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Aug 19, 2015 - Brain Uptake of Tetrahydrohyperforin and Potential Metabolites after Repeated Dosing in Mice. Claudia Fracasso,. †. Renzo Bagnati,. â...
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Brain Uptake of Tetrahydrohyperforin and Potential Metabolites after Repeated Dosing in Mice Claudia Fracasso,† Renzo Bagnati,† Alice Passoni,† Giovanna Guiso,† Lavinia Cantoni,† Antonella Riva,‡ Paolo Morazzoni,‡ and Marco Gobbi*,† †

IRCCS - Istituto di Ricerche Farmacologiche “Mario Negri”, 20156 Milan, Italy Indena S.p.A., 20139 Milan, Italy

‡

S Supporting Information *

ABSTRACT: Tetrahydrohyperforin (IDN-5706) is a semisynthetic derivative of hyperforin, one of the main active components of Hypericum perforatum extracts. It showed remarkable positive effects on memory and cognitive performances in wild-type mice and in a transgenic mouse model of Alzheimer’s disease, but little was known about the concentrations it can reach in the brain. The investigations reported herein show that repeated treatment of mice with tetrahydrohyperforin (20 mg/kg intraperitoneally, twice daily for 4 days and once on the fifth day) results in measurable concentrations in the brain, up to 367 ng/g brain (∼700 nM) 6 h after the last dose; these concentrations have significant effects on synaptic function in hippocampal slices. The other main finding was the identification and semiquantitative analysis of tetrahydrohyperforin metabolites. In plasma, three hydroxylated/dehydrogenated metabolites were the largest (M1−3) and were also formed in vitro on incubation of tetrahydrohyperforin with mouse liver microsomes; the fourth metabolite in abundance was a hydroxylated/deisopropylated derivative (M13), which was not predicted in vitro. These metabolites were all detected in the brain, with peak areas from 10% (M1) to ∼1.5% (M2, M3, and M13) of the parent compound. In summary, repeated treatment of mice with tetrahydrohyperforin gave brain concentrations that might well underlie its central pharmacological effects. We also provide the first metabolic profile of this compound.

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One of them, tetrahydrohyperforin (IDN-5706), obtained by reduction of the keto groups at the 1- and 10-positions (Int. Publication Number WO 03/091194), showed a striking effect in improving spatial memory in wild-type mice12 and in rescuing cognitive performance in mice modeling an advanced stage of AD after subchronic intraperitoneal treatment.13,14 Few data are available regarding the pharmacokinetics and the actual brain uptake of tetrahydrohyperforin, all of them obtained after single treatment. Rozio et al.15 used HPLC-UV to analyze plasma and brain tissue after an oral dose of 30 mg/ kg in rats. The Cmax of tetrahydrohyperforin in plasma was 211 ng/mL, reached within 60−120 min, but concentrations in the brain were below the limit of quantitation of 20 ng/g tissue. Hatanaka et al.16 used a more sensitive HPLC with an electrochemical detector method to investigate plasma and brain concentrations of hyperforin at different times after a

t. John’s wort (SJW, Hypericum perforatum) extracts are believed to have important biological activities, and hyperforin, a prenylated phloroglucinol component, may well be responsible for some of them.1 These include antiinflammatory, antibacterial, and antitumoral effects,2 as well as central nervous system effects resulting in antidepressant3,4 and memory-enhancing properties.5 Intrahippocampal injection of hyperforin prevents the spatial memory impairments induced in rats by amyloid-β fibrils,6 suggesting that it might be therapeutic in Alzheimer’s disease (AD).7 Hyperforin is also involved in the clinically relevant pharmacokinetic interactions described when SJW extracts are given together with many conventional drugs.8,9 Hyperforin induces some cytochrome P450 enzymes,10,11 raising concern about the benefit/risk ratio in therapeutic use. Moreover, hyperforin is easily degradable and very prone to oxidation. These critical features have led to the semisynthesis and pharmacological evaluation of hyperforin derivatives. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 24, 2015

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corresponding levels in the plasma of the same animals. Assuming that cerebral blood accounts for 2% of total blood,18−20 it follows that these levels cannot be accounted for by the residual cerebral blood. Brain levels were highest at longer times (4−6 h) than the tmax in plasma (1 h), resembling a previous report of the plasma and brain concentrations of hyperforin after a single oral dose of SJW extracts.16 Different drug-related or tissue-related factors might underlie the delayed accumulation in the brain; their elucidation requires further investigation. It has long been debated whether or not the antidepressant effects of hyperforin4,21−24 are due to central action25−28 since pharmacokinetic data did not support this.29,30 The present data and those of Hatanaka et al.16 suggest it might be useful to re-evaluate, under more suitable experimental conditions (e.g., longer time-course, highly sensitive MS instruments), the concentrations of hyperforin reached in the brain after repeated treatment, to see whether they are high enough to allow interaction with the relevant receptors.30 The highest brain levels of tetrahydrohyperforin (Figure 1) correspond to a concentration of ∼700 nM, which might explain the positive effects on memory and cognitive performances in wild-type mice12 and in AD mice.14 It was reported12 that 1 μM tetrahydrohyperforin had significant effects on synaptic function in mouse hippocampal slices: it increased basal neuronal activity and completely antagonized the toxic effects of amyloid-β oligomers. Both these effects were completely abolished in the presence of SKF96365, an inhibitor of canonical transient receptor potential channels (TRPC). Moreover, SKF96365 prevented the positive effects on spatial memory induced in wild-type mice by chronic treatment with 6 mg/kg tetrahydrohyperforin. This finding suggested that the activity of tetrahydrohyperforin may be mediated by TRPC and is consistent with previous data31 showing that 100−300 nM of the congener hyperforin induced neurite outgrowth in PC12 cells through direct activation of TRPC6 channels, which are the nonselective Ca2+ channels on excitatory synapses needed for the formation of dendritic spines, plasticity, and memory.32 In silico studies confirmed strong pharmacophore similarity of tetrahydrohyperforin and other TRPC6 activators.12 Semiquantitative Analysis of Tetrahydrohyperforin Metabolites in Mouse Plasma and Brain. Metabolites of tetrahydrohyperforin were identified using a high-resolution mass spectrometer (LTQ-Orbitrap XL), which greatly facilitated the detection of MS/MS fragments and the chemical structures of metabolites. Analyses were carried out in undiluted plasma samples of mice treated as described above,

single oral dose of SJW extracts in mice (5.2 mg hyperforin/ kg): plasma levels peaked at 60 min (∼100 ng/mL), declining rapidly to ∼17 and ∼10 ng/mL at 3 and 6 h, respectively; however, the time-course was completely different in the brain, with levels of 2.5, 4.9, and 4.3 ng/g at 1, 3, and 6 h. The main aim of the present study was to assess, using a highly sensitive HPLC-MS/MS method, whether tetrahydrohyperforin reaches the brain after repeated intraperitoneal injections in mice, i.e., the conditions showing improvement of cognitive functions in different experimental models.12−14 It was also investigated for the first time the formation of tetrahydrohyperforin metabolites in vivo in plasma and brain and in vitro by incubating tetrahydrohyperforin with mouse liver microsomes.

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RESULTS AND DISCUSSION Quantitative Analysis of Tetrahydrohyperforin in Plasma and Brain of Treated Mice. Mice received an intraperitoneal injection of 20 mg/kg tetrahydrohyperforin (10 mL/kg) twice daily for 4 days and once on the fifth day. On day 5, mice were injected, then euthanized by decapitation after 1, 2, 4, and 6 h. One group of mice was killed without this last dose and is indicated as t = 0 (i.e., before the last injection, 14 h from the previous one). The levels of tetrahydrohyperforin in plasma and brain of treated mice are reported in Figure 1. Plasma concentrations ranged from 1340 ± 284 ng/mL at t = 0 to 4270 ± 1234 ng/ mL 1 h after treatment, with an increase of ∼2900 ng/mL. They then declined slowly, with an estimated half-life of elimination (determined on the last three points) of about 10 h, similar to the value previously shown for hyperforin.17 Brain concentrations were 62 ± 35 ng/g at t = 0, remained stable for 2 h after treatment, and then rose to 256 ± 112 at 4 h and 367 ± 77 ng/g at 6 h, corresponding to 12% and 17% of the

Figure 1. Pharmacokinetic profiles of tetrahydrohyperforin in the plasma and brain of mice. Mice were given an intraperitoneal injection with 20 mg/kg tetrahydrohyperforin twice daily for 4 days and once on the fifth day and were killed 1, 2, 4, and 6 h after the last dose. One group of mice was killed without receiving the last dose (t = 0, 14 h from the eighth dose). Each value is the mean ± SE of 3 or 4 animals. B

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using pools from all time points after the ninth dose on the fifth day. The metabolites found in the tetrahydrohyperforin-treated, but not in untreated mice, are indicated in Table 1; their chemical structures and the corresponding chromatograms and spectra are shown in the Supporting Information (Appendices 1 and 2, respectively).

normalized peak areas of the metabolites were much lower than for the parent compound (data not shown): at t = 6 h, they were respectively 2.6%, 0.5%, 0.5%, and 0.1% for M1, M2, M3, and M13 in plasma and 9.7%, 1.7%, 1.1%, and 1.4% in brain, suggesting that metabolites account for only a small fraction of the parent compound. According to these percentages, it can be estimated that metabolite concentrations in the low nanomolar range are present in the brain 6 h after treatment. These might be conservative estimates because the conditions used (extraction procedure and collision energies) were optimized for tetrahydrohyperforin and may not have been optimal for the metabolites. Of note, the brain levels of M13 were similar to those of M2 and M3, despite much lower plasma levels. Unless there were differences in the recovery, this finding may indicate preferential formation of M13 in the brain or suggest a better brain uptake of M13. If this latter possibility is true and M13 maintains the same pharmacological properties as the parent compound, these data may guide the development of an active tetrahydrohyperforin derivative with a superior pharmacokinetic profile. Further studies with purified M13 are planned to clarify these points. In Vitro Metabolism of Tetrahydrohyperforin by Mouse Liver Microsomes. Figure 3 shows the relative amounts (normalized on the IS) of tetrahydrohyperforin and its main metabolites formed after incubation of the parent compound (100 μM) for 60 min at 37 °C with or without mouse liver microsomes. M1, M2, and M3 were formed only in the presence of the microsomal fraction, suggesting that their formation was cytochrome P-450-dependent, consistent with the in vitro metabolism of hyperforin by mouse and human liver microsomes.33,34 At the end of the incubation M1, M2, and M3 accounted for about 30%, 7%, and 1.3% of the parent compound, respectively. M13 was formed comparably with and without microsomes and was about 0.15% of the parent compound at the end of incubation. The microsome-independent formation of M13 was then investigated. Incubation of tetrahydrohyperforin at 37 °C in phosphate buffer resulted in time-dependent formation of M13, but only when incubation was in open, but not closed, tubes (Figure 4), suggesting this nonenzymatic transformation of the parent compound needs oxygen. There was also no M13 formation after incubation of tetrahydrohyperforin in human plasma at 37 °C, even in open tubes. This suggests that this blood fraction does not contain enzymatic systems that can form M13 and/or that plasma components prevent its nonenzymatic formation. This plasma component is unlikely to be albumin, since in the present study in buffer 1% albumin was used to dissolve tetrahydrohyperforin. The small but detectable levels of M13 in the plasma of treated mice might be due to other metabolic routes present in whole blood or other tissues, but this calls for further investigation. In summary, the investigations reported herein show that repeated intraperitoneal treatment of mice with tetrahydrohyperforin results in brain concentrations (∼700 nM) that might well underlie the central pharmacological effects of the compound. The other main finding was the identification and semiquantitative analysis of tetrahydrohyperforin metabolites, whose brain concentrations were, however, much lower than the brain levels of the parent compound. Thus, unless the metabolites have a much higher potency (not yet proved), it still remains likely that the in vivo pharmacological activity of

Table 1. LC/MS Data Obtained for Tetrahydrohyperforin and Its Metabolites in Plasmaa compound tetrahydrohyperforin M1 1× hydroxylation M2 2× hydroxylation M3 1× hydroxylation +1× dehydrogenation M4 2× hydroxylation +1× dehydrogenation M5 2× hydroxylation +2× dehydrogenation M6 1× dehydrogenation M7 3× hydroxylation M8 3× hydroxylation + 1× dehydrogenation M9 3× hydroxylation + 2× dehydrogenation M10 sulfate M11 glucuronate M12 1× demethylation M13 1× hydroxylation + 1× deisopropylation

m/z calc

m/z found

539.4106 555.4055 571.4004 553.3898

539.4103 555.4060 571.4021 553.3904

569.3848

569.3856

567.3691

567.3692

537.3949 587.3953 585.3797

not detected 587.3943 585.3793

583.3640

583.3645

619.3674 701.4270 525.3949 513.3585

not detected not detected not detected 513.3587

a

Analysis was done with an LTQ-Orbitrap XL mass spectrometer on a pool of plasma from mice treated with tetrahydrohyperforin.

Consistent with previous data on hyperforin,33,34 the present study found hydroxylated and/or dehydrogenated metabolites, but one unexpected metabolite, M13, was also found. Three possible structures of M13, consistent with its mass, are shown in the Supporting Information (Appendix 1), indicating that it might be formed by oxidation or hydroxylation, associated with deisopropylation. The presence of more than one hydroxyl group and isobutylic chain in the parent compound makes it difficult to establish univocally the actual structure of M13. An API-3000 triple quadrupole mass spectrometer was used for the semiquantitative determination of metabolites. The conditions for multiple reaction monitoring (MRM) determination and MRM chromatograms are reported in the Supporting Information (Appendices 3 and 4, respectively). Plasma samples were diluted 100-fold, and in these conditions, only the most abundant metabolites, M1, M2, M3, and M13, could be detected. Since pure metabolites for the calibration curves were not available, it was not possible to determine their absolute tissue levels, and the data are expressed as peak areas, always normalized for the peak area of the internal standard (IS). These normalized peak areas were used to estimate the relative abundances of the metabolites versus the parent compound, with the assumption of similar LC/MS responses between all these molecules.34 In plasma (Figure 2, left panels) the time-courses differed for the parent compound and the main metabolites (M1, M2, M3, and M13), the latter showing a Tmax at about 2 h, with no decline in the next 4 h. The time-courses in the brain (Figure 2, right panels) were similar for tetrahydrohyperforin and the metabolites, with the highest levels 6 h after injection. The C

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Figure 2. Pharmacokinetic profiles of tetrahydrohyperforin main metabolites in the plasma (left panels) and brain (right panels) of mice after repeated treatment (20 mg/kg intraperitoneally, twice daily for 4 days and once on the fifth day). The data are expressed as peak areas, normalized for the peak area of the internal standard, since pure metabolites were not available for calibration purposes. Each value is the mean ± SE of 3 or 4 animals. XenoTech (Lenexa, KS, USA). These microsomes are characterized by protein content, total cytochrome P450 and cytochrome b5, oxidoreductase activity, and cytochrome P4501A and cytochrome P4503A isozyme activity. For these studies, tetrahydrohyperforin was dissolved in methanol at a final concentration of 10 mM, then further diluted to 200 μM in phosphate buffer (100 mM), pH 7.4, containing 2% bovine serum albumin.33 These conditions give a clear solution. In a first study, tetrahydrohyperforin (100 μM) was incubated for 60 min at 37 °C with microsomes together with appropriate cofactors (Dglucose 6-phosphate sodium salt, NADP sodium salt, NADH reduced disodium salt, glucose-6-phosphate dehydrogenase);33 in parallel, control samples were prepared with no incubation (t = 0) or no microsomes. In a second study, tetrahydrohyperforin (100 μM) was incubated for 60−240 min at 37 °C in phosphate buffer (100 mM, pH

tetrahydrohyperforin in wild-type mice and in a transgenic mouse model of AD12,14 are due to the parent compound.

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EXPERIMENTAL SECTION

Chemicals and Reagents. Tetrahydrohyperforin (IDN-5706, chemical purity 98%) and hyperforin dicyclohexylammonium (hyperforin-DCHA), used as IS, were obtained from Indena S.P.A., Milan, Italy. HPLC-grade methanol, acetonitrile, and formic acid were obtained from Carlo Erba, Milan, Italy. HPLC-grade water was obtained from Milli-Ro60 Water Systems, Millipore, Milford, MA, USA. Solutol (Solutol-HS15, Macrogol 15 Hydroxystearate Ph.Eur. Polyoxyl 15 Hydroxystearate USP) was from BASF, Limburgerhof, Germany. In Vitro Studies with Mouse Liver Microsomes. Liver microsomes from pooled male CD-1 mice were purchased from D

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Figure 3. In vitro metabolism of tetrahydrohyperforin by mouse liver microsomes. Tetrahydrohyperforin (100 μM) was incubated for 60 min at 37 °C with or without mouse liver microsomes. The bars indicated as t = 0 refer to samples not incubated. Levels of the parent compound and metabolites at the end of incubation are expressed as the ratios of the peak area of the compound of interest to that of the internal standard. Each value is the mean ± SD of three replications. **p < 0.01 versus corresponding t = 0; §§p < 0.01 versus corresponding group without microsomes; Sidak’s multiple comparison test following two-way ANOVA (GraphPad Prism version 6.03 for Windows, GraphPad Software, La Jolla, CA, USA). Broken lines indicate the limit of detection. involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (D.L. n. 116, G.U., suppl. 40, 18 febbraio 1992, Circolare No. 8, G.U., 14 Luglio 1994) and international laws and policies (EEC Council Directive 86/609, OJL 358, 1, Dec 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996). This study was carried out under the approved institutional protocol number 42/01-C. Tetrahydrohyperforin was freshly dissolved (2 mg/mL) in 6% Solutol HS15 in sterile water, before each treatment. Since the procedure included heating at 37 °C and stirring for up to 120 min, the purity and stability of tetrahydrohyperforin in the solution were verified at the end of this procedure by HPLC-MS/MS, immediately before use. Mice received an intraperitoneal injection of 20 mg/kg tetrahydrohyperforin (10 mL/kg) twice daily for 4 days and once on the fifth day. On day 5, mice were injected, then euthanized after 1, 2, 4, and 6 h. One group of mice was killed without this last dose and is indicated in the text as t = 0 (i.e., before the last injection, 14 h from the previous one). Mice were anesthetized with isoflurane before collecting blood from the heart, which was followed by decapitation and dissection of the brain. Sample Preparation and Analysis. The analysis of tetrahydrohyperforin concentrations in plasma and brain tissue was conducted with slight modifications of an HPLC/MS/MS method developed for hyperforin.33 Tetrahydrohyperforin and hyperforin

Figure 4. Microsome-independent formation of M13. Tetrahydrohyperforin (100 μM) was incubated at 37 °C in phosphate buffer (filled symbols) or in human plasma (open symbols), in open (continuous lines) or closed (broken lines) tubes. Data are expressed as the ratios of the peak area of M13 to that of the internal standard. Each value is the mean ± SD of three (plasma) or six (buffer) replications. 7.4) or plasma from healthy subjects, in open or closed Eppendorf tubes. In Vivo Studies. Male CD1 mice (22−25 g, 4−5 weeks old) were used (Charles River, Calco, Como, Italy). Animals were housed four per cage with controlled room temperature (23 °C) under a 12 h light−dark cycle with standard diet and water ad libitum. Procedures E

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dicyclohexylammonium, which was used as IS, were prepared as 1 mg/ mL stock solutions in methanol. Working solutions were prepared before each analytical run by dilution in methanol. Plasma samples were diluted 100 times with control plasma, and 100 μL of this solution was mixed with 10 μL of IS (final concentration 10 ng/mL) and 200 μL of CH3CN. After vortexing for 30 s, the mixture was centrifuged at 4 °C for 10 min at 10 000 rpm. The supernatant was recovered, and 10 μL was injected into the HPLC-MS/MS system. Brain tissue (1 g) was homogenized in 2 mL of 70% acetonitrile and diluted 1:10 with the same solvent; 200 μL was mixed with 10 μL of IS working solution (final concentration 5 ng/mL). After vortexing the mixture was centrifuged at 4 °C for 10 min at 10 000 rpm. The supernatant was recovered, and 10 μL was injected into the HPLCMS/MS system. The HPLC system consisted of two Series 200 pumps and a Series 200 autosampler (PerkinElmer, Norwalk, CT, USA). The MS system was an API-3000 triple quadrupole mass spectrometer, equipped with a turbo ion spray source (AB Sciex, Thornhill, Ontario, Canada). HPLC separation was obtained with a Luna C5 column (50 × 2 mm; 5 μm particle size), using an elution mixture composed of solvent A (0.05% acetic acid in water) and solvent B (acetonitrile). The injection volume was 10 μL, and the flow rate 200 μL/min. The elution gradient was from 30% to 98% of solvent B in 12 min, hold at 98% for 5 min, and re-equilibration for 7 min at 30% solvent B. The mobile phase was introduced directly into the ion source, which was operated with turbo ion gas at 400 °C. The retention times were respectively 15 and 17 min for tetrahydrohyperforin and IS. Mass spectrometric analyses were done using negative ionization and MRM mode, measuring the fragmentation products of the deprotonated pseudomolecular ions of tetrahydrohyperforin (539.4 → 467.4, collision energy −44 eV) and the IS (535.6 → 383.3, collision energy −42 eV). The choice of fragmentation products for tetrahydrohyperforin and the IS and the optimization of collision-induced dissociation energies were done in continuous-flow mode, using standard solutions at concentrations of about 1 ng/μL. Tetrahydrohyperforin was quantified using the IS as reference. Sixpoint calibration curves were generated by spiking control plasma with different amounts of tetrahydrohyperforin: 0, 0.1, 0.25, 0.5, 1.0, 5.0, and 25 ng/mL and 10 ng/mL of IS. Control brain homogenate was spiked with 0, 20, 50, 100, 200, 500, and 1000 pg of tetrahydrohyperforin, and 1000 pg of IS, in 200 μL of homogenate. The first point (0) was used as an instrumental blank. Calibration curves were constructed using the weighted (1/x) linear regression of peak area ratios (tetrahydrohyperforin/IS) versus concentration. Analysis of two replicates of quality control samples at 0.25, 1.0, and 10.0 ng/mL for plasma and 50, 200, and 1000 pg/100 mg for brain indicated an accuracy [(calculated concentration − nominal concentration)/nominal concentration)] × 100) always higher than 80%. The detection limit and quantification limit for tetrahydrohyperforin were determined using the lowest standard samples. The detection limit (i.e., the concentration giving peaks with a signal-tonoise ratio of 3) was 0.03 ng/mL plasma or 0.03 ng/g brain. The quantification limit (i.e., the concentration giving peaks with a signalto-noise ratio of 10) was 0.1 ng/mL plasma or 0.1 ng/g brain. Identification of Metabolites by LTQ-Orbitrap XL. Tetrahydrohyperforin metabolites were identified using an LTQ-Orbitrap XL mass spectrometer (Thermo Scientific Inc., Waltham, MA, USA) coupled to a 1200 series capillary pump and autosampler (Agilent Technologies, Santa Clara, CA, USA), on the basis of previous studies investigating hyperforin metabolites.33,34 The ion source was a desorption electrospray ionization Omni Spray (Prosolia, Indianapolis, IN, USA), used in nanoelectrospray mode for negative ions. HPLC separation was obtained with a Zorbax SB-C 18 column (100 × 0.5 mm; 5 μm particle size), using an elution mixture composed of solvent A (0.05% acetic acid in water) and solvent B (acetonitrile). The injection volume was 1 μL, and the flow rate was 10 μL/min. The elution gradient was from 20% to 100% solvent B in 40 min, hold at 100% for 6 min, and re-equilibration for 8 min at 20% solvent B. Samples were injected directly into the HPLC column, which was directly coupled to the ion source spray capillary by a liquid junction.

A complete scan at 60 000 resolving power, looking for molecules within the m/z range of 250−1200, was done on undiluted plasma samples from tetrahydrohyperforin-treated or untreated mice. MS/MS spectra were acquired simultaneously on the same samples, using a data-dependent method, which selected the most abundant ions of each MS scan in real time.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00302. Appendix 1: chemical structures of tetrahydrohyperforin, its metabolites, and internal standard (hyperforinDCHA). Appendix 2: chromatograms and spectra of tetrahydrohyperforin and its metabolites in plasma and brain samples obtained with LTQ-ORBITRAP XL. Appendix 3: analyzer conditions for MRM determination of tetrahydrohyperforin and metabolites. Appendix 4: HPLC-MRM chromatograms of tetrahydrohyperforin and its metabolites in plasma and brain samples (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +39 02 3901 4570. Fax: +39 02 3901 4744. E-mail: [email protected]. Notes

The authors declare the following competing financial interest(s): This research was funded by Indena S.p.A., Milan, Italy. A. Riva and P. Morazzoni are employed with INDENA S.p.A.

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ACKNOWLEDGMENTS The authors wish to thank J. Lucchetti and M. Canovi for their help with treatment of the mice. This research was funded by Indena S.p.A., Milan, Italy.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.5b00302 J. Nat. Prod. XXXX, XXX, XXX−XXX