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Methoxylated flavones were recently shown to be promising cancer chemopreventive agents. Their high metabolic stability compared with the hydroxylated analogs was shown in our laboratory using the human hepatic S9 fraction with cofactors for glucuronidation, sulfation, and oxidation. In the present study, the resistance of methoxylated flavones toward oxidative metabolism was investigated with human liver microsomes and recombinant cytochrome P450 (P450) isoforms. Among 15 methoxylated flavones investigated, the two partially methylated compounds, tectochrysin and kaempferide, were among the most susceptible to microsomal oxidation (Cl(int) 283 and 82 ml/min/kg). Of the fully methylated compounds, 5,7-dimethoxyflavone and 5-methoxyflavone were the most stable (Cl(int) 13 and 18 ml/min/kg, respectively), whereas 4'-methoxyflavone, 3'-methoxyflavone, 5,4'-dimethoxyflavone, and 7,3'-dimethoxyflavone were the least stable (Cl(int) 161, 140, 119, and 92 ml/min/kg, respectively), emphasizing the importance of the positions of the methoxy substituents in the flavone ring system. Among the five P450 isoforms tested, CYP1A1 showed the highest rate of metabolism of fully methylated compounds, followed by CYP1A2 and CYP3A4. CYP2C9 and CYP2D6 gave minimal disappearance of the parent compound. Finally, in incubations with hepatic S9 fraction with cofactors for oxidation and both conjugation reactions, partially methylated flavones, as expected, were much less metabolically stable than fully methylated flavones, confirming that oxidative demethylation is the rate-limiting metabolic reaction for fully methylated flavones only. In summary, the rate of oxidative metabolism of methoxylated flavones, mainly involving CYP1A1 and CYP1A2, varied widely, even between compounds with very similar structures.

Oral cancer, originating from smoking-induced lesions of the basal cells in the complex stratified oral epithelium, is difficult to treat. Early detection of premalignant lesions, e.g., leukoplakia, has suggested the possibility of chemopreventive measures, such as topical application of antimutagenic/antiproliferative dietary or pharmaceutical agents. As an extension of a study in human oral epithelial cell monolayers, we determined the carcinogen, i.e., benzo[a]pyrene (BaP), transport, bioactivation, and DNA binding in a bioengineered human gingival epithelial tissue construct and the chemopreventive effects of dietary polyphenols. Short-term experiments showed that both types of compounds can traverse this tissue as well as be effectively taken up by the tissue. The model cigarette smoke carcinogen BaP very slowly, but to a great extent, accumulated in the tissue with maximal uptake at 24 h. Such exposure clearly resulted in DNA binding of BaP by the tissue. This DNA binding was associated with BaP-induced CYP1B1 as well as CYP1A1 expression, as evidenced by mRNA measurements. Cotreatment of the oral tissue with dietary polyphenols, including resveratrol and quercetin, and BaP, resulted in significant inhibition of the BaP-DNA binding. Using fluorescence microscopy as well as simultaneous autoradiography, we also demonstrated that quercetin indeed penetrates the entire stratified tissue layer, but that quercetin was also oxidized within the cells. Thus, this bioengineered oral tissue construct opens up improved ways of understanding and preventing/treating smoking-induced oral cancer.

The objective of this study was to examine the ability of dietary polyphenols to inhibit cytochrome P450 (CYP) 1A1 expression and activity and benzo[a]pyrene (BaP) DNA binding, with the main emphasis on prevention of chemical-induced hepatic carcinogenesis. For this purpose we used Hep G2 cells, a good model of the normal human hepatocyte for CYP1A1 cell signaling. First, when these cells were exposed to a low concentration (1 microM) of BaP, DNA binding occurred, which dramatically increased after 6 h of treatment. BaP also dramatically induced CYP1A1 activity, protein expression and mRNA levels, the likely reason for the marked increase in DNA binding. Second, we screened 25 polyphenols with highly varying chemical structures for maximum ability to inhibit CYP1A1 activity in the Hep G2 cells. Highly varying responses were obtained, ranging from a 10-fold induction by some polyphenols to almost complete inhibition, in particular by 5,7-dimethoxyflavone (DMF), a flavonoid found in some tropical plants. Third, we examined the ability of DMF to inhibit DNA binding of BaP and the mechanisms involved. DMF (2-20 microM) inhibited BaP-induced DNA binding. DMF also inhibited BaP-induced CYP1A1 activity, CYP1A1 protein expression and mRNA levels. Moreover, DMF directly inhibited the catalytic activity of recombinant CYP1A1 with an IC50 of 0.8 microM. In conclusion, DMF was a highly potent inhibitor of BaP-induced DNA binding and CYP1A1 protein expression and activity in the Hep G2 cells. These properties may make DMF an effective chemoprotectant in chemical-induced liver cancer.

Increasing epidemiological evidence supports the view that dietary flavonoids have protective roles in oral diseases, including cancer. However, the dietary forms of flavonoids, the flavonoid glycosides, must first be hydrolyzed to the aglycones, which is thought to occur mainly in the intestine. In the present study we tested whether this hydrolytic activity occurs in the oral cavity. Saliva was collected from human subjects, incubated with flavonoid glycosides, and analyzed for aglycone formation by HPLC. When quercetin 4'-glucoside or genistein 7-glucoside was incubated with human saliva, hydrolysis to quercetin and genistein, respectively, was detected within minutes. Studies of additional flavonoid glycosides demonstrated that glucose conjugates were rapidly hydrolyzed, but not conjugates with other sugars, i.e., rutin, quercitrin, and naringin. In a limited study of 17 subjects, the interindividual variability in the hydrolysis of genistein 7-glucoside was >20-fold. This supports the contention that salivary hydrolysis of certain flavonoid glucosides may be important in some individuals but not in others. Support for a bacterial contribution to this hydrolysis was obtained from the inhibitory effect of antibacterials in vivo and in vitro and from experiments with subcultured oral bacterial colonies. However, cytosol isolated from oral epithelial cells was also capable of effective hydrolysis. Dietary flavonoid glucosides may thus be hydrolyzed in the oral cavity by both bacteria and shedded epithelial cells to deliver the biologically active aglycones at the surface of the epithelial cells. The aglycones quercetin and genistein both potently inhibited proliferation of oral cancer cells. The large interindividual variability in this hydrolytic activity may be a factor that should be taken into consideration in future studies.

Quercetin is an abundant flavonoid in the human diet with numerous biological activities, which may contribute to the prevention of human disease but also may be potentially harmful. Quercetin is oxidized in cells to products capable of covalently binding to cellular proteins, a process that may be important for its biological activities. In the present study, using radiolabeled drug and quantifying the products after electrophoretic separation, proteins to which oxidized quercetin is binding irreversibly were identified. The binding of quercetin to human serum albumin (HSA) in human blood and the effect of stimulation of neutrophilic myeloperoxidase on this binding were also measured. The in vitro binding of quercetin to eight proteins in the presence of catalytic amounts of horseradish peroxidase and hydrogen peroxide was highly selective for HSA. For all proteins the binding was dramatically decreased by reduced L-glutathione. In the blood samples, the release of neutrophilic myeloperoxidase by phorbol ester caused a 3-fold increase in the binding of quercetin to HSA. This study shows that quercetin in the presence of peroxidase/hydrogen peroxide covalently links to proteins with a particularly high affinity for HSA and that this also may occur in vivo after exposure to quercetin. This provides further insights into the complex behavior of this major dietary flavonoid.

Resveratrol is a dietary constituent suggested to have protective effects against cancer as well as cardiovascular disease. The purpose of the study was to learn whether this agent could be absorbed in man and enter the systemic circulation. This was examined by measuring transport and metabolism of resveratrol (5–40 μM) by the human intestinal epithelial cell line Caco-2 cultured in Transwells. Transport across the Caco-2 monolayer occurred in a direction-independent manner with Papp values of ≅ 7 times 10−6 cm s−1, much higher than for the paracellular transport marker mannitol (≅ 0.4 times 10−6 cms−1), suggesting efficient absorption in-vivo. At the highest resveratrol concentration, the absorption increased, possibly due to saturation of metabolism. In sharp contrast to previous findings in the rat, the metabolism of resveratrol in Caco-2 cells involved mainly sulfation and, to a minor extent, glucuronidation. At low resveratrol concentrations, most of the sulfate conjugate was exported to the apical side, presumably by MRP2, which is well expressed in these cells. At high concentrations, there was a shift towards the basolateral side, possibly involving MRP3, which was recently shown also to be expressed in Caco-2 cells. These results indicate that absorption of resveratrol in-vivo may be high but with limited bioavailability due to efficient sulfate conjugation. Extensive accumulation of resveratrol in the Caco-2 cells, demonstrated in additional experiments, suggests enterocytes as a major target site for this cancer preventive agent.

Recent studies in our laboratory in the human hepatic and intestinal cell lines Hep G2 and Caco-2 have demonstrated induction of UGT1A1 by the flavonoid chrysin (5,7-dihydroxyflavone) using catalytic activity assays and Western and Northern blotting. In the present study, we examined which features of the flavonoid structures were associated with induction of UGT1A1 and whether common drug-metabolizing enzyme inducers also produce this induction. We also determined whether flavonoid treatment affected sulfate conjugation and CYP1A1 activity. We used intact Hep G2 cells for these studies, with chrysin as the model substrate. Both glucuronidation and sulfation were measured. Hep G2 cells were pretreated for 3 days with 25 microM concentrations of 22 flavonoids (n = 4-12). Only four flavonoids demonstrated induction of glucuronidation similar to that of chrysin (i.e., 3-5-fold in the intact cells). These were acacetin, apigenin, luteolin, and diosmetin, all of which, like chrysin, are 5,7-dihydroxyflavones with varying substituents in the B-ring. 5-Hydroxy-7-methoxyflavone and 5-methyl-7-hydroxyflavone produced a modest 1.5 to 2-fold induction, whereas all other flavonoids examined were without effect. None of the flavonoids caused more than a modest change in sulfation activity (60-140% of control). In contrast, all tested 5,7-dihydroxyflavones and -flavonols induced CYP1A1 activity (ethoxyresorufin deethylation). Of seven common drug-metabolizing enzyme inducers only 3-methylcholanthrene and oltipraz showed modest induction of chrysin glucuronidation but not 2,3,7,8-tetrachlorodibenzo-p-dioxin or phenobarbital. Together, these results strongly suggest that the flavonoid induction of UGT1A1 is through a novel nonaryl hydrocarbon receptor-mediated mechanism.

Purpose. Dietary flavonoids, present in fruits, vegetables and beverages have been demonstrated to be protective in cancer. Recently, we showed that the flavonoid chrysin induced UDP-glucuronosyl- transferase (UGT) activity and expression in the human intestinal cell line Caco-2. In the present study, we determined the specific UGT isoform(s) induced and whether this induction facilitates glucuronidation and potential detoxification of the colon carcinogen 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP).

Flavonoids, dietary components in vegetables, fruits and beverages, may protect against coronary heart disease, stroke and cancer. However, the bioavailability of these compounds is questionable. A previous study in ileostomy patients of the most abundant flavonoid, quercetin, suggested a 52% absorption of its major dietary forms, monoglucoside (QMG) and diglucoside (QDG), from an onion meal. However, this was based on indirect measurements after acid hydrolysis. Because human intestinal Caco-2 cell monolayers showed minimal absorption of the glucosides, we repeated the study in ileostomy patients, using molecularly specific analytical methodology for the intact glucosides and quercetin. The onion meal had high concentrations of both QMG and QDG with only trace amounts of quercetin. The intake of QMG and QDG in four patients ranged from 10.9 to 51.6 mg. No QMG or QDG was detected in the ileostomy fluid. In contrast, the amounts of the aglycone quercetin were substantial, 2.9-11.3 mg. This corresponded to 19.5-35.2% of total quercetin glucosides ingested, implying absorption of 64.5-80.7%. These findings suggest a different interpretation than that from the previous study, i.e., that both QMG and QDG are efficiently hydrolyzed in the small intestine by beta-glucosidases to quercetin, most of which is then absorbed.

Cooked-food mutagens formed when frying meat have been suggested to contribute to the etiology of colon, breast and prostate cancer. The most prevalent of these mutagens is 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which after absorption is bioactivated by both phase I and phase II enzymes. Although available data suggest absorption of PhIP in humans, the extent and mechanism of absorption are unknown. In the present study we examined the transport of [(3)H]PhIP through the human Caco-2 intestinal epithelial cell monolayer, a well-accepted model of human intestinal absorption. The influx, or absorption, was extensive and linear for 2 h and up to a PhIP concentration of 5 microM. Still, the basolateral to apical efflux [apparent permeability coefficient (P(app)) 54.2 +/- 0.7x10(-6) cm/s, mean +/- SEM, n = 24] was 3.6 times greater than the apical to basolateral influx (P(app) 15.1 +/- 0.6x10(-6) cm/s, n = 21, P < 0.0001). Equilibrium exchange experiments demonstrated the efflux to be a true active process. Preincubations with verapamil, an inhibitor of P-glycoprotein-mediated transport, or MK-571, an inhibitor of multidrug resistance-associated protein-mediated transport, stimulated influx and reduced efflux of PhIP, suggesting that PhIP is a substrate for both of these transporters. These findings should be considered when determining exposure to the cooked food mutagens.

Although the bioflavonoids, a large group of polyphenolic natural products, exert chemopreventive effects in cardiovascular disease and cancer, there is little information about the disposition of these dietary components in man. The objective of this study was to investigate the plasma-protein binding of the most abundant bioflavonoid, quercetin, using 14C-labelled quercetin. An ultracentrifugation assay (170 000 g for 16 h at 20°C) was shown to sediment plasma proteins. Binding of quercetin to normal plasma was extensive (99.1 ± 0.5%, mean ± s.d., n = 5). The unbound fraction varied as much as 6-fold, 0.3–1.8%, between subjects. This high binding was independent of quercetin concentration over the range 1.5–15 μM (0.5–5 μg mL−1). Human serum albumin was the primary protein responsible for the binding of quercetin in plasma (99.4 ± 0.1%). Binding by α1-acid glycoprotein (39.2 ± 0.5%) and very-low-density lipoproteins (< 0.5% of total quercetin) did not make substantial contributions to overall plasma binding. The equilibrium association constant for the binding of quercetin to serum albumin was 267 ± 33 times 103 M−1 (n=15). Thermodynamic data for the binding of quercetin to serum albumin indicated spontaneous, endothermic association. Displacement studies suggested that in man the ‘IIA’ subdomain binding site of human serum albumin was the primary binding site for quercetin. Association of quercetin with erythrocytes was significantly (P < 0.001) reduced by plasma protein binding. These data indicate poor cellular availability of quercetin because of its extensive binding to plasma proteins.

Little is known about the cellular distribution of paclitaxel in humans. In the present study we examined the distribution of [3H]-paclitaxel in human blood. When 1 microM paclitaxel was incubated with fresh blood at 37 degrees C, the platelet/plasma concentration ratio was 240 +/- 17 (mean +/- SEM), whereas the red blood cell (RBC)/plasma concentration ratio was only 0.59 +/- 0.05. In kinetics experiments using platelet-rich plasma, we observed that the platelet accumulation of paclitaxel was highly temperature- and concentration-dependent. Scatchard analysis of the 37 degrees C uptake data demonstrated a dissociation constant (Kapp) of 0.80 +/- 0.10 microM and a maximal binding capacity of 672 +/- 102 pmol/10(9) platelets. It is proposed that the platelet accumulation of paclitaxel reflects binding to microtubules and may serve as a useful model for binding to less accessible cellular sites.

Selective extraction of sulfate conjugates of basic drugs from biological matrices has been difficult because of their highly polar nature. Immunoaffinity isolation may be the best solution to this analytical problem. This was tested for a model compound, the metabolite 4'-hydroxypropranolol sulfate (HOPS), which was effectively extracted from plasma by a column containing antibodies to the parent drug propranolol. The specificity was very high, giving little interference from the biological material in subsequent high-performance liquid chromatographic analysis with fluorometric detection. The method for HOPS was highly reproducible and provided a sensitivity of 1 ng/ml plasma. The technique was applied to measurements of HOPS in plasma after therapeutic doses of propranolol as well as to the individual enantiomers after chiral derivatization.

Dietary polyphenols, including flavonoids, have been implied to have cancer preventive properties. Suggested mechanisms include inhibition of carcinogen-activating cytochrome P450 (CYP) transcription and activities. These studies have focused mainly on CYP1A1. However, CYP1B1 has recently been shown to be of particular importance in smoking-induced oral and oesophageal cancer. Previous observations in our laboratory demonstrated that methoxylated flavonoids may be effective inhibitors of CYP1A1 transcription and activity as well as being orally bioavailable. In this study, an initial screening of 19 methoxylated flavones, using the ethoxyresorufin de-ethylation assay in human oral squamous cell carcinoma SCC-9 cells pretreated with 1μM benzo[a]pyrene, identified six strongly inhibitory compounds for further studies. The effect of these flavones on CYP1B1 mRNA expression was measured with quantitative branched DNA methodology. Four of the compounds −3′,4′-dimethoxyflavone and 5,7,4′-trimethoxyflavone and, in particular, 7,3′-dimethoxyflavone and 7,4′-dimethoxyflavone — were potent inhibitors of CYP1B1 mRNA expression. Two of the more common unmethylated polyphenols — curcumin and quercetin — were also potent inhibitors. Whereas most unmethylated polyphenols, such as curcumin and quercetin, have very poor bioavailability, the high metabolic stability of the methoxylated flavones studied here suggests that these CYP1B1 inhibitors may also be effective in-vivo.

Dietary flavonoids and other polyphenols have the potential to be developed as effective food supplements as well as drugs for the prevention, as well as treatment of, cancer and other disease conditions. However, their very poor oral bioavailability, mainly due to extensive conjugation by glucuronidation and sulfation, is a severe limiting factor. First, this review shows the use of a simple, commercially available model system, the human hepatic S9 fraction, by which metabolic stability can be assessed effectively and accurately. Second, permethylation of the polyphenols effectively blocks the metabolic conjugation reactions, thereby dramatically increasing both metabolic stability and intestinal absorption, while maintaining or even increasing the biologic activities. Thus, permethylated polyphenols may have a future as chemoprotective agents.

A recent study in human hepatic preparations demonstrated that a methylated flavone, 5,7-dimethoxyflavone (DMF), abundant in certain plants, such as the leaves of Piper caninum, has high metabolic ...

Poor oral bioavailability has been a major limitation for the successful use of dietary flavonoids as cancer chemopreventive agents. In this study, we examined fully methylated flavones as promising improved agents. In the human oral SCC-9 cancer cells, 5,7-dimethoxyflavone and 5,7,4'-trimethoxyflavone were both 10 times more potent inhibitors of cell proliferation (IC(50) values 5-8 microM) than the corresponding unmethylated analogs chrysin and apigenin. Flow cytometry indicated that both methylated flavones arrested the SCC-9 cells in the G1 phase with a concomitant decrease in the S phase, dramatically different from the unmethylated analogs, which promoted G2/M phase arrest. Both methylated compounds inhibited the proliferation of two other cancer cell lines with very little effect on two immortalized normal cell lines. Examination of additional flavone structures indicated that methylated flavones in general have antiproliferative properties. Finally, we demonstrated that 5,7-dimethoxyflavone, in contrast to its unmethylated analog chrysin, was well absorbed and had high oral bioavailability as well as tissue accumulation in vivo in the rat. Thus, fully methylated flavones appear to have great potential as cancer chemopreventive/chemotherapeutic agents, in particular in oral cancer.

Epidemiologic evidence supports the view that dietary flavonoids exert protective effects in oral diseases, including cancer. However, the dietary forms of flavonoids, the flavonoid glycosides, are thought to be inactive, thus they must first be hydrolysed to their active aglycones. This may occur in the saliva in the oral cavity. We have examined if the flavonoid glycosides directly could affect cell proliferation, using the human oral squamous carcinoma SCC-9 cells. The cellular uptake and hydrolysis of the glycosides were assessed also. The four flavonoid glycosides tested each behaved differently. Genistin, the 7-glucoside of genistein, showed clear and consistent inhibition of cell proliferation, which appeared to be the result of rapid cellular uptake of the glucoside and hydrolysis to genistein. Spiraeoside, the 4′-glucoside of quercetin, showed a similar inhibition of cell proliferation, which also appeared to be associated with its hydrolysis to quercetin. Diosmin, the 7-rutinoside of diosmetin, surprisingly, was more potent and effective than diosmetin. In contrast, quercitrin, the 3-rhamnoside of quercetin, showed no effect and only minimal cellular uptake and no hydrolysis. In summary, dietary flavonoid glycosides may exert cellular effects in the oral cavity, but this varies greatly with the nature of the glycoside.

The dietary polyphenol resveratrol has been shown to have chemopreventive activity against cardiovascular disease and a variety of cancers in model systems, but it is not clear whether the drug reaches the proposed sites of action in vivo after oral ingestion, especially in humans. In this study, we examined the absorption, bioavailability, and metabolism of 14C-resveratrol after oral and i.v. doses in six human volunteers. The absorption of a dietary relevant 25-mg oral dose was at least 70%, with peak plasma levels of resveratrol and metabolites of 491 +/- 90 ng/ml (about 2 microM) and a plasma half-life of 9.2 +/- 0.6 h. However, only trace amounts of unchanged resveratrol (

Phloridzin, a glucoside of the flavonoid-like polyphenol phloretin, has long been known to be a specific nontransportable inhibitor of the sodium-dependent glucose transporter SGLT1. The objective of this study was to determine whether efflux by multidrug resistance-associated protein (MRP) transporters might have masked the absorption by SGLT1 in previous studies. Various cells used as transport models were incubated with phloridzin (50 microM) in the absence and presence of 50 microM 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK-571), a highly selective MRP1/MRP2 inhibitor, and the cellular uptake of phloridzin was measured by high performance liquid chromatography. The uptake of phloridzin by SGLT1-transfected Chinese hamster ovary (CHO) (G6D3) cells was 1.7-fold higher than that by parent CHO cells (p < 0.01). In the presence of MK-571, the uptake of phloridzin by CHO cells increased 3.7-fold (p < 0.001). MK-571 caused an 8.0-fold increase in the uptake of phloridzin by G6D3 cells (p < 0.0001). Thus, in the absence of MRP1 efflux, transport of phloridzin by SGLT1 was clearly demonstrated. Similar results were obtained for the glycosides of the flavonoids quercetin, genistein, and diosmetin. A significantly lower accumulation of phloridzin in MRP2-transfected Madin-Darby canine kidney (MDCK) cells compared with parent MDCK cells demonstrated that phloridzin was a substrate also for MRP2 (p < 0.05). This conclusion was further strengthened when MK-571 increased the uptake by MRP2-MDCK cells as much as 3.6-fold (p < 0.01). These results demonstrate that phloridzin, in contrast to previous notions, is transported by SGLT1. In addition, they demonstrate that this and other flavonoid glycosides unexpectedly are efficiently effluxed by both MRP1 and MRP2.

Quercetin-rich foods have the potential to prevent human disease. However, knowledge of its biological fate and mechanism of action is limited. This study extends previous observations of the oxidation of quercetin by peroxidases to quinone/quinone methide intermediates and, for the first time, demonstrates covalent binding of [14C]quercetin to macromolecules. This was first demonstrated using horseradish peroxidase and hydrogen peroxide with human liver microsomal protein to trap the intermediates. To extend this observation to the cellular level, human intestinal Caco-2 cells and hepatic Hep G2 cells were incubated for up to 2hr with [14C]quercetin, and cellular DNA and protein were isolated. The cellular uptake of [14C]quercetin was rapid, and the covalent binding of [14C]quercetin to DNA and protein was determined by liquid scintillation spectrometry after extensive purification. Both cell types demonstrated DNA binding with a maximum level of 5-15pmol/mg DNA. The level of covalent binding to protein was considerably higher in both cell types, 75-125pmol/mg protein. To determine potential specificity in the protein binding, Hep G2 cells were treated with [14C]quercetin, and the cell lysate was subjected to SDS-PAGE followed by staining and autoradiography. Several distinct radiolabeled protein bands did not correspond to the major Coomassie blue stained cellular proteins. We propose that this specific binding may mediate part of the antiproliferative and other cellular actions of quercetin.

A previous study in ileostomy patients indicated that dietary glucosides of the flavonoid quercetin are hydrolyzed efficiently in the intestinal lumen, followed by absorption of a large fraction of the quercetin aglycone. To determine the fate of quercetin, we administered 1.85 MBq (50 microCi) of (14)C-quercetin both orally (100 mg, 330 micromol) and intravenously (iv; 0.3 mg, 1 micromol) to healthy volunteers. Serial plasma samples, urines and stools were collected for 72 h. Total radioactivity was determined by liquid scintillation spectrometry directly in plasma and urine and after repeated methanol extraction of stool homogenate samples. The oral absorption, based on total radioactivity, was surprisingly high, ranging from 36.4 to 53.0%. The biological half-life was very long, ranging from 20 to 72 h. The urinary recovery of total radioactivity ranged from 18.4 to 26.8% after the iv dose and from 3.3 to 5.7% after the oral dose. The corresponding fecal recoveries were only 1.5-5.0% and 1.6-4.6%, respectively. Thus, the total recovery of the (14)C-quercetin doses, in particular after oral administration, was very low. In search for the unaccounted for fraction of the (14)C-quercetin dose, we performed (14)CO(2) recovery studies in three volunteers (3 iv and 3 oral doses). At timed intervals, (14)CO(2) in expired air was trapped in hyamine hydroxide/thymolphthalein and analyzed for radioactivity. As much as 23.0-81.1% of the quercetin dose was recovered as (14)CO(2) in the expired air from these volunteers, after both oral and iv doses. The disposition of quercetin in humans is thus highly complex, requiring further studies.

1 The metabolism of (+)-, (-)- and (±)-salbutamol by sulphoconjugation was determined in vitro using human lung cytosol and bronchial epithelial BEAS-2B cell homogenate. 2 For the lungs the intrinsic clearance (Vmax/Km) value for the pharmacologically active (-)-salbutamol (0.49 ± 0.32 ml min--1 g-1 protein) exceeded that of (+)-salbutamol (0.046 ± 0.028 ml min-1 g-1 protein) by 11-fold. This was mainly due to a difference in Km value, which was 16 times higher for (+)-salbutamol (1300 ± 170 μM) than for (-)-salbutamol (83 ± 12 μM). 3 The stereoselectivity of sulphoconjugation of salbutamol was very similar in the BEAS-2B cells, although the absolute activity was considerably lower. 4 The enzyme catalyzing this reaction both in the lungs and in the BEAS-2B cells was the monoamine (M) form phenolsulphotransferase. 5 These observations emphasize that the smooth muscle of the bronchi most likely are exposed to considerably higher concentrations of the potentially toxic (+)-enantiomer than of the bronchodilating (-)-enantiomer during therapy with (±)-salbutamol.

The natural product quercetin was a potent inhibitor of the human P-form phenolsulfo-transferase with an IC50 value of 0.10 +/- 0.03 microM (mean +/- SEM; N = 5), which was three to four orders of magnitude more potent than its inhibition of other human sulfotransferases. The inhibition was noncompetitive with a Ki value of 0.10 microM. The potency and mechanism of this inhibition appear similar to those of the current standard P-form inhibitor, 2,6-dichloro-4-nitrophenol. Among other flavonoids examined, kaempferol was found to have an IC50 value of 0.39 +/- 0.07 microM, naringenin 10.6 +/- 1.6 microM and naringin 265 +/- 90 microM (N = 3). These observations suggest the potential for clinically important pharmacologic and toxicologic interactions by flavonoid-containing foods and beverages.

The objective of this study was to determine the potential role of circulating testosterone and estradiol in regulation of the activity of the sex-dependent pathways of propranolol metabolism (i.e., α-naphthoxylactic acid and propranolol glucuronide). The pharmacokinetics of a single 80 mg oral dose of propranolol and the plasma levels of the sex steroid hormones were therefore determined in normal volunteers. In 33 young men there was a positive correlation between the testosterone levels and the propranolol clearances through both α-naphthoxylactic acid (p < 0.001) and propranolol glucuronide (p < 0.002), as well as the total clearance (p < 0.05), but not through aromatic ring hydroxylation. Testosterone cypionate administration led to an increased clearance of propranolol through α-naphthoxylactic acid in nine of the 11 men studied, further supporting a stimulatory effect of testosterone on propranolol metabolism. In 23 young women there was no significant association between the circulating levels of either estradiol or testosterone and any of the clearances of propranolol. These observations may be clinically relevant for propranolol therapy and may provide improved insight into the influence of gender and circulating gonadal hormones on drug metabolism in humans. Clinical Pharmacology and Therapeutics (1994) 56, 127–132; doi:10.1038/clpt.1994.115

Isolated hepatocyte cultures have become a frequently used model system for investigating drug metabolism. Although rat and hamster hepatocytes are frequently used for this purpose, metabolism in these species differs in many respects from human metabolism. A species with a metabolism more closely resembling that of humans might be more useful. Our in vivo experiments demonstrated that the metabolism of propranolol in the rabbit is more similar to that in humans than in rats or hamsters. We therefore examined the usefulness of rabbit parenchymal liver cells for studies of propranolol metabolism. A detailed method is presented for their preparation and culture, along with data on their viability, structure and protein synthesizing capability. One- or two-day-old hepatocyte cultures were exposed to 10 μmol/L 3H-propranolol from 30 min to hr; metabolites were identified by gas chromatography-mass spectrometry and quantitated by high-performance liquid chromatography. Propranolol metabolism was linear over 1 hr, with 15% of the substrate metabolized during this time. The cytochrome P-450 pathways, which result in ring oxidation and sidechain oxidation, were expressed in a reproducible fashion similar to that found in vivo in humans. The arylhydrocarbon hydroxylase inhibitor α-naphthoflavone (100 μmol/L) inhibited side-chain oxidation of propranolol by 90% without affecting ring oxidation. In contrast, cholorpromazine (100 μmol/L) was shown to inhibit ring oxidation of propranolol by 85% without affecting sidechain oxidation. Cimetidine (250 μmol/L) inhibited both pathways by about 50%. These observations suggest that rabbit hepatocyte metabolic pathways more closely resemble human cell metabolism and thus may be a more useful adjunct that rat or hamster hepatocytes in the study of drug-drug interactions when combined with in vivo studies in humans. (HEPATOLOGY 1990;11:394–400.)

The extensive therapeutic use of racemates of chiral drugs, wherein one enantiomer contains most, if not all of the biological activity, raises questions about the usefulness of pharmacokinetic parameters based on measurements of racemic drug. Not only do individual enantiomers behave as distinctly different chemicals with respect to their pharmacologic and toxicologic actions but also with respect to their fate in the body. Therefore, it is critically important, as Thomas Walle and U. Kristina Walle point out, to realize that blood concentrations and pharmacokinetics of racemates often do not reflect those of the active enantiomers.

The disposition of the pharmacologically active 4-hydroxypropranolol (HO-P), its glucuronic acid conjugate (HO-P-G), and propranolol were compared after single intravenous and oral doses of propranolol in 6 normal subjects and after long-term therapy in 32 patients with hypertension or coronary artery disease. The areas under the plasma concentration/time curves (AUC∞, ng · hr/ml) after 4-mg intravenous doses of propranolol were 6.6 ± 2.2 (mean ± SEM) for HO-P and 55 ± 11 for propranolol. After 20- and 80-mg oral doses the AUC∞ for HO-P were 59 ± 9 and 162 ± 21 and for propranolol were 72 ± 9 and 306 ± 46. Peak HO-P concentrations were reached at 1 to 1.5 hr after the oral doses. Although there was a rapid decline in plasma HO-P between 1.5 and 3 hr when HO-P-G was still rising to levels above HO-P levels 3.5- to 5-fold, the apparent half-lifes (t½s) after 3 hr were in the same range for HO-P, HO-P-G, and propranolol (3.0 to 4.2 hr). While during long-term therapy plasma HO-P rose over the whole dose range (40 to 960 mg daily) in an apparently linear fashion, the HO-P/propranolol plasma level ratio fell from 1.07 ± 0.13 at 40 mg daily to only 0.09 ± 0.01 at 640 mg daily. Plasma HO-P-G rose exponentially with dose and demonstrated significant cumulation. HO-P and HO-P-G in urine accounted for about 9% of long-term propranolol doses. This study suggests a significant contribution of HO-P to pharmacologic effects, in particular at low single and long-term oral doses of propranolol and saturation of naphthalene ring oxidation as a main determinant of propranolol bioavailability. Clinical Pharmacology and Therapeutics (1980) 27, 22–31; doi:10.1038/clpt.1980.4

The relative contributions of the debrisoquin and mephenytoin isozymes of hepatic cytochrome P-450 to the stereoselective metabolism of propranolol have been studied in a panel of volunteers of known oxidative phenotypes. Six subjects were extensive metabolizers of both debrisoquin and mephenytoin (EM). Four subjects were poor metabolizers of debrisoquin but rapid for mephenytoin (PMD). Five subjects were poor metabolizers of mephenytoin but rapid for debrisoquin (PMM), and one individual had a deficiency for both test compounds (PMD/M). Partial metabolic clearances of each propranolol enantiomer to 4-hydroxypropranolol (4-OH-P), the sulfate, and glucuronide conjugates of 4-OH-P, naphthoxylactic acid (NLA) and propranolol glucuronide, were estimated after a single oral dose of racemic propranolol (80 mg). The partial metabolic clearance of both enantiomers to total 4-OH-P in the PMD group was 75% less than in the EM and PMM groups, indicating the contribution of the debrisoquin isozyme to this route of metabolism. The R/S ratios for the clearance to 4-OH-P were similar between EM and PMD (2.5 +/- 0.5 vs 2.5 +/- 0.4, respectively), implying that the different enzymes involved in ring hydroxylation (i.e., the debrisoquin isozyme and other hydroxylases) have similar stereoselective preferences. The partial metabolic clearance to NLA was 55% less in the PMM group than in the EM and PMD groups, indicating that S-mephenytoin 4-hydroxylase contributes to the metabolic conversion of propranolol to NLA. The R/S ratios for the clearance to NLA were close to unity in all groups. The partial metabolic clearance to propranolol glucuronide also did not exhibit stereoselectivity and was similar in all groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of a meal on the disposition and metabolism of oral propranolol was examined in six normal subjects. The meal induced a mean 53% increase in propranolol bioavailability (range 2% to 92%; P less than 0.01) without affecting time to maximum concentration, half-life, or the amount of unchanges drug in urine. There was no effect on the plasma concentrations of 4-hydroxypropranolol or four other metabolites. The increased bioavailability was linearly related to the protein content of the meal (r = 0.884, P less than 0.02) above a threshold content of about 7 gm.

This study determined the total clearance of propranolol and the partial clearances through each of its three primary metabolic pathways after administration of an 80 mg single oral dose in 28 young, white subjects (13 women; 15 men). The oral clearance of propranolol was significantly higher (63%, p less than 0.02) in the men (65.7 +/- 7.7 ml/min/kg; mean +/- SE) than in the women (40.2 +/- 6.2 ml/min/kg). This sex difference was mainly attributable to a 137% higher clearance through the P-450-mediated side-chain oxidation in the men (p less than 0.001). There was also a 52% higher clearance through glucuronidation in the men (p less than 0.02). In contrast, the clearance through the P-450-mediated ring oxidation was not different between men and women. After administration of simultaneous intravenous doses of hexadeuterium-labeled drug (0.1 mg/kg) to 11 of the subjects, there were no differences between men and women in volume of distribution or half-life. Moreover, there were no sex differences in plasma and blood binding of propranolol. This study thus demonstrates that higher plasma levels of propranolol occur in women than in men after oral doses and suggests that some drug metabolizing enzymes, but not others, are regulated by sex hormones in human beings.

The comparative disposition of propranolol glucuronide (PG) and propranolol was determined in 35 patients with hypertension or coronary artery disease during initiation of propranolol therapy, during steady-state conditions, and after discontinuation of propranolol (dose range, 40 to 960 mg daily, every 6 hr). The 2.3-fold PG cumulation in plasma was identical to propranolol cumulation. PG plasma levels were about 4 times as high as propranolol levels over the whole dose range. Unexpectedly slow terminal elimination rate of propranolol (t1/2 approximately 16 to 24 hr) on discontinuation of propranolol appeared to be related to equally slow PG elimination. PG and propranolol could be detected in plasma and urine up to 3 to 5 days after propranolol discontinuation. The PG formed in man was deconjugated to propranolol in the dog after intravenous administration, suggesting that PG may serve as a storage pool for propranolol. Observations consistent with systemic and enteric deconjugation of PG, including enterohepatic recirculation, may, at least in part, explain the observed propranolol cumulation as well as the slow elimination of propranolol after its discontinuation. PG renal clearance (29 to 70 ml/min) and PG plasma levels were highly dependent on glomerular filtration rate, suggesting that PG may cumulate abnormally in patients with severe renal disease.

Our objective was to determine the kinetics of (+)- and (-)-propranolol after intravenous doses of racemic drug. Five normal subjects received 0.1 mg/kg of a pseudoracemate of propranolol that consisted of deuterium-labeled (+)-propranolol and unlabeled (-)-propranolol. Plasma concentrations of (+)- and (-)-propranolol as measured by gas chromatography-mass spectrometry demonstrated enantiomeric differences in systemic clearance (Cls) [(+)-propranolol, 1.21 +/- 0.15 l/min; (-)-propranolol, 1.03 +/- 0.12 l/min; P less than 0.01] and apparent volume of distribution (Vd) [(+)-propranolol, 4.82 +/- 0.34 l/kg; (-)-propranolol, 4.08 +/- 0.33 l/kg; P less than 0.001], but no difference in distribution or elimination t1/2s (t1/2 beta 3.5 hr). The higher Cls of (+)-propranolol suggests stereoselective hepatic elimination. The higher apparent Vd of (+)-propranolol is mainly related to its lower plasma binding [(+)-propranolol, 20.3 +/- 0.8% unbound; (-)-propranolol, 17.6 +/- 0.7% unbound; P less than 0.001]. There was no stereoselective uptake by red blood cells. These findings demonstrate that multiple stereoselective mechanisms are involved in the disposition of propranolol and determine the access of the drug to active sites.

Only limited information is available on the stereochemistry of the in vivo distribution of beta-receptor-blocking drugs. In this study we determined the levels of the propranolol enantiomers in plasma, cerebrospinal fluid (CSF) and central nervous system (CNS), and peripheral tissues in the dog following an intravenous dose of a deuterium-labeled pseudoracemate. The appearance of the propranolol enantiomers in the CSF was rapid and nonstereoselective, with maximum concentrations reached at 15 min after dosing. The levels of the enantiomers in both CSF and plasma then declined in a parallel biphasic fashion, with a terminal t1/2 of about 125 min. Except for an early high CSF/plasma concentration ratio of 0.35, the CSF propranolol levels corresponded to the unbound concentration in plasma, CSF/plasma 0.20. All areas of the brain showed a similar uptake of propranolol, with a tissue concentration exceeding that in plasma about 10-fold during the terminal phase of elimination. The uptake of propranolol by peripheral tissues varied widely, ranging from a 50-fold accumulation by the lungs compared to plasma to no accumulation by adipose tissue. However, as for the CSF, there was no evidence of stereoselective uptake of propranolol by any CNS or peripheral tissue except for the liver. A significantly higher level of (+)-vs. (-)-propranolol in liver tissue presumably was a reflection of stereoselective hepatic metabolism of (-)-propranolol by this tissue. The slight stereoselectivity in plasma binding of propranolol known to exist in the dog had no significant influence on tissue or CSF distribution.

The metabolism of propranolol, alprenolol and oxprenolol was studied in the dog and rat; propranolol in five additional species, including man. Basic, phenolic and neutral metabolites were extracted from urine at pH 9.6 after enzymatic hydrolysis. Separation and identification of parent drug and seven metabolites each for propranolol, alprenolol and oxprenolol in the dog were accomplished by gas chromatography mass spectrometry as the trifluoroacetyl derivatives. A very uniform and predictable fragmentation pattern was observed for all 24 compounds. Seven new metabolites were identified. The metabolism of all three drugs was qualitatively the same, including N-dealkylation followed by N-methylation or deamination of the primary amines. The parent drugs as well as all of their sidechain metabolism products were also partially ring hydroxylated. N-Methylation was only found in the dog and is a minor metabolic pathway. The stereochemical composition of N-methyldesisopropylpropranolol and its immediate precursor N-desisopropylpropranolol showed a marked enrichment of the (+)-isomer.

The objective of this study was to determine whether changes in dietary protein and carbohydrate influence the oral clearance of propranolol, a high-clearance drug, and theophylline, a low-clearance drug. Six normal subjects studied in a clinical research center each received a single oral dose of propranolol, 80 mg, and theophylline, 5 mg/kg, after having been on each of two well-defined diets for a period of 10 days. When the diet was altered from high carbohydrate/low protein to low carbohydrate/high protein, the oral clearance of propranolol increased by 74% +/- 20% (mean +/- SE; range 9% to 156%; P less than 0.01) with no change in plasma half-life or plasma binding. This dietary change resulted in an increase in theophylline clearance of 32% +/- 6% (range 18% to 50%; P less than 0.02) and a corresponding decrease in plasma half-life of 26% +/- 6% (range 6% to 42%; P less than 0.05) with no alteration in the apparent volume of distribution. These observations reemphasize the importance of diet in drug disposition and suggest that the clearance of high-clearance drugs like propranolol is more susceptible than the clearance of low-clearance drugs to dietary manipulations, effects that may have to be considered in drug therapy.

The influence of a high-protein meal as compared to fasting on the disposition of simultaneous intravenous and oral doses of propranolol, as well as on indocyanine green clearance, was examined in six normal subjects. The intravenous dose (0.1 mg/kg) was unlabeled propranolol and the oral dose (80 mg) was a stereospecifically deuterium-labeled pseudoracemate of propranolol. Systemic clearance of propranolol increased 38%, from 1005 +/- 57 to 1384 +/- 115 ml/min (mean +/- SE; P less than 0.05) as a result of the meal, with no change in t1/2 or apparent volume of distribution. A 12% decrease in oral clearance occurred with the meal but was not statistically significant (3717 +/- 185 ml/min, fasting; 3245 +/- 498 after meal), whereas bioavailability increased 67% (27.2% +/- 1.7% fasting; 45.5% +/- 4.3% after meal; P less than 0.01). Estimated hepatic blood flow, as measured by indocyanine green clearance, rose 34% 60 minutes after the meal (1719 +/- 155 ml/min fasting; 2304 +/- 218 ml/min after meal; P less than 0.02). A difference was observed in the oral clearance of the propranolol enantiomers in the fasting state, but this difference was unaffected by the meal. These alterations in propranolol disposition, as the result of a high-protein meal, are consistent with a transient increase in hepatic blood flow.

The intravenous and oral dose kinetics of propranolol were studied in the dog both in a fasted state and immediately after a meal consisting of 100 g of cooked beef liver. Fifty microCi of 3H-propranolol was administered intravenously simultaneously with a 40-mg oral dose of unlabeled propranolol. Plasma 3H-propranolol was measured by specific extraction and liquid scintillation spectrometry, and unlabeled plasma propranolol was determined by gas chromatography-mass spectrometry. Feeding significantly reduced (25%) the elimination half-life and increased (52%) the systemic clearance of intravenous propranolol. The increase in the systemic clearance of propranolol after feeding was mostly due to an increase (60%) in apparent hepatic blood flow, which appeared to remain elevated for 5-7 hr. The meal had no influence on the apparent volume of distribution or plasma binding. Feeding did not affect the area under the concentration-time curve of oral propranolol, but significantly delayed the rate of oral propranolol absorption, shifting the time to reach peak plasma levels from 60 to 158 min. The results of this study suggest that feeding alters the disposition of propranolol in the dog by producing a sustained increase in hepatic blood flow.

The relative importance of presystemic and systemic glucuronidation of propranolol was examined in normal subjects given single oral and intravenous doses of propranolol. The areas under the plasma concentration--time curves (AUCs) of propranolol glucuronide (PG), 41 +/- 15 ng . hr/ml, and propranolol, 48 +/- 15 ng . hr/ml, were of the same order after the intravenous dose (0.05 mg/kg). After oral doses of 20 and 80 mg, the AUCs of PG were 302 +/- 105 and 1,398 +/- 409 ng . hr/ml; these were 7 times the AUCs of propranolol, 44 +/- 15 and 220 +/- 38 ng . hr/ml. The time lapse to peak concentration, 1.5 to 3.0 hr, and the plasma half-life, 3.2 to 3.7 hr, were the same for PG and propranolol. These results demonstrate glucuronidation as an important determinant of propranolol bioavailability.