Evaluation of the Intestinal Absorption Mechanism of Casearin X in

Article pubs.acs.org/jnp

Evaluation of the Intestinal Absorption Mechanism of Casearin X in Caco‑2 Cells with Modified Carboxylesterase Activity Rodrigo Moreira da Silva,† Sheela Verjee,‡ Cristiane Masetto de Gaitani,† Anderson Rodrigo Moraes de Oliveira,§ Paula Carolina Pires Bueno,⊥ Alberto José Cavalheiro,⊥ Norberto Peporine Lopes,∥ and Veronika Butterweck*,‡ †

Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, 14040-903 Ribeirao Preto, Brazil ‡ Institute for Pharma Technology, School of Life Sciences, University of Applied Sciences Northwestern Switzerland, 4132 Muttenz, Switzerland § Department of Chemistry, Faculty of Philosophy, Sciences and Letters of Ribeirao Preto, University of Sao Paulo, 14040-901 Ribeirao Preto, Brazil ⊥ Nucleus of Bioassays, Biosynthesis and Ecophysiology of Natural Products, Institute of Chemistry, Department of Organic Chemistry, Sao Paulo State University, 14801-970 Araraquara, Brazil ∥ Nucleus of Research for Natural Products and Synthetics, Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, 14040-903 Ribeirao Preto, Brazil ABSTRACT: The clerodane diterpene casearin X (1), isolated from the leaves of Casearia sylvestris, is a potential new drug candidate due to its potent in vitro cytotoxic activity. In this work, the intestinal absorption mechanism of 1 was evaluated using Caco-2 cells with and without active carboxylesterases (CES). An LC-MS method was developed and validated for the quantification of 1. The estimation of permeability coefficients was possible only under CESinhibited conditions in which 1 is able to cross the Caco-2 cell monolayer. The mechanism is probably by active transport, with no significant efflux, but with a high retention of the compound inside the cells. The enzymatic hydrolysis assay demonstrates the susceptibility of 1 to first-pass metabolism as substrate for specific CES expressed in human intestine. due to its high in vitro/in vivo correlation.22,23 Over time, cultured Caco-2 cells can spontaneously differentiate into a polarized monolayer of enterocytes with microvilli on the apical membrane, tight junctions between cells, and transepithelial electrical resistance similar to human colon.21 Caco-2 cells also can express influx and efflux transporter proteins24 and metabolic enzymes responsible for first-pass metabolism, such as cytochrome P45025 and carboxylesterase (CES) isoenzymes.26 However, due to the high lipophilicity and ester-containing character of 1, problems with low stability can be expected during the transport across Caco-2 cells, and a significant fraction of the compound can elude detection.27 Therefore, the intestinal absorption mechanism of 1 was evaluated using Caco2 cell monolayers in the presence or absence of the bis-pnitrophenyl phosphate (BNPP), a specific inhibitor of CES. In addition, a mass balance study was performed encompassing

Casearia sylvestris (Salicaceae) is a plant that occurs naturally throughout Brazil and other countries of South and Central America.1 The crude extract of the leaves of C. sylvestris is used in folk medicine, and a variety of pharmacological activities have been confirmed such as anti-inflammatory,2−4 antihyperlipidemic,5 antiprotozoal,6 antiophidic,7−11 and antimutagenic.12,13 Activity against tumor cells at low concentrations has been extensively explored.14−16 These extracts contain a group of clerodane diterpenes known as casearins.17 Among these, casearin X (1), the major constituent of C. sylvestris, is the most selective and cytotoxic.18 Despite promising pharmacodynamic properties,14,19 no reports about the bioavailability of 1 exist in the literature. The most desirable and convenient route for administration of therapeutic agents is oral, in which the drug has to be taken up from the gastrointestinal tract into the bloodstream. The human colon adenocarcinoma cell line Caco-2 is the most established in vitro model to predict drug absorption20,21 and is considered the standard method for predicting permeability of drugs in the Biopharmaceutical Classification System (BCS) © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 22, 2015

A

DOI: 10.1021/acs.jnatprod.5b01139 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Caco-2 cell monolayers with and without active CES hydrolysis. The transepithelial electrical resistance (TEER) of cultured cells on Transwell inserts was monitored before and after each permeation experiment. The TEER values above 250 Ω cm2 confirm that the monolayers were intact during transport assays (Table 3). A significant amount of 1 appeared to be metabolized during the absorptive transport experiments in intact Caco-2 cells. However, since a LC-MS equipped with a single quadrupole detector was used for the current experiments, it was not possible under these conditions to identify particular metabolites. Table 2 shows that after 120 min of incubation the mean of total recovery was only 12%. This was expected due to the ester-containing character of 1. On the other hand, the total recovery of 1 from monolayers with CES inhibited remained constant over 85%. The absorptive permeability profiles of 1 in Caco-2 cells with and without CES activity are shown in Figure 2A and B, and the amounts of 1 distributed in apical and basolateral compartments, inside cells, nonspecifically bounded to the plastic material, and lost are shown in Figure 3A and B. The concentrations of 1 decreased rapidly from the apical compartment during the first 10 min in a similar manner for both situations (Figure 2A and B). According to the distribution of 1 presented in Figure 3B for monolayers with CES inhibited, the major amount in the first 10 min was transported to the intracellular compartment, indicating that the lipophilicity of 1 has a high impact in the kinetics of transport. A summary of the physicochemical properties of 1 are listed in Table 4. The amount found inside cells remained approximately 20% until the last quantification at 120 min (Figure 3B). This result confirms that the intracellular compartment should be taken into account in transport studies of highly lipophilic compounds, in order to have a reliable estimate of the permeability coefficients.27 From 60 to 120 min, in the absorptive direction, there was a decrease in the concentration of 1 in the basolateral compartment of intact monolayers, which contrasts with the increase observed for monolayers with CES inhibited (Figure 2A and B). With the inhibition of the intracellular hydrolysis, 1 was better transported to the basolateral compartment, reaching approximately 40% of the total amount of 1 found in the system after 120 min (Figure 3 B). Table 5 shows the permeability coefficients obtained in transport experiments. The apparent permeability (Papp) value for absorptive transport (A → B) in Caco-2 cells under CESinhibited conditions was (66.9 ± 2.3) × 10−6 cm/s. According to Ye et al. (1997),30 this value means that the compound is well absorbed (70−100%). Compound 1 exhibited high permeability in the absorptive direction when CES was inhibited by treatment of the Caco-2 cell monolayers with BNPP. It was not possible to calculate Papp under CES uninhibited conditions due to the low recovery of 1, as mentioned above. Secretory transport was also evaluated in Caco-2 cells with and without CES activity inhibited by BNPP. The secretory permeability profiles were not markedly different under CES inhibited and noninhibited conditions (Figure 2C and D). However, the total recovery of 1, considering the intracellular compartment and the amount nonspecifically bound, was higher in monolayers with CES inhibited (Table 2). The distribution of 1 in each compartment of the experiments in the secretory direction was different compared

intracellular accumulation and nonspecific binding by adsorption. Since it has been reported that the CES isoforms expressed in Caco-2 cells are different from those in human small intestine,26 an enzymatic hydrolysis assay of 1 with the specific CES expressed in human intestinal cells was also performed to improve its in vitro permeability.

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RESULTS AND DISCUSSION Due to the high cytotoxicity of clerodane diterpenes, it was necessary to evaluate which casearin X (1) concentrations could be employed for in vitro permeability assays with Caco-2 cells without damage to the monolayers. The viability of Caco-2 cells was determined by the MTT assay after incubation of 1− 10 μM casearin X (1) for 5 h at 37 °C. Figure 1 shows that only

Figure 1. Dose-dependent cytotoxicity of casearin X (1) in Caco-2 cells after incubation at 37 °C for 5 h. Statistical significance: ***p < 0.05 vs control (n = 18).

concentrations up to 2 μM casearin X (1) were not toxic to Caco-2 cells. There was no significant difference in viability of Caco-2 cells comparing the control assay with the treatments with 1 and 2 μM casearin X (1) (98.9 ± 8.8% and 97.9 ± 11.1%, respectively). Subsequently, an analytical method for quantification of 1 was developed and validated in accordance with current international guidelines.29 The recovery of the sample preparation was approximately 100%. There was no interference from transport buffers in extract ion chromatograms of the compound, and carryover from subsequent injections was not observed. All other validation parameters are listed in Table 1. The in vitro permeability of 1 was evaluated in both absorptive (A → B) and secretory (B → A) directions, using B



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Table 1. Validation Parameters of LC-MS Quantification Method of Casearin X (1) in Transport Buffers Employed in in Vitro Permeability Experiments with Caco-2 Cellsa,b concentrations evaluated (μM) transport buffer 0.01 precision (CV%) accuracy (RSE%) stability (CV%)

matrix effect (CV%) calibration curve a

within-run between-run within-run between-run autosampler 37 °C/2h 3 freezing cycles

0.03

1.6 2.2 1.0 −1.6

6.7 9.7 −6.4 1.6 −4.2 −1.4 −1.4 6.3 y = 740227x + 1698.5 0.9995

equation correlation coefficient

2.00

0.01

1.5 1.5 1.9 1.5

2.2 2.2 0.6 0.7 −1.7 −2.5 0.4 5.6

2.5 3.3 −5.8 −6.7

0.03

1.8 2.9 −6.9 −5.9 −1.2 4.3 −1.9 5.6 y = 823306x + 14581 0.0994

1.00

2.00

0.8 1.6 1.7 0.6

4.0 3.4 1.7 0.7 2.0 −2.4 −5.2 6.2

CV: coefficient of variation expressed as percentage (n = 5). bRSE: relative standard error expressed as percentage (n = 5).

transporter (PEPT) and the organic anion transporting polypeptide (OATP). Both are able to transport substrates according to their concentration gradient.33,34 Further experiments using specific inhibitors are necessary to confirm the involvement of uptake transporters in the absorption of 1. On the other hand, the EfR calculated by the equation Papp(B → A)/Papp(A → B) was 0.1, which suggests that there is no involvement of efflux transporters. Values of EfR greater 2 mean that the compound is a substrate for apical efflux transporters, whereas values between 1 and 2 have been considered ambiguous and need additional assays with specific inhibitors.32 Therefore, with the EfR of 0.1, casearin X (1) should not be considered a substrate for efflux transporters, such as P-glycoprotein (P-gp), multidrug resistance protein 2 (MRP2), and breast cancer resistance protein (BCRP), which utilize ATP as energy source to transport substrates against a concentration gradient.34 Although this finding demonstrated that 1 was rapidly transported in the absorptive direction and with no significant efflux, this was possible only using Caco-2 cell monolayers with the CES activity inhibited by pretreatment with BNPP. Thus, in order to obtain a better prediction of the human bioavailability of 1, a hydrolysis assay using the purified isoenzyme carboxylesterase 2 (hCE2) was performed. The serine esterase human carboxylesterase 1 (hCE1) and hCE2 are both involved in drug metabolism, and both are present in several organs. Both esterases have different substrate specificity; while hCE1 hydrolyzes preferentially substrates with small alcohol groups and large acyl groups, hCE2 hydrolyzes substrates with large alcohol and small acyl groups. The expression pattern of hCE1 and -2 is different in Caco-2 cells compared to the human small intestine.35 The CES isoenzyme expressed by the human gastrointestinal tract is hCE2, whereas Caco-2 cells have a mutant gene that results in expression of carboxylesterase 1

Table 2. Total Recovery of Casearin X (1) (%) Obtained in Permeability Assays Employing Caco-2 Cells with and without CES Inhibitiona absorptive transport secretory transport a

4% FBS transport buffer

1.00

CES noninhibited CES inhibited CES noninhibited CES inhibited

10 min

60 min

120 min

68.4(4.7)

33.3(0.7)

12.0(0.9)

85.4(3.7) 74.4(1.8)

87.2(3.7) 46.5(0.7)

87.4(4.1) 27.6(1.5)

93.9(6.1)

89.3(4.4)

92.9 (6.7)

Mean(SD) for three independent replicates.

with absorptive transport studies. As shown in Figure 3C and D, the amounts recovered from the donor compartments were always higher than those of the receiver compartment. This is expected in the case of passive diffusion, probably due to the lower surface contact between the medium and the cellular membrane on the basal side.27,31 Interestingly, the amounts of 1 found inside cells under CES inhibited conditions were higher in the secretory direction compared to the absorptive direction (Figure 3B and D). It is therefore speculated that transporters are involved in the permeability of 1. To elucidate the absorption mechanisms involved in the transport of 1, Papp values for secretory transport (B → A) were calculated. In addition, the efflux ratio (EfR) was determined (Papp(B → A)/Papp(A → B)). The Papp(B → A) in Caco-2 cells with CES inhibited was (7.5 ± 0.6) × 10−6 cm/s. The difference in values between Papp(A → B) and Papp(B → A) was almost 9-fold, which suggests that the mechanism involved in the absorption of 1 could not be by passive diffusion. In this case, the transport in both directions should be symmetric.32 This result supports the involvement of uptake mechanisms in the absorptive direction by influx transporters. Caco-2 cells can express a variety of influx transporters; the most common ones are the peptide

Table 3. TEER Values (Ω cm2) of Caco-2 Cell Monolayers at Time Point Zero and after 2 h with Casearin X (1)a CES noninhibited absorptive direction (A→B) secretory direction (B→A) a

CES inhibited

0h

2h

0h

2h

289.5(4.7) 301.3(2.7)

275.5(4.7) 286.4(2.7)

272.6(5.4) 283.8(2.7)

264.6(2.7) 272.4(5.4)

Mean(SD) for three independent replicates. C



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Figure 2. Permeability profiles of casearin X (1) (2 μM) after transport experiments in Caco-2 cell monolayers using transport buffer in apical compartment and 4% FBS transport buffer in basolateral compartment. (A) Absorptive direction with CES noninhibited; (B) absorptive direction with CES inhibited; (C) secretory direction with CES noninhibited; (D) secretory direction with CES inhibited (n = 3).

Figure 3. Distribution of casearin X (1) in different compartments of Caco-2 cell monolayer systems for (A) absorptive direction with CES noninhibited; (B) absorptive direction with CES inhibited; (C) secretory direction with CES noninhibited; (D) secretory direction with CES inhibited (n = 3).

°C. The metabolic half-life of 1 was 49.5 min, and the specific activity of hCE2 in this assay was 55.8 ± 3.2 pM/min/mg of protein. This result is in accordance with the differences in the CES substrate specificity. Only substrates with smaller acyl and

(hCE1).36 Therefore, hCE2 was used in the present experiments to better reflect the human gastrointestinal tract. Figure 4 shows that approximately 30% of the initial concentration of 1 added to the reaction medium containing purified hCE2 was hydrolyzed after 30 min of incubation at 37 D



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Table 4. Physicochemical Properties of Casearin X (1)a molecular weight (g/mol) 532.67

log P

mass solubility (g/L)

molar solubility (mol/L)

pKa

polar surface area (Å2)

5.672

0.014

2.6 × 10−5

13.84

108

cell culture medium was supplemented with 10% (v/v) FBS, 1.8 mM L-glutamine, and 1% (v/v) MEM. The permeability studies were carried out in transport buffer, containing DMEM base powder (without D-glucose, L-glutamine, phenol red, sodium pyruvate, and sodium bicarbonate) (8.6 g/L) supplemented with D-glucose (25 mM), L-glutamine (6 mM), sodium chloride (34 mM), and HEPES (20 mM) dissolved in purified water. The medium was adjusted to a pH 7.4 and sterile filtered (Supor-200, 0.2 μm pore size, Pall Corporation, Port Washington, NY, USA).38 The substances were purchased from Sigma-Aldrich Chemie GmbH (Buchs, Switzerland). Petri dishes (56.7 cm2) were purchased from Nunc A/S (Roskilde, Denmark), and Petri dishes (21 cm2) and sixwell polycarbonate membrane Transwell plates with an insert area of 4.7 cm2 and 0.4 μm pore size were ordered from Costar (Corning Incorporated, Corning, NY, USA). The inhibition of CES in Caco-2 cell monolayers was carried out by treatment with the inhibitor BNPP (Sigma-Aldrich Chemie GmbH) dissolved in Hank’s balanced salt solution (HBSS) (with Ca2+, Mg2+) (Gibco) supplemented with D-glucose (19.5 mM) and HEPES (11 mM). The pH was adjusted to 7.4 with NaOH, and the solution was sterile filtered. Casearin X (1) isolated from Casearia sylvestris leaves was provided by Prof. Dr. Alberto José Cavalheiro (NUBBE, Institute of Chemistry, Sao Paulo State University, Araraquara, Brazil). The purified human carboxylesterase 2 enzyme used in the hydrolysis assay and the dimethyl sulfoxide (DMSO) used as solvent for solutions of 1 were bought from Sigma-Aldrich Chemie GmbH. Caco-2 Cell Culture. The Caco-2 cells were cultivated in Petri dishes of 56.7 cm2 (Nunc A/S) using culture medium at 37 °C in a water-saturated atmosphere of 8% CO2 (Sorvall Heraeus, Heracell, Kendro Laboratory Products, Zurich, Switzerland). The cells were subcultured after reaching 70−90% confluence to a split ratio of 1:8− 1:12 by treatment with 0.25% trypsine and 2.4 mM EDTA. The passage numbers of 60−65 were used for every assay. Cell Viability Assay. The MTT test (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) was employed to evaluate the toxicity of 1 and determine the concentration for Caco-2 cell transport experiments. Growing cells were harvested and seeded at a density of 1.14 × 105 cells/cm2 into 96-well microplates. After 24 h, culture medium was replaced for transport buffer containing casearin X (1) at 1, 2, 3, 4, 5, and 10 μM, followed by incubation for 5 h at 37 °C and 8% CO2. Cells were then washed with DPBS, and 100 μL of fresh MTT solution (5 mg/mL in DPBS, diluted 1:10 in transport buffer) was added and incubated for 2 h. MTT solution was removed, and formazan crystals were dissolved with 100 μL of DMSO. After 5 min on an orbital shaker at 200 rpm (KS 250 Basic, Ika Labortechnik, Staufen, Germany), optical density was read at 570 and 630 nm as a reference on a microplate reader (SpectraMax M2e, Molecular Devices, Sunnyvale, CA, USA). A blank experiment consisting of cells incubated with 1% DMSO in transport buffer for detection of cell-free background absorbance was also performed in parallel. Treatment of Caco-2 Cells with BNPP. The Caco-2 cells were seeded at a density of 1.14 × 105 cells/cm2 in six Transwell plates and incubated for 19−21 days. The medium was changed every other day. The treatment of cell monolayers with BNPP was carried out using the method described by Ohura (2009)39 with few modifications. Caco-2 cell monolayers were gently washed twice with HBSS, followed by incubation of both apical and basolateral sides with 200 μM BNPP dissolved in HBSS for 40 min at 37 °C. The monolayers were then washed once with DMEM and incubated with fresh DMEM in both the apical and basolateral sides for 40 min at 37 °C to remove BNPP nonspecifically bound to the Caco-2 cell monolayer. Finally, the cell monolayers were washed one more time with fresh DMEM before the transport assays. The intact cell monolayers used for transport experiments were submitted at the same conditions, but without the CES inhibitor BNPP. Transport Experiments. The Caco-2 cell monolayers treated with BNPP or sham-treated with HBSS were equilibrated for 20 min in the cell culture incubator, and the integrity of monolayers in Transwell plates was verified by measurement of the transepithelial electrical

a 2

Å : angströms squared, 1 Å = 0.1 nm. Values predicted by Advanced Chemistry Development (ACD/Laboratories) Software V11.02 (source: SciFinder/American Chemical Society).

Table 5. Permeability Coefficients Obtained in Transport Experiments with Casearin X (1) in Caco-2 Cell Monolayers with and without Inhibition of CES Activity Caco-2 cell monolayers CES noninhibited

CES inhibited

NDd ND ND

66.9 ± 2.3 7.5 ± 0.6 0.1

Papp(A→B) (×10−6 cm/s)a Papp(B→A) (×10−6 cm/s)b EfRc

a Papp(A→B): apparent permeability in absorptive direction. bPapp(B→ A): apparent permeability in secretory direction. cEfR: efflux ratio. d ND: not determined.

Figure 4. Hydrolysis profile of casearin X (1) after incubation at 37 °C for 30 min, with the hCE2 isoform of carboxylesterase (n = 3).

larger alcohol groups, such as 1, can be recognized by hCE2, whereas hCE1 prefers the opposite. However, hCE1 also can hydrolyze substrates containing either large or small acyl groups.37 Since 1 is a substrate for both hCE1 in Caco-2 cells and purified hCE2, we can expect that the extent of hydrolysis can significantly lower its bioavailability due to intestinal first-pass metabolism. Therefore, successful development of 1 as an estercontaining drug candidate needs further detailed analysis of its metabolism. Such experiments including metabolite identification are currently under way. The outcome of these experiments will provide important information to guide chemical modifications and potential alterations in the hydrolytic susceptibility of 1.

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EXPERIMENTAL SECTION

Chemicals. The human colon adenocarcinoma cell line Caco-2 was a gift of Prof. H. P. Hauri (Biocenter, University of Basel, Basel, Switzerland) originated from the American Type Culture Collection (ATCC, Rockville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM with L-glutamine, high glucose, without sodium pyruvate), Dulbecco’s phosphate-buffered saline (without Ca2+, Mg2+) (DPBS), MEM nonessential amino acids solution (without L-glutamin), Lglutamine 200 mM, fetal bovine serum (FBS), and 0.5% trypsin/4.8 mM EDTA solution were purchased from Gibco (Paisley, UK). The E



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resistance. An EVOM resistance meter equipped with an EndOhm24SNAP chamber (World Precision Instruments, Sarasota, FL, USA) was tempered at 37 °C with 4.6 mL of transport buffer for TEER measurements before and after the assays. Caco-2 monolayers with TEER values above 250 Ω cm2 were used for transport experiments. A casearin X (1) (1 mg/mL) stock solution was prepared in DMSO, and a work solution was prepared in transport buffer at a concentration of 2 μM (final DMSO concentration of 1%). The transport buffer containing 1 was added to the apical compartment (1.6 mL) to investigate the absorptive transport (apical-to-basal direction) or basolateral compartment (2.8 mL) to investigate the secretory transport (basal-to-apical direction). The remaining compartment was filled with blank transport buffer. The basolateral transport buffer was always added with 4% FBS to closely mimic physiological conditions.40 Plates were shaken at 120 rmp on an orbital shaker (KS 15, Edmund Bühler GmbH, Germany) under a water-saturated atmosphere at 37 °C. The permeability of 1 across Caco-2 cell monolayers was monitored by taking samples of 25 μL from both compartments at 0, 5, 10, 20, 30, 45, 60, 90, and 120 min, in triplicate. An equal volume of acetonitrile was added to the samples collected, mixed on a vortex for 5 s (Ika Genius 3, Huber & Co AG, Reinach, Switzerland), and centrifuged for 10 min at 16100g (Eppendorf 5415R, Hamburg, Germany). The supernatant was analyzed by LC-MS. The volume of transport buffer removed was replaced only at the end of the experiment for the final TEER measurements. Apparent permeability (Papp) values were calculated for both absorptive (Papp(A → B)) and secretory (Papp(B → A)) experiments. The determined concentration of 1 appearing in the receiver compartment was plotted against the sampling time, and curve fitting using linear regression parameters in GraphPad Prism (version 5.01, GraphPad Software Inc., San Diego, CA, USA) was used to give the rate of compound transported. The following equation was used to calculate the apparent permeability coefficients, Papp = (ΔQ/Δt)ν/AC0, where ΔQ/Δt is the rate of the drug transport (μM/s), ν is the volume of the receiver compartment (cm3), A is the surface area of the membrane (cm2), and C0 is the initial donor concentration (μM). The efflux ratio was calculated by the equation Papp(B → A)/Papp(A → B), which is a quotient of the apparent permeability in the secretory to that in the absorptive direction. Mass Balance. At times of 10, 60, and 120 min one plate with three inserts was used for the evaluation of the amount of 1 inside cells and nonspecifically binding to the membrane and plastic material of the insets and Transwell plates. For cell extraction, the monolayers were washed twice with cold DPBS. The inserts were transferred to a Petri dish (22.1 cm2), 300 μL of 0.25% trypsin/2.4 mM EDTA solution was added to each insert, and the dishes were shaken on an orbital shaker at 75 rpm and 37 °C for 15 min. After adding 1 mL of transport buffer, cells were removed from the membrane using a cell scraper (BD Falcon, BD Biosciences Discovery Labware, Bedford, MA, USA) and transferred to a 1.5 mL tube. The suspension was spun 5 min at 100g, and the supernatant was discarded. The pellet was ressuspended in 500 μL of transport buffer at 37 °C. At this stage positive controls of 1 were added to blank cell pellets previously treated with BNPP to correct the recovery in the samples from transport experiments. The suspension was vortexed for 5 s, frozen at −80 °C for 15 min, and then thawed at 37 °C under shaking at 1400 rpm (Thermomixer Comfort, Eppendorf AG, Hamurg, Germany). The samples were supplemented with 500 μL of acetonitrile, vortexed for 5 s, and put into an ice bath for 20 min. The tubes were shaken again for 10 min in the thermomixer at 37 °C and 1400 rpm, followed by a centrifugation for 10 min at 16100g. The buffer−acetonitrile (1:1) supernatant was transferred to another tube and stored in the refrigerator at 4 °C. The pellet was extracted twice with 500 μL of acetonitrile and disintegrated with six pulses of an ultrasonic disintegrator (Branson Sonifier 250, Branson Ultrasonic Corporation, Danbury, CT, USA, instrument settings: output control 2, duty cycle 30%). Samples were mixed for 10 min in a thermomixer (37 °C and 1400 rpm) and centrifuged for 10 min (16100g). The

supernatants of the second and third extraction were united and evaporated to dryness under nitrogen flow. Each residue was dissolved with its supernatant of the first extraction, mixed for 10 min (37 °C and 1400 rpm), and centrifuged for 10 min (16100g), and the supernatant was analyzed by LC-MS. For plastic extraction, wells and inserts were washed twice with cold DPBS, and acetonitrile was added (1.6 mL apical; 2.8 mL basolateral). The plates were sealed with three layers of Parafilm and incubated in an orbital shaker at 37 °C for 45 min. Samples were taken from the apical or basolateral side and diluted in an equal volume of transport buffer or 4% FBS transport buffer, respectively. After vortexing for 5 s and centrifugation at 16100g for 10 min, the supernatant was analyzed by LC-MS. An additional experiment was performed to determine the recovery of 1 from the plastic, employing a transport assay in Transwell plates without cells. Hydrolysis Experiments. Hydrolysis of 1 was performed in 0.2 μg/mL purified human hCE2 diluted in 1 mL of 90 mM KH2PO4, 40 mM KCl, pH 7.3. After preincubation for 5 min at 37 °C, the reaction was started by adding 1 μM casearin X (1) dissolved in DMSO. The final concentration of DMSO in the hydrolyzing incubation was maintained at 0.1%, which had no effect on hydrolase activity. The degradation of 1 was monitored every 5 min until 30 min of incubation, in triplicate. The reaction was terminated by adding an equal volume of ice-cold acetonitrile and mixing in a vortex for 5 s. After centrifugation of the reaction mixture at 16100g for 10 min, the supernatant was analyzed by LC-MS. The metabolic half-life was calculated dividing ln 2 by the terminal elimination rate constant, which is the slope of the linear regression from natural log percentage substrate remaining versus incubation time. An additional experiment was performed using inhibited hCE2 by BNPP (200 μM) preincubated for 10 min. LC-MS Analysis. Casearin X (1) was analyzed by LC-MS with an Agilent Series 1200 equipped with a G1379B degasser, a G1312A binary pump, a G1367B autosampler, a G1330B thermostat, a G1316A column oven, and a G6130A single quadrupole MS detector. A Zorbax Eclipse XDB-C18 reversed-phase column, 5 μm, 2 × 125 mm, and a mobile phase consisting of acetonitrile−water (80:20, v/v) in an isocratic flow of 0.3 mL/min were used. The samples were maintained at 20 °C, and the column was heated to 32 °C. The ions were generated by atmospheric pressure electrospray ionization, and the MS detector was run in scan (m/z 100−700) and SIM modes, at positive polarity with a capillary voltage of 4000 V, 240 V fragmentor, 9 mL/ min drying gas flow, 350 °C drying gas temperature, and 40 psi nebulizer pressure. Casearin X (1) was detected at m/z 555, corresponding to the sodium adduct. The method for quantification of 1 in transport buffer and in 4% FBS transport buffer was validated according to the Validation of Analytical Procedures (ICH).28 Data Analysis. One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test was carried out for statistical analysis using the software GraphPad Prism (version 5.01), and p values < 0.05 were considered significant.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +41 61 467 46 89. Fax: +41 61 467 47 01. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge FAPESP (Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo), CNPq (Conselho ́ Nacional de Desenvolvimento Cientifico e Tecnológico), and ́ CAPES (Coordenaçaõ de Aperfeiçoamento de Pessoal de Nivel Superior) for financial support. F



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