Tingenone, a pentacyclic triterpene, induces peripheral antinociception due to opioidergic activation

Abstract!

Plants belonging to the genus Maytenus are rou-tinely used in folk medicine for the treatment ofpain diseases. Our previous phytochemical studyof the roots of Maytenus imbricata resulted in theisolation and characterization of tingenone, apentacyclic triterpene. Natural triterpenoids areof growing interest because they have several bio-logical activities, including analgesic properties.The present study assessed the involvement ofthe opiodergic pathway in the tingenone-inducedantinociceptive effect against hyperalgesia in-duced by prostaglandin E2 (2 µg) in the peripheralpathway.We evaluated the effect of several antag-

onists to opioid receptors using the mouse pawpressure test. Tingenone administered into theright hind paw induced a local antinociceptive ef-fect that was antagonized by naloxone, a non-selective antagonist to opioid receptors. Clocinna-mox, naltrindole, and nor-binaltorphimine areselective antagonists to µ, δ, and κ receptors, re-spectively, which reverted the peripheral antino-ciception induced by tingenone. Bestatine acts asan inhibitor of aminopeptidase, an enzyme thatdegrades endogenous opioid peptides, and wasshown to intensify the antinociceptive effect oftingenone. The results suggest that the opioider-gic system participates in the peripheral antinoci-ception induced by tingenone.

Tingenone, a Pentacyclic Triterpene, Induces PeripheralAntinociception Due to Opioidergic Activation

Authors Clarice de Carvalho Veloso1, Vanessa Gregório Rodrigues2, Renata Cristina Mendes Ferreira1, Lucienir Pains Duarte2,Andre Klein1, Igor Dimitri Duarte1, Thiago Roberto Lima Romero1, Andrea de Castro Perez1

Affiliations 1 Department of Pharmacology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte,Minas Gerais, Brazil

2 Department of Chemistry, Institute of Exact Sciences, UFMG, Belo Horizonte, Minas Gerais, Brazil

Key wordsl" Maytenus imbricatal" Celastraceael" tingenonel" pentacyclic triterpenel" opioid receptorsl" peripheral antinociception

received May 7, 2014revised Sept. 12, 2014accepted Sept. 14, 2014

BibliographyDOI http://dx.doi.org/10.1055/s-0034-1383147Published online October 22,2014Planta Med 2014; 80:1615–1621 © Georg ThiemeVerlag KG Stuttgart · New York ·ISSN 0032‑0943

CorrespondenceAndrea de Castro PerezDepartment of PharmacologyInstitute of Biological SciencesFederal University of MinasGerais (UFMG)Av Antonio Carlos 662731270–100 Belo Horizonte,Minas GeraisBrazilPhone: + 55313409272Fax: + [email protected]

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Introduction!

The development of new analgesics for clinicaluses without side effects is the main goal of stud-ies regarding pain.While opioids are themost po-tent and effective drugs in relieving severe pain,their utilization has several effects, such as respi-ratory depression, nausea, constipation, physicaldependence, and tolerance [1].Natural substances derived from plants play animportant role in the development of new analge-sic drugs. Some antinociceptive secondary me-tabolites discovered include alkaloids, terpenoids,and flavonoids [2,3]. The isolation and identifica-tion of triterpenes with antinociceptive effectshave been shown in many studies [4–6].Maytenus is a genus of the Celastraceae family,and its species are used in traditional medicinefor the treatment of gastric disorders, inflamma-tory diseases, and pain as well as other disorders[7–9]. For example, the hexane and ethyl acetateextracts of Maytenus ilicifolia inhibited formalde-hyde-induced nociception and paw edema inmice and carrageenan-induced paw edema in rats[10], while the chloroform extract of Maytenus

Veloso CC et al. T

senegalensis reduced edema induced by crotonoil in mice [8]. The antinociceptive effect of May-tenus rigida [11] and the anti-inflammatory effectof Maytenus heterophylla [12] were also demon-strated.Our previous phytochemical study of Maytenusimbricata roots resulted in the isolation and char-acterization of pentacyclic triterpenes, includingtingenone [13]. The antinociceptive effect of oral-ly administered tingenone in the second phase ofthe formalin test, which is characterized by in-flammatory pain, was demonstrated [14].Pain can be effectively diminished by various en-dogenousmechanisms in the central nervous sys-tem (CNS) and in the terminals of primary affer-ent neurons, such as by endogenous opioids inthe inflamed peripheral tissue [15]. The aim ofthis study was, thus, to investigate whether tinge-none induces a peripheral antinociceptive effectvia the activation of the opioidergic system thatis free from major side effects.

ingenone, a Pentacyclic… Planta Med 2014; 80: 1615–1621

Fig. 1 The effects of tingenone on prostaglandinE2-induced hyperalgesia in mice. Tingenone (Ting)was injected in the third hour after the local ad-ministration of PGE2 (2 µg). The antinociceptive re-sponse was measured by the paw pressure test, asdescribed in Material and Methods. Each columnrepresents the mean ± the standard deviation of Δof the measurement of the nociceptive thresholdexpressed in grams (g) for four animals. * Indicatesthe statistical significance p < 0.05 when comparedto the PGE2 2 µg + V control group. V is 20% DMSO+ 1% Tween 20 in saline and Et is ethanol. ΔDenotesthe difference between the nociceptive thresholdobtained in the beginning of the experiment (basalvalue) before any injection (time zero) and thethreshold measured 10min after the third hour fol-lowing the injection of PGE2 (at which time themaximum antinociceptive effect of tingenone wasobserved).

Fig. 2 Exclusion of the antinociceptive effects oftingenone on the outside paw. PGE2 (2 µg) was ad-ministered in both the right (R) and left (L) hindpaws. Tingenone (200 µg/paw) was administered3 h after PGE2 to the right hind paw. Its vehicle wasadministered to the left paw. Each column repre-sents the mean ± the standard deviation of Δ of themeasurement of the nociceptive threshold ex-pressed in grams (g) regarding four animals. * Indi-cates the statistical significance p < 0.05 whencompared to the PGE2 2 µg + V (R Paw) controlgroup and #p < 0.05 when compared to thePGE2 2 µg + V (L Paw) group. V is 20% DMSO + 1%Tween 20 in saline. Δ Denotes the difference be-tween the nociceptive threshold obtained in thebeginning of the experiment (basal value) beforeany injection (time zero) and the threshold mea-sured 10min after the third hour following the in-jection of PGE2 (at which time the maximum anti-nociceptive effect of tingenone was observed).

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Results!

The intraplantar administration of dosages of 50, 100, and200 µg/paw of tingenone in the third hour after the injection ofprostaglandin E2 (PGE2; 2 µg) induced a peripheral antinocicep-tive effect. Tingenone (25 µg) showed no statistically significantdifference when compared to the group treated with PGE2 + ve-hicle (V; 20% DMSO + 1% Tween 20) (l" Fig. 1).Intraplantar administration of 200 µg tingenone did not alter thenociceptive threshold when injected in the absence of PGE2. Thesolutions of 2% ethanol (vehicle for PGE2) and 20% DMSO + 1%Tween 20 (vehicle of tingenone) did not induce antinociceptionor hyperalgesia when injected alone (l" Fig. 1).To exclude any possible systemic effects, PGE2 was administeredin the right paw and left paw of the animals, while tingenone(200 µg) was administered only to the right paw and its vehiclewas administered to the left paw. The evaluation of the nocicep-tive threshold showed that this dosage of tingenone induced anantinociceptive effect restricted to the treated paw because itdid not alter the hyperalgesia induced by PGE2 in the contralater-

Veloso CC et al. Tingenone, a Pentacyclic… Planta Med 2014; 80: 1615–1621

al paw, suggesting a peripheral site of action (l" Fig. 2). The samedosage was used in subsequent experiments.The intraplantar administration of naloxone (NX; 25, 50, and100 µg/paw), a nonselective antagonist to opioid receptors, re-verted the peripheral antinociception induced by 200 µg/pawdoses of tingenone in a dosage-dependent manner (l" Fig. 3).To investigate the selective involvement of the opioid receptorsubtypes in the antinociceptive effect observed, clocinnamox,naltrindole, and nor-binaltorphimine, selective antagonists to µ,δ, and κ receptors, respectively, were administered. Clocinnamox(Cloc; 10, 20, and 40 µg/paw) and naltrindole (NTD; 15, 30, 60,and 120 µg/paw) reverted the peripheral antinociception in-duced by 200 µg/paw doses of tingenone in a dosage-dependentmanner (l" Figs. 4 and 5, respectively).The intraplantar administration of nor-binaltorphimine (Nor-bin; 100 µg/paw) did not antagonize the antinociceptive effect oftingenone, but this effect was partially antagonized by the localadministration of Nor-bin (200 µg) (l" Fig. 6). Bestatine is an in-hibitor of aminopeptidase, an enzyme that degrades endogenousopioid peptides, and was shown to intensify the antinociceptiveeffect of tingenone when administered in a lower dosage (50 µg/

Fig. 5 Effects of the intraplantar administration ofnaltrindole on the peripheral antinociceptive effectinduced by tingenone. NTD was administered30min before tingenone. Each column representsthe mean ± the standard deviation of Δ of the mea-surement of the nociceptive threshold expressed ingrams (g) for four animals. * Indicates the statisticalsignificance p < 0.05 when treated groups werecompared to the PGE2 2 µg + V1 + V2 control groupand # p < 0.05 when compared to the PGE2 2 µg +V1 + Ting 200 µg group. V1 is saline, V2 is 20%DMSO + 1% Tween 20 in saline, and Et is ethanol.Δ Denotes the difference between the nociceptivethreshold obtained in the beginning of the experi-ment (basal value) before any injection (time zero)and the threshold measured 10min after the thirdhour following the injection of PGE2 (at which timethe maximum antinociceptive effect of tingenonewas observed).

Fig. 4 Effects of the intraplantar administration ofclocinnamox on the peripheral antinociceptive ef-fect induced by tingenone. Cloc was administered30min before tingenone. Each column representsthemean ± the standard deviation of Δ of themea-surement of the nociceptive threshold expressed ingrams (g) regarding four animals.* Indicates thestatistical significance p < 0.05 when compared tothe PGE2 2 µg + V1 + V2 control group and # p < 0.05when compared to the PGE2 2 µg + V1 + Ting 200 µggroup. V1 is saline, V2 is 20% DMSO + 1% Tween 20in saline, and Et is ethanol. ΔDenotes the differencebetween the nociceptive threshold obtained in thebeginning of the experiment (basal value) beforeany injection (time zero) and the thresholdmea-sured 10min after the third hour following the in-jection of PGE2 (at which time themaximum anti-nociceptive effect of tingenone was observed).

Fig. 3 Effects of the intraplantar administration ofnaloxone on the peripheral antinociceptive effectinduced by tingenone. NX was administered 30minbefore tingenone. Each column represents themean± the standard deviation of Δ of themeasurement ofthe nociceptive threshold expressed in grams (g) re-garding four animals. * Indicates the statistical sig-nificance p < 0.05 when compared to the PGE2 2 µg+ V1 + V2 control group and # p < 0.05 when com-pared to the PGE2 2 µg + V1 + Ting 200 µg group. V1is saline, V2 is 20% DMSO + 1% Tween 20 in saline,and Et is ethanol. ΔDenotes the difference betweenthe nociceptive threshold obtained in the beginningof the experiment (basal value) before any injection(time zero) and the thresholdmeasured 10min afterthe third hour following the injection of PGE2 (atwhich time themaximum antinociceptive effect oftingenone was observed).

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paw). When each of the antagonists (NX, Cloc, NTD) and besta-tine were administered alone in the highest dosage, no antinoci-

ception or hyperalgesia was induced (l" Fig. 3, 4, 5, and 7, respec-tively).

Veloso CC et al. Tingenone, a Pentacyclic… Planta Med 2014; 80: 1615–1621

Fig. 7 Effects of the intraplantar administration ofbestatine on the peripheral antinociceptive effectinduced by tingenone. Bestatine (Best) was admin-istered 30min before tingenone. Each column rep-resents the mean ± the standard deviation of Δ ofthe measurement of the nociceptive threshold ex-pressed in grams (g) for four animals. * Indicatesthe statistical significance (p < 0.05) when treatedgroups were compared to the PGE2 2 µg + V1 + V2control group and # p < 0.05 indicates significancewhen the treated groups were compared to thePGE2 2 µg + V1 + Ting 50 µg group. V1 is saline, V2is 20% DMSO + 1% Tween 20 in saline, and Et isethanol. Δ Denotes the difference between the no-ciceptive threshold obtained in the beginning of theexperiment (basal value) before any injection (timezero) and the threshold measured 10min after thethird hour following the injection of PGE2 (at whichtime the maximum antinociceptive effect of tinge-none was observed).

Fig. 6 Effects of the intraplantar administration ofnor-binaltorphimine on the peripheral antinocicep-tive effect induced by tingenone. Nor-bin was ad-ministered 30min before tingenone. Each columnrepresents themean± the standard deviation ofΔ ofthemeasurement of the nociceptive threshold ex-pressed in grams (g) for four animals.* Indicates thestatistical significance p < 0.05 when treated groupswere compared to the PGE2 2 µg + V1 + V2 controlgroup and # p < 0.05 when compared to the PGE22 µg +V1+ Ting 200 µggroup. V1 is saline, V2 is 20%DMSO + 1% Tween 20 in saline, and Et is ethanol.ΔDenotes the difference between the nociceptivethreshold obtained in the beginning of the experi-ment (basal value) before any injection (time zero)and the threshold measured 10min after the thirdhour following the injection of PGE2 (at which timethemaximum antinociceptive effect of tingenonewas observed).

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Discussion!

Natural products have greatly contributed to the development ofimportant drugs that are used in contemporary medicine. One ofthe most important analgesic drugs employed in the clinic is thealkaloid morphine, and other substances with antinociceptive ef-fects include flavonoids and terpenes [2].Tingenone, a pentacyclic triterpene, presents an antinociceptiveeffect in the second phase of the formalin test for inflammatorypainwhen administered orally [14]. To study themechanisms in-volved in this effect, we chose a model in which mechanic hyper-algesia is induced by the intraplantar injection of exogenous PGE2into mice.In the present study, the utilization of peripherally administeredPGE2 (2 µg/paw) induced hyperalgesiawith a maximum intensityin the third hour after its application. These results are in agree-ment with a previous study [16]. Tingenone (200 µg/paw) in-duced a peripheral antinociceptive effect because the intraplan-tar administration of this triterpene in the right paw did not alterthe hyperalgesia induced by PGE2 observed in the contralateralpaw. Thus, this dose was utilized in subsequent experiments.

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The result showed once again that triterpenes are capable of in-ducing central [17] and peripheral [4,18,19] antinociceptive ef-fects.We further demonstrated the peripheral antinociceptive ef-fect of tingenone as well as its mechanism of action.Pain can be effectively mitigated by several endogenous mecha-nisms in the CNS and in first-order afferent neuron terminals.For example, endogenous opioids in the inflamed peripheral tis-sue can induce analgesia [15]. Opioid peptides such as β-endor-phin and Met-enkephalin were detected in immune cells in theinflamed subcutaneous tissues of rat paws, indicating that thesepeptides are synthetized by resident andmigratory immune cellssuch as neutrophils, lymphocytes, monocytes, and macrophages[15,20,21]. Opioid peptides are also synthetized by keratinocytes[20] and peripheral sensory neurons [1].The pretreatment with orally administered tingenone was dem-onstrated to induce an antinociceptive effect in the second phaseof the formalin test for inflammatory pain. However, a lack ofanti-inflammatory effects was observed in the paw edema testand in the recruitment of total leukocytes and neutrophils in thepleural cavity test, both induced by carrageenan. The absence ofantinociceptive effects in the tail flick and in the first phase of the

Fig. 8 Structure of tin-genone, a natural pen-tacyclic triterpene iso-lated from the roots ofM. imbricata.

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formalin test was also observed [14]. Thus, the peripheral antino-ciceptive action of tingenone in the second phase of the formalintest does not suggest an anti-inflammatory action, but instead in-dicates the activation of other intracellular signaling pathways,such as the opioid receptors present in the peripheral nerve end-ings. Previous studies have shown that the activation of peripher-al and central opioidergic receptors result in a reduction in thelicking time during the second stage of the formalin test [22,23].Earlier studies have also shown that µ opioid receptors are upre-gulated in the peripheral sensory nerve endings during the in-flammation of the rat paw in addition to an increase in the num-ber of immune cells containing β-endorphin [24], an opioid pep-tide that is responsible for antinociception in inflammatory pain[25]. Therefore, we suggest that tingenone activates the opioider-gic pathway during the second phase of the formalin test ratherthan during the first phase. Tingenone may not be acting directlyon opioid receptors but instead on immune cells, which in turnrelease endogenous opioid peptides. Considering that immunecells migrate with effects during the second phase (which corre-sponds to inflammatory pain), it is possible that the antinocicep-tive effects of tingenone are dependent on the release of opioidsfrom immune cells during the second phase, which may also belinked to the absence of an anti-inflammatory effect. In otherwords, tingenone does not interfere with cell migration, butthose cells are still important in the process of pain modulation.Thus, to evaluate the involvement of the opioidergic pathway inthe peripheral antinociceptive effects of tingenone, paws werepretreated with NX, a nonselective antagonist for opioid recep-tors. NX reverted the antinociceptive effects of the triterpene ina dosage-dependent manner. We chose the following selectiveantagonists for the opioid receptors: Cloc, a selective antagonistof the µ receptor; NTD, a selective antagonist of the δ receptor;and Nor-bin, a selective antagonist of the κ receptor.Anatomical, molecular, and electrophysiological studies haveshown that all three opioid receptors (µ, δ, and κ) are expressedby sensory neurons. These receptors have been found on cell bod-ies in the dorsal root ganglia (DRG) and on peripheral terminalsof primary afferent neurons in both animals and humans [26].According to our results, it seems that the peripheral antinoci-ceptive mechanism of tingenone involves the µ, δ, and κ recep-tors. The endogenous opioid peptides β-endorphin and enkepha-lins play their roles via the µ and δ receptors, while dynorphinacts through the κ opioid receptor. Endorphins 1 and 2 have ahigh selectivity for the µ opioid receptor [27]. Opioid peptidesoriginating from immune cells may activate the opioidergic re-ceptors present in the peripheral nerve endings of inflamed cells[24,25]. In a previous study, it was demonstrated that the periph-eral administration of µ, δ, and κ opioid receptor agonists pro-duced analgesic effects in inflamed tissues but not in non-in-flamed tissues, suggesting that peripheral opioid receptors canbe functionally activated during inflammation [28]. The inhibi-tion of peptidases is used as a pharmacological strategy to max-imize the effects of the endogenously released opioid peptides.Aminopeptidase N, which is responsible for inactivating endoge-nous opioid peptides, is a transmembrane peptidase expressedon leukocytes and neurons that contains a large extracellular do-main containing the active site [1,29]. A previous study hasshown that leukocytes and peripheral sensory neurons are a ma-jor source of aminopeptidase N and that blockage of this enzymeintensifies the action of opioids in peripheral receptors, which lo-cally inhibits inflammatory pain [30]. In this study, we used bes-tatine to inhibit aminopeptidase N in order to evaluate the in-

volvement of endogenous opioid peptides in modulating theantinociceptive peripheral effects of tingenone administered inlow doses. The intraplantar administration of bestatine intensi-fied the peripheral antinociceptive effect of tingenone, suggest-ing that endogenous opioid peptides participate in this effect.Therefore, we suggest that the endogenous opioid peptides in-volved in tingenone peripheral antinociception are derived fromthe migratory immune cells that contain and secrete opioid pep-tides because they are found in peripheral inflamed tissues andnot in non-inflamed tissues [31].In conclusion, tingenone presents a peripheral antinociceptiveeffect, evidencing the involvement of selective endogenousopioid peptides for receptors µ, δ, and κ. Tingenone has the po-tential to be used as a newanalgesic drug with no systemic collat-eral effects.

Materials and Methods!

Plant materialThe roots of M. imbricata were carefully collected to preventdamage to the specimen. The collection area was the Ouro Pretomunicipality, Minas Gerais state, Brazil. The plant material wasidentified by the botanists Rita M. de Carvalho Okano, BotanicDepartment of the Federal University of Viçosa, and M. CristinaTeixeira Braga Messias, Botanic Department of the Federal Uni-versity of Ouro Preto. A voucher specimen (number 27780) wasdeposited in the Herbarium collection at the Botanic Departmentof the Federal University of Viçosa, Brazil.TheM. imbricata roots were dried at room temperature and pow-dered in a mill. The powder (1.5 kg) was submitted to extractionwith the organic solvent hexane/ethyl ether (1 :1) in a Soxhletapparatus. The solvent was removed in a rotator evaporator. Thequantity of the residue obtained was 16.1 g in the hexane/ethylether (1 :1) extract. From this extract, 1.5 g of tingenone(l" Fig. 8) was isolated and characterized as previously described[13].

AnimalsMale Swiss mice (30–35 g) obtained from the Bioterism Center ofFederal University of Minas Gerais (CEBIO/UFMG) were used inthe experiments (n = 4 per group). Themice were housed in stan-dard cages and kept at a constant temperature of 23°C with a 12-h light-dark cycle and free access to food and tapwater. All testingprocedures were conducted in accordancewith the ethical guide-lines of the International Association for the Study of Pain (IASP)[32] and approved by the Ethics Committee in Animal Experi-

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mentation at the Federal University of Minas Gerais (protocol115/2012).

Measurement of hyperalgesiaHyperalgesia was induced by subcutaneous injection of PGE2(2 µg) into the plantar surface of the hind paw. Hyperalgesia wasmeasured according to the rat paw pressure test [33], which hasbeen adapted to mice [34]. An analgesimeter equipped with acone-shaped paw-presser with a rounded tip was used (Ugo-Ba-sile) to apply a linearly increasing force to the hind paw. Theweight in grams (g) required to elicit the nociceptive response ofpaw flexion was determined to be the nociceptive threshold. Acutoff value of 160 g was used to reduce the possibility of damageto the paws. The nociceptive threshold (Δ) was calculated as thedifference between the nociceptive threshold obtained in the be-ginning of the experiment (basal value) before any injection(time zero) and the threshold measured 10min after the thirdhour following PGE2 injection (at which time the maximum anti-nociceptive effect of tingenone was observed). A nociceptivethreshold value of Δ > 0 indicated hyperalgesia induced by PGE2injection, whereas decreases in this value indicated the antihy-peralgesic effect of the tested drug.

Drugs administrationAll drugs were administered using an injected volume of 20 µL/paw. Tingenone (purity > 98%) was dissolved in 20% DMSO and1% Tween 20 in saline. NX (purity > 98%; Sigma), Cloc (Tocris),NTD (purity > 99%; Tocris), Nor-bin (purity > 98%; Tocris), andbestatine (purity > 99%; Tocris) were dissolved in isotonic saline.PGE2 (Sigma) was dissolved in 2% ethanol in saline.Abbreviations of the solvents are: V is 20% DMSO + 1% Tween 20in saline, Et is ethanol (l" Fig. 1), V1 is saline, and V2 is 20% DMSO+ 1% Tween 20 in saline as shown in the figure legends.

Experimental protocolTingenone was administered subcutaneously in the right hindpaw 3 h after a local injection of PGE2. To determine whether tin-genone was acting outside the injected paw, PGE2 was injectedinto both hind paws, while tingenone was administered to theright paw and the vehicle was administered to the left paw. After10min (corresponding to the peak action of tingenone), the noci-ceptive threshold was measured in both hind paws. NX, Cloc,NTD, Nor-bin, and bestatine were administered 30min prior tothe administration of tingenone.It should be noted that the protocols concerning the doses andtimes of administration of each drug used in this study were ob-tained from the literature data [35] and pilot experiments.

Statistical analysisThe obtained results were analyzed using Graph Pad Prism 3.0and expressed as mean ± SD. Statistical differences betweengroups were calculated by one-way ANOVA followed by the Bon-ferroni test. Statistical significance was set at p < 0.05.

Acknowledgements!

This work was supported by CAPES and CNPq (Brazil).

Veloso CC et al. Tingenone, a Pentacyclic… Planta Med 2014; 80: 1615–1621

Conflict of Interest!

The authors declare that there are no conflicts of interest in rela-tion to this study.

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