Andreas Koeberle

Hinnak Northoff

Dr Oliver Werz
email: oliver.werz@uni-tuebingen.de

Identification of 5-lipoxygenase and microsomal prostaglandin E 2 synthase-1 as functional targets of the anti-inflammatory and anti-carcinogenic garcinol

Keywords: AA, arachidonic acid, COX, cyclooxygenase, cPL, cytosolic phospholipase, ERK, extracellular signal-regulated kinase, FLAP, 5-lipoxygenase-activating protein, 12-HHT, 12

Garcinol (camboginol) from the fruit rind of Guttiferae species shows anti-carcinogenic and anti-inflammatory properties, but the underlying molecular mechanisms are unclear. Here we show that garcinol potently interferes with 5-lipoxygenase (EC 7.13.11.34) and microsomal prostaglandin (PG)E 2 synthase (mPGES)-1 (EC 5.3.99.3), enzymes that play pivotal roles in inflammation and tumorigenesis. In cell-free assays, garcinol inhibited the activity of purified 5-lipoxygenase and blocked the mPGES-1-mediated conversion of PGH 2 to PGE 2 with IC 50 values of 0.1 and 0.3 µM, respectively. Garcinol suppressed 5-lipoxygenase product formation also in intact human neutrophils and reduced PGE 2 formation in interleukin-1βstimulated A549 human lung carcinoma cells as well as in human whole blood stimulated by lipopolysaccharide. Moreover, garcinol interfered with isolated cyclooxygenase (COX)-1 (EC 1.14.99.1, IC 50 = 12 µM) and with the formation of COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and thromboxane B 2 in human platelets. In contrast, neither Ca 2+ -ionophore (A23187)-induced arachidonic acid release in neutrophils nor COX-2 activity in A549 cells or whole blood, measured as formation of 6-keto PGF 1α , or isolated human recombinant COX-2 were significantly affected by garcinol (≤ 30 µM). Together, the high potency of garcinol to selectively suppress PGE 2 synthesis and 5-lipoxygenase product formation provides a molecular basis for the anti-inflammatory and anti-carcinogenic effects of garcinol and rationalizes its therapeutic use.

A c c e p t e d M a n u s c r i p t

Introduction

Garcinol (Fig. 1A) is a polyprenylated acylphloroglucinole contained in the rind of Guttiferae (Garcinia indica, G. huillkensis and G. cambogia), named Kokum, which is used as culinary spice and garnish for curry. In India, Kokum is used in ayurvedic medicine for the treatment of inflammatory and infectious diseases [START_REF] Krishnamurthy | On the structures of garcinol, isogarcinol and camboginol[END_REF]. In rats, garcinol protects against 4-nitroquinoline-1-oxide-induced tongue carcinogenesis [START_REF] Yoshida | Dietary garcinol inhibits 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in rats[END_REF] and azoxymethane-induced colon carcinogenesis [START_REF] Tanaka | Prevention of colonic aberrant crypt foci by dietary feeding of garcinol in male F344 rats[END_REF]. Functional investigations have revealed anti-oxidative [START_REF] Yamaguchi | Antioxidative and anti-glycation activity of garcinol from Garcinia indica fruit rind[END_REF], anti-proliferative [START_REF] Hong | Effects of garcinol and its derivatives on intestinal cell growth: Inhibitory effects and autoxidation-dependent growth-stimulatory effects[END_REF], proapoptotic [START_REF] Pan | Induction of apoptosis by garcinol and curcumin through cytochrome c release and activation of caspases in human leukemia HL-60 cells[END_REF], anti-invasive [START_REF] Liao | Garcinol modulates tyrosine phosphorylation of FAK and subsequently induces apoptosis through down-regulation of Src, ERK, and Akt survival signaling in human colon cancer cells[END_REF], and anti-inflammatory properties of garcinol [START_REF] Pan | Induction of apoptosis by garcinol and curcumin through cytochrome c release and activation of caspases in human leukemia HL-60 cells[END_REF][START_REF] Liao | Suppression of inducible nitric oxide synthase and cyclooxygenase-2 in downregulating nuclear factor-kappa B pathway by Garcinol[END_REF][START_REF] Matsumoto | Cytotoxic benzophenone derivatives from Garcinia species display a strong apoptosis-inducing effect against human leukemia cell lines[END_REF].

Garcinol reduced the expression or activation of kinases involved in cell proliferation (i.e., focal adhesion kinase, extracellular signal-regulated kinase (ERK)-1/2, p38 mitogen-activated protein kinase [START_REF] Liao | Suppression of inducible nitric oxide synthase and cyclooxygenase-2 in downregulating nuclear factor-kappa B pathway by Garcinol[END_REF][START_REF] Hong | Modulation of arachidonic acid metabolism and nitric oxide synthesis by garcinol and its derivatives[END_REF]), blocked the expression of enzymes involved in the development and progression of cancer and inflammation (i.e., cyclooxygenase (COX)-2 (EC 1.14.99.1) and inducible nitric oxide synthase (iNOS)), and suppressed nuclear factor (NF)κB signalling [START_REF] Liao | Suppression of inducible nitric oxide synthase and cyclooxygenase-2 in downregulating nuclear factor-kappa B pathway by Garcinol[END_REF][START_REF] Hong | Modulation of arachidonic acid metabolism and nitric oxide synthesis by garcinol and its derivatives[END_REF]). However, molecular targets of garcinol are less defined, and thus far, only histone acetyltransferase p300 (IC 50 = 7 µM) [START_REF] Balasubramanyam | Polyisoprenylated benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression[END_REF], P300/CBP-associated factor (IC 50 = 5 µM) [START_REF] Balasubramanyam | Polyisoprenylated benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression[END_REF], and acetylcholine esterase [START_REF] Lenta | Leishmanicidal and cholinesterase inhibiting activities of phenolic compounds from Allanblackia monticola and Symphonia globulifera[END_REF] (IC 50 = 0.7 µM) were identified.

Certain COX-1/2-derived prostanoids and 5-lipoxygenase (5-LO)-derived leukotrienes (LTs) initiate and maintain inflammatory and allergic reactions (for review see [START_REF] Funk | Prostaglandins and leukotrienes: advances in eicosanoid biology[END_REF]), and pharmacological intervention with their biosynthesis (i.e., utilizing COX-and 5-LO (EC 7.13.11.34) inhibitors) is applied in therapy of respective diseases. COX-2 and/or 5-LO are also overexpressed in various cancer cells (e.g., prostate, breast, lung, and colon) [START_REF] Zha | Cyclooxygenases in cancer: progress and perspective[END_REF][START_REF] Gupta | Lipoxygenase-5 is overexpressed in prostate adenocarcinoma[END_REF][START_REF] Werz | Therapeutic options for 5-lipoxygenase inhibitors[END_REF], and pre-clinical studies indicate tumor-preventive effects of COX inhibition by non steroidal antiinflammatory drugs (NSAIDs) [START_REF] Gasparini | Inhibitors of cyclo-oxygenase 2: a new class of anticancer agents?[END_REF]. Moreover, pharmacological suppression of the 5-LO pathway, using 5-LO inhibitors or inhibitors of the 5-LO-activating protein (FLAP), causes A c c e p t e d M a n u s c r i p t 5 apoptosis of various tumor cells [START_REF] Werz | Therapeutic options for 5-lipoxygenase inhibitors[END_REF][START_REF] Chen | Five-lipoxygenase pathway of arachidonic acid metabolism in carcino-genesis and cancer chemoprevention[END_REF]. Prostaglandin (PG)E 2 is the most prominent COX product in inflammation and tumorigenesis and is formed from PGH 2 by PGE 2 synthases. The microsomal PGE 2 synthase (mPGES)-1 (EC 5.3.99.3) is an inducible isoform functionally coupled to COX-2. Accumulating evidence suggests that mPGES-1 is responsible for massive PGE 2 formation associated with pathologies [START_REF] Samuelsson | Membrane prostaglandin E synthase-1: a novel therapeutic target[END_REF]. Therefore, mPGES-1 may represent an alternative drug target for treatment of inflammation and cancer, which is supported by data from mPGES-1 deficient mice and animal studies with selective mPGES-1 inhibitors [START_REF] Samuelsson | Membrane prostaglandin E synthase-1: a novel therapeutic target[END_REF][START_REF] Cote | Substituted phenanthrene imidazoles as potent, selective, and orally active mPGES-1 inhibitors[END_REF][START_REF] Xu | MF63 {2-(6-chloro-1H-phenanthro[9,10-d]imidazol-2-yl)isophthalonitrile}, a selective microsomal prostaglandin E synthase 1 inhibitor, relieves pyresis and pain in preclinical models of inflammation[END_REF].

Despite the anti-inflammatory and anti-tumorigenic potential of garcinol, its effects on the formation of eicosanoids were rarely analyzed. Hong et al. recently showed that garcinol modulates arachidonic acid (AA) metabolism in lipopolysaccharide-stimulated macrophages by blocking cytosolic phospholipase (cPL)A 2 (EC 3.1.1.4) activation due to inhibition of ERK1/2-mediated phosphorylation [START_REF] Hong | Modulation of arachidonic acid metabolism and nitric oxide synthesis by garcinol and its derivatives[END_REF]. However, modulation of PG and/or LT biosynthesis by garcinol or effects on the catalytic activity of the key enzymes involved (e.g., COX-1/2, PG synthases, 5-LO) have not been addressed yet. Here, we demonstrate that garcinol inhibits prostanoid and LT formation in cellular and cell-free assays by preferential and potent interference with mPGES-1 and 5-LO.

A c c e p t e d M a n u s c r i p t 6

Materials and Methods

Reagents

Garcinol (Biomol, Hamburg, Germany) was dissolved in dimethyl sulfoxide (DMSO) and kept in the dark at -20 °C, and freezing/thawing cycles were kept to a minimum. The thromboxane synthase inhibitor CV4151 [START_REF] Kato | Thromboxane synthetase inhibitors (TXSI). Design, synthesis, and evaluation of a novel series of omegapyridylalkenoic acids[END_REF] and anti-6-keto PGF 1α antibody were generous gifts by Dr. S. Laufer (University of Tuebingen, Germany) and Dr. T. Dingermann (University of Frankfurt, Germany), respectively. Materials used: DMEM/High Glucose (4.5 g/l) medium, penicillin, streptomycin, trypsin/EDTA solution, PAA (Coelbe, Germany);

PGH 2 , Larodan (Malmö, Sweden); 11β-PGE 2 , PGB 1 , MK-886 (3-[1-(4-chlorobenzyl)-3-t- butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid), 6-keto PGF 1α , human
recombinant COX-2, ovine COX-1, Cayman Chemical (Ann Arbor, MI); [5, 6, 8, 9, 11, 12, 14, 15-3 H]arachidonic acid ([ 3 H]AA), BioTrend Chemicals GmbH (Cologne, Germany); Ultima Gold TM XR, Perkin Elmer (Boston, MA). All other chemicals were obtained from Sigma-Aldrich (Deisenhofen, Germany) unless stated otherwise.

Cells and cell viability assay

A549 cells were cultured in DMEM/High glucose (4.5 g/l) medium supplemented with heatinactivated fetal calf serum (10%, v/v), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 °C in a 5% CO 2 incubator. After 3 days, confluent cells were detached using 1  trypsin/EDTA solution and reseeded at 2  10 6 cells in 20 ml medium in 175 cm 2 flasks. Cell viability was assessed by trypan blue staining and light microscopy. A549 cells (4 × 10 5 ) were plated in a 75 cm 3 cell culture flask and incubated at 37 °C and 5% CO 2 for 72 h. Then, garcinol was added and the incubation was continued for 5 or 24 h before cell viability was A c c e p t e d M a n u s c r i p t 7 determined. Treatment with garcinol (10 or 30 µM) for 5 h did not significantly reduced cell viability, excluding acute cytotoxic effects in the cellular assays used in this study. However, prolonged incubation (24 h) with 10 or 30 µM garcinol led to a strong increase in the ratio of dead cells to total number of cells (50% and 71% dead cells, respectively). Blood cells were freshly isolated from leukocyte concentrates obtained at the Blood Center of the University Hospital Tuebingen (Germany) as described [START_REF] Albert | Hyperforin is a dual inhibitor of cyclooxygenase-1 and 5-lipoxygenase[END_REF]. In brief, venous blood was taken from healthy adult donors that did not take any medication for at least 7 days, and leukocyte concentrates were prepared by centrifugation (4000g, 20 min, 20 °C). Cells were immediately isolated by dextran sedimentation and centrifugation on Nycoprep cushions (PAA, Coelbe, Germany). Platelet-rich-plasma was obtained from the supernatants, mixed with phosphate-buffered saline (PBS) pH 5.9 (3:2 v/v), centrifuged (2100g, 15 min, room temperature), and the pelleted platelets were resuspended in PBS pH 5.9/0.9% NaCl (1:1, v/v). Washed platelets were finally resuspended in PBS pH 7.4 and 1 mM CaCl 2 . Neutrophils were immediately isolated from the pellet after centrifugation on Nycoprep cushions, and hypotonic lysis of erythrocytes was performed as described [START_REF] Werz | Activation of 5lipoxygenase by cell stress is calcium independent in human polymorphonuclear leukocytes[END_REF]. Cells were finally resuspended in PBS pH 7.4 (PBS) containing 1 mg/ml glucose and 1 mM CaCl 2 (PGC buffer) (purity > 96-97%).

Release of arachidonic acid in neutrophils

Freshly isolated human neutrophils (3 × 10 7 /ml PGC buffer) were pre-incubated with the test compounds for 10 min prior to stimulation with Ca 2+ -ionophore A23187 (2.5 µM). After 5 min at 37 °C, the reaction was stopped by addition of 1 ml of methanol. After adjusting pH 3 and addition of magarinic acid (11.1 nmol) as internal standard, released AA was separated by solid phase extraction (RP-18 material and elution with methanol) and coupled to 2,4- Derivatized AA was analyzed by RP-HPLC (gradient: 71% methanol / 29% water to 100% methanol in 20 min, detection at 272 nm).

Determination of COX-1 product formation in washed platelets

Freshly isolated platelets (10 8 /ml PBS containing 1 mM CaCl 2 ) were pre-incubated with the indicated agents for 5 min at room temperature. After addition of 5 µM AA and further incubation for 5 min at 37 °C, the COX-1-derived TxB 2 was quantified using a TxB 2 High Sensitivity EIA Kit (Assay Designs, Ann Arbor, MI). 12(S)-Hydroxy-5-cis-8,10-transheptadecatrienoic acid (12-HHT, non-enzymatically formed from PGH 2 ) was extracted and analyzed by HPLC as described [START_REF] Albert | Hyperforin is a dual inhibitor of cyclooxygenase-1 and 5-lipoxygenase[END_REF].

Determination of PGE 2 and 6-keto PGF 1α formation in intact A549 cells

Cellular PGE 2 and 6-keto PGF 1α formation was determined according to [START_REF] Koeberle | Licofelone suppresses prostaglandin E2 formation by interference with the inducible microsomal prostaglandin E2 synthase-1[END_REF]. A549 cells (2  10 6 cells) were incubated for 16 h at 37 °C and 5% CO 2 , the medium was changed, and the cells were stimulated with interleukin-1β (1 ng/ml) for 48 h. After trypsination, cells were washed with PBS twice. For determination of PGE 2 , 4  10 6 cells per ml PBS containing CaCl 2 (1 mM) were pre-incubated with the indicated compounds at 37 °C for 10 min, and PGE 2 formation was started by addition of ionophore A23187 (2.5 µM), AA (1 µM), and For determination of 6-keto PGF 1α , 1  10 6 cells, resuspended in 1 ml PBS containing CaCl 2 (1 mM), were pre-incubated with the indicated compounds for 15 min at 37 °C, and 6-keto PGF 1α formation was initiated by addition of AA (30 µM). After 15 min at 37 °C, the reaction was stopped by cooling on ice. Cells were centrifuged (300g, 5 min, 4 °C), and the amount of released 6-keto PGF 1α was assessed by ELISA as described [START_REF] Koeberle | Licofelone suppresses prostaglandin E2 formation by interference with the inducible microsomal prostaglandin E2 synthase-1[END_REF].

Activity assays of isolated COX-1 and -2

Inhibition of the activities of isolated ovine COX-1 and human COX-2 was performed as described [START_REF] Koeberle | Licofelone suppresses prostaglandin E2 formation by interference with the inducible microsomal prostaglandin E2 synthase-1[END_REF]. Briefly, purified COX-1 (ovine, 50 units) or COX-2 (human recombinant, 20 units) were diluted in 1 ml reaction mixture containing 100 mM Tris buffer pH 8, 5 mM glutathione, 5 µM haemoglobin, and 100 µM EDTA at 4 °C and pre-incubated with the test compounds for 5 min. Samples were pre-warmed for 60 sec at 37 °C, and AA (5 µM for COX-1, 2 µM for COX-2) was added to start the reaction. After 5 min at 37 °C, the COXderived 12-HHT was extracted and then analyzed by HPLC as described [START_REF] Albert | Hyperforin is a dual inhibitor of cyclooxygenase-1 and 5-lipoxygenase[END_REF].

Preparation of crude mPGES-1 in microsomes of A549 cells and determination of PGE 2 synthase activity

Preparation of A549 cells and determination of mPGES-1 activity was performed as described [START_REF] Koeberle | Licofelone suppresses prostaglandin E2 formation by interference with the inducible microsomal prostaglandin E2 synthase-1[END_REF]. In brief, cells were incubated for 16 h at 37 °C and 5% CO 2 , the medium was replaced, A c c e p t e d M a n u s c r i p t 10 interleukin-1β (1 ng/ml) was added and cells were incubated for another 48 h. Cells were harvested, frozen in liquid nitrogen, and ice-cold homogenisation buffer (0.1 M potassium phosphate buffer pH 7.4, 1 mM phenylmethanesulphonyl fluoride, 60 µg/ml soybean trypsin inhibitor, 1 µg/ml leupeptin, 2.5 mM glutathione, and 250 mM sucrose) was added. After 15 min, cells were sonicated on ice (3  20 sec) and the homogenate was subjected to differential centrifugation at 10,000g for 10 min and 174,000g for 1 h at 4 °C. The pellet (microsomal fraction) was resuspended in 1 ml homogenization buffer and the total protein concentration was determined by Coomassie protein assay [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. Microsomal membranes were diluted in potassium phosphate buffer (0.1 M, pH 7.4) containing 2.5 mM glutathione. Test compounds or vehicle were added and after 15 min at 4 °C, the reaction (100 µl total volume) was initiated by addition of PGH 2 (20 µM, final concentration). After 1 min at 4 °C, the reaction was terminated using stop solution (100 µl; 40 mM FeCl 2 , 80 mM citric acid, and 10 µM of 11β-PGE 2 ). PGE 2 was separated by solid phase extraction on reversed phase (RP)-C18 material using acetonitrile (200 µl) as eluent and analyzed by RP-HPLC (30% acetonitrile / 70% water + 0.007% TFA (v/v)) with UV detection at 195 nm. 11β-PGE 2 was used as internal standard to quantify PGE 2 product formation by integration of the area under the peaks.

Determination of PGE 2 , 6-keto PGF 1α , and 12-HHT formation in human whole blood

Peripheral blood from healthy adult volunteers, who had not received any medication for at least two weeks under informed consent, was obtained by venipuncture and collected in syringes containing heparin (20 U/ml). For determination of PGE 2 and 6-keto PGF 1α , aliquots of whole blood (0.8 ml) were mixed with the thromboxane synthase inhibitor CV4151 (1 µM) and with aspirin (50 µM). A total volume of 1 ml was adjusted with sample buffer (10 mM A c c e p t e d M a n u s c r i p t 11 potassium phosphate buffer pH 7.4, 3 mM KCl, 140 mM NaCl, and 6 mM D-glucose). After pre-incubation with the indicated compounds for 5 min at room temperature, the samples were stimulated with lipopolysaccharide (10 µg/ml) for 5 h at 37 °C. Prostanoid formation was stopped on ice, the samples were centrifuged (2300g, 10 min, 4 °C), and 6-keto PGF 1α was quantified in the supernatant using a 6-keto PGF 1α High Sensitivity EIA Kit (Assay Designs, Ann Arbor, MI) according to the manufacturer's protocol. PGE 2 was determined as described [START_REF] Koeberle | Licofelone suppresses prostaglandin E2 formation by interference with the inducible microsomal prostaglandin E2 synthase-1[END_REF]. In brief, the supernatant was acidified with citric acid (30 µl, 2 M), and after centrifugation (2300g, 10 min, 4 °C), solid phase extraction, and HPLC, analysis of PGE 2 was performed to isolate PGE 2 . The PGE 2 peak (3 ml), identified by co-elution with the authentic standard, was collected, and acetonitrile was removed under a nitrogen stream. The pH was adjusted to 7.2 by addition of 10  PBS buffer pH 7.2 (230 µl) before PGE 2 contents were quantified using a PGE 2 High Sensitivity EIA Kit (Assay Designs, Ann Arbor, MI) according to the manufacturer's protocol.

For determination of 12-HHT, human whole blood (2 ml) was pre-incubated with the indicated compounds at 37 °C for 5 min, and formation of 12-HHT was initiated by addition of 30 µM Ca 2+ -ionophore A23187 and 100 µM AA. After 10 min at 37 °C, the reaction was stopped on ice, and the samples were centrifuged (600g/10 min/4 °C). Aliquots of the resulting plasma (500 μl) were then mixed with 2 ml of methanol, and 200 ng of PGB 1 was added as internal standard. The samples were placed at -20 °C for 2 h and centrifuged again (600g/15 min/4 °C). The supernatants were collected and diluted with 2.5 ml PBS and 75 μl HCl 1 N. Formed 12-HHT was extracted and analyzed by HPLC as described [START_REF] Albert | Hyperforin is a dual inhibitor of cyclooxygenase-1 and 5-lipoxygenase[END_REF].

Expression of human recombinant 5-lipoxygenase in E.coli, preparation of homogenates and 100,000g supernatants, and semi-purification of 5-lipoxygenase

A c c e p t e d M a n u s c r i p t 12 protein E.coli MV1190 was transformed with pT3-5-LO plasmid, and recombinant 5-LO protein was expressed at 27 °C as described [START_REF] Fischer | Phosphorylation-and stimulus-dependent inhibition of cellular 5-lipoxygenase activity by nonredox-type inhibitors[END_REF]. Cells were lysed in 50 mM triethanolamine/HCl pH 8.0, 5 mM EDTA, soybean trypsin inhibitor (60 µg/ml), 1 mM phenylmethanesulphonyl fluoride, and lysozyme (500 µg/ml), homogenized by sonication (3  15 sec), and centrifuged at 100,000g for 70 min at 4 °C. The 100,000g supernatant (S100) was applied to an ATPagarose column to partially purify 5-LO as described previously [START_REF] Burkert | The C2-like beta-barrel domain mediates the Ca2+-dependent resistance of 5-lipoxygenase activity against inhibition by glutathione peroxidase-1[END_REF]. S100 or semi-purified 5-LO were immediately used for activity assays.

Determination of 5-lipoxygenase product formation in cell-free systems

Aliquots of S100 or semi-purified 5-LO were diluted with ice-cold PBS containing 1 mM EDTA, and 1 mM ATP was added. Samples were pre-incubated with the test compounds as indicated. After 10 min at 4 °C, samples were pre-warmed for 30 sec at 37 °C, and 2 mM CaCl 2 plus 20 µM AA was added to start 5-LO product formation. The reaction was stopped after 10 min at 37 °C by addition of 1 ml ice-cold methanol, and the formed metabolites were analyzed by RP-HPLC as described [START_REF] Werz | Activation of 5lipoxygenase by cell stress is calcium independent in human polymorphonuclear leukocytes[END_REF]. 5-LO products include the all-trans isomers of LTB 4 and 5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid.

Determination of 5-lipoxygenase product formation in intact cells

Freshly isolated neutrophils (5 × 10 6 /ml PGC buffer) were pre-incubated with the test compounds for 15 min at room temperature and 5-LO product formation was started by addition of 2.5 µM ionophore A23187 plus 20 µM AA. After 10 min at 37 °C, the reaction was stopped with 1 ml of methanol and 30 µl of 1 N HCl, and then, 200 ng PGB 1 and 500 µl PBS were added. Formed 5-LO metabolites were extracted and analyzed by HPLC as described [START_REF] Werz | Activation of 5lipoxygenase by cell stress is calcium independent in human polymorphonuclear leukocytes[END_REF]. 5-LO products include LTB 4 and its all-trans isomers, and 5(S)-

Effects of garcinol on arachidonic acid release from human neutrophils

Previously, it was suggested that garcinol interferes with eicosanoid formation at the level of gene expression (COX-2) [START_REF] Liao | Suppression of inducible nitric oxide synthase and cyclooxygenase-2 in downregulating nuclear factor-kappa B pathway by Garcinol[END_REF], or ERK1/2 phosphorylation resulting in reduced AA release in lipopolysaccharide-stimulated RAW264.7 murine macrophages [START_REF] Hong | Modulation of arachidonic acid metabolism and nitric oxide synthesis by garcinol and its derivatives[END_REF]. We investigated the effects of garcinol on cellular cPLA 2 -mediated AA release in human neutrophils using the Ca 2+ -ionophore A23187 as stimulus that acts by substantial elevation of [Ca 2+ ] i , independent of cellular signalling pathways. Neutrophils, pre-incubated with garcinol (3 to 33 µM), were stimulated with Ca 2+ -ionophore (2.5 µM), and the released AA was derivatized with 2,4dimethoxyaniline and analyzed by RP-HPLC. Garcinol failed to significantly suppress the release of AA up to 33 µM (Fig. 1B), whereas the cPLA 2 α-inhibitor (control) potently inhibited AA liberation.

Effects of garcinol on cellular prostanoid formation

Next, we investigated the effects of garcinol on the transformation of AA to prostanoids in intact cells. Prostanoid formation was initiated by addition of exogenous AA in order to circumvent endogenous AA supply, and thus, to exclude inhibition of prostanoid formation at the level of PLA 2 -mediated substrate release. For determination of COX-1 inhibition, human platelets were pre-incubated with garcinol (1 to 33 µM), and formation of COX-1-derived TxB 2 and 12-HHT (non-enzymatically formed from PGH 2 in platelets [START_REF] Hamberg | Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets[END_REF]) was initiated by AA (5 µM). Garcinol significantly suppressed TxB 2 (Fig. 1C) and 12-HHT (Fig. 1D) formation at 10 µM with an IC 50 of 16 and 11 µM, respectively. To investigate inhibition of cellular COX-2 activity, we assessed the formation of PGE 2 and the stable PGI 2 degradation A c c e p t e d M a n u s c r i p t 15 product 6-keto PGF 1α (as biomarker for PGI 2 formation) in A549 cells. For measuring 6-keto PGF 1α , higher concentrations of AA (30 instead of 1 µM) were required than for determination of PGE 2 to get detectable amounts of 6-keto PGF 1α (detection limit: 14 ng/ml at a signal to noise ratio of 3). A contribution of COX-1 can be excluded in interleukin-1βstimulated A549 cells because COX-1 is neither detectable on the protein nor on the RNA level [START_REF] Asano | Prostaglandin G/H synthase-2 is the constitutive and dominant isoform in cultured human lung epithelial cells[END_REF][START_REF] Thoren | Coordinate up-and down-regulation of glutathionedependent prostaglandin E synthase and cyclooxygenase-2 in A549 cells. Inhibition by NS-398 and leukotriene C4[END_REF]. COX-2-mediated synthesis of PGE 2 was significantly inhibited at 10 µM with an apparent IC 50 of approx. 10 µM (Fig. 2A), whereas the synthesis of 6-keto PGF 1α was not significantly reduced by garcinol up to 33 µM (Fig. 2B). PGE 2 formation is inhibited by garcinol within a narrow concentration range (3 to 10 µM), and higher concentrations of garcinol failed to completely block PGE 2 synthesis. Also MK-886 (33 µM), a mPGES-1 inhibitor, did not interfere with 6-keto PGF 1α synthesis and failed to entirely suppress PGE 2 formation [START_REF] Koeberle | Licofelone suppresses prostaglandin E2 formation by interference with the inducible microsomal prostaglandin E2 synthase-1[END_REF]. In contrast, the COX-2 inhibitor celecoxib (5 µM) efficiently suppressed both formation of PGE 2 (Fig. 2A) and 6-keto PGF 1α (Fig. 2B), respectively.

Effects of garcinol on the activity of COX-1/2 in cell-free assays

We determined whether garcinol may directly interfere with the catalytic activity of isolated COX-1 and/or COX-2. The formation of 12-HHT, the major COX-1/2-derived product under the experimental conditions chosen [START_REF] Capdevila | The catalytic outcomes of the constitutive and the mitogen inducible isoforms of prostaglandin H2 synthase are markedly affected by glutathione and glutathione peroxidase(s)[END_REF], was initiated by addition of AA (5 µM for COX-1, 2 µM for COX-2). Garcinol concentration-dependently and significantly inhibited COX-1mediated 12-HHT formation at 10 µM with an IC 50 = 12 µM, which is in good agreement with the efficacy in intact platelets (IC 50 = 11 µM, see above). In contrast, COX-2 activity was not significantly reduced up to 33 µM (Fig. 3A). Indomethacin (10 µM, for COX-1) and celecoxib (5 µM, for COX-2), used as reference compounds, potently inhibited 12-HHT formation, as expected (Fig. 3B).

A c c e p t e d M a n u s c r i p t 16

Effects of garcinol on the activity of mPGES-1

Because garcinol failed to inhibit COX-2 in cell-free assays and did not suppress 6-keto PGF 1α synthesis in A549 cells but reduced PGE 2 formation, it appeared reasonable that garcinol might selectively suppress PGE 2 formation via inhibition of PGE 2 synthases downstream of COX-2. Microsomal preparations of interleukin-1β-stimulated A549 cells, used as source of mPGES-1 [START_REF] Jakobsson | Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target[END_REF], were pre-incubated with garcinol or vehicle (DMSO) for 15 min, and then, PGE 2 formation was initiated by addition of 20 µM PGH 2 . In agreement with the literature [START_REF] Koeberle | Licofelone suppresses prostaglandin E2 formation by interference with the inducible microsomal prostaglandin E2 synthase-1[END_REF][START_REF] Claveau | Microsomal prostaglandin E synthase-1 is a major terminal synthase that is selectively up-regulated during cyclooxygenase-2-dependent prostaglandin E2 production in the rat adjuvant-induced arthritis model[END_REF], the mPGES-1 inhibitor MK-886 (used as control) inhibited PGE 2 formation with an IC 50 of 2.1 µM (data not shown). Garcinol was slightly superior over MK-886 and concentration-dependently inhibited PGE 2 formation with an IC 50 of 1.2 µM (Fig. 4A). Decreasing the PGH 2 concentration to 1 µM even enhanced the potency of garcinol to suppress mPGES-1 activity (IC 50 = 0.3 µM, Fig. 4B) suggesting a substrate competitive inhibitory mechanism.

To investigate whether garcinol inhibits PGE 2 synthesis in a reversible manner, microsomal preparations of A549 cells were pre-incubated with garcinol and MK-886, and wash-out experiments were carried out. MK-886 and garcinol failed to efficiently block PGE 2 synthesis at 0.3 µM, whereas PGE 2 formation was efficiently inhibited at 3 µM (Fig. 4C). However, 10-fold dilution of the samples containing 3 µM garcinol and MK-886 restored mPGES-1 activity (Fig. 4C) implying a reversible mode of inhibition.

Effects of garcinol on prostanoid formation in human whole blood

Many potent inhibitors of prostanoid biosynthesis in cell-free assays or in intact isolated cells lose their efficacy under physiologically relevant conditions such as in whole blood.

A c c e p t e d

M a n u s c r i p t 17 Therefore, we investigated whether garcinol could affect prostanoid biosynthesis also in a human whole blood assay. To assess inhibition of COX-1, freshly drawn human venous blood was pre-incubated with the test compounds or vehicle (DMSO) for 5 min and then treated with Ca 2+ -ionophore plus AA (100 µM). Formation of 12-HHT, which mainly derives from constitutively expressed COX-1 under these experimental conditions, was not significantly affected by garcinol up to 33 µM (Fig. 5A). In control experiments, indomethacin (20 µM) almost completely blocked 12-HHT synthesis.

For determination of COX-2 inhibition, heparinized blood was pre-incubated with garcinol or vehicle (5 min) and stimulated with lipopolysaccharide for 5 h. Then, the levels of PGE 2 and 6-keto PGF 1α were determined in the corresponding plasma by ELISA. For measuring PGE 2 levels, PGE 2 was first separated from other AA metabolites by RP-HPLC [START_REF] Koeberle | Licofelone suppresses prostaglandin E2 formation by interference with the inducible microsomal prostaglandin E2 synthase-1[END_REF]. In agreement with the results obtained with A549 cells, garcinol suppressed PGE 2 formation in whole blood in a concentration-dependent manner starting at 3 µM with an apparent IC 50 value of 30 µM (Fig. 5B). Significant inhibition of PGE 2 formation was only reached at 30 µM for both garcinol and the mPGES-1 reference inhibitor MK-886, and also prolonged stimulation of whole blood with LPS up to 24 h did not increase the potency of garcinol. Neither garcinol nor MK-886 reduced 6-keto PGF 1α formation up to 30 µM under the same conditions, suggesting a selective interference of garcinol with PGE 2 synthesis (Fig. 5C). Note that garcinol as well as MK-886 were less efficient in inhibiting PGE 2 formation as compared to indomethacin or celecoxib.

Inhibition of 5-lipoxygenase product formation by garcinol

To determine the effects of garcinol on 5-LO product formation in intact cells, freshly isolated human neutrophils were pre-incubated with garcinol for 15 min and 5-LO product synthesis A c c e p t e d M a n u s c r i p t 18 was initiated by Ca 2+ -ionophore A23187 (2.5 µM) plus exogenous AA (20 µM). 5-LO product formation was concentration-dependently inhibited by garcinol with an IC 50 = 1.9 µM (Fig. 6A). Differences in the potency of garcinol to inhibit the formation of LTB 4 (formed via LTA 4 hydrolase) and 5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid (IC 50 = 1.5 and 1.8 µM, respectively) were not observed, excluding an interference of garcinol with LTA 4 hydrolase at these concentrations. The efficiency of garcinol was somewhat improved in a cell-free assay using 100,000×g supernatant of homogenized neutrophils as source of enzyme (IC 50 = 0.5 µM, Fig. 6B) and was even higher for semi-purified recombinant 5-LO (IC 50 = 0.1 µM, Fig. 6C). In contrast, the iron-ligand type 5-LO inhibitor BWA4C (1 µM, used as control) was equally effective independent of the assay conditions (data not shown).

A c c e p t e d M a n u s c r i p t 19

Discussion

Animal studies have revealed an anti-carcinogenic potential of garcinol [START_REF] Yoshida | Dietary garcinol inhibits 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in rats[END_REF][START_REF] Tanaka | Prevention of colonic aberrant crypt foci by dietary feeding of garcinol in male F344 rats[END_REF], and in vitro studies have demonstrated potent growth-inhibitory and apoptotic [START_REF] Hong | Effects of garcinol and its derivatives on intestinal cell growth: Inhibitory effects and autoxidation-dependent growth-stimulatory effects[END_REF][START_REF] Pan | Induction of apoptosis by garcinol and curcumin through cytochrome c release and activation of caspases in human leukemia HL-60 cells[END_REF] as well as antioxidant [START_REF] Yamaguchi | Antioxidative and anti-glycation activity of garcinol from Garcinia indica fruit rind[END_REF] and anti-inflammatory effects [START_REF] Liao | Suppression of inducible nitric oxide synthase and cyclooxygenase-2 in downregulating nuclear factor-kappa B pathway by Garcinol[END_REF][START_REF] Hong | Modulation of arachidonic acid metabolism and nitric oxide synthesis by garcinol and its derivatives[END_REF]. Interference with lipopolysaccharideinduced NFκB signalling, lowering COX-2 and iNOS expression, may explain the long-term anti-inflammatory and anti-carcinogenic effects of garcinol [START_REF] Liao | Suppression of inducible nitric oxide synthase and cyclooxygenase-2 in downregulating nuclear factor-kappa B pathway by Garcinol[END_REF], whereas the short term effects were ascribed to impaired cPLA 2 activation through inhibition of ERK-1/2 phosphorylation restricting the supply of AA for eicosanoid formation [START_REF] Hong | Modulation of arachidonic acid metabolism and nitric oxide synthesis by garcinol and its derivatives[END_REF]. Although suppression of eicosanoid formation may be a crucial mode of action of garcinol, direct inhibitory effects on enzymes involved in eicosanoid biosynthesis have not been investigated yet.

Our data demonstrate that garcinol is a direct and potent inhibitor of purified 5-LO and cellfree mPGES-1 with IC 50 = 0.1 and 0.3 µM, respectively, and also a direct inhibition of COX-1 by garcinol is evident. Inhibition of other molecular targets (i.e., histone acetyltransferase p300 [START_REF] Balasubramanyam | Polyisoprenylated benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression[END_REF], P300/CBP-associated factor [START_REF] Balasubramanyam | Polyisoprenylated benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression[END_REF], and acetylcholine esterase [START_REF] Lenta | Leishmanicidal and cholinesterase inhibiting activities of phenolic compounds from Allanblackia monticola and Symphonia globulifera[END_REF]) required higher concentrations of garcinol (IC 50 = 0.7 to 7 µM). However, because of the different experimental settings applied for analysis of the interference of garcinol with these targets, direct comparison of garcinol´s potencies is difficult. 5-LO and mPGES-1 are crucial enzymes in the biosynthesis of key mediators in inflammatory processes [START_REF] Werz | Therapeutic options for 5-lipoxygenase inhibitors[END_REF][START_REF] Samuelsson | Membrane prostaglandin E synthase-1: a novel therapeutic target[END_REF], providing a rationale for the anti-inflammatory effects of garcinol. Moreover, 5-LO and mPGES-1 are overexpressed in many tumors, and PGE 2 as well as 5-LO products play important roles in tumorigenesis [START_REF] Werz | Therapeutic options for 5-lipoxygenase inhibitors[END_REF][START_REF] Chen | Five-lipoxygenase pathway of arachidonic acid metabolism in carcino-genesis and cancer chemoprevention[END_REF][START_REF] Samuelsson | Membrane prostaglandin E synthase-1: a novel therapeutic target[END_REF]. Together, we conclude that the interference of garcinol with 5-LO and mPGES-1 provides a molecular basis for its documented anti-carcinogenic and antiinflammatory properties.

A c c e p t e d M a n u s c r i p t 20 Inhibition of 5-LO and mPGES-1 is evident also in intact cell assays and in human whole blood. On the other hand, inhibition of COX-1-derived 12-HHT formation in human whole blood was minute and not significant up to 33 µM, although COX-1 activity is obviously inhibited in intact platelets and in the cell-free assay (IC 50 = 11 and 12 µM, respectively). This loss of potency in whole blood might be related to albumin-binding of garcinol but also to competition with high AA concentrations (that are required in whole blood for analysis of 12-HHT formation) for binding to the active site of COX-1.

The experimental settings (stimuli used, availability of respective substrates, and products determined) of these cell-based assays allowed us to attribute the effects of garcinol to the interference with the putative targets and to exclude other points of attack. In particular, garcinol may simply block the supply of AA by interference with cPLA 2 activity , an enzyme, whose activation and cellular functionality has been previously proposed to be affected by garcinol [START_REF] Hong | Modulation of arachidonic acid metabolism and nitric oxide synthesis by garcinol and its derivatives[END_REF]. Hong et al. showed that in lipopolysaccharide-stimulated RAW264.7 murine macrophages garcinol blocked AA release by impairing the activation of cPLA 2 . This was ascribed to the interference with defined signalling steps (i.e., ERK signalling, phosphorylation of cPLA 2 ) [START_REF] Hong | Modulation of arachidonic acid metabolism and nitric oxide synthesis by garcinol and its derivatives[END_REF]. To circumvent such signalling pathways for cPLA 2 activation, we challenged human neutrophils with the Ca 2+ -ionophore A23187, where the massive increase in intracellular Ca 2+ essentially stimulates cPLA 2 . Garcinol failed to block AA liberation under these experimental conditions, indicating that PLA 2 -mediated AA release is not generally inhibited by garcinol. Note that in our cell-based assays analyzing AA transformations by COX-1/2 or by 5-LO, AA was exogenously supplemented to avoid effects on eicosanoid formation due to inhibition of PLA 2 . On the other hand, the effects of garcinol on PGE 2 and 6-keto PGF 1α formation in whole blood were analyzed under conditions (lipopolysaccharide challenge) where AA was supplied by endogenous sources. In contrast to PGE 2 formation, the synthesis of 6-keto PGF 1α was unaffected by garcinol, excluding general A c c e p t e d M a n u s c r i p t 21 suppression of AA and suggesting PGE 2 synthase (i.e., mPGES-1) as molecular target of garcinol. However, our data cannot exclude that garcinol also inhibits other PGE 2 synthases (such as cytosolic PGE 2 synthase or mPGES-2) in addition to mPGES-1. PGE 2 and 6-keto PGF 1α are synthesized via the COX-2 pathway in lipopolysaccharidestimulated whole blood [START_REF] Patrignani | Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases[END_REF]. Because 6-keto PGF 1α formation in whole blood was unaffected by garcinol, our data imply that garcinol-mediated suppression of PGE 2 synthesis in the same assay is independent of an interference with COX-2. Also, garcinol failed to inhibit isolated COX-2. Co-expression studies have revealed a preferred functional coupling of COX-2 and mPGES-1 [START_REF] Murakami | Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2[END_REF] suggesting that mPGES-1 is the major contributing PGE 2 synthase for PGE 2 synthesis in lipopolysaccharide-stimulated blood. Nevertheless, in analogy to other mPGES-1 inhibitors [START_REF] Koeberle | Licofelone suppresses prostaglandin E2 formation by interference with the inducible microsomal prostaglandin E2 synthase-1[END_REF], garcinol failed to completely suppress PGE 2 synthesis in whole blood and the remaining PGE 2 synthesis of approx. 40% might be attributable to other PGE 2 synthases (i.e., cytosolic PGE 2 synthase or mPGES-2) [START_REF] Koeberle | Licofelone suppresses prostaglandin E2 formation by interference with the inducible microsomal prostaglandin E2 synthase-1[END_REF].

For inhibition of purified 5-LO (IC 50 = 0.1 µM) and cellular 5-LO product formation (IC 50 = 1.9 µM), a discrepancy in the potencies of garcinol was obvious. Interestingly, a similar pattern was found for the structurally related hyperforin [START_REF] Albert | Hyperforin is a dual inhibitor of cyclooxygenase-1 and 5-lipoxygenase[END_REF] that possesses an unique inhibitory mode of action by interfering with the membrane-binding region of 5-LO's C2-like domain (Feißt et al., 2009, manuscript submitted). However, garcinol is an effective antioxidant [START_REF] Yamaguchi | Antioxidative and anti-glycation activity of garcinol from Garcinia indica fruit rind[END_REF] and may act as redox-type inhibitor of 5-LO reducing the active site iron. Such reduction might be more vigorous for cell-free 5-LO as compared to intracellular enzyme, a common phenomenon observed for many redox-active 5-LO inhibitors [START_REF] Werz | Inhibition of 5-lipoxygenase product synthesis by natural compounds of plant origin[END_REF].

Despite the obvious inhibition of PGE 2 and LT formation by garcinol in vitro, the question whether the interference of garcinol with mPGES-1 and 5-LO accounts for its anti- Unpublished studies, mentioned by Hong et al. [START_REF] Hong | Modulation of arachidonic acid metabolism and nitric oxide synthesis by garcinol and its derivatives[END_REF], showed that garcinol inhibited phorbol myristate acetate-induced mouse ear edema accompanied by decreased PGE 2 and LTB 4 production. Of interest, in these experiments the peak plasma levels of garcinol (12 µM) obtained after oral application of garcinol were in a concentration range, where cellular inhibition 5-LO and mPGES-1 is evident in our study. Therefore, one may speculate that garcinol, after oral administration, could achieve plasma levels sufficient to markedly suppress PGE 2 and LT biosynthesis under inflammatory conditions. PGE 2 is one of the mostestablished key players in the initiation and progression of inflammation [START_REF] Smith | The eicosanoids and their biochemical mechanisms of action[END_REF], and NSAIDs exert their beneficial effects in vivo essentially by repressing PGE 2 formation [START_REF] Funk | Prostaglandins and leukotrienes: advances in eicosanoid biology[END_REF]. Animals deficient in COX-2 or mPGES-1 clearly show reduced inflammatory symptoms [START_REF] Samuelsson | Membrane prostaglandin E synthase-1: a novel therapeutic target[END_REF][START_REF] Langenbach | Cyclooxygenase-deficient mice. A summary of their characteristics and susceptibilities to inflammation and carcinogenesis[END_REF].

Moreover, LTB 4 is a potent chemotactic and chemokinetic agent that recruits proinflammatory cells towards sites of inflammation [START_REF] Claesson | Asthma and leukotrienes: antileukotrienes as novel antiasthmatic drugs[END_REF]. But also tumorigenesis, particularly of colon, breast, prostate, and lung carcinoma has been associated with excessive PGE 2 and LT formation [START_REF] Werz | Therapeutic options for 5-lipoxygenase inhibitors[END_REF][START_REF] Chen | Five-lipoxygenase pathway of arachidonic acid metabolism in carcino-genesis and cancer chemoprevention[END_REF][START_REF] Samuelsson | Membrane prostaglandin E synthase-1: a novel therapeutic target[END_REF]. COX-2 selective inhibitors have shown a chemopreventive potential [START_REF] Rao | NSAIDs and chemoprevention[END_REF], and mPGES-1 is overexpressed in various cancers such as non-small cell lung cancer, invasive breast cancer, colorectal cancer, and gastric cancer [START_REF] Samuelsson | Membrane prostaglandin E synthase-1: a novel therapeutic target[END_REF]. Also 5-LO and its helperprotein FLAP are overexpressed in cancer cells, and 5-LO products promote cell proliferation and/or inhibit apoptosis, whereas 5-LO or FLAP inhibitors caused cell death [START_REF] Chen | Five-lipoxygenase pathway of arachidonic acid metabolism in carcino-genesis and cancer chemoprevention[END_REF].

In conclusion, we have identified human mPGES-1 and 5-LO as molecular targets of garcinol, and we showed that garcinol potently interferes with the catalytic activity of these enzymes in cell-free and cell-based assays. Inhibition of mPGES-1 and 5-LO may contribute to the anti-carcinogenic and anti-inflammatory effectiveness of garcinol observed in previous studies. Our data indicate that garcinol exerts a promising pharmacological profile combining potent inhibition of PGE 2 and LT formation without marked inhibition of COX isoenzymes.

  0.14 mg, 0.75 µmol) using N-Ethyl-N'-(3dimethylaminopropyl)carbodiimide (1.46 mg, 9.4 µmol) as coupling reagent for 1 h at 37 °C.

[ 3 H

 3 ]AA(18.4 kBq). The reaction was stopped after 15 min at 37 °C, and the samples were put on ice. After centrifugation (800g, 5 min, 4 °C), the supernatant was acidified (pH 3) by addition of citric acid (20 µl, 2 M), and the internal standard 11β-PGE 2 (2 nmol) was added. Radiolabeled PGE 2 was separated and analyzed by RP-18 solid phase extraction and HPLC A c c e p t e d M a n u s c r i p t 9 analysis. The amount of 11β-PGE 2 was quantified by integration of the area under the eluted peaks. For quantification of radiolabeled PGE 2 , fractions (0.5 ml) were collected and mixed with Ultima Gold TM XR (2 ml) for liquid scintillation counting in a LKB Wallac 1209 Rackbeta Liquid Scintillation Counter.

  A c c e p t e d M a n u s c r i p t 13 hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid. Cysteinyl-LTs C 4 , D 4 , and E 4 were not detected, and oxidation products of LTB 4 were not determined.2.12. StatisticsData are expressed as mean ± S.E. IC 50 values are approximations determined by graphical analysis (linear interpolation between the points between 50% activity). The program Graphpad Instat (Graphpad Software Inc., San Diego, CA) was used for statistical comparisons. Statistical evaluation of the data was performed by one-way ANOVAs for independent or correlated samples followed by Tukey HSD post-hoc tests. A P value of <0.05 (*) was considered significant.

A

  c c e p t e d M a n u s c r i p t 22 inflammatory and anti-tumorigenic properties in vivo, can not be clearly answered yet.

Fig. 1 .

 1 Figure Legends

Fig. 2 .

 2 Fig. 2. Effects of garcinol on cellular arachidonic acid release and prostanoid formation.

Fig. 3 .

 3 Fig. 3. Effects of garcinol on the activity of isolated COX-1 and -2.

Fig. 4 .

 4 Fig. 4. Effects of garcinol on the activity of mPGES-1.

Fig. 5 .

 5 Fig. 5. Effects of garcinol on prostanoid formation in human whole blood.

Fig. 6 .

 6 Fig. 6. Inhibition of 5-lipoxygenase activity by garcinol.

  Fig. 1

Fig. 5 6

 5 Fig. 5

Acknowledgments

The authors thank Bianca Jazzar and Gertrud Kleefeld for expert technical assistance. The financial support by the Deutsche Forschungsgemeinschaft is acknowledged.


Abbara

Noël-Hudson Ms

Thomas -Bourgneuf

Bonhomme-Faivre

Céline Chu
email: celinechu82@yahoo.fr

Disposition of everolimus in mdr1a-/1b-mice and after a pre-treatment of lapatinib in Swiss mice

Keywords: lapatinib, everolimus, P-glycoprotein, pharmacokinetics, mice Category of article: Pharmacokinetics and Drug Metabolism

M a n u s c r i p t

Lapatinib increases 2.5-fold everolimus AUC in mice. A 38.5% decrease of P-gp expression was observed in duodenum with a lapatinib pre-treatment. An inhibition of CYP 450 could not be excluded.

 [START_REF] Ruiz | Use of the new proliferation signal inhibitor everolimus in renal transplant patients in Spain: preliminary results of the EVERODATA registry[END_REF][START_REF] Rothenburger | Calcineurin inhibitor-free immunosuppression using everolimus (Certican) in maintenance heart transplant recipients: 6 months' follow-up[END_REF]This drug belongs to the family of inhibitors of the mTOR protein (mammalian target of rapamycin) which is a regulatory protein of cells metabolism, growth and proliferation. Since mTOR protein is present both in hematopoietic cells (T cells or B) and in non hematopoietic cells (such as smooth muscle cell), everolimus will inhibits growth factors stimulated proliferation. Several studies showed the interest of everolimus as an anti-tumor agent. An increase of survival is observed in patients in post liver transplantation neoplasms who received everolimus as compared to patients who received only an inhibitor of calcineurine (ciclosporin or tacrolimus) with steroids at 6, 12, and 24 months (100%, 90%, 72% vs. 50%, 29%, 14%) [START_REF] Gomez-Camarero | Use of Everolimus as a rescue immunosuppressive therapy in liver transplant patients with neoplasms[END_REF]. Everolimus anti-tumor properties could also be explained by the presence of the protein mTOR in many tumors, an inhibition of tumor growth and angiogenesis was observed in treated human tumor cells in ovarian cancer resistant cisplatine [START_REF] Mabuchi | RAD001 inhibits human ovarian cancer cell proliferation, enhances cisplatin-induced apoptosis, and prolongs survival in an ovarian cancer model[END_REF].

Everolimus is highly metabolized in intestine and liver. Everolimus absolute oral bioavaibility in rats is about 16% [START_REF] Crowe | Absorption and intestinal metabolism of SDZ-RAD and rapamycin in rats[END_REF]. Everolimus is substrate of P-glycoprotein (P-gp) and also of P450 3A4, 3A5 and 2C8 cytochromes [START_REF] Laplante | Interaction of immunosuppressive agents rapamycin and its analogue SDZ-RAD with endothelial PgP[END_REF][START_REF] Kovarik | Everolimus drug interactions: application of a classification system for clinical decision making[END_REF][START_REF] Kirchner | Clinical pharmacokinetics of everolimus[END_REF]. Cytochromes P450 3A (CYP3A) and P-glycoprotein (P-gp) are located in enterocytes and hepatocytes. The CYP3A/P-gp system contributes to the first-pass metabolism of many drugs, resulting in a limited bioavailability [START_REF] Wacher | Role of P-glycoprotein and cytochrome P450 3A in limiting oral absorption of peptides and peptidomimetics[END_REF]. Cytochrome P450 isoenzymes are parts of the phase I metabolizing enzymes. The CYP3A subfamily is predominant and is involved in the metabolism of more than 70% of currently administered drugs. The CYP 3A represent almost 30% of the cytochromes in the liver and 70% of the cytochromes in the small intestine [START_REF] Fakhoury | Localization and mRNA expression of CYP3A and P-glycoprotein in human duodenum as a function of age[END_REF]. Many studies have demonstrated that inhibitors of cytochromes 3A are leading to an increase of AUC of drug substrates with a prolongation of half time elimination [START_REF] Saari | Voriconazole and fluconazole increase the exposure to oral diazepam[END_REF][START_REF] Kovarik | Blood concentrations of everolimus are markedly increased by ketoconazole[END_REF]. P-gp is part of a large family of efflux transporters which is A c c e p t e d M a n u s c r i p t 4 Linking powered by eXtyles expressed in the gonads, kidneys, biliary system, intestinal epithelium, brain capillaries and lymphocytes. P-gp is coded by the multidrug resistance MDR1 genes in humans and the mdr1a and mdr1b in mice and rats. These genes encode drug-transporting protein that can cause multidrug resistance in tumor cells by decreasing intracellular drug levels. Enhanced expression of P-gp is considered to be a major mechanism of chemotherapeutics resistance [START_REF] Schinkel | Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug -transporting) Pglycoprotein[END_REF]. Conversely, inhibitors of P-gp may increase the oral absorption of drugs transported by P-gp. For example, verapamil enhanced bioavaibility of etoposide in rats [START_REF] Piao | Effects of verapamil on etoposide pharmacokinetics after intravenous and oral administration in rats[END_REF]. In vitro study using human intestinal cell line (Caco-2) has shown that everolimus is a potent substrate for P-gp-like mediated efflux and this efflux was completely inhibited by verapamil. [START_REF] Crowe | In vitro and in situ absorption of SDZ-RAD using a human intestinal cell line (Caco-2) and a single pass perfusion model in rats: comparison with rapamycin[END_REF].

Modifications of pharmacokinetics of drugs substrates of cytochromes or P-gp by many inhibitors of these proteins are leading to therapeutic drug monitoring. As everolimus is substrate of cytochromes and P-gp, it is conceivable that pharmacokinetics parameters of this drug could be modified after a pre-treatment by P-gp inhibitor.

Lapatinib is an inhibitor of tyrosine kinase receptors (HER 1 and HER 2) which is indicated in combination with capecitabine for the treatment of patients with advanced or metastatic breast cancer whose tumors overexpress HER2 [START_REF] Hegde | Delineation of molecular mechanisms of sensitivity to lapatinib in breast cancer cell lines using global gene expression profiles[END_REF][START_REF] Geyer | Lapatinib plus capecitabine for HER2-positive advanced breast cancer[END_REF]. Lapatinib was chosen in this pharmacological study for several reasons. First, in vitro and in vivo studies showed that lapatinib decreases the expression of P-gp and inhibits cytochrome 3A4 and BCRP [START_REF] Collins | Lapatinib inhibits Pglycoprotein (P-GP) and synergistically enhances taxane cytotoxicity in PgP + cell lines[END_REF][START_REF] Polli | The role of efflux and uptake transporters in N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (GW572016, lapatinib) disposition and drug interactions[END_REF][START_REF] Molina | Evaluation of lapatinib and topotecan combination therapy: tissue culture, murine xenograft, and phase I clinical trial data[END_REF].

Thus, everolimus pharmacokinetic parameters could be modified after a lapatinib pretreatment. Second, it is relevant to study the pharmacological interaction of these two drugs acting by two different anti-tumor mechanisms (inhibition of mTor protein and inhibition of tyrosine kinases receptor) as they could be combined for an anti-tumor treatment. As the matter of fact, a Phase I Study evaluates the combination of lapatinib and everolimus in patients with advanced solid tumors or non-Hodgkin's lymphoma [21].

The present pharmacological study was to thus undertaken with a double purpose: a) to A c c e p t e d M a n u s c r i p t 5 Linking powered by eXtyles document in vivo the effect of intestinal P-gp on everolimus disposition, b) to investigate the effect of lapatinib pre-treatement on everolimus pharmacokinetics and its influence on intestinal P-gp expression. Therefore, we have investigated the pharmacokinetics of everolimus and digoxin (substrate test of P-gp) after oral administration in mice with naturally deficient in P-gp (mdr 1a-/1b-) compared to the wild type (Fvb mice). In a second time, everolimus pharmacokinetic parameters were determined after oral administration either alone or after lapatinib pretreatment in Swiss mice.

2-MATERIALS AND METHODS

2-1. Drugs

Digoxin, (digoxine nativelle ® ) was purchased from Procter Gamble Pharmaceuticals (Asnières-sur-Seine, France). 100 μL of suspension (50 µg/mL) was diluted in 900 μL of sterile water to obtain a stock solution at 5 µg/mL. Everolimus, (Certican ® ) was purchased from Novartis Pharma SAS (Rueil-Malmaison, France). The oral preparation was obtained by dispersion of a tablet (0.25 mg) in 10 mL of sterile water (0.025 mg/mL). Lapatinib, (Tyverb ® ) was purchased from GlaxoSmith Kline (Marly-le-Roi, France). The oral preparation was obtained by dispersion of a tablet (250 mg) in 25 mL of sterile water (10 mg/mL).

2-2. Animals

Two strains of mice were used in this study. The first strain of mice was Fvb (sensitive to the B strain of friend leukemia) mice. The Fvb mice were used in digoxin and everolimus pharmacocinetics studies to document the effect of P-gp on everolimus disposition. For each drug, the wild type was compared to mdr1a-/1b-mice (mice naturally deficient of P-gp). The second strain was Swiss mice OF1. OF1 mice were used to study the influence of lapatinib pretreatment on everolimus disposition.

All mice used in the study were female mice (20-30 g) and provided by Charles River Laboratories (Orléans, France). They were caged under standard laboratory conditions, with tap water and regular chow provided ad libitum in a 12 hours/12 hours light/dark cycle at a temperature of 21-23°C. Anesthesia was induced with 5% isoflurane and maintained with 2.5% isoflurane in air. The animals were treated in accordance with the European committee standards concerning the care and use of laboratory animals.

2-3. Digoxin Pharmacokinetic Study

Fvb and mdr1a-/b-mice in each group orally received 0.03 mg/kg of digoxin. They were anesthetized with isoflurane and the blood samples were collected by cardiac puncture at 15, 30 min and 1, 2, 3, 4, 6, 8 and 24 hr after the administration of digoxin on heparinazed tubes.

The samples were centrifuged at 12.000 g for 8 min and the plasma was harvested into clean tubes. For digoxin quantification, an automated method was used. Plasma digoxin concentrations were measured with Fluorescent Polarization Assay (FPIA) on TDx ® using Digoxin II ® reagent kit (Abbott Diagnostic, Rungis, France). This method was linear up to 5 ng/mL with a limit of quantification at 0.2 ng/mL. All plasma samples were assayed together with calibration standard and quality control.

2-4. Everolimus Pharmacokinetic Study

Fvb and mdr1a-/b-mice in each group orally received 0.25 mg/kg of everolimus. The mice were anesthetized with isoflurane and the blood samples were collected by cardiac puncture at 30 min and 1, 2, 3, 5, 8 and 24 hr after the administration of everolimus on EDTA tubes. For everolimus quantification, an automated method was used. Blood everolimus concentrations were measured with Fluorescent Polarization Assay (FPIA) on TDx ® using INNOFLUOR ® CERTICAN ® kit (Biomedical diagnostics, Marne la Vallée, France). This method was linear up to 40 ng/mL with a limit of quantification at 2 ng/mL. All blood samples were assayed together with calibration standard and quality control.

2-5. Everolimus Pharmacokinetic After Lapatinib Pre-Treatment

Swiss mice were divided into two groups. The first one orally received 0.25 mg/kg of everolimus, the second one orally received 100 mg/kg of lapatinib twice a day for two days and 0.25 mg/kg of everolimus on the third day. The collection of everolimus blood samples A c c e p t e d M a n u s c r i p t 8 Linking powered by eXtyles and the quantification of everolimus blood concentration were done in the same conditions described for everolimus pharmacokinetic studies in Fvb and mdr1a-/1b-mice.

2-6. Data Analysis. Pharmacokinetic studies

As each animal provided only one sample of blood, data from animals of the same group were pooled using a naïve averaging data approach [START_REF] Burtin | Sparse sampling for assessment of drug exposure in toxicological studies[END_REF]. The noncompartmental analysis was performed using Winonlin ® professional version 4.1 software (Pharsight, Mountain View, CA). Data were used to estimate the mean maximal concentration (C max ) and the time necessary to reach maximal concentration (T max ). In addition, for each treatment, the elimination rate constant (k e ) and the area under the mean concentration-time curve (AUC 0→Tlast ) were estimated. The elimination rate constant (k e ) was estimated as the slope of the log-linear terminal portion of the plasma concentration vs. time curve, determined using unweighted linear least-squares regression analysis.

Additionally, from the estimated parameters, the terminal elimination half-life (t 1/2 ) for each group was derived and calculated as t 1/2= ln2/k e .

2-7. Semiquantitative Determination of P-gp Expression by Western Blot Analysis

P-gp expression was measured in three segments of intestine (duodenum, jejunum, and ileum) by Western Blot analysis in two groups of mice: (1) 3 female Swiss mice non-pretreated, (2) 3 female Swiss mice with a lapatinib pre-treatment. For last group, mice orally received 100 mg/kg of lapatinib twice a day for 2 days and 100 mg/kg of lapatinib on the third day. The intestines were removed 6 hours after the last administration of lapatinib on the third day.

Tissues were cut into small pieces and suspended in three volumes of 0.25 M sucrose, 50 mM phosphate buffer, pH 7.20, 1 mM EGTA, 2 mM MgCl 2 , and protease inhibitors: 5 µg/ml leupeptin, 0.15 mg/ml benzamidine, 5 µg/ml aprotinin, 1 µg/ml pepstatin, and 16 mg/ml A c c e p t e d M a n u s c r i p t 9 Linking powered by eXtyles phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA). The suspension was then homogenized in a glass tube. The homogenate was centrifuged at 500 g for 15 min. The supernatant obtained was then centrifuged at 3,000 g for 15 min. The pellet was washed twice with three volumes of 0.25 M sucrose, 50 mM phosphate buffer, pH 7.20, 2 mM MgCl 2 , and protease inhibitors and centrifuged at 3,000 g for 15 min. Protein concentrations were determined using the colorimetric bicinchoninic assay kit (Sigma-Aldrich, St. Louis, MO, USA), with bovine serum albumin (BSA;Sigma-Aldrich) as a standard. Proteins (40 µg) were separated by SDS-polyacrylamide gel electrophoresis on an 8% polyacrylamide gel and transferred onto a nitrocellulose membrane (Amersham, Orsay, France). The nitrocellulose membranes were then incubated with a primary antibody (C219, diluted to 1:100; DakoCytomation Denmark A/S, Glostrup, Denmark), washed, and finally incubated with a peroxidase-conjugated anti-mousse IgG secondary antibody. The immunoreactive bands were visualized by the enhanced Chemi-luminescent system (Perkin Elmer life science, Boston, USA). The nitrocellulose membranes were also incubated with anti-β actin (dilted to 1:5000; Sigma-Aldrich, St. Louis, USA) to normalize P-gp expression.

The autoadiographs of P-gp and β actin proteins were scanned by densitometry using the image J software to obtain a quantitative evaluation of protein levels in the three segments of intestines.

2-8. Statistical Analysis

Pharmacokinetic Studies: The mean AUC 0→Tlast estimated for each study group after administration of treatment were compared using Bailer's method [START_REF] Bailer | Testing for the equality of area under the curves when using destructive measurement techniques[END_REF].

The test for the equality of the mean AUC between each study group after administration of treatment was performed using the standard Wald statistic. Under the null hypothesis that Western Blot Studies: In the Western blot analysis of the P-gp expression, the intensity P-gp/β actin ratio was compared using Wilcoxon's test with a significant level fixed at 0.05.

3-RESULTS

3-1. Digoxin Pharmacokinetic Study

The results of the non-compartimental analysis in Fvb mice group and in mdr1a-/1b-mice group after the administration of digoxin are summarized in Table 1.

As shown in Fig. 1, the oral administration of digoxin to the mdr1a-/1b-mice group resulted in a mean C max 3 fold higher than the mean C max in the Fvb group. Also, AUC 0→Tlast of the mdr1a-/1b-group is 5.9 fold greater than AUC 0→Tlast of the Fvb group (p<0.00001, Bailer's method). The half-life of elimination were comparable (5.7 vs. 5.5 h).

3-2. Everolimus Pharmacokinetic Study

The results of the non-compartimental analysis in Fvb mice group and mdr1a-/1b-mice group after the administration of everolimus are summarized in Table 2.

As shown in Fig. 2, the oral administration of everolimus to the mdr1a-/1b-mice group resulted in a mean C max 1.3 fold greater than the mean C max in the Fvb group. Also, AUC 0→Tlast of the mdr1a-/1b-group is 1.3 fold greater than AUC 0→Tlast of the Fvb group (p<0.001, Bailer's method). The half-life of elimination were comparable (9.3 vs. 11.3 h).

3-3. Everolimus Pharmacokinetic After Lapatinib Pre-Treatment

The results of the non-compartimental analysis after the administration of everolimus alone 3. As shown in Fig. 3, the oral administration of everolimus following a pre-treatment of lapatinib in Swiss mice resulted in a mean C max 1.7 fold greater than the mean C max in everolimus alone group. In addition, the AUC 0→Tlast of the lapatinib pretreated Swiss mice group is 2.6 fold greater than AUC 0→Tlast of everolimus alone group (p<0.001, Bailer's method). The half-time of elimination were comparable (4.0 vs. 5.3 h).

3-4. Semiquantitative Determination of P-gp expression by Western Blot Analysis in intestine

Western blot analysis of P-gp expression was performed on three parts of intestine (duodenum, jejunum, ileon) from three control mice and three mice pretreated with lapatinib (100 mg/kg of lapatinib twice a day for 2 days and 100 mg/kg of lapatinib on the third day). A 38.5% significant decrease was observed in duodenum segment in lapatinib pre-treated group as compared with control group (0.298 ± 0.08 vs. 0.183 ± 0.05; p<0.05, Wilcoxon test ) (see Fig. 4 and Fig. 5). Nevertheless, in jejunum and ileum segment, no significant difference of Pgp expression was observed (0.58 ± 0.43 vs. 0.6 ± 0.2 and 0.47 ± 0.11 vs. 0.25 ± 0.68 respectively) (data not shown).

A c c e p t e d M a n u s c r i p t 4-DISCUSSION

The aim of this study was to document in vivo the effect of intestinal P-gp on everolimus disposition and to investigate the effect of lapatinib pre-treatment on everolimus pharmacokinetics and on intestinal P-gp expression.

In order to study the effect of P-gp on the disposition of everolimus, pharmacokinetics after oral administration of this drug was characterized in Fvb mice and mdr1a-/1-b mice. At the same time, the pharmacokinetics of digoxin, which is known to be a P-gp substrate [START_REF] Bonhomme-Faivre | Disposition of Delta(9) tetrahydrocannabinol in CF1 mice deficient in mdr1a P-glycoprotein[END_REF], was realized.

The administration of the two molecules in mdr1a-/1b-mice resulted in higher C max and AUC 0→Tlast as compared to those obtained in wild type mice. The application of Bailer's method has shown that the oral bioavaibility of both molecules was significantly higher in the mdr1a-/1b-group than in the wild type group. In addition, half-lives of elimination in each group appear to be comparable. All these findings, added to the results obtained previously [START_REF] Crowe | Absorption and intestinal metabolism of SDZ-RAD and rapamycin in rats[END_REF][START_REF] Laplante | Interaction of immunosuppressive agents rapamycin and its analogue SDZ-RAD with endothelial PgP[END_REF], suggest that everolimus is transported by intestinal P-gp in mice like digoxin. However, the ratio of AUC 0→Tlast in everolimus group is lower than the ratio of AUC 0→Tlast in digoxin group. Unlike digoxin, everolimus is highly metabolized by cytochromes [START_REF] Kovarik | Everolimus drug interactions: application of a classification system for clinical decision making[END_REF][START_REF] Kirchner | Clinical pharmacokinetics of everolimus[END_REF]. Thus, intestinal cytochromes could play a role in everolimus absorption and affect its AUC 0→Tlast after oral administration.

In order to document the influence of lapatinib on disposition of everolimus, pharmacokinetics study of this drug was carried in Swiss mice.

The first group of mice received everolimus alone (0.25 mg/kg). The second group of mice received everolimus after a lapatinib pre-treatment for two days (100 mg/kg, twice a day). A 2.6 fold increase of AUC 0→Tlast of everolimus was observed in mice pretreated by lapatinib as compared to everolimus alone. A 1.7 fold increase of everolimus C max was also observed. The A c c e p t e d M a n u s c r i p t 13 Linking powered by eXtyles elimination half-lifes were comparable (4.0 vs. 5.3 h). In vitro and in vivo studies had shown that lapatinib is a substrate and an inhibitor of the P-gp as well as cytochromes 3A and 2C8 [START_REF] Collins | Lapatinib inhibits Pglycoprotein (P-GP) and synergistically enhances taxane cytotoxicity in PgP + cell lines[END_REF][START_REF] Polli | The role of efflux and uptake transporters in N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (GW572016, lapatinib) disposition and drug interactions[END_REF][START_REF] Molina | Evaluation of lapatinib and topotecan combination therapy: tissue culture, murine xenograft, and phase I clinical trial data[END_REF].

Kovarick et al. have shown that when everolimus is coadmistrated with verapamil in 16 healthy subjects, 2.3-fold increase of everolimus C max (21 ± 8 vs 47 ± 18 ng/ml) was observed and accompanied by 3.5 fold increase of AUC 0→Tlast (115 ± 45 vs 392 ± 142 ng/ml.h). On the other hand, a slight prolongation of elimination half life has been noticed (32 ± 6 vs. 37 ± 6 h) [START_REF] Kovarik | Pharmacokinetic interaction between verapamil and everolimus in healthy subjects[END_REF]. When everolimus was coadministred with ketoconazole, a potent cytochrom 3A4 inhibitor, in 12 healthy volonteers, a 15-fold AUC 0→Tlast increase has been observed, as compared to everolimus group alone. Also, a 1.9-fold prolongation of elimination half-life (30 ± 4 vs 56 ± 5 h) was shown [START_REF] Kovarik | Blood concentrations of everolimus are markedly increased by ketoconazole[END_REF]. Verapamil is described like an important inhibitor of P-gp and less important inhibitor of cytochrom 3A while ketoconazole is both a potent inhibitor of P-gp and cytochrom 3A. In our results, an increase of 2.6-fold everolimus AUC 0→Tlast after a lapatinib pre-treatment was observed and accompanied with an slight prolongation of elimination half-life. Our results, when compared to those obtained in the previous study, suggest that lapatinib could have the same type of inhibition of P-gp and cytochromes as verapamil.

The determination of P-gp expression in intestines segments by Western Blot analysis showed that P-gp expression decreased only in duodenum in the lapatinib pretreated group as compared with the control group. No significant difference of P-gp expression was observed in jejunum and ileum. The concept of anatomical region-dependant modification of P-gp was already proposed to account for the inhibiting effect of some drugs on intestinal P-gp. For instance, IFN-α substantially inhibits P-glycoprotein expression in rats jejunum and ileum [START_REF] Ben-Reguiga | Modification of the P-glycoprotein dependent pharmacokinetics of digoxin in rats by human recombinant interferon-α[END_REF]. Quinidine increases ileal and jejunal digoxin absorption by inhibiting P-gp in rats [START_REF] Su | Inhibition of the intestinal digoxin absorption and exsorption by quinidine[END_REF].

An inhibition of duodenal P-gp expression by lapatinib could explain a decrease of the protein

A c c e p t e d M a n u s c r i p t 14 Linking powered by eXtyles activity. Further investigations are needed to understand which regulators are implicated in the decrease of P-gp expression. However, it has been demonstrated that a HER-2 over expression leads to an induction of NF-κB, a transcription factor controlling expression of genes which promote cell growth and survival [START_REF] Pianetti | Her-2/neu Linking powered by eXtyles overexpression induces NF-κB via a PI3-kinase/ Akt pathway involving calpain-mediated degradation of IκB-α that can be inhibited by the tumor expressor PTEN[END_REF]. In addition, it has been showed that NF-κB induces P-gp expression [START_REF] Bentires-Alj | NF-kappaB transcription factor induces drug resistance through MDR1 expression in cancer cells[END_REF]. These findings lead us to think that one of the mechanisms of the decrease of the P-gp expression by the lapatinib could be due to an inhibition of HER-2 leading to a decrease of induction of NF-κB which could result in an inhibition of P-gp expression. Other mechanism of P-gp inhibition could be implicated. In vitro study showed that lapatinib could directly inhibit the function of P-gp by binding to their ATP-binding sites in order to stimulate the ATPase activity for its own transport [START_REF] Dai | Lapatinib (Tykerb, GW572016) reverses multidrug resistance in cancer cells by inhibiting the activity of ATP-binding cassette subfamily B member 1 and G member 2[END_REF]. However, the increase of everolimus AUC 0→Tlast after a lapatinib pre-treatment can not be explained by a competition of the two drugs transport by P-gp, as lapatinib last dose was administered 16 hours before the administration of everolimus. Thus, it is conceivable that lapatinib decreases P-gp expression, and so, enhances everolimus AUC 0→Tlast.

It had been demonstrated that cyclosporine inhibits the first-pass extraction of everolimus by the intestine, rather than the liver in rats. Everolimus was administered 10 minutes after ciclosporin administration [START_REF] Yokomasu | Effect of intestinal and hepatic first-pass extraction on the pharmacokinetics of everolimus in rats[END_REF]. An inhibitory effect of lapatinib on the first-pass metabolism of everolimus by cytochromes P450 in the intestine could be lead to an increase of everolimus AUC 0→Tlast , and C max without modifying the elimination half-life as noticed in our pharmacokinetic study. Nevertheless, as we discussed above, a combination of lapatinib and everolimus is necessary to evaluate a competition of the two drugs on proteins. If lapatinib was administered 16 hours before the administration of everolimus, a competition of the two drugs on cytochromes P450 couldn't be observed. However, an inhibition of cytochromes by lapatinib in mice liver could not be excluded. In order to confirm any inhibition of cytochromes, metabolite quantification should be done. Also, in vitro studies showed that A c c e p t e d M a n u s c r i p t 15 Linking powered by eXtyles lapatinib inhibits BCRP transporter [START_REF] Polli | The role of efflux and uptake transporters in N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2methylsulfonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (GW572016, lapatinib) disposition and drug interactions[END_REF]. However, no study has documented the influence of BCRP on everolimus disposition. Thus, the results obtained from our pharmacokinetic study and from the Western Blot analysis concerning everolimus and lapatinib interaction, suggest that lapatinib could enhance everolimus disposition by decreasing P-gp expression.

Administration of everolimus in Fvb mice resulted in an increase of 4.1 fold of AUC 0→Tlast and 1.8 fold of C max higher than in Swiss mice. Obviously, absorption is enhanced, but also the elimination process appears to be different as half times of elimination are not comparable in the two strains. Many hypotheses could explain these results: physiological changes, differences in enzymatic arsenal, and protein expression according to the strains of mice.

In conclusion, this study showed first that everolimus is transported by P-gp in mice. In addition, a 2.6 fold significant increase of everolimus AUC was noticed after a lapatinib pretreatment.

These results show the necessity of a therapeutic monitoring of everolimus when it is administered with an inhibitor of P-gp and/or cytochromes like lapatinib in an anti-tumor treatment. Moreover, it could be relevant to document the pharmacological interaction of these two drugs in combination in order to observe an eventually competition on P-gp and cytochromes and therefore, an increase of a toxicity effect. Finally, everolimus and lapatinib are two drugs with anti-tumor properties, acting on two different pathogenesis way. Thus, lapatinib may increase the risk of toxicity associated with a higher concentration of everolimus in blood but equally the efficiency. Further investigations are necessary to document the pharmacodynamic effect of a pre-treatment of lapatinib on the anti-tumor activity of everolimus.

5-ACKNOWLEDGEMENT

  Everolimus (Certican ® ), 40-O-(2-hydroxyethyl) -rapamycine, is a new immunosuppressant used in cardiac and renal transplantation

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  c c e p t e d M a n u s c r i p t 10 Linking powered by eXtyles mean AUCs are equal, this statistic follows a normal distribution. The null hypothesis was rejected if │Z obs │was greater than 1.96.

  eXtyles and after lapatinib pretreatment in Swiss mice are summarized in Table

A c c e p t e d M a n u s c r i p t

Figure 1 :

 1 Figure 1: Plot of time course of mean plasma digoxin concentrations (SD) after oral

Figure 2 :

 2 Figure 2: Plot of time course of mean plasma everolimus concentrations (SD) after oral

Figure 3 :

 3 Figure 3: Plot of time course of mean plasma everolimus concentrations (SD) after oral

Figure 4 :

 4 Figure 4: Histogram of median P-gp / β actin ratio ± S.D. values from mouse duodenum (n =

Figure 5 :

 5 Figure 5: P-gp immunodetection from the duodenum of mice treated with 2 days of lapatinib

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 4 Figure 4

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1 Table 1 :

 11 Main pharmacokinetic parameters of digoxin after oral administration (0.03 mg/kg) obtained by a non-compartimental analysis in Fvb wild type and mdr1a-/1b-mice. Ratio AUC 0  Tlast = AUC 0  Tlast mdr1a-1/1b-group / AUC 0  Tlast wild type group b Ratio C max = C max mdr1a-1/1b-group / C max wild type group

	Table
			Fvb wild type	mdr1a-/1b-
	Half -life (hr)		5.72		5.45
	AUC 0  Tlast (ng.hr/ml)	42.13		250.58
	Ratio AUC 0  Tlast T max (hr) C max (ng/ml) Ratio C max b	a	5.9 0.25 6.3 3.0	4 18.8 a n u s c r i p t M
	A c c e p t e d
					2

a

Table 2 :

 2 Main pharmacokinetic parameters of everolimus after oral administration (0.25 mg/kg) obtained by a non-compartimental analysis in Fvb wild type and mdr1a-/1b-mice. Ratio AUC 0  Tlast = AUC 0  Tlast mdr1a-1/1b-group / AUC 0  Tlast wild type group b Ratio C max = C max mdr1a-1/1b-group / C max wild type group

			Fvb wild type	mdr1a-/1b-
	Half -life (hr)		9.3	11.3
	AUC 0  Tlast (ng.hr/ml)	515.9	709.9
	Ratio AUC 0  Tlast	a	1.3
	T max (hr)		3	3
	C max (ng/ml)		36.5	48.1
	Ratio C max b		1.3

a

Table 3 :

 3 Main pharmacokinetic parameters of everolimus (0.25 mg/kg) after oral administration (0.25 mg/kg) obtained by a non-compartimental analysis in Swiss mice. Ratio AUC 0  Tlast = AUC 0  Tlast evero + lapa group / AUC 0  Tlast evero group b Ratio C max = C max evero+ lapa group / C max evero group

			everolimus	everolimus + lapatinib
	Half -life (hr)		4.0	5.3
	AUC 0  Tlast (ng.hr/ml)	128.3	332.6
	Ratio AUC 0  Tlast	a	2.6
	T max (hr)		3	3
	C max (ng/ml)		19.9	34.2
	Ratio C max b		1.7

a