Inhibitory effects of apigenin and kaempferol on the essential solute carrier transporters

Abstract

AIM: To evaluate the inhibitory effects of apigenin and kaempferol on the uptake of several important solute carrier (SLC) transporters.

METHODS: Various SLC transporters including the essential human organic anion transporter 1 (OAT1), OAT2, OAT3 and OAT4 as well as the important organic cation transporter 1 (OCTN1) and OCTN2, were over-expressed in human embryonic kidney (HEK)-293 cells, a well-established cell model of transporter studies. Transport uptake assay was performed 24 h after the transfection. The transport activity was assessed with the uptake of previously determined transporter model substrates and the inhibitory effect of apigenin and kaempferol was evaluated with the substrate uptake in the presence of 10 μmol/L of each compound. Uptake measurements with varying concentrations of inhibitors (ranged from 0.0001 to 50 μmol/L) were performed to further characterize the inhibitory potency of apigenin and kaempferol. The IC₅₀ value (the concentration that inhibits 50% of the transporter function) of each compound was then calculated by the nonlinear regression model of Graphpad Prism 6.0 software.

RESULTS: Our data indicated that apigenin could potently inhibit the uptake of estrone-3-sulfate (ES) mediated by the HEK-293 cells expressing OAT2, OAT3 and OAT4 as well as the L-ergothioneine uptake via OCTN1-expressing HEK-293 cells. Among these transporters, the most prominent inhibition of apigenin was observed in the case of OAT3. Kaempferol showed significant inhibitory effects on the uptake of ES mediated through OAT2 and OAT3. Impaired L-ergothioneine uptake due to the presence of kaempferol was also observed in OCTN1-expressing HEK-293 cells. Similar to apigenin, kaempferol showed the most potent inhibitory effect on OAT3 as well. To further assess the inhibitory potencies of these two compounds on the uptake of ES mediated by OAT3-expressing HEK-293 cells, their IC₅₀ values were then determined. Both chemicals showed pronounced inhibitory potencies on OAT3 with the IC₅₀ values of 1.7 ± 0.1 and 1.0 ± 0.1 μmol/L (P < 0.01) for apigenin and kaempferol, respectively.

CONCLUSION: Both apigenin and kaempferol are potent inhibitors of OAT3; precautions will be necessary when co-administrating them with drugs that are substrates of OAT3.

Key Words: Apigenin, Kaempferol, Organic anion transporters, Organic cation transporters, Pharmacokinetics, Drug-drug/herb interactions

Core tip: Our study showed that both apigenin and kaempferol could significantly inhibit various solute carrier transporters, in particular organic anion transporter 3, at their clinical doses. Inhibition on these transporters can greatly impact on pharmacokinetic performance of drugs that are substrates of these transporters by altering their absorption, distribution and excretion. Precautions should be implemented when co-administering apigenin and kaempferol with drugs that are substrates of these specific solute carrier transporters, in order to maximise desired therapeutic outcomes and avoid unexpected toxicities. This overall enhances our understanding of drug-drug/herb interactions related to apigenin and kaempferol and provides critical information to improve future multi-drug therapies.

INTRODUCTION

Herbal preparations, such as ginkgo biloba, are currently used with increasing popularity for their broad range of pharmacological effects. Apigenin and kaempferol are flavonoids found in ginkgo biloba as well as in a wide range of vegetables and fruits like tea, beans, broccoli and apples[1,2]. Apigenin and kaempferol have been considered to be clinically important for their anti-oxidative and neuroprotective effects[3,4] as well as chemoprotective properties[2,5]. However, when co-administered with other agents, they could largely impact on the pharmacokinetic performance of other drugs, mainly due to drug-drug/herb interactions[4,6]. The common pathways for such interactions are through competing for specific metabolising enzymes[7-10] or the influx/efflux transporters in charge of the cellular entry/exit of various drugs in body[11-13]. Impaired biotransformation and/or cellular entry/exit can lead to unsatisfactory clinical outcomes and/or increased risk of side effects.

The solute carrier (SLC) gene super family is responsible for transporting both endogenous and exogenous substrates across the cell membrane[14,15]. SLCO genes coding the organic anion transporting polypeptides (OATPs) as well as SLC22A genes coding the organic anion transporters (OATs) and organic cation transporters (OCTs/OCTNs) are the two most important SLC subfamilies involved with drug transport[16]. These organic ion transporters mediate the influx of a wide range of commonly prescribed therapeutic drugs including antibiotics, cholesterol-lowering medications and anti-virals[16,17]. They are expressed in various tissues including intestines, liver and kidney, which are the key organs involved with drug performance in body. Any alteration in the function of these transporters may consequently alter the efficacy and/or inducing toxicity of these agents[18,19].

Up till now, apigenin and kaempferol have been found to be inhibitors of a few SLC transporters, in particular OATP1A2, 1B1, 1B3, 2B1 and OCT2[20-22], which effect may impact on the pharmacokinetics or pharmacodynamics of co-administered drugs[20]. However, no data has yet been shown to demonstrate the relationship of apigenin and kaempferol with the OATs and OCTNs.

Apigenin and kaempferol are structurally similar to naringin, an active constituent found in grapefruit (Figure 1). It has reported that naringin could interact with multiple drugs through competing for OATPs[23]. In addition, naringin is an potent inhibitor of OAT3[24]. Therefore, it is reasonable to postulate that, apigenin and kaempferol may also exhibit inhibitory effects on OATs as well as OCTNs. In this study, we investigated the potential relationship of apigenin and kaempferol with a number of important OATs and OCTNs on their uptake of specific substrates, which could assist our understanding of the potential drug-drug/herb interactions between these flavonoids and other co-administered drugs.

Figure 1 Chemical structures of apigenin, kaempferol and naringin.

MATERIALS AND METHODS

Chemicals

[³H]-estrone-3-sulfate (ES) (57.3 Ci/mmol) was purchased from PerkinElmer (Melbourne, VIC, Australia). [³H]-4-Aminohippuric acid (PAH, 60 Ci/mmol), [³H]-L-ergothioneine (1.7 Ci/mmol) and [¹⁴C]-L-carnitine (56 mCi/mmol) were purchased from BioScientific Pty. Ltd., Gymea, NSW, Australia. Culture media were purchased from Invitrogen (Mount Waverley, VIC, Australia). Apigenin, kaempferol and all the other chemicals were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).

As described previously[25], the plasmids containing the full-length human OCTN1 (reference sequence: NM-003059) and OCTN2 (reference sequence: NM_003060) cDNAs were obtained from Gene-Ethics (Asia) Pte Ltd., Singapore. The plasmids cloned with the ORFs of human OAT1 (reference sequence: NM-004790.4), OAT2 (reference sequence: NM-006672.2) and OAT3 (reference sequence: NM-004254.2) were purchased from Australian Biosearch, Balcatta, Western Australia. The plasmids of human OAT4 was cloned in house as described previously[26].

Expression of SLC transporters in human embryonic kidney-293 cells

Human embryonic kidney (HEK)-293 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% heat-deactivated fetal calf serum at 37 °C and 5% CO₂. Cells were transfected with plasmid DNA using Lipofectamine 2000 Reagent (Invitrogen, Mount Waverley, VIC, Australia) following the manufacturer’s instructions. Twenty-four hours after transfection, transport activities were measured.

Transport assays

Uptake of radio-labelled typical substrate of each SLC transporter was initiated in phosphate-buffered saline (PBS), pH 7, containing 5 mmol/L glucose, 1mM CaCl₂ and MgCl₂. Substrate concentrations and time used in the study were compatible with the previously determined time- and concentration-dependence studies of these transporters: 0.3 μmol/L [³H]-ES for OAT3, OAT4[26,27]; 0.5 μmol/L [³H]-ES for OAT2 (pH 5.5)[28]; 1 μmol/L [³H]-PAH for OAT1[29]; 1 μmol/L [³H]-L-ergothioneine for OCTN1[30]; 5 μmol/L [¹⁴C]-L-carnitine for OCTN2[31], respectively. The uptake was terminated at 8 min intervals by rapidly washing the cells in ice-cold PBS. As our experiments indicated, the initial rates of transporter-mediated substrate uptake in HEK293 cells were linear over at least 8 min (data not shown). The cells were then solubilized in 0.2 mol/L NaOH and neutralized with 0.2 mol/L HCl, and the intracellular accumulation of activity was determined through liquid scintillation counting. Uptake count was standardized to the amount of protein in each well and all uptake data were subtracted with the background counts of vector transfected cells. Data are presented as mean ± SE (n = 3).

A range of uptake measurements with varying concentrations (ranged from 0.0001 to 50 μmol/L) were performed to assess the inhibitory effect of each compound. The IC₅₀ value (the concentration that inhibits 50% of the transporter) of each compound was then calculated by nonlinear regression models in GraphPad Prism 6.0 (GraphPad Inc, LaJolla, CA, United States). Each experiment was repeated for three times with triplicate wells for each data group in every experiment.

Statistical analysis

The Student’s t test was used for differences between two data sets. The criterion of significance was taken to be P < 0.01 and P < 0.001.

RESULTS

Inhibitory effects of apigenin and kaempferol on the uptake of the specific substrates of OATs and OCTNs

HEK293 cells are well established in over-expressing genes, which was also widely used in a number of transporter studies[25,31-34]. The uptake of the specific substrate for human OAT1, OAT2, OAT3, OAT4, OCTN1 and OCTN2 with the presence of apigenin at 10 μmol/L was measured to examine the potential inhibitory effect of this compound. As shown in Figure 2A, apigenin led to a significant inhibition of the uptake of ES in OAT2, OAT3 and OAT4. The inhibition of L-ergothioneine uptake via OCTN1 was also observed (Figure 2A). Among these transporters, OAT3 showed the most prominent inhibition (greater than 80%).

Figure 2 Inhibitory effects of apigenin and kaemperol on the uptake of the specific substrates of organic anion transporters and organic cation transporters. A: Uptake of each radio-labelled substrate was measured in the absence (white bars) and presence of 10 μmol/L apigenin (black bars); B: Uptake of each radio-labelled substrate was measured in the absence (white bars) and presence of 10 μmol/L kaemperol (black bars). The specific substrates used in this experiment were 1 µmol/L [³H]-PAH for OAT1; 500 nmol/L [³H]-ES for OAT2 (pH 5.5); 300 nmol/L [³H]-ES for OAT3 and OAT4; 1 μmol/L [³H]-L-ergothioneine for OCTN1; 5 μmol/L [¹⁴C]-L-carnitine for OCTN2. The transporter-mediated uptake of each substrate was calculated by subtracting the uptake of vector control and expressed as a percentage of the uptake to the control (without apigenin or kaemperol). Values were mean ± SE (n = 3) of triplicate repeats in three independent experiments. ^bP < 0.01, vs absence group. OAT: Organic anion transporter; ES: Estrone-3-sulfate; OCTN: Organic cation transporter.

The inhibitory effect of kaempferol was also evaluated on the OATs and OCTNs mentioned above (Figure 2B). The uptake measurement was performed with the presence of kaempferol (10 μmol/L) and two of the transporters showed significant reductions in the uptake of their specific substrate, in particular OAT2 and OAT3. An inhibition on L-ergothioneine uptake was also observed for OCTN1-expressing cells. Interestingly, similar to apigenin, kaempferol also showed the most potent inhibitory effect on OAT3.

Inhibitory potency of apigenin and kaempferol on OAT3

To further assess the inhibitory potency of these two compounds on the uptake of ES via OAT3, the IC₅₀ values were determined as per described in the methods. The IC₅₀ values for all other transporters were considered to be not clinically significant, since the inhibition on their substrate uptake was less than 50% with the presence of apigenin and kaempferol at the concentration of 10 μmol/L. As shown in Figure 3, both chemicals showed similar potency with the IC₅₀ values of 1.7 and 1.1 μmol/L for apigenin and kaempferol, respectively.

Figure 3 Inhibitory potency of apigenin and kaempferol on the uptake of estrone-3-sulfate mediated by organic anion transporter 3. A: IC₅₀ of apigenin inhibition on OAT3; B: IC₅₀ of kaemperol inhibition on OAT3. The OAT3 mediated uptake of [³H]-estrone-3-sulfate with the presence of a range of varying concentrations of apigenin or kaempferol (0.0001 to 50 µmol/L) were measured. The transporter-mediated uptake was calculated by subtracting the uptake of vector control and expressed as a percentage of the uptake for the control (without presence of apigenin or kaemperol). Values were mean ± SE (n = 3) of triplicate repeats in three independent experiments. The IC₅₀ value of each compound was then calculated by nonlinear regression models in GraphPad Prism 6.0. OAT3: Organic anion transporter 3.

DISCUSSION

Apigenin and kaempferol are two active flavonoids found in many commonly used herbal preparations such as ginkgo biloba, which is mostly used for its neuroprotective effect to improve memory and concentration problems. They are also enriched in a wide range of daily diet products[35-37]. More importantly, both apigenin and kaempferol have gained popularity in recent years due to their potential chemoprotective effects with low intrinsic toxicity[38-43]. Due to their increased use in the community, potential drug-drug/herb interactions between these compounds and other therapeutic drugs might occur through competing for metabolising enzymes and/or transporters, which may reduce efficacy and increase toxicity in these combination therapies. As shown in previous literature and the current study (Figure 2A), apigenin has significantly reduced the specific substrate uptake of OAT2, OAT3, OAT4, OCTN1 as well as OATPs and OCTs[20-22]. According to the tissue localisation and physiological roles of these transporters, co-administration of apigenin with drugs that are substrates of these transporters, could likely lead to an impaired pharmacokinetic performance of the specific drugs, which consequently could result in unsatisfactory clinical outcomes and/or elevated toxicities.

Similarly, inhibitory effect was also demonstrated with kaempferol on various SLC transporters by literature and this study (Figure 2B). Previous studies reported that kaempferol is a potent inhibitor of OATP1B1 and OATP2B1[20,21], which transporters are highly liver specific. Additionally, in a previous study conducted by Mandery et al[20,21], similar inhibition was also observed in OATP1A2 and OATP1B3 with a different substrate. In the current study, we have demonstrated a significant inhibitory effect of kaempferol on OAT2, OAT3 and OCTN1 for the first time (Figure 2B). Because these transporters are highly expressed in the kidney and liver, co-administration of kaempferol with drugs that are substrates of these transporters could possibly result in poor metabolism and elimination of drugs as well as largely impact on the pharmacological response of such agents.

Furthermore, in our study, both apigenin and kaempferol were observed to be potent inhibitors of human OAT3 with IC₅₀ values of 1.7 and 1.1 μmol/L, respectively (Figure 3). OAT3 is mainly expressed in the kidney. At the basolateral membrane of the renal proximal tubules, it is responsible for taking up organic anions from the blood into the proximal tubule cells to assist the secretion of substances like steroid hormones and prostaglandins[44]. In addition, OAT3 has also been found to transport a range of different exogenous drugs including cholesterol lowering medications, antibiotics, antihistamines, anti-metabolites and anti-retrovirals[19] (Table 1). Previous studies confirmed that inhibition of OAT3 could lead to accumulation of specific drugs due to its role in the excretion and clearance of exogenous drugs[45-48]. In the study conducted by Yasui-Furukori et al[45], it was also found that co-administration of probenecid (a potent inhibitor of OAT3) caused a 50% increase in the AUC and a 70% reduction in the clearance of fexofenadine (a substrate of OAT3) in healthy patients. A similar drug-drug interaction was also found between gemfibrozil (a known inhibitor of OAT3) and pravastatin (a substrate of OAT3), where co-administration of both drugs resulted in a 43% reduction in the renal clearance of pravastatin in healthy patients[47,48]. More importantly, previous in vivo studies have found that with 100 mg oral administration of kaempferol, 0.6 mmol/L of maximum concentration was reached in rats, which was significantly higher than the IC₅₀ value found in this study[49]. Similarly this was also observed for apigenin where 17 mg oral administration (recommended daily dosage range from 50-150 mg) of apigenin reached a Cmax of 1.27 μmol/L[50]. Based on the information gathered previously and in the current study, it is very likely that both apigenin and kaempferol can inhibit OAT3 transport function under their standard clinical doses. This inhibition can significantly reduce the uptake of co-administered drugs into the renal tubular cells, which may lead to an impaired renal clearance of such agents. Thus drug-drug/herb interactions involved with apigenin and kaempferol, could be clinically significant, especially for drugs with narrow therapeutic window like methotrexate, a known substrate of OAT3[51].

Table 1 Tissue localization and known substrates of organic anion transporter 3.

Tissue localization[52]	Endogenous substrates[52]		Exogenous substrates/Inhibitors[44,52]
Renal proximal tubule cells	Second messengers	cAMP, cGMP	ACE Inhibitors	Captopril, quinapril, enalapril
	Bile salts	Cholate, taurocholate	Angiotensin II receptor blockers	Candesartan, losartan, olmesartan, pratosartan, telmisartan, valsartan
	Hormones	Cortisol, DHEA and ES	Antibiotics	Benzylpenicillin, cefadroxil, cefamandol, cefazolin, cefoperazone, cefotaxim, ceftriaxone, cephalexin, cephaloridine, cephalotin, tetracycline
	Prosta-glandins	E2 and F2α	Antivirals	Acyclovir, ganciclovir, valacyclovir, zidovudine
			H2 antagonists	cimetidine, famotidine, ranitidine
			Antiepileptic	Valproate
			Cytostatics	Azathiopurine, methotrexate
			Diuretics	Acetazolamide, bumetanide, chlorothiazide, cyclothiazide, ethacrynate, furosemide, hydrochlorothiazide, methazolamide,trichlormethiazide
			NSAIDs	Acetylsalicylate, diclofenac, flufenamate, ibuprofen, indomethacin, ketoprofen, loxoprofen, mefenamate, naproxen, phenacetin, phenylbutazone, piroxicam, salicylate, sulindac
			Statins	Atorvastatin, fluvastatin, pravastatin, rosuvastatin, simvastatin
			Uricosuric	Probenecid

cAMP: Cyclic adenosine monophosphate; cGMP: Cyclic guanosine monophosphate; DHEA: Dehydroepiandrosterone; ACE: Angiotensin-converting-enzyme inhibitor; ES: Estrone-3-sulfate; NSAIDs: Non-steroidal anti-inflammatory drugs.

In summary, the current study assessed the inhibitory effects of two bioactive flavonoids, apigenin and kaempferol, on a number of essential OATs and OCTNs. Our results indicated that both apigenin and kaempferol can largely influence the transport function of several important organic ion transporters including OAT2, OAT4, OCTN1, and particularly OAT3. As these compounds are widely used in our daily life, precautions should be taken when co-administering such compounds together with other drugs that are substrates of these transporters, so as to achieve desired therapeutic outcomes as well as minimise unexpected toxicities.

COMMENTS

Background

Apigenin and kaempferol are both commonly used flavonoids in the community for their potential pharmacological effects. They are both commercially available and are found in our everyday diet. Previous studies have found that both of these compounds may interact with clinically important drugs through competing for transporters or metabolism enzymes. Solute carrier (SLC) transporters [includes anion transporting polypeptides (OATPs), organic anion transporters (OATs), organic cation transporters (OCTs/OCTNs) and OCTNs] are important transporters that mediate the cellular entry of many drugs in different tissues including the intestines, liver and kidney. Up till now, no studies have yet been conducted to explore the effect of apigenin and kaempferol on OATs and OCTNs.

Research frontiers

As herbal preparations have gained their popularity in the community in the recent years, their pharmacological effect and potential drug-drug/herb interactions became the hot topics in this area. As one of the key mediator of such drug-drug/herb interactions, SLC transporters have also been extensively investigated in regards to their transport mechanism and structural function. Most importantly, the interaction between flavonoids and SLCs becomes the center of focus due to its great influence on the overall therapeutic outcome.

Innovations and breakthroughs

Their study was the first to report that apigenin and kaempferol can both inhibit OATs and OCTNs, in particular OAT3 at their clinical doses. Due to the physiological role and tissue localization of these transporters, such information significantly contribute to better understanding of unsatisfied pharmacological effects and/or unexpected adverse events when co-administering apigenin and kaempferol with other drugs that are substrates of these transporters.

Applications

This study enhanced our knowledge of the inhibitory effects of apigenin and kaempferol on SLC transporters, which allows the advancement of optimizing combination therapy in clinical settings. Dosages should be adjusted accordingly to minimize the chance of toxicity.

Terminology

Drug-drug/herb interaction: a drug affects the activity of another drug when both are administered together. This can also apply to the interactions between drugs and herbal preparations.

Peer review

This interesting study explored the inhibitory effects of apigenin and kaempferol on several SLC transporters. The study design was well thought and the results were properly analysed. The potent inhibitory effect of both compounds on OAT3 is with great clinical significance, since this transporter is responsible for the elimination of a wide range of front-line agents. Precautions should be prioritized to optimize the concurrent therapies involving apigenin and kaempferol.