Pre-systemic transport and metabolism

The second class of interactions discussed considers interactions that alter the absorption of a drug and mostly occur before the drug appears in the systemic circulation. Such interactions may be facilitated by modulation of the activity of membrane transporters or enzymes involved in xenobiotic metabolism or drug binding⁽ Reference Chan ^{24 )} and occur on the level of the pre-systemic transport and metabolism.

Vitamin E and pre-systemic drug transport

Epithelial efflux transporters, such as the multidrug resistance proteins (for example, P-glycoprotein (P-gp), also known as ATP-binding cassette transporter B1 (ABCB1) and multidrug resistance protein 1 (MDR1))⁽ Reference Roninson, Chin and Choi ^{26 )}, may limit the intestinal absorption of drugs. Intestinal P-gp is expressed on the apical surface of enterocytes⁽ Reference Thiebaut, Tsuruo and Hamada ^{27 )} and limits the bioavailability and ultimately the activity of its substrates by increasing their efflux back into the intestinal lumen⁽ Reference Mayer, Wagenaar and Beijnen ^{28 )}. A prominent example for a P-gp-mediated drug interaction is the herbal remedy St John's wort, which induces P-gp and thereby reduces the plasma concentrations of the orally administered heart medication digoxin⁽ Reference Dürr, Stieger and Kullak-Ublick ^{29 )}.

All four T₃, but none of the T, induced P-gp mRNA expression via activation of the transcription factor pregnane X receptor (PXR; also known as steroid and xenobiotic sensing nuclear receptor) in the human colorectal adenocarcinoma cell line LS180 (incubated with 10 μmol/l of the respective T or T₃ for 24 h)⁽ Reference Zhou, Tabb and Sadatrafiei ^{30 )}. Consistent with the effects on mRNA, P-gp protein expression and export of the P-gp substrate rhodamine 123 were increased in LS180 cells incubated with γT₃ (25 μmol/l; 48 h)⁽ Reference Abuznait, Qosa and O'Connell ^{31 )}. No studies on effects of any of the remaining vitamin E congeners on intestinal P-gp protein expression or activity have been reported in the literature.

These in vitro studies suggest that, albeit at very high concentrations, T₃ may potentially enhance the transfer activity of P-gp in intestinal cells. However, the scientific literature does not contain reports from animal or human trials suggesting that intestinal P-gp-mediated interactions of vitamin E with drugs may actually occur. Thus, controlled in vivo trials are now warranted to elucidate the biological relevance of this potential mechanism.

Vitamin E and pre-systemic drug metabolism

The intestine is responsible for the uptake of nutrients, but also serves as a barrier against ingested xenobiotics⁽ Reference Kaminsky and Zhang ^{32 )}. Intestinal phase I enzymes, such as cytochrome P₄₅₀ mixed-function mono-oxygenases (CYP), and phase II enzymes, such as UDP-glucuronosyltransferases (UGT), glutathione-S-transferases (GST) or NAD(P)H:quinone oxidoreductase (NQO1), to name only a few, play a key role in the first-pass metabolism of drugs, which determines their and their metabolites’ plasma concentrations. CYP3A4 is a phase I enzyme involved in the metabolism of approximately 50 % of all prescription drugs⁽ Reference Guengerich ^{33 )} and the most abundant CYP in the intestine⁽ Reference Benet and Cummins ^{34 )}. Inhibition of CYP3A4, for example, by drinking a glass of grapefruit juice, reduces the oral availability of the sedative and CYP3A4 substrate⁽ Reference Paine, Shen and Kunze ^{35 )} midazolam in humans⁽ Reference Kupferschmidt, Ha and Ziegler ^{36 )}.

In LS180 colon cells, neither T nor T₃ (10 μmol/l; 24 h) altered the mRNA expression of CYP3A4⁽ Reference Zhou, Tabb and Sadatrafiei ^{30 )}. In agreement with these in vitro findings, the intestinal protein expression of CYP3A4 did not differ between guinea-pigs fed RRR-αT at normal dietary (20 mg/kg diet) or pharmacological doses (250 mg/kg diet) for 6 weeks⁽ Reference Podszun, Grebenstein and Hofmann ^{37 )}.

During phase I metabolism, reactive quinones and epoxides may be generated. These reactive metabolites are inactivated by phase II enzymes, including NQO1, which reduces quinones to less reactive hydroquinones, and GST, which conjugates electrophiles with glutathione. In the colon cancer cell line Colo205, αT dose-dependently (0·0001–10 μmol/l; 48 h) increased NQO1 activity, whereas GST activity remained unchanged⁽ Reference Wang and Higuchi ^{38 )}.

The phase II enzyme UGT1A conjugates lipophilic substances with UDP-glucuronic acid. UGT1A1 is the quantitatively most important UGT in the small intestine in humans and its activity in intestinal microsomes is higher than in hepatic microsomes. Hence, intestinal glucuronidation contributes significantly to the first-pass metabolism and low bioavailability of UGT1A1 substrates⁽ Reference Fisher, Vandenbranden and Findlay ^{39 )}. In LS180 cells, UGT1A1 mRNA increased at most 2-fold upon incubation with αT₃, βT₃ or δT₃ (10 μmol/l; 24 h), while neither γT₃ nor any of the T altered UGT1A1 mRNA⁽ Reference Zhou, Tabb and Sadatrafiei ^{30 )}. Such small increases in UGT1A1 mRNA, however, are unlikely to have a significant impact on UGT1A1 protein expression and/or activity. In agreement with this notion, the mean total UGT activity in the small and large intestine of rats fed 200 mg αT/kg diet for 2 weeks was unchanged relative to control animals⁽ Reference Van der Logt, Roelofs and van Lieshout ^{40 )}.

Thus, there is no evidence from in vivo studies to suggest that T or T₃ may alter the intestinal metabolism of drugs even though modest effects on NQO1 and UGT1A1 activity have been observed in cultured colonic adenocarcinoma cells.

Vitamin E and hepatic uptake of drugs

Transport proteins in the plasma membrane are involved in the uptake of many drugs into hepatocytes and strongly determine their overall hepatic clearance, regardless of other subsequent reactions⁽ Reference Shitara, Sato and Sugiyama ^{41 )}. In humans, the organic anion transporting polypeptide (OATP C, also known as solute carrier organic anion transporter family member 1B1 (OATP1B1) or OATP2) is a liver-specific transporter facilitating the uptake of various endogenous (for example, 17β-glucuronosyl oestradiol)⁽ Reference König, Cui and Nies ^{42 )} and xenobiotic substances (for example, the drug pravastatin)⁽ Reference Hsiang, Zhu and Wang ^{43 ,} Reference Hagenbuch and Meier ^{44 )} from the blood into the liver.

In rats, daily subcutaneous injections with 100 mg RRR-αT/kg body weight for 1 week, a mode of administration that was chosen to load the liver with extremely high concentrations of αT, reduced hepatic OATP C mRNA compared with control⁽ Reference Traber, Labut and Leonard ^{45 ,} Reference Farley, Leonard and Labut ^{46 )}. In humans, subcutaneous injection of αT, which has been used to treat retinopathy (retrolental fibroplasia) in prematurely born infants⁽ Reference Bougle, Boutroy and Heng ^{47 )}, as well as intramuscular⁽ Reference Guggenheim, Ringel and Silverman ^{48 )} and intravenous injections of vitamin E⁽ Reference Brion, Bell and Raghuveer ^{49 )} are rare modes of administration and thus of minor relevance in the context of vitamin E–drug interactions. Also, changes in mRNA expression often do not mirror changes in protein concentration and/or activity. In a model that more closely reflects the co-ingestion of nutrients and drugs observed in human subjects, feeding guinea-pigs for 6 weeks with either 20 or 250 mg RRR-αT/kg diet in the presence or absence of the OATP C substrate atorvastatin (300 mg/kg diet) did not alter hepatic OATP C protein expression, atorvastatin plasma concentrations (reflecting OATP C activity) or the lipid-lowering activity of the drug⁽ Reference Podszun, Grebenstein and Hofmann ^{37 )}.

In summary, these studies suggest that subcutaneous and potentially also intramuscular and intravenous injections with high doses of αT may carry a limited risk of altering OATP C-mediated hepatic drug uptake, but there is currently no in vivo evidence for the biological importance of this potential mechanism of vitamin E–drug interactions. Also the oral ingestion of high doses of αT does not appear to affect OATP C-mediated drug transport and efficacy in vivo. None of the other vitamin E congeners has been studied for impact on the hepatic uptake of drugs yet.

Vitamin E and hepatic phase I metabolism

During phase I metabolism in the liver, cytochrome P₄₅₀ enzymes (CYP) and other phase I enzymes oxidise, reduce or hydrolyse xenobiotics (such as drugs) and thereby activate them for the subsequent phase II conjugation reactions. All vitamin E congeners undergo phase I metabolism and are ω-hydroxylated at the terminal methyl group in the side chain by a CYP, in humans probably CYP4F2⁽ Reference Sontag and Parker ^{50 ,} Reference Bardowell, Duan and Manor ^{51 )}. Another member of the cytochrome family, CYP3A4, is involved in the metabolism of many drugs and can either contribute to the formation of the active agent from a parent drug or to the generation of metabolites destined for elimination⁽ Reference Guengerich ^{33 )}. Since the metabolism of all vitamin E congeners and of many drugs is catalysed by the same enzyme family, it is feasible that interactions on the level of hepatic metabolism may occur.

The impact of vitamin E on CYP3A4 expression and activity has been extensively studied in numerous in vitro and in vivo models. The expression of CYP3A4 is controlled by the nuclear receptor PXR⁽ Reference Lehmann, McKee and Watson ^{52 )}. In a reporter gene assay with HepG2 cells, T₃ at 10 and 50 μmol/l activated PXR (5- and 10-fold relative to untreated control cells), while T at these concentrations were much less potent activators (maximum 2-fold induction relative to control)⁽ Reference Zhou, Tabb and Sadatrafiei ^{30 ,} Reference Landes, Pfluger and Kluth ^{53 )}. We, on the other hand, did not observe an induction of PXR at all in a HEK293 reporter gene assay with concentrations of up to 200 μmol/l RRR- or all rac-αT⁽ Reference Podszun, Grebenstein and Spruss ^{54 )}. In primary human hepatocytes, 10 μmol/l T₃ induced CYP3A4 mRNA 3- to 5-fold, whereas T were ineffective⁽ Reference Zhou, Tabb and Sadatrafiei ^{30 )}. Incubation of HepG2 cells with increasing concentrations (0–300 μmol/l) of either RRR- or all rac-αT for 7 d dose-dependently increased mRNA expression of the orphan CYP20A1 only, but not of any of the other thirteen CYP (including CYP3A4) expressed in these cells⁽ Reference Hundhausen, Frank and Rimbach ^{55 )}.

A number of in vivo studies investigated the effects of high-dose supplementation as well as vitamin E deficiency on the expression of hepatic CYP, especially CYP3A4. In mice, feeding of a vitamin E-deficient diet (2 mg RRR-αT/kg diet) for 3 months reduced, whereas RRR-αT supplementation up to 9 months (200 mg/kg diet) did not alter hepatic mRNA expression of Cyp3a11 (the murine homologue of the human CYP3A4) compared with control (20 mg/kg diet)⁽ Reference Kluth, Landes and Pfluger ^{56 )}. In a similar study, feeding rats a diet deficient or adequate in vitamin E ( < 2 or 60 mg RRR-αT) for up to 9 months did not change the mRNA of any of the thirty-three hepatic CYP expressed (including CYP3A4)⁽ Reference Hundhausen, Frank and Rimbach ^{55 )}. On the other hand, feeding of high doses of all rac-αT acetate (1000 mg/kg diet) for 4 months induced the hepatic mRNA expression of Cyp3a11 approximately 1·3- to 4-fold (depending on the analytical method used) compared with control mice on a standard diet (35 mg/kg diet)⁽ Reference Mustacich, Gohil and Bruno ^{57 )}. However, 1000 mg/kg diet of synthetic all rac-αT acetate is a supraphysiological dose, which corresponds to about 7–10 g/d for an average human, and might explain the differences between the latter and the aforementioned mouse and rat studies. Although T₃ have a higher potential to induce CYP3A4 in reporter gene assays as well as in primary hepatocytes, hepatic mRNA expression of the murine homologue Cyp3a11 remained unchanged after oral administration of mice with γT₃ (250 μg/d for 7 d)⁽ Reference Kluth, Landes and Pfluger ^{56 )}.

Although, as mentioned earlier, changes in mRNA may not necessarily result in similar changes in protein expression and activity, none of the above studies investigated the effects of vitamin E on CYP protein expression and activity. In an attempt to close this gap, we fed guinea-pigs with 20 or 250 mg RRR-αT/kg diet for 6 weeks and studied the metabolism of atorvastatin, a drug that is metabolised by CYP3A4, and its lipid-lowering efficacy. The hepatic protein expression of CYP3A4 and CYP20A1 and the serum concentrations of the CYP3A4 products of atorvastatin, para- and ortho-hydroxy-atorvastatin were similar in both groups. In agreement with this, the cholesterol-lowering activity of atorvastatin was unaltered by high-dose αT feeding⁽ Reference Podszun, Grebenstein and Hofmann ^{37 )}. This is in agreement with studies in human subjects addressing the effects of αT on CYP3A4-mediated drug metabolism. Patients receiving either lovastatin or simvastatin (both CYP3A4 substrates) were supplemented with 400 mg/d RRR-α-tocopheryl acetate for 8 weeks and CYP3A4 activity was determined by quantification of urinary cortisol and its CYP metabolite 6β-hydroxycortisol. In these patients, vitamin E supplementation neither altered CYP3A4 activity (urinary 6β-hydroxycortisol), nor the lipid-lowering activity of the statins⁽ Reference Leonard, Joss and Mustacich ^{58 )}. In another human intervention trial, healthy volunteers were intravenously injected with 1 mg of the CYP3A4 substrate midazolam and its area under the plasma concentration–time curve (AUC) was determined before and after a 3-week intervention with 750 IU (503 mg) RRR-αT/d orally or placebo (six subjects per group). By using intravenous injection of the test compound, the investigators were able to circumvent intestinal phase I metabolism and to investigate the effects of vitamin E on hepatic phase I metabolism only. The plasma AUC for midazolam and its metabolite hydroxymidazolam did not differ between the placebo and vitamin E groups⁽ Reference Clarke, Burnett and Wu ^{59 )}.

In summary, vitamin E–drug interactions due to altered phase I metabolism have not been reported and appear unlikely based on the available evidence. Even at high oral doses, neither T nor T₃ appear to significantly alter CYP activity in animals or humans.

Vitamin E and hepatic phase II metabolism

Phase II of xenobiotic metabolism, which takes place to a significant extent in the liver, comprises the conjugation of xenobiotics or their phase I metabolites with polar groups, such as glucuronic acid, sulfate, glutathione or amino acids, to enable rapid biliary and urinary elimination. The inhibition of phase II enzymes, such as the UGT and GST, results in increased blood and tissue concentrations of their substrates. The induction of phase II enzymes, on the other hand, may increase the clearance and thereby reduce the activity of drugs.

None of the eight vitamin E congeners (incubation with 10 μmol/l for 24 h) altered UGT1A1 mRNA in primary human hepatocytes⁽ Reference Zhou, Tabb and Sadatrafiei ^{30 )} and feeding rats 200 mg αT/kg diet for 2 weeks had no effect on hepatic UGT activity⁽ Reference Van der Logt, Roelofs and van Lieshout ^{40 )}. A small increase (1·76– to 2·68-fold, depending on assay) in hepatic GST activity was observed in rats fed very high doses of αT (2500 mg/kg diet) for 10 d⁽ Reference Manson, Ball and Barrett ^{60 )}. This dose, however, would be equal to more than 17 g/d of αT for a 70 kg human. Similarly, feeding mice with 1000 mg/kg diet of all rac-αT acetate for 4 months led to a small increase (maximum 1·9-fold) in the hepatic mRNA expression of the murine Gstm3 compared with mice on a standard diet (35 mg all rac-αT acetate/kg diet)⁽ Reference Mustacich, Gohil and Bruno ^{57 )}. In disagreement with the above-described effects in vivo, studies with isolated human GST P1-1 suggest that the concentrations at which T and T₃ inhibit GST activity by 50 % (IC₅₀) are: αT, 0·7; δT, 0·8⁽ Reference Van Haaften, Evelo and Penders ^{61 )}; αT₃, 1·8; and γT₃, 0·7 μmol/l⁽ Reference van Haaften, Haenen and Evelo ^{62 )}. However, direct interactions of vitamin E with enzymes in artificial in vitro systems may differ significantly from cellular systems and the situation in vivo, where vitamin E resides in membranes and may thus not have direct access to these soluble cytosolic proteins.

The mRNA expression of another phase II enzyme, sulfotransferase 2A, was significantly induced (11-fold) in the aforementioned mouse study, where animals were fed 1000 compared with 35 mg/kg diet all rac-αT acetate for 4 months⁽ Reference Mustacich, Gohil and Bruno ^{57 )}.

Overall, there is no evidence to support the notion that T or T₃ ingested at normal dietary or supplemental doses may alter the activity of hepatic phase II metabolism in the living organism.

Vitamin E and blood coagulation

The best-known vitamin E–drug interactions are those with drugs that affect platelet aggregation and blood coagulation, which are presently the only interactions listed by the US Food and Drug Administration (FDA) in their consumer health information^{( 64 )}. Blood coagulation is the process during which blood is (locally) converted from a liquid to a gel and is the body's answer to injury ensuring that ruptured blood vessels are rapidly sealed off to minimise blood loss.

In brief, coagulation is initiated when collagen, which is normally buried underneath the epithelium, is exposed and platelets adhere to it via collagen-specific surface receptors (glycoprotein Ia/IIa). Platelet adhesion and the release of signalling molecules (including tissue factor and von Willebrand factor) from both the injured endothelium and the platelets activate further platelets and the intrinsic and extrinsic coagulation pathways. These coagulation cascades include a number of enzymes that are synthesised as precursors in the liver, where they undergo vitamin K-dependent carboxylation by γ-glutamyl-carboxylase, and are secreted as mature (carboxylated) proteins into the bloodstream (Fig. 3). The adhesion and aggregation of platelets at the site of injury result in the formation of a patch that is stabilised by the deposition and maturation of the fibrous protein fibrin (also known as factor Ia). Fibrin is formed by polymerisation of its precursor fibrinogen through the activity of the enzyme thrombin, which itself is activated by cleavage of prothrombin (factor II). Activated platelets secrete granules containing ADP, serotonin, platelet-activating factor, von Willebrand factor, platelet factor 4 and thromboxane A₂ (TXA₂) that induce platelet aggregation through cross-linkage of activated glycoprotein IIb-IIa and fibrinogen. TXA₂ is synthesised from arachidonic acid through the cyclo-oxygenase (COX) and hydroperoxidase catalytic activities of PG synthase, via the intermediate PGH₂. The COX reaction is the rate-limiting step in the synthesis of prostaglandins and thromboxanes and is inhibited by drugs, such as acetylsalicylic acid, which thereby reduce platelet aggregation.

Fig. 3 (a) The precursors of mature factor II, VII, IX and X are carboxylated by γ-glutamyl-carboxylase to their mature forms. The cofactor (vitamin K) for this reaction is oxidised and then regenerated by vitamin K epoxidase. The inhibition of vitamin K epoxidase by, for example, warfarin decreases the recycling of oxidised vitamin K and results in a shortage of the cofactor for the carboxylation reaction. (b) Upon endothelial injury, platelets bind to sub-endothelial collagen (via glycoprotein Ia/IIa (GPIa/IIa) and von Willebrand factor (vWF)). Tissue factor (TF) is also released to activate the extrinsic pathway. Interaction between GPVI and collagen induces cyclo-oxygenase (COX) and triggers the formation of thromboxane A₂ (TXA₂), which cross-links platelets via GPIIb-IIIa and fibrinogen. Protein kinase C (PKC) activates, among other things, TXA₂ formation and granule secretion. (A colour version of this figure can be found online at www.journals.cambridge.org/nrr).

Platelet activation is a complex regulatory pathway with multiple agonists, including ADP, TXA₂, collagen, adrenaline and many more. Ex vivo platelet aggregation can be activated by either endogenous agonist or phorbol myristate actetate (PMA), a protein kinase C (PKC) activator. PKC is a family of enzymes catalysing the phosphorylation and thus controlling the activity of many regulatory proteins. PKC activation stimulates, among other things, platelet granule secretion, TXA₂ synthesis and thereby the aggregation of platelets⁽ Reference Harper and Poole ^{65 )}.

Vitamin E and platelet adhesion, activation and aggregation

Platelets obtained from healthy volunteers and pre-incubated with RRR-αT for 5 min (0·016–16·22 μmol/l) exhibited a dose-dependent reduction in the metabolism of arachidonic acid to TXA₂ and PGD₂. The authors suggested that this may be due to an inhibition of COX activity in αT-treated platelets⁽ Reference Ali, Gudbranson and McDonald ^{66 )}. Incubation of platelets from healthy human subjects with supraphysiological concentrations (500 μmol/l) of RRR-αT or RRR-α-tocopheryl acetate, but not all rac-αT, inhibited arachidonic acid- or PMA-induced platelet aggregation and PKC-mediated protein phosphorylation⁽ Reference Freedman, Farhat and Loscalzo ^{67 ,} Reference Freedman and Keaney ^{68 )}. Although 500 μmol/l αT is a supranutritional dose, the authors observed that the cellular content of αT in the incubated platelets was comparable with that of platelets isolated from human subjects receiving 800 mg αT/d for 14 d⁽ Reference Freedman, Farhat and Loscalzo ^{67 )}. In the latter study, platelets isolated from human subjects supplemented for 14 d with 267, 533 or 800 mg αT/d, PMA-induced platelet aggregation was dose-dependently reduced, arachidonic acid-induced aggregation was reduced in the 800 mg/d group only, and ADP-induced aggregation was not altered by αT⁽ Reference Freedman, Farhat and Loscalzo ^{67 )}. PKC-mediated phosphorylation was determined in two subjects and was inhibited after 2 weeks’ supplementation with 800 mg RRR αT/d compared with baseline⁽ Reference Freedman, Farhat and Loscalzo ^{67 )}. In another study where healthy volunteers were given placebo, 100 mg all rac-αT acetate, or mixed T (20 mg RRR-αT, 100 mg RRR-γT and 40 mg RRR-δT) daily for 8 weeks, only mixed T significantly reduced ADP-induced, but not PMA-induced platelet aggregation, although phosphorylation of PKC was significantly inhibited by both vitamin E interventions⁽ Reference Liu, Wallmon and Olsson-Mortlock ^{69 )}. In yet another study, platelet adhesion to collagen was dose-dependently reduced in twelve healthy subjects supplemented with increasing doses of all rac-αT acetate (400, 800 and 1200 mg/d for 2 weeks, each; 6 weeks supplementation in total) compared with eleven healthy control subjects. Platelet aggregation (induced with collagen, adrenaline or ADP) was only modestly decreased in these subjects and only collagen-induced platelet aggregation was significantly reduced (P< 0·05) in women, but not men⁽ Reference Steiner ^{70 )}. The same study also analysed the combination of all rac-αT with the analgesic, antipyretic and anti-inflammatory drug acetylsalicylic acid (aspirin)⁽ Reference Steiner ^{70 )}, which itself inhibits the COX-catalysed formation of TXA₂ from arachidonic acid⁽ Reference Vane and Botting ^{71 )}. The combined oral administration of all rac-αT acetate in doses up to 1200 mg/d with 300 mg aspirin every other day had no effect on collagen-, adrenaline- or ADP-induced platelet aggregation or platelet adhesion to collagen compared with vitamin E or acetylsalicylic acid alone⁽ Reference Steiner ^{70 )}. In a subset of the Alpha-Tocopherol Beta-Carotene Cancer Prevention Study, male smokers aged 50–59 years who received a combination of 100–3200 mg acetylsalicylic acid and 50 mg all rac-αT acetate/d and were followed for 5–7 years showed a significant increase in oral ‘bleeding on probing’ (gingival bleeding) compared with subjects receiving acetylsalicylic acid alone⁽ Reference Liede, Haukka and Saxen ^{72 )}. Accordingly, in patients who previously suffered from an ischaemic event, the combination of acetylsalicylic acid (325 mg) and all rac-αT acetate (400 mg/d) for up to 2 years (n 52), compared with acetylsalicylic acid alone (n 48), was more effective in the prevention of transient ischemic attacks, suggesting a synergistic activity of αT with aspirin⁽ Reference Steiner, Glantz and Lekos ^{73 )}.

The mechanism for the reduction in ex vivo platelet aggregation observed in the above studies still remains to be fully elucidated. The inhibition of PKC by αT may be an important factor, since platelet aggregation induced by the PKC activator PMA was reduced by vitamin E in most studies concomitantly with a reduction in PKC activity⁽ Reference Freedman, Farhat and Loscalzo ^{67 –} Reference Liu, Wallmon and Olsson-Mortlock ^{69 )}. Interactions with vitamin K-dependent coagulation cascades, however, may be part of the explanation, as discussed in the next section.

Vitamin E, vitamin K and the extrinsic and intrinsic coagulation pathways

As mentioned earlier, the coagulation factors II, VII, IX and X undergo post-translational carboxylation by γ-glutamyl-carboxylase (Fig. 3), an enzyme that requires vitamin K as a cofactor, in the liver. Vitamin K is oxidised in the carboxylation reaction and requires subsequent reduction by vitamin K epoxide reductase in order to take part in another cycle of carboxylation⁽ Reference Suttie ^{74 )}. Some anticoagulants, such as warfarin, act by reducing the vitamin K epoxide reductase-catalysed recycling of oxidised vitamin K, thereby limiting the carboxylation of the precursors of factors II (prothrombin), VII, IX and X to their mature forms (Fig. 3)⁽ Reference Hirsh, Dalen and Anderson ^{75 )}.

In rats with decreased concentrations of reduced vitamin K (induced by intraperitoneal injections of warfarin), intramuscular injections of RRR-αT acetate (1000 mg/kg body weight/d for 7 d) reduced mature prothrombin coagulant activity compared with animals treated with warfarin only, but not the amount of the prothrombin precursor produced in the liver⁽ Reference Corrigan and Ulfers ^{76 )}. Since αT influences the formation of the mature prothrombin rather than the production of the precursor, this might be caused by a vitamin E-induced aggravation of vitamin K deficiency. Prothrombin activity in warfarin-treated patients supplemented with vitamin E (800 or 1200 IU vitamin E (congener not specified)/d, n 13; or 100 or 400 IU all rac αT-acetate/d, n 6) for 4 weeks did not differ significantly from that of placebo-treated patients⁽ Reference Corrigan and Ulfers ^{76 ,} Reference Kim and White ^{77 )}. Although these trials are limited due to their small sample size, they suggest that doses up to 1200 IU vitamin E have no clinical effects in patients on warfarin therapy. Nevertheless, vitamin E may interfere with vitamin K metabolism to some extent. Human subjects supplemented with 1000 IU RRR-αT/d (12 weeks) showed an increase in PIVKA-II (protein induced by vitamin K absence factor II) plasma concentrations⁽ Reference Booth, Golly and Sacheck ^{78 )}, which is a marker of subclinical vitamin K deficiency⁽ Reference Widdershoven, Van Munster and De Abreu ^{79 )}. Interactions between the metabolism of vitamins E and K are feasible, since both appear to share the same metabolic fate and may be ω-hydroxylated by the same enzyme(s) (inter alia CYP4F2)⁽ Reference Sontag and Parker ^{50 ,} Reference Bardowell, Duan and Manor ^{51 ,} Reference Edson, Prasad and Unadkat ^{80 )}. However, vitamin K ω-hydroxylation was neither affected in cells incubated with, nor in animals fed high doses of, vitamin E⁽ Reference Farley, Leonard and Labut ^{46 ,} Reference Farley, Leonard and Taylor ^{81 )}. Thus, the mechanism of vitamin E-induced changes in vitamin K status remains unclear and warrants further investigation.

In summary, reduced blood coagulation and increased bleeding have been observed in some cases in animals and human subjects treated with aspirin or warfarin simultaneously with high doses of vitamin E (mainly αT). Such a synergistic activity may be desirable, for example in patients at risk for CVD or stroke, or undesirable, as in the case of increased (gastrointestinal) bleeding, a known side effect of aspirin⁽ Reference Shiotani, Kamada and Haruma ^{82 )}. Vitamin K status and coagulation activity should be monitored in patients supplementing vitamin E while on warfarin therapy (or taking other medication interfering with vitamin K-dependent activation of coagulation factors) to detect and counteract changes in haemostasis.

Vitamin E may impair tamoxifen drug activity in breast cancer patients

Oestrogen-responsive cancer may be treated with the selective oestrogen receptor modulator tamoxifen⁽ Reference Fisher, Redmond and Legault-Poisson ^{83 )}, but most patients eventually develop resistance to the drug⁽ Reference Shou, Massarweh and Osborne ^{84 )}. Tamoxifen rapidly increases intracellular concentrations of the second messenger Ca^{2+ (} Reference Chang, Huang and Wang ^{85 )}. Cell-culture studies suggest that vitamin E may alter the activity of tamoxifen. In oestrogen-responsive mammary carcinoma cells (MCF-7, T47D), incubation with tamoxifen in the presence of αT (10 μmol/l; 24 h) inhibited the tamoxifen-induced rise in intracellular Ca²⁺ and reduction in cell proliferation⁽ Reference Peralta, Viegas and Louis ^{86 )}. In a small prospective study with seven breast cancer patients on tamoxifen therapy, supplementation with 400 mg/d αT acetate for 30 d reduced tamoxifen blood concentrations in five patients and increased markers of tamoxifen resistance in six of the seven patients. In four of these patients, tamoxifen blood concentrations dropped to below the minimum therapeutic concentration⁽ Reference Peralta, Brewer and Louis ^{87 )}.

In summary, the limited evidence from cell-culture and human intervention trials suggests that it might be undesirable for women on tamoxifen therapy to consume dietary supplements containing high doses of αT, as they may impair drug efficacy. However, before valid conclusions can be drawn, sufficiently powered randomised clinical trials investigating the interactions of αT with tamoxifen pharmacokinetics and pharmacodynamics are required.

High-dose vitamin E supplementation may alter the pharmacokinetics of cyclosporine A

Cyclosporine A is an immunosuppressant administered to prevent graft rejection in organ transplant recipients. One of the side effects of cyclosporine A is an increased generation of reactive oxygen species, which was suggested to be involved in cyclosporine A-induced nephrotoxicity⁽ Reference Baliga, Ueda and Walker ^{88 )}. To investigate if supplementation with antioxidants may be beneficial in cyclosporine A-treated renal transplant recipients, ten patients were supplemented with an antioxidant cocktail (800 IU vitamin E (αT), 1000 mg vitamin C and 6 mg β-carotene per d) or placebo for 6 months in a cross-over study with a 6-month washout period⁽ Reference Blackhall, Fassett and Sharman ^{89 )}. All cyclosporine A trials reported in this paragraph used αT in the dietary supplements (ML Blackhall, RG Fassett, JE Sharman, DP Geraghty and JS Coombes, personal communication). The antioxidant cocktail did not alter any of the markers of oxidative stress, but decreased cyclosporine A concentrations in blood by 24 %⁽ Reference Blackhall, Fassett and Sharman ^{89 )}. In a comparable trial, kidney transplant recipients on cyclosporine A therapy were supplemented for 3 months with placebo (n 28) or 1000 mg vitamin C plus 300 mg vitamin E (αT; n 25) per d and reduced cyclosporine A blood concentrations were reported for the vitamin-supplemented group⁽ Reference de Vries, Oterdoom and Gans ^{90 )}. Similar observations were made retrospectively in heart transplant patients; compared with baseline, cyclosporine A blood concentrations decreased by 30 % when patients were supplemented with 1000 mg vitamin C and 800 IU vitamin E (αT) per d⁽ Reference Lake, Aaronson and Gorman ^{91 )}. However, in all studies, vitamin E was administered in combination with vitamin C, which makes it impossible to discern the contribution of each individual vitamin.

The effect of vitamin E alone on cyclosporine A pharmacokinetics was studied earlier in healthy volunteers before and after supplementation with 800 IU vitamin E (αT) per d for 6 weeks. Vitamin E supplementation had no impact on the glomerular filtration rate or peak plasma concentration, but reduced the AUC of a single oral dose of cyclosporine A (5 mg) by 21 % compared with baseline⁽ Reference Barany, Stenvinkel and Ottosson-Seeberger ^{92 )}.

The mechanism underlying the interaction of vitamin E with cyclosporine A is currently unknown. In the trials discussed above, vitamin E and cyclosporine A were administered separately, which eliminates the possibility of a direct interaction. Cyclosporine A is a substrate for intestinal P-gp and CYP3A4 as well as hepatic CYP3A4 and CYP3A5⁽ Reference Hebert ^{93 )}, which makes pre-systemic interactions on the level of drug absorption and intestinal and systemic interactions possible. Induction of intestinal and hepatic metabolism would both decrease the plasma concentration and AUC of cyclosporine A. However, as discussed above, orally administered αT does not induce the expression of CYP or membrane transporters⁽ Reference Podszun, Grebenstein and Hofmann ^{37 ,} Reference Hundhausen, Frank and Rimbach ^{55 ,} Reference Kluth, Landes and Pfluger ^{56 ,} Reference Leonard, Joss and Mustacich ^{58 )}, rendering this mode of interaction rather unlikely.

Since vitamin E supplementation at daily doses of ≥ 300 mg affected the pharmacokinetics of cyclosporine A in four independent human trials, patients treated with this drug should abstain from the consumption of dietary supplements containing such high doses of vitamin E or at least have the blood concentration and efficacy of the drug tested regularly by their physician.

α-Tocopherol supplementation reverses the overexpression of the fatty acid translocase CD36

The potential interaction of vitamin E with ritonavir described below differs from the cases discussed above in that it relates to a potential adverse interaction in the case of vitamin E deficiency rather than under vitamin E supplementation. Ritonavir is a drug that specifically inhibits the HIV 1 protease and thereby HIV replication⁽ Reference Vacca and Condra ^{94 )} and is used for the treatment of HIV-infected patients. Adverse effects observed upon long-term treatment with ritonavir include hyperlipidaemia and accelerated atherosclerosis, which may in part be caused by an overexpression of the scavenger receptor CD36 protein in macrophages⁽ Reference Dressman, Kincer and Matveev ^{95 )}.

In THP1 monocytes, ritonavir-induced CD36 (over-) expression was reversed by incubation with RRR-αT (50 μmol/l; 24 h)⁽ Reference Munteanu, Zingg and Ricciarelli ^{96 )}. Further evidence to support a role of vitamin E in the expression of CD36 came from two studies in rats fed vitamin E-deficient (1·7 mg αT acetate; < 1 mg total T/kg diet, respectively) compared with vitamin E-sufficient (60 mg αT acetate; 12 mg RRR-αT and 24 mg RRR-γT/kg, respectively) diets for 6 and up to 9 months, respectively, in which an up-regulation of hepatic CD36 mRNA was observed in the deficient animals from the third month onwards⁽ Reference Barella, Muller and Schlachter ^{97 ,} Reference Gaedicke, Zhang and Schmelzer ^{98 )}. In guinea-pigs fed a high-fat diet (21 % fat by weight), CD36 protein expression in the liver was almost twice that observed in control animals on a 5 % fat diet. Inclusion of high doses of RRR-αT (250 mg/kg diet) in the high-fat diet reversed the increase in hepatic CD36 expression back to the expression level observed in animals fed the 5 % fat control diet⁽ Reference Podszun, Grebenstein and Spruss ^{54 )}.

Taken together, these data suggest that vitamin E deficiency might increase CD36 expression and could thus potentially aggravate ritonavir-induced hyperlipidaemia. Therefore, a sufficient intake (and perhaps even low-dose supplementation) of vitamin E may be beneficial in ritonavir-treated patients. However, there are presently no in vivo data available to corroborate that such an interaction may be biologically relevant in humans and further studies addressing the potential benefit as well as the safety of a co-administration of ritonavir and vitamin E are necessary before valid recommendations can be given.