular concentration of Saq significantly increased, whereas that of Dar significantly decreased (Figs. 1A and 1B). When Rito was present in the incubation medium, concentrations of both drugs in the hepatocytes significantly increased compared to the reference values. The results obtained with both hepatic models are therefore consistent with those obtained previously with cancerous HepG2 cells,13) where an identical influence of AGE on Saq and Dar efflux transporters was noted. However, in the previous study13) it was impossible to evaluate CYP3A4 metabolism and metabolite transport rates because HepG2 cells do not express CYP enzymes. Using rat liver slices and isolated rat hepatocytes enabled us to assess the impact of AGE on these processes too, although the interpretation of the results is much more complex compared to the uptake experiments with HepG2 cells. The activities of hepatic Oatp transporters in the presence of AGE or its constituents were not evaluated in this study. Since their participation in the hepatic distribution of HIV-PIs has been established,23) their impact on in vitro Saq distribution in this study could not be overlooked, especially since our previous in vitro permeability studies indicated significantly increased intestinal Oatp-mediated transport of statins in the presence of AGE.12) However, HepG2 cells do not express OATPs, and since the results of this and of our previous study12) are in accordance, the short-term impact of AGE on the activity of hepatic SLC transporters

and thus on the uptake of both tested HIV-PIs was most probably negligible. Therefore, we assumed that the distribution changes observed in this study were induced by altered activities of efflux ABC transporters and CYP3A4 enzymes. The transport rates (Table 5) of two metabolites for each HIV-PI (Saq-M1, Saq-M3 and Dar-M1, Dar-M4) and their final concentrations (Figs. 1C and 1D) in extracts and in the lysates were measured. AGE induced a significant decrease of the final intracellular concentrations of all metabolites (Figs. 1B, 1D) in the liver slices and in the isolated hepatocytes. Metabolite transport rates from hepatocytes into the incubation medium significantly decreased too (Table 5). Comparing the results obtained in the presence of Rito for both in vitro hepatic models, one can observe that no metabolites were detected inside isolated rat hepatocytes or in the incubation medium. However, in the rat liver slices some metabolites were still formed in the presence of Rito. Because of these differences, it is clear that the preservation of liver architecture and participation of non-parenchymal hepatic cells are also important in HIVPI metabolism.

Discussion Before reaching the systemic circulation, a drug distributes into the liver, undergoes hepatic metabolism (with phase I and II enzymes) and then the parent drug and its metabolites are transported either to the bile or plasma. Parent drug plasma concentrations are thus strongly dependent on the rate and extent of its metabolism and transport. The hepatic distributions of Saq and Dar depend on the activities of efflux ABC (Pgp, MRP-2) and influx SLC [OATP1A2 (OATP-A), 1B1 (OATP-C) and 1B3 (OATP-8)] transporters, which affect

528

Katja BERGINC, et al.

their exposure to CYP3A4 and consequently influence therapeutic outcome.16,22,23) Previous studies with the hepatic HepG2 cell line, which expresses sufficient levels of Pgp and MRP-2 but lacks CYP enzymes and uptake OATP transporters, enabled the evaluation of AGE impact only on ABC efflux hepatic transporters.13) In this study, the uptake of both HIV-PIs into rat liver slices and into isolated rat hepatocytes changed in the presence of AGE; the intracellular concentrations of Saq significantly increased and those of Dar significantly decreased (Table 5), indicating that the results of this study are in accordance with a previous study with HepG2 cells.13) Since the cancerous HepG2 cell line lacks OATPs, the effect AGE exerted on the hepatic distribution of HIV-PIs could only be explained by modified activities of Pgp and/or MRP-2, whose protein structures allow simultaneous binding of multiple substrates.24) Because garlic phytochemicals also bind to both efflux transporters,12) their impact on Saq/Dar transport depends on whether the binding sites for garlic phytochemicals and HIV-PIs are the same. Therefore, in the case of Saq, phytochemicals from AGE most probably competed for the same binding site, resulting in Saq displacement from its efflux transporter and thus in higher intrahepatic retention. However, Dar most probably bound to a different binding site than garlic phytochemicals, which caused communication between multiple binding sites on transporters, a phenomenon known as the positive cooperative effect.24) A positive-cooperative effect induced by allosteric modifications12) upon binding of garlic phytochemicals led to the transition of other binding site's affinity (HIVPI binding site) from low to high and to increased drug transport. Because of this, the efflux of Dar increased and its intracellular concentrations in hepatocytes decreased. This clearly indicated that Saq and Dar bound to different binding sites on Pgp and/or MRP-2 efflux transporters. Besides HIV-PI distribution into rat hepatocytes, we were also able to evaluate CYP3A4 metabolism of both drugs and transport of the investigated metabolites (Table 5), which was impossible to study with HepG2 cells. Because AGE inhibits CYP3A4 (Table 2) and affects Pgp and MRP-2 transporter activities,12,13) the final intracellular metabolite concentrations and their transport rates from hepatocytes into the incubation medium significantly decreased. Although we did not confirm that the metabolites of Saq and Dar are Pgp and MRP-2 substrates, we assumed that they could be transported by these proteins because of structural similarities between metabolites and their parent drugs and because, in the presence of Rito (potent CYP3A4, Pgp and MRP-2 inhibitor),20) a significant inhibition of metabolite formation and transport rate was noted. Despite the numerous compounds present in AGE, we made an attempt to evaluate the effects of some garlic

phytochemicals on the activities of Pgp (Table 1) and CYP3A4 (Table 2). These data helped us to identify important phytochemicals and ascertain the interaction mechanisms that contributed to the observed changes of Saq and Dar distribution and metabolism in the presence of AGE. Pharmacologically active garlic flavonoids (tangeretin, nobiletin, rutin, quercetin)8) and organosulfur compounds (DADS, AMS, SAC)7,12) were tested, and grapefruit flavonoids (naringin, naringenin and bergamottin) were included in the set as positive controls. All mentioned phytochemicals significantly inhibited Pgp (Table 1) and CYP3A4 (Table 2), except for SAC, DADS and AMS.12) These last three compounds increased Pgp activity, but SAC had no impact on CYP3A4. Because of SAC's pharmacokinetic quiescence towards CYP3A4, this sulfur compound, which exerts antioxidative, hepatoprotective and anticarcinogenic properties and participates in lowering plasma cholesterol levels,25) could pose less threat in terms of altering HIV-PI drug plasma profiles and therapeutic outcome than other investigated phytochemicals, if consumed in food supplements concomitantly with prescribed HIV-PIs. After acquiring this data, the influence of the abovementioned phytochemicals on Saq's hepatic distribution and metabolism was also evaluated (Table 4). Saq was used because, for this HIV-PI, important pharmacokinetic changes during concomitant consumption with garlic supplements have been witnessed in a clinical study during long-term garlic supplementation.14) Categorization of tested substances into 4 groups showed that there are distinct differences in the binding of garlic phytochemicals to the multiple sites on efflux transporters (Pgp, MRP-2). While some phytochemicals bound specifically to the Saq-M1 binding site on efflux transporters, others nonspecifically occupied both Saq-M1 and Saq-M3 binding sites. Some garlic phytochemicals could thus selectively occupy the Saq-M1 transport site, which led to competitive inhibition between that garlic phytochemical and Saq-M1, resulting in slower Saq-M1 transport (group C). Simultaneously, the same binding also induced allosteric changes in the Saq-M3 transport site, resulting in a positive cooperative effect between multiple binding sites in the transporter.24) This caused an increase of the transporter affinity for Saq-M3 and therefore resulted in faster Saq-M3 transport. However, in group B (tangeretin, nobiletin and rutin) only Saq-M1 and not Saq-M3 transport rate was affected. Since the positive cooperative effect depends on the concentration of flavonoid and its affinity towards the transporter, these garlic constituents, bound to Saq-M1 transport site, could be present in too low concentration to significantly affect the structure and/or affinity of the Saq-M3 transport site. In contrast, the binding of bergamottin, naringenin and quercetin (group D) was most probably nonspecific to both Saq-M1 and Saq-M3 binding sites, leading to sig-

Garlic Phytochemicals and HIV-PI Hepatic Pharmacokinetics

nificantly slower transport of both metabolites. One can also assume that the intensities of Pgp and CYP3A4 inhibition varied depending on the phytochemical used. If the intensity of Pgp inhibition exceeded that of CYP3A4 inhibition, intracellular concentrations of Saq-M1 and Saq-M3 increased (observed with nobiletin, rutin, naringin, naringenin, bergamottin, quercetin), whereas, if CYP3A4 inhibition exceeded that of Pgp inhibition, the concentration of substrates in the extracts decreased (observed with tangeretin). The obtained results clearly indicated the preference of some flavonoids to nonspecifically occupy multiple binding sites on efflux transporters. From the viewpoint of the pharmaceutical industry, these phytochemicals could represent therapeutically useful Pgp inhibitors, because they would definitely inhibit the transport of Pgp substrates, regardless of a drug's binding site on the efflux protein. Overcoming low bioavailability or high intersubject variability due to Pgp and MRP-2 involvement in the drug first-pass metabolism by inhibiting these transporters with phytochemicals that do not differentiate between multiple binding sites (Pgp has 4 and MRP-2 has 2) would thus represent a patient-friendly approach. To ascertain which garlic phytochemicals could be responsible for the effect of AGE on the hepatic pharmacokinetics of Saq and Dar, one can conclude that the hepatic distribution of Dar is presumably affected by lipophilic organosulfur garlic phytochemicals because they increase Pgp activity and therefore promote the efflux of Dar. However, in the case of Saq, inhibition of its hepatic transport is mostly due to flavonoids that (un)specifically occupy binding sites in membrane proteins (MRP-2, Pgp). Because concentrations of the tested garlic phytochemicals were selected according to the sparse available literature, the in vivo intraluminal or plasma concentrations of these constituents could significantly fluctuate depending on many variables, such as the type of administered garlic supplement, supplement batch-tobatch differences, quality of garlic used, the processing methods, etc. Furthermore, the tested set of garlic phytochemicals was very small compared to the numerous and diverse mixture of phytochemicals in natural herbal products, thus enabling us to only partially assess the effects of garlic.

5)

6)

7)

8)

9) 10)

11)

12)

13)

14)

15)

16)

17) 18)

19)

References 1)

2) 3) 4)

Lelekis, I.: Alternative medicines update: A focus on the most common products your patients are using. PharmaNote, 23: 1–7 (2008). Neustadt, J.: Herb-drug interactions: What clinicians need to know. Integr. Med., 5: 16–26 (2006). Williamson, E. M.: Drug interactions between herbal and prescription medicines. Drug Saf., 26: 1075–1092 (2003). Izzo, A. A., Di Carlo, G., Borrelli, F. and Ernst, E.: Cardiovascular pharmacotherapy and herbal medicines: the risk of drug in-

20)

21)

22)

529

teractions. Int. J. Cardiol., 98: 1–14 (2005). Barnes, J., Anderson, L. A. and Phillipson, J. D.: Garlic. In Herbal medicines. 3rd ed. Pharmaceutical Press, London Chicago, 2007. Nagpurkar, A., Peschell, J. and Holub, B. J.: Garlic constituents and disease prevention. In Mazza, G., Oomah, B. D. (eds.): Herbs, botanicals & teas, CRC Press, 2000. Shord, S. S., Shah, K. and Lukose, A.: Drug-botanicals interactions: a review of the laboratory, animal and human data for 8 common botanicals. Integr. Cancer Ther., 8: 208–227 (2009). Aszalos, A.: Role of ATP-binding cassette (ABC) transporters in interactions between natural products and drugs. Curr. Drug Metab., 9: 1010–1018 (2008). Matsuura, H.: Saponins in garlic as modifiers of the risk of cardiovascular disease. J. Nutr., 131: 1000S–1005S (2001). Cermak, R.: Effect of dietary flavonoids on pathways involved in drug metabolism. Expert Opin. Drug Metab. Toxicol., 4: 17–35 (2008). Bansal, T., Jaggi, M., Khar, R. K. and Talegaonkar, S.: Emerging significance of flavonoids as P-glycoprotein inhibitors in cancer chemotherapy. J. Pharm. Pharmaceut. Sci., 12: 46–78 (2009). Berginc, K., ½Zakelj, S. and Kristl, A.: In vitro interactions between aged garlic extract and drugs used for the treatment of cardiovascular and diabetic patients. Eur. J. Nutr. accepted for publication (DOI 10.1007/s00394-010-0095-x) (2010). Berginc, K., Trontelj, J. and Kristl, A.: The influence of aged garlic extract on the uptake of saquinavir and darunavir into HepG2 cells and rat liver slices. Drug Metab. Pharmacokin., 25: 307–313 (2010). Piscitelli, S. C., Burstein, A. H., Welden, N., Gallicano, K. D. and Falloon, J.: The effect of garlic supplements on the pharmacokinetics of saquinavir. Clin. Infect. Dis., 34: 234–238 (2002). Maclean, C., Moenning, U., Reichel, A. and Fricker, G.: Closing the gaps—A full scan of the intestinal expression of Pgp, Bcrp and Mrp2 in male and female rats. Drug Metab. Dispos., 36: 1249–1254 (2008). Fitzsimmons, M. E. and Collins, J. M.: Selective biotransformation of the human immunodeficiency virus protease inhibitor saquinavir by human small-intestinal cytochrome P4503A4. Drug Metab. Dispos., 25: 256–266 (1996). Milisav, I., Nipi ¾c, D. and ½Suput, D.: The riddle of mitochondrial caspase-3 from liver. Apoptosis, 14: 1070–1075 (2009). Brand, W. et al.: Flavonoid-mediated inhibition of intestinal ABC transporters may affect the oral bioavailability of drugs, foodborne toxic compounds and bioactive ingredients. Biomed. Pharmacother., 60: 508–519 (2006). de Castro, W. V., Mertens-Talcott, S., Derendorf, H. and Butterweck, V.: Effect of grapefruit juice, naringin, naringenin, and bergamottin on the intestinal carrier-mediated transport of talinolol in rats. J. Agric. Food Chem., 56: 4840–4845 (2008). Gupta, A., Zhang, Y., Unadkat, J. D. and Mao, Q.: HIV protease inhibitors are inhibitors but not substrates of the human breast cancer resistance protein (BCRP/ABCG2). J. Pharmacol. Exp. Ther., 310: 334–341 (2004). Coecke, S., et al.: The use of long-term hepatocyte cultures for detecting induction of drug metabolism enzymes: the current status. ATLA, 27: 579–638 (1999). Vermeir, M., et al.: Absorption, metabolism, and excretion of

530

23)

Katja BERGINC, et al.

darunavir, a new protease inhibitor, administered alone and with low-dose ritonavir in healthy subjects. Drug Metab. Dispos., 37: 809–820 (2009). Hartkoorn, R. C., et al.: A. HIV protease inhibitors are substrates for OATP1A2, OATP1B1 and OATP1B3 and lopinavir plasma concentrations are influenced by SLCO1B1 polymorphisms. Pharmacogenet. Genomics, 2: 112–120 (2010).

24)

25)

Martin, C., Berridge, G., Higgins, C. F., Mistry, P., Charlton, P. and Callaghan, R.: Communication between multiple drug binding sites on P-glycoprotein. Mol. Pharmacol., 58: 624–632 (2000). Amagase, H., Petesch, B. L., Matsuura, H. and Kasuga, S.: Intake of garlic and its bioactive components. J. Nutr., 131: 955S–962S (2001).