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Acid diterpenes from Copaiba oleoresin (Copaifera langsdorffii): Chemical and plasma stability and intestinal permeability using Caco-2 cells
Journal of Ethnopharmacology 235 (2019) 183–189
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Acid diterpenes from Copaiba oleoresin (Copaifera langsdorffii): Chemical and plasma stability and intestinal permeability using Caco-2 cells
T
M. Mauroa, R.A. De Grandisa, M.L. Camposb, A. Bauermeisterc, R.G. Peccininia, F.R. Pavana, ⁎ N.P. Lopesc, N.V. De Moraesa, a
São Paulo State University (UNESP), School of Pharmaceutical Sciences, Araraquara, SP ZIP 14801-902, Brazil Federal University of Mato Grosso, Sinop, MT, Brazil c University of Sao Paulo (USP), NPPNS, School of Pharmaceutical Sciences of Ribeirao Preto, Ribeirao Preto, SP ZIP 14040-903, Brazil b
ARTICLE INFO
ABSTRACT
Keywords: Copalic acid Kaurenoic acid Caco-2 Intestinal absorption Viability
Ethnopharmacological relevance: Copaiba oleoresin has been used in folk medicine in the treatment of bronchitis, syphilis, skin diseases and ulcers due to its anti-inflammatory and antiseptic activities, but there is no information about major compounds oral absorption to support the traditional use. Aim of study: Considering the potential of copalic (CA) and kaurenoic acid (KA) – major biological activity (in vitro) diterpenes found in the oleoresin, this study aimed to evaluate the intestinal permeability of CA and KA using Caco-2 cells model as predictive test for oral drug absorption. Materials and methods: Chemical stability at pH 1.2 and 7.4 and plasma stability were evaluated to mimic physiological conditions of the gastrointestinal tract. The intestinal permeability of CA and KA was evaluated in Caco-2 cells in the presence and absence of the P-glycoprotein inhibitor verapamil. Results: CA and KA were rapidly degraded at pH 1.2 (0.2 M Clark-Lubs buffer). At pH 7.4 (0.1 M phosphate buffer), CA was stable for up to 24 h and KA for up to 6 h. In human plasma, CA and KA can be considered stable for 24 h and 12 h at 37 °C, respectively. Caco-2 cells were considered viable when incubated with CA or KA in the range of 3.9–250 μM for 24 h. CA and KA exhibited moderate apparent permeability (Papp) of 4.67 ( ± 0.08) × 10−6 cm/s and 4.66 ( ± 0.04) × 10−6 cm/s, respectively. Simultaneous incubation with verapamil showed that P-glycoprotein does not play a relevant role on CA and KA oral absorption, with Papp of 4.48 ( ± 0.26) × 10−6 cm/s and 5.37 ( ± 0.72) × 10−6 cm/s observed for CA and KA, respectively. Conclusion: The oral absorption of both CA and KA is driven by mainly passive permeability, is not limited by pglycoprotein, but enteric-coated dosage forms should be used to avoid chemical instability in the gastric pH.
1. Introduction
lesions, copaiba oleoresin has also been used orally in folk medicine in the treatment of bronchitis and syphilis due to anti-inflammatory and antiseptic activities (Albuquerque et al., 2017; Vargas et al., 2015; Tincusi et al., 2002). Natives also apply the oleoresin as mosquito repellent, and currently investigations allowed the development patented formulations for the use based on the traditional knowledge (Pohlit et al., 2011a, 2011b). Copalic acid (CA) and kaurenoic acid (KA) (Fig. 1) are two major diterpenes of copaiba oleoresin (Veiga Junior et al., 2007, 2005). CA presents in vitro antibacterial activity (da Trindade et al., 2018; Souza et al., 2018), antifungal activity against dermatophytes, (Nakamura
Copaifera species, popularly known as "copaibeiras" or "copaíba", are of great economic and ecological interest in Amazon forest. It is estimated that Copaiba oleoresin has been used in popular medicine for more than 500 years (Pieri et al., 2009). Gaspar Barléu observed that animals rubbed themselves in the trunk of Copaifera to heal their wounds (Barléu, 1974). It is assumed that by observing this behaviour, natives began to use the oleoresin with the same intention of healing (Pieri et al., 2009; Veiga Junior and Pinto, 2002). Beyond the use for healing several skin wounds such as ulcers scarring and leishmania skin
⁎ Correspondence to: São Paulo State University (UNESP), School of Pharmaceutical Sciences, Rodovia Araraquara-Jaú, km 01, Araraquara, SP ZIP 14801-902, Brazil. E-mail addresses: [email protected] (M. Mauro), [email protected] (R.A. De Grandis), [email protected] (M.L. Campos), [email protected] (A. Bauermeister), [email protected] (R.G. Peccinini), [email protected] (F.R. Pavan), [email protected] (N.P. Lopes), [email protected] (N.V. De Moraes).
https://doi.org/10.1016/j.jep.2019.02.017 Received 30 November 2018; Received in revised form 4 February 2019; Accepted 10 February 2019 Available online 11 February 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
Journal of Ethnopharmacology 235 (2019) 183–189
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analyzed by gas chromatography and the identity of CA and KA were confirmed by the GC library, and by NMR, respectively (Supplementary material; Ohsaki et al., 1994). Resazurin sodium salt (CAS 62758-13-8), dimethyl sulfoxide (DMSO, CAS 67–68–5), HEPES (CAS 7365-45-9), fluvastatin sodium (≥98% purity; CAS 93957-55-2), verapamil hydrochloride (≥99% purity; CAS 152–11-4) and fluorescein (CAS 232107-5) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS) and Hank's salt solution were purchased from Gibco (Paisley, United Kingdom) and antibiotic mixture (5 mg of penicillin, 5 mg of streptomycin and 10 mg of neomycin per mL) were obtained from Gibco (Paisley, Reino Unido). Acetonitrile (HPLC grade) was purchased from J.T. Baker (Center Valley, PA, EUA) and methyl-tert butyl ether (HPLC grade) from Panreac (Darmstadt, Germany). All other reagents were analytical grade. Water was purified by a Milli-Q system (Millipore).
Fig. 1. Chemical structures of kaurenoic acid (A) and copalic acid (B).
et al., 2017) and antitrypanosomal activity (da Trindade et al., 2018). KA has shown in vitro antimicrobial, anti-inflammatory, anti-allergic, immunosuppressive and apoptosis-inducing activities (Kian et al., 2018). Due to its various pharmacological properties and because the oleoresin is commercially available to several countries, this is considered a major renewable natural product (Medeiros and Vieira, 2008). The pharmacological interests on natural products of Copaifera spp. stimulated, in the 80's, pharmaceutical companies to develop products based on traditional knowledge. However, the absence of pharmacokinetics and toxicological studies led to the withdrawal of these products from the Brazilian market. Therefore, candidates for oral drug development from copaíba require studies to better understand their pharmacokinetic properties, such as absorption, distribution, metabolism and excretion (ADME). In vitro ADME data can be further used in physiology-based pharmacokinetic (PBPK) models to predict pharmacokinetic processes in humans through in vitro-in vivo extrapolation. This approach will advance further steps on drug development to achieve a safe and effective performance by design and optimization of dosage regimens, predicting drug-drug interactions and avoiding drug toxicity (Li, 2001; Rostami-Hodjegan, 2012; Rostami-Hodjegan and Tucker, 2007; Fan and de Lannoy, 2014). This study aimed to evaluate the intestinal permeability of CA and KA using Caco-2 – the human colon adenocarcinoma cells - as a predictive test for human oral drug absorption and oral bioavailability (Chung et al., 2001; Ungell and Artursson, 2009; Gertz et al., 2010; Kostewicz et al., 2014). Caco-2 cells can differentiate spontaneously in vitro, mimicking the human intestinal epithelium and can be used to rapidly screen oral drug candidates (Ungell and Artursson, 2009). This in vitro model has the advantage of evaluating not only passive permeability, but also active transport across membranes (Lin et al., 2011). P-glycoprotein (P-gp) is a major efflux drug transporter located at the apical membrane of enterocytes and limits the oral absorption of several drugs and xenobiotics (Wang et al., 2007; Dezani et al., 2017). The intestinal permeability of both diterpenes CA and KA was assayed using the P-gp inhibitor verapamil to evaluate the role of the efflux transporter on oral absorption (Bansal et al., 2009). The chemical stability of CA and KA at pH 1.2, pH 7.4 and the stability in plasma were also evaluated to simulate different physiological conditions that could affect the oral drug disposition.
2.2. In vitro aqueous buffer stability Chemical stability was tested at pH 1.2 (Clark-Lubs buffer, 0.2 M) and pH 7.4 (phosphate buffer, 0.1 M), in triplicates at the concentration of 0.05 μM. The solutions were kept under stirring (70 rpm) at 37 °C in an orbital shaker (Ethik Technology model 430 - RDB T5, São Paulo, Brazil). Samples (100 μL) were collected at 0; 0.5; 1; 2; 4; 6; 12 and 24 h and analyzed using the analytical method developed and validated in high-performance liquid chromatography (HPLC) coupled to mass spectrometry (HPLC-MS). 2.3. In vitro plasma stability The experimental protocol of this study was approved by the Committee of Ethics in Research of the School of Pharmaceutical Sciences of Araraquara - UNESP (CAAE 65707517.6.0000.5426). Healthy volunteers of both gender, 18–60 years, body weight > 50 kg, were invited to donate blood at Hemonúcleo Regional of Araraquara "Professor Clara Pechmann Mendonça”. All participants attended the procedures of the blood donation service. Only the plasma was obtained for research and other blood components were screened for donation. Plasma samples were spiked with CA and KA at concentrations of 0.8 and 1 μM, respectively, in triplicates. The samples were vortexed and kept under constant agitation (70 rpm), at 37 °C (Incubator Ethik Technology model 430 - RDB T5, São Paulo, Brazil). Aliquots of 100 μL were collected at times 0; 0.5; 1; 2; 4; 6; 12 and 24 h and analyzed by HPLC-MS. Results were presented as percentages of the compounds remaining in human plasma. 2.4. HPLC-MS analysis The HPLC-MS system (Perkin Elmer, Shelton, USA) consisted of a binary pump (Flexar LC Pump), degasser (Flexar solvent manager), oven (Flexar LC column oven), automatic injector (Flexar LC Autosampler) and a single quadrupole mass spectrometer (Flexar SQ 300 MS). Data acquisition and quantification of samples were performed using Chromera software, version 3.4.0.5712 (Perkin Elmer, Shelton, USA). CA and KA were resolved on a reversed phase Poroshell 120 EC-C18 endcapped column (100 × 4.6 mm), with 2.7 µm particle size (Agilent Technologies, Santa Clara, United States), kept at 22 °C and mobile phase consisting of acetonitrile: water (90:10, v/v) containing 5 mM ammonium formate at a flow rate of 0.5 mL/min using an isocratic mode. The mass spectrometry detection was performed on negative ion mode. The deprotonated molecular ions [M-H]- with the mass-to-charge ratio (m/z) 303 and 301 were used for monitoring of CA and KA, respectively. The injection volume was 20 μL and the chromatographic run time was 14 min. The method was developed and validated according to current international guidelines.
2. Material and methods 2.1. Chemicals and reagents Copalic acid (CA) and kaurenoic acid (KA) were isolated from copaiba oleoresin obtained from a cooperative center (Rio Branco, AC, Brazil) as previously described (Nakamura et al., 2017). In 1951, the American botanist John Duncan Dwyer described Copaifera langsdorffii var. krukovii Dwyer in materials from the State of Acre (Brazil), including the large densities of these species in Tarauacá municipality. This data is used by the community for a long term commercialization process. After isolation and purification procedures, samples were 184
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2.5. Plasma sample preparation
Relative error (RE , %) Experimental average concentration = Nominal value
Stock solutions of CA (2000 µM) and KA (2500 µM) were prepared separately in DMSO. Working solutions were prepared by diluting the stock solutions in acetonitrile to reach the concentrations 0.25; 0.5; 1; 2, 5; 16 and 20 μM for KA and 0.2; 0.4; 0.8; 1.6; 4.0; 12.85 and 16 μM for CA. The internal standard (IS) fluvastatin was prepared at the concentration of 1 μM in acetonitrile. All solutions were stored at −20 °C until analysis. Aliquots of 100 μL of plasma were spiked with 20 μL of each working solution, 20 μL of the IS solution (1 μM fluvastatin), 20 μL of 1 M HCl and 300 μL of methyl tert-butyl ether as extractor solvent. The samples were agitated for 20 min using a horizontal reciprocating shaker (130 ± 10 cycles/min) and centrifuged for 10 min (15,000×g, 5 °C). The organic phase (270 μL) of each sample was transferred to 1.5 mL microtubes and evaporated to dryness at 40 °C, using a vacuum sample concentrator (Genevac ™ Centrifugal Duo, Ipswich UK). Residues were reconstituted in 90 μL of mobile phase, vortexed for 1 min and 20 μL were injected into the chromatographic system.
× 100
Stability of the analytes was assessed in the following conditions: a) short-term stability at room temperature (23 ± 2 °C) for 1 h; b) postprocessed stability for 7 h at 18 °C; c) after 3 cycles of freeze (−20 °C) and thaw. 2.7. Cell viability The Caco-2 human colon adenocarcinoma cell line (BCRJ, n° 0059) was purchased from Rio de Janeiro Cell Bank. Caco-2 cells were cultured in DMEM supplemented with 4.5 g/L D-glucose, 20% of FBS, 1% antibiotic mixture containing 5 mg of penicillin, 5 mg of streptomycin and 10 mg of neomycin per mL at 37 °C in an atmosphere of 5% CO2 saturation. The resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide sodium) assay was performed to evaluate the in vitro cell viability (de Grandis et al., 2017). In summary, cells were seeded at a density of 7.5 × 104 cells mL−1 in a 96-well plate and incubated for 24 h (37 °C under 5% CO2). On the next day, CA and KA solutions containing no more than 1% DMSO in DMEM were added to the wells. Dilutions were prepared to obtain CA and KA concentrations ranging from 3.91 to 500 µM (3.91; 7.81; 15.62; 31.25; 62.5; 125; 250 and 500 µM) in DMEM and six replicates of each concentration were evaluated. A solvent control containing 1% DMSO in DMEM was used to assess the viability of cells using only this solvent. DMEM samples were used as negative controls and 15% DMSO (v/v) in DMEM was used as a positive control. After 24 h of incubation (37 °C under 5% CO2), 50 μL of 0.01% resazurin solution was added to each well and plates were incubated for 2 h. Absorbance was evaluated in a microplate reader Synergy H1 (BioTek®) using excitation and emission filters at wavelengths of 530 and 590 nm, respectively. Cell viability was calculated by the following equation:
2.6. Bioanalytical method validation Selectivity was performed to check if unrelated compounds from the biological matrix could interfere with the ability to measure the analytes of interest. Blank plasma samples from six different sources, being four normal, one with in vitro hemolysis (hemolyzed plasma) and one highly concentrated in lipids (lipemic plasma) were evaluated. The absence of interfering components was defined when responses close to the analyte retention time were less than 20% of the lower limit of quantification (LLOQ) and less than 5% for IS. The carry-over effect was evaluated by placing blank plasma samples, one before and two after the calibration standard at the upper quantification limit. Carry-over in a blank sample after a high concentration standard should not be higher than 20% of the LLOQ response and 5% of the IS. The matrix effect was evaluated by comparing peak areas of the analytes and IS in solution with peak areas resulting from plasma samples spiked after extraction. To evaluate the matrix effect, eight different sources of blank plasma were used, being six normal samples, one lipemic and one haemolysed. The coefficients of variation of the ISnormalized matrix factor (NMF) were calculated for both low and high concentration quality controls, by the equation:
Cell viability (%) =
Fluorescence intensity of sample × 100 Fluorescence intensity of solventcontrol
2.8. Permeability across Caco-2 cells Drug permeability was evaluated in Caco-2 cells in line with Hubatsch et al. (2007). To evaluate if CA and KA are P-gp substrates, the transport across Caco-2 monolayer was investigated using verapamil as a P-gp inhibitor (Jin et al., 2013). Cells were cultured at a seeding density of 5 × 104 cells cm−2 in translucent membrane inserts for 12-well plates (ThinCert™, 0.4 µm pore size, Greiner Bio-One, Kremsmünster, Áustria) for 21 days to reach confluence and differentiation. Plates were incubated in an atmosphere of 5% CO2, 95% relative humidity and 37 °C before cell permeability experiments. The integrity of the monolayer was examined by measuring the transepithelial electrical resistance (TEER) with an epithelial voltammeter Millicell-ERS (Millipore Corporation, Bedford, MA) and by using verapamil (44 µM) and fluorescein (45 µM) as high and low permeability controls, respectively (Kratz et al., 2011; Fernandes et al., 2014). Only cell monolayers with a TEER above 200 Ω × cm2 were used for the transport assays (Fernandes et al., 2014; de Grandis et al., 2017). Drug solutions containing 3.9 µM for CA and KA were prepared in Hank's buffer at pH 7.4 containing 200 mM of HEPES. Both apical to basolateral and basolateral to apical transport across the monolayer was evaluated by adding drug solutions in apical and basolateral compartments, respectively. Drug permeability was assessed with and without the P-gp inhibitor verapamil (Jin et al., 2013). Cells were incubated with 50 µM verapamil for 30 min before the incubation with substrates (Lin et al., 2007, 2011). The plates were placed at 37 °C in an orbital shaker (25 rpm) (Ethik Technology model 430 – RDB T5, São Paulo, Brazil) and aliquots of 200 μL were sampled at times 0, 15, 30, 60, 120
NMF =
Nominal value
response of analyte inmatrix / response of internal standardin matrix response of analyte insolution/ response of internal standardin solution
The absence of matrix effect was defined by the relative standard of deviation (RSD, %) of the NMF not greater than 15%. Calibration curves were prepared by spiking the blank matrix (100 μL) with working solutions (20 μL) to reach the range of final plasma concentrations of 0.05–4 μM for KA and 0.04–3.2 μM for CA. The criteria for acceptance of the calibration curve was that recommended by the current guidelines on bioanalytical method validation set by the European Medicines Agency (EMA, 2012) (EMA) and the USA Food and Drug Administration (US FDA, 2018). Precision and accuracy were determined in the same run (withinrun) and in three different runs (between-run) using four quality control levels: LLOQ, low-quality control (LQC), medium-quality control (MQC), and high-quality control (HQC). The method can be considered precise when the RSD does not exceed 15%, except for the LLOQ for which RSD up to 20% is acceptable. Accuracy was evaluated by the relative error (RE), which should be less than ± 15% in terms of the nominal value, except for the LLOQ, for which values less than ± 20% are allowed (EMA, 2012; US FDA, 2018). The RE was calculated according to the equation: 185
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and 180 min. After each sampling, the same volume of a fresh permeability buffer at 37 °C was replaced, in order to keep the volume in the compartment. The apparent permeability (Papp) was calculated according to the following equation.
Papp =
Table 1 Validation parameters of copalic acid (CA) and kaurenoic acid (KA) in human plasma by HPLC-MS.
Inter-assay precision (RSD%, n = 3) LOQ LQC MQC HQC Intra-assay precision (RSD%, n = 5) LOQ LQC MQC HQC Inter-assay accuracy (RE%, n = 3) LOQ LQC MQC HQC Intra-assay accuracy (RE%, n = 5) LOQ LQC MQC HQC Stability (RE%, n = 3) Short duration stability (1 h à 23 °C) LQC HQC Post processing stability (7 h à 16 °C) LQC HQC Freeze/thaw stability (1 cycle) LQC HQC
VR dC × (A × C0) dT
where VR is the volume of the receiver compartment, A is the monolayer surface area (in cm2), C0 is the initial drug concentration in the donor compartment, and dC/dt represents the change of concentration over time. The efflux ratio (ER) was defined as Papp(B-A)/Papp(A-B). The Papp (A-B) of verapamil > 12.2 × 10−6 cm/s and of fluorescein < 3.7 × 10−6 cm/s were used as indicators of a functional Caco-2 monolayer (Park et al., 2015; Hoffmann et al., 2018). The transporter substrate index (TSIA-B) was calculated for P-glycoprotein as in the equation:
TSIapp (A
B)
=
P iapp (A
B)
Papp (A
Papp (A
B)
B)
× 100
Piapp
where (A-B) is measured in the presence of the specific P-gp inhibitor verapamil (Lin et al., 2011). 3. Results and discussion The oral administration of drugs is preferred route in pharmacological therapy due to the convenience, safety and cost for patients, health professionals and the public health system (Skolnik et al., 2010; Eek et al., 2016; Benjamin et al., 2012). In vitro assays to evaluate the oral absorption and bioavailability of drug candidates have shown to minimize the costs of both drug discovery and drug development. By increasing the use of these in vitro ADME assays, the number of withdrawals in drug development reduced from 40% in 1991 to 14% in 2000 (Wang et al., 2007). Thus, in order to evaluate CA and KA as oral drug candidates, stability in the acidic stomach and basic intestinal pH, stability in plasma as well as intestinal permeability using Caco-2 were evaluated. The bioanalytical method was fully validated using human plasma as matrix and considering the parameters selectivity, residual effect, matrix effect, linearity, precision, accuracy and stability (Table 1). Selectivity was demonstrated by the lack of interfering peaks in the retention time of analytes and IS. The absence of matrix effect was shown by the coefficient of variation of IS-normalized matrix factor, which was below 15% at both low and high concentration levels (Supplementary material). The method was linear over the concentration range of 0.05–4 µM for KA and 0.04–3.2 µM for CA, using the 1/x2 weight. Precision and accuracy did not exceed 15% of relative standard deviation and relative error, respectively, except for the LLOQ for which values up to 20% are acceptable. No carry-over effect was observed for CA, KA or the IS. The validated method was slightly modified for the analysis of samples obtained in permeability assays, considering calibration curve in the range of concentrations of 0.0125–5 μM for KA and CA. Methods reporting the analysis of CA and KA in plant material or biological matrix have been reported in the literature by using HPLC with UV detection (Costa et al., 2015; de Matos et al., 2018) or coupled to mass spectrometry (MS) (Gasparetto et al., 2015; Bardají et al., 2016; Jiang et al., 2019), gas chromatography (GC) with flame ionization detection (Tappin et al., 2004) or GC-MS (Gelmini et al., 2013; Miyazaki et al., 2015; Xavier-junior et al., 2017). The bioanalytical methods using HPLC with UV detection (de Matos et al., 2018) have reported LLOQ of 2.48 µM for KA using 100 μL of rat plasma. In the present study, the LLOQ of 0.05 and 0.04 µM for KA and CA, respectively, were observed using 100 μL of human plasma. Other methods using UPLC-MS/MS – which is a technique with better sensitivity than HPLC-MS - have reported lower LLOQ (0.016 µM), using either 100 μL
CA
KA
18.0 6.6 14.0 6.7
13.3 9.9 9.9 8.6
6.6 2.6 2.3 2.9
9.9 5.8 7.9 3.8
−8.5 −3.0 −7.5 −13.2
−7.2 14.9 9.1 −4.8
0.4 1.9 3.9 −7.4
9.9 5.8 7.9 1.9
−12.3 −14.9
−11.8 −14.4
−10.3 −13.1
−12.1 −13.7
−5.8 −13.6
9.7 −12.1
RSD: relative standard deviation or coefficient of variation = [(standard deviation/mean) × 100]; RE: relative error (%) = [(observed concentration – nominal concentration)/nominal concentration] × 100; LOQ: limit of quantification (CA: 0,04 µM; KA: 0.05 µM); LQC: Low concentration quality control (CA: 0.08 µM; KA: 0.10 µM); MQC: Medium concentration quality control (CA: 0.32 µM; KA: 0.40 µM); HQC: High concentration quality control (CA: 2.57 µM; KA: 3.2 µM).
(Jiang et al., 2019) or 200 μL of plasma (Gasparetto et al., 2015). Up to date, this is the first bioanalytical method reporting the determination of both CA and KA in the same chromatographic run and the first reporting CA analysis in plasma. Considering that CA and KA are the main bioactive diterpenes of copaiba oleoresin, this method can be applied in future ADME studies to reduce expenses with solvents and other consumables and to be time-saving when evaluating these two diterpenes. Stability evaluations have shown that CA and KA were rapidly degraded at pH 1.2 with statistical disappearance (p < 0.05) after 0.5 h. After 1 h, concentrations were below the LLOQ of the analytical method, demonstrating that both compounds are unstable at simulated stomach pH. At pH 7.4, CA was stable for up to 24 h and KA for up to 6 h, with statistically significant disappearance (p < 0.05) only after 12 h (Fig. 2). These results are in agreement with the recent findings of pharmacokinetics of KA in rats after oral administration. Non-detectable plasma levels were reported after oral administration of 50 mg/kg of KA (de Matos et al., 2018). Maximum plasma concentrations (Cmax) of only 12.6, 44.7 and 48.0 ng/mL were observed after single oral doses of 10, 20 or 40 mg/kg in rats (Jiang et al., 2019) and these low plasma concentrations can be at least partially explained by the instability of KA in acidic stomach pH. However, it is required to understand if the compounds are converted in the stomach to bioactive compounds. It is already known that oral administration of KA or the oleoresin in rats showed anti-inflammatory and antilipoperoxidative activities (Paiva et al., 2003. Lima-Silva et al., 2009). By the present data, it is suggested that KA in vivo activities could be achieved using lower doses and enteric-coated dosage forms. The cytotoxicity of CA and KA towards Caco-2 cells was evaluated 186
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concentration that caused 50% inhibition (IC50) for CA and KA in Caco2 cells were 237.4 and 154.0 μM, respectively, and the concentration of 3.9 µM was selected for permeability assays to ensure the viability of Caco-2 cells (Fig. 3). The transepithelial electrical resistance (TEER) was measured at 10, 15 and 21 days of cell cultivation on the insert filters as an indicator of the membrane integrity formed by Caco-2 cells (Fernandes et al., 2014; Srinivasan et al., 2015) (Supplementary material). Only cell monolayers with a TEER above 200 Ω × cm2 were used for the transport assays. The membrane integrity was also assessed by determining the permeability of high and low permeability standards, such as verapamil and fluorescein, respectively (Kratz et al., 2011; Bonetti et al., 2018). The Papp (A-B) for verapamil was 115 × 10−6 cm/s. For fluorescein, concentrations in the basolateral compartment at all times sampled were below the limit of quantification of the method. These results are in agreement with the literature in terms of the very low permeability of fluorescein and the high permeability of verapamil (Engman et al., 2003; Koljonen et al., 2006; Chen et al., 2016), and indicates the integrity and viability of Caco-2 cell monolayers during the permeability experiments. Apparent permeability values (Papp) were determined from the amount permeated through the Caco-2 cell membranes at both apicalbasolateral (A-B) and basolateral-apical (B-A) direction. CA and KA exhibited Papp (A-B) values of 4.67 ( ± 0.08) × 10−6 cm/s and 4.66 ( ± 0.04) × 10−6 cm/s, respectively. Compounds with Papp value < 2 × 10−6 cm/s are considered as poorly absorbed; moderately absorbed when the Papp value is between 2 and 10 × 10−6 cm/s; and Papp values higher than 10 × 10−6 cm/s defines compounds with high in vivo intestinal permeability (Flynn and Vohra, 2018). Thus, although requiring enteric coating polymers to protect against gastric fluids, CA and KA shows moderate intestinal permeability and are promising oral drug candidates. In terms of basolateral-apical permeability, CA and KA shows Papp (B-A) of 1.68 ( ± 0.50) × 10−6 cm/s and 2.82 ( ± 0.20) × 10−6 cm/s, respectively. The assessment of transport in both directions can show whether the compounds undergo active efflux. The efflux ratio (ER) is used to evaluate the impact of the efflux on oral permeability. ER values ≥3 indicate predominant efflux activity and ER ˂2 characterize passive transport. When ER is between 2 and 3, oral permeability shows moderate efflux (Skolnik et al., 2010). For CA and KA, ER was < 2, suggesting that passive transport is the main mechanism involved in their oral absorption. The permeability through the Caco-2 cells membranes at the apical-basolateral direction in the presence of the Pglycoprotein inhibitor verapamil (Table 2) shows that this efflux drug transporter does not influence KA and CA oral absorption. Both CA and KA can be considered non-substrates of P-glycoprotein, since TSIPapp of both were < 25% (Lin et al., 2011). Using the four-zone graphical model proposed by Lin et al. (2011), it can be concluded that passive permeability drives both KA and CA in vitro permeability and in vivo oral absorption (Lin et al., 2011). CA and KA can be considered stable in human plasma for 24 h and 12 h at 37 °C, respectively (Fig. 2). For CA, no statistical differences were observed after incubation for up to 24 h. The remaining KA showed a statistical difference in comparison to the basal
Fig. 2. Stability of copalic acid (о) and kaurenoic acid (■) at 37 °C in pH 1.2, pH 7.4 and in plasma. *means statistically significant compared to time 0 by repeated measures ANOVA followed by the Tukey test (p < 0.05). *means statistically significant compared to time 0 by repeated measures ANOVA followed by the Tukey test (p < 0.05).
before the permeability assays to design a protocol using nontoxic concentrations (de Grandis et al., 2017; McMillian et al., 2002). DMSO control, containing no more than 1% of DMSO in DMEM, demonstrated cell viability of approximately 90% in relation to DMEM only. According to the international guide ISO 10993-5, the cytotoxic effect is defined by cell viability lower than 70% (ISO/EN10993-5, 2009). The
Table 2 Apparent drug permeability in Caco-2 cells in the presence and absence of 50 μM verapamil as P-glycoprotein inhibitor. Compound
Copalic acid Kaurenoic acid Verapamil Fluorescein
Papp (A-B) (× 10−6 cm/s)
4.67 ± 0.08 4.66 ± 0.04 115 N.D.
Papp (B-A) (× 10−6 cm/s)
1.68 ± 0.50 2.82 ± 0.20
Efflux ratio (ER) 0.36 0.60
Papp (B Papp (A
A) B)
Piapp(A-B)a
4.48 ± 0.26 5.37 ± 0.72
TSIPapp(A-B)
i (A Papp
B)
Papp (A
Papp (A
B)
B)
×100
−4.07 15.24
Values presented as average ± standard deviation of triplicates; N.D.: not determined (concentrations in the basolateral compartment were bellow the LLOQ). i indicates that experiments were conducted in the presence of 50 μM verapamil. 187
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Fig. 3. Viability of kaurenoic acid (A) and copalic acid (B) in Caco-2 cells after incubation for 24 h at 37 °C, using resazurin assay. The solvent control containing no more than 1% of DMSO in DMEM only was used to determine the relative cell viability.
concentrations only after 24 h of incubation. In rats, a slow elimination has been reported for KA after oral administration (Jiang et al., 2019). However, further studies on KA and CA metabolism and renal excretion are required to identify the main elimination pathways. In conclusion, CA and KA showed moderate intestinal permeability, and passive diffusion is the main mechanism driving its oral absorption, supporting the popular use. Although P-glycoprotein does not impair their oral bioavailability, other intestinal efflux drug transporters were not investigated and this is a limitation of the present findings. Further in vitro studies focusing on the hepatic and intestinal metabolism of both diterpenes should be conducted to better understand their oral bioavailability. As oral drug candidates, CA and KA require entericcoated dosage forms to avoid chemical instability in stomach pH.
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Acknowledgments The authors are grateful to São Paulo Research Foundation (FAPESP, Brazil), Grant no. 14/50265-3. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) – Finance code 001. Author contributions NVM and NPL conceived and planned the study. AB and NPL isolated the compounds. MM carried out all stability and permeability experiments. RGP, FRP and RAG contributed to the permeability experiments. MLC contributed to the stability experiment. MM, NPL and NVM wrote the manuscript. All authors approved and gave critical contributions to the manuscript. Declaration of interest All authors declare no conflict of interest. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jep.2019.02.017. References Albuquerque, K.C.O., da Veiga, A.S.S., Silva, J.V.S., Brigido, H.P.C., Ferreira, E.P.R., Costa, E.V.S., Marinho, A.M.R., Percário, S., Dolabela, M.F., 2017. Brazilian Amazon traditional medicine and the treatment of difficult to heal leishmaniasis wounds with
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Acid diterpenes from Copaiba oleoresin (Copaifera langsdorffii): Chemical and plasma stability and intestinal permeability using Caco-2 cells
T
M. Mauroa, R.A. De Grandisa, M.L. Camposb, A. Bauermeisterc, R.G. Peccininia, F.R. Pavana, ⁎ N.P. Lopesc, N.V. De Moraesa, a
São Paulo State University (UNESP), School of Pharmaceutical Sciences, Araraquara, SP ZIP 14801-902, Brazil Federal University of Mato Grosso, Sinop, MT, Brazil c University of Sao Paulo (USP), NPPNS, School of Pharmaceutical Sciences of Ribeirao Preto, Ribeirao Preto, SP ZIP 14040-903, Brazil b
ARTICLE INFO
ABSTRACT
Keywords: Copalic acid Kaurenoic acid Caco-2 Intestinal absorption Viability
Ethnopharmacological relevance: Copaiba oleoresin has been used in folk medicine in the treatment of bronchitis, syphilis, skin diseases and ulcers due to its anti-inflammatory and antiseptic activities, but there is no information about major compounds oral absorption to support the traditional use. Aim of study: Considering the potential of copalic (CA) and kaurenoic acid (KA) – major biological activity (in vitro) diterpenes found in the oleoresin, this study aimed to evaluate the intestinal permeability of CA and KA using Caco-2 cells model as predictive test for oral drug absorption. Materials and methods: Chemical stability at pH 1.2 and 7.4 and plasma stability were evaluated to mimic physiological conditions of the gastrointestinal tract. The intestinal permeability of CA and KA was evaluated in Caco-2 cells in the presence and absence of the P-glycoprotein inhibitor verapamil. Results: CA and KA were rapidly degraded at pH 1.2 (0.2 M Clark-Lubs buffer). At pH 7.4 (0.1 M phosphate buffer), CA was stable for up to 24 h and KA for up to 6 h. In human plasma, CA and KA can be considered stable for 24 h and 12 h at 37 °C, respectively. Caco-2 cells were considered viable when incubated with CA or KA in the range of 3.9–250 μM for 24 h. CA and KA exhibited moderate apparent permeability (Papp) of 4.67 ( ± 0.08) × 10−6 cm/s and 4.66 ( ± 0.04) × 10−6 cm/s, respectively. Simultaneous incubation with verapamil showed that P-glycoprotein does not play a relevant role on CA and KA oral absorption, with Papp of 4.48 ( ± 0.26) × 10−6 cm/s and 5.37 ( ± 0.72) × 10−6 cm/s observed for CA and KA, respectively. Conclusion: The oral absorption of both CA and KA is driven by mainly passive permeability, is not limited by pglycoprotein, but enteric-coated dosage forms should be used to avoid chemical instability in the gastric pH.
1. Introduction
lesions, copaiba oleoresin has also been used orally in folk medicine in the treatment of bronchitis and syphilis due to anti-inflammatory and antiseptic activities (Albuquerque et al., 2017; Vargas et al., 2015; Tincusi et al., 2002). Natives also apply the oleoresin as mosquito repellent, and currently investigations allowed the development patented formulations for the use based on the traditional knowledge (Pohlit et al., 2011a, 2011b). Copalic acid (CA) and kaurenoic acid (KA) (Fig. 1) are two major diterpenes of copaiba oleoresin (Veiga Junior et al., 2007, 2005). CA presents in vitro antibacterial activity (da Trindade et al., 2018; Souza et al., 2018), antifungal activity against dermatophytes, (Nakamura
Copaifera species, popularly known as "copaibeiras" or "copaíba", are of great economic and ecological interest in Amazon forest. It is estimated that Copaiba oleoresin has been used in popular medicine for more than 500 years (Pieri et al., 2009). Gaspar Barléu observed that animals rubbed themselves in the trunk of Copaifera to heal their wounds (Barléu, 1974). It is assumed that by observing this behaviour, natives began to use the oleoresin with the same intention of healing (Pieri et al., 2009; Veiga Junior and Pinto, 2002). Beyond the use for healing several skin wounds such as ulcers scarring and leishmania skin
⁎ Correspondence to: São Paulo State University (UNESP), School of Pharmaceutical Sciences, Rodovia Araraquara-Jaú, km 01, Araraquara, SP ZIP 14801-902, Brazil. E-mail addresses: [email protected] (M. Mauro), [email protected] (R.A. De Grandis), [email protected] (M.L. Campos), [email protected] (A. Bauermeister), [email protected] (R.G. Peccinini), [email protected] (F.R. Pavan), [email protected] (N.P. Lopes), [email protected] (N.V. De Moraes).
https://doi.org/10.1016/j.jep.2019.02.017 Received 30 November 2018; Received in revised form 4 February 2019; Accepted 10 February 2019 Available online 11 February 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
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analyzed by gas chromatography and the identity of CA and KA were confirmed by the GC library, and by NMR, respectively (Supplementary material; Ohsaki et al., 1994). Resazurin sodium salt (CAS 62758-13-8), dimethyl sulfoxide (DMSO, CAS 67–68–5), HEPES (CAS 7365-45-9), fluvastatin sodium (≥98% purity; CAS 93957-55-2), verapamil hydrochloride (≥99% purity; CAS 152–11-4) and fluorescein (CAS 232107-5) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS) and Hank's salt solution were purchased from Gibco (Paisley, United Kingdom) and antibiotic mixture (5 mg of penicillin, 5 mg of streptomycin and 10 mg of neomycin per mL) were obtained from Gibco (Paisley, Reino Unido). Acetonitrile (HPLC grade) was purchased from J.T. Baker (Center Valley, PA, EUA) and methyl-tert butyl ether (HPLC grade) from Panreac (Darmstadt, Germany). All other reagents were analytical grade. Water was purified by a Milli-Q system (Millipore).
Fig. 1. Chemical structures of kaurenoic acid (A) and copalic acid (B).
et al., 2017) and antitrypanosomal activity (da Trindade et al., 2018). KA has shown in vitro antimicrobial, anti-inflammatory, anti-allergic, immunosuppressive and apoptosis-inducing activities (Kian et al., 2018). Due to its various pharmacological properties and because the oleoresin is commercially available to several countries, this is considered a major renewable natural product (Medeiros and Vieira, 2008). The pharmacological interests on natural products of Copaifera spp. stimulated, in the 80's, pharmaceutical companies to develop products based on traditional knowledge. However, the absence of pharmacokinetics and toxicological studies led to the withdrawal of these products from the Brazilian market. Therefore, candidates for oral drug development from copaíba require studies to better understand their pharmacokinetic properties, such as absorption, distribution, metabolism and excretion (ADME). In vitro ADME data can be further used in physiology-based pharmacokinetic (PBPK) models to predict pharmacokinetic processes in humans through in vitro-in vivo extrapolation. This approach will advance further steps on drug development to achieve a safe and effective performance by design and optimization of dosage regimens, predicting drug-drug interactions and avoiding drug toxicity (Li, 2001; Rostami-Hodjegan, 2012; Rostami-Hodjegan and Tucker, 2007; Fan and de Lannoy, 2014). This study aimed to evaluate the intestinal permeability of CA and KA using Caco-2 – the human colon adenocarcinoma cells - as a predictive test for human oral drug absorption and oral bioavailability (Chung et al., 2001; Ungell and Artursson, 2009; Gertz et al., 2010; Kostewicz et al., 2014). Caco-2 cells can differentiate spontaneously in vitro, mimicking the human intestinal epithelium and can be used to rapidly screen oral drug candidates (Ungell and Artursson, 2009). This in vitro model has the advantage of evaluating not only passive permeability, but also active transport across membranes (Lin et al., 2011). P-glycoprotein (P-gp) is a major efflux drug transporter located at the apical membrane of enterocytes and limits the oral absorption of several drugs and xenobiotics (Wang et al., 2007; Dezani et al., 2017). The intestinal permeability of both diterpenes CA and KA was assayed using the P-gp inhibitor verapamil to evaluate the role of the efflux transporter on oral absorption (Bansal et al., 2009). The chemical stability of CA and KA at pH 1.2, pH 7.4 and the stability in plasma were also evaluated to simulate different physiological conditions that could affect the oral drug disposition.
2.2. In vitro aqueous buffer stability Chemical stability was tested at pH 1.2 (Clark-Lubs buffer, 0.2 M) and pH 7.4 (phosphate buffer, 0.1 M), in triplicates at the concentration of 0.05 μM. The solutions were kept under stirring (70 rpm) at 37 °C in an orbital shaker (Ethik Technology model 430 - RDB T5, São Paulo, Brazil). Samples (100 μL) were collected at 0; 0.5; 1; 2; 4; 6; 12 and 24 h and analyzed using the analytical method developed and validated in high-performance liquid chromatography (HPLC) coupled to mass spectrometry (HPLC-MS). 2.3. In vitro plasma stability The experimental protocol of this study was approved by the Committee of Ethics in Research of the School of Pharmaceutical Sciences of Araraquara - UNESP (CAAE 65707517.6.0000.5426). Healthy volunteers of both gender, 18–60 years, body weight > 50 kg, were invited to donate blood at Hemonúcleo Regional of Araraquara "Professor Clara Pechmann Mendonça”. All participants attended the procedures of the blood donation service. Only the plasma was obtained for research and other blood components were screened for donation. Plasma samples were spiked with CA and KA at concentrations of 0.8 and 1 μM, respectively, in triplicates. The samples were vortexed and kept under constant agitation (70 rpm), at 37 °C (Incubator Ethik Technology model 430 - RDB T5, São Paulo, Brazil). Aliquots of 100 μL were collected at times 0; 0.5; 1; 2; 4; 6; 12 and 24 h and analyzed by HPLC-MS. Results were presented as percentages of the compounds remaining in human plasma. 2.4. HPLC-MS analysis The HPLC-MS system (Perkin Elmer, Shelton, USA) consisted of a binary pump (Flexar LC Pump), degasser (Flexar solvent manager), oven (Flexar LC column oven), automatic injector (Flexar LC Autosampler) and a single quadrupole mass spectrometer (Flexar SQ 300 MS). Data acquisition and quantification of samples were performed using Chromera software, version 3.4.0.5712 (Perkin Elmer, Shelton, USA). CA and KA were resolved on a reversed phase Poroshell 120 EC-C18 endcapped column (100 × 4.6 mm), with 2.7 µm particle size (Agilent Technologies, Santa Clara, United States), kept at 22 °C and mobile phase consisting of acetonitrile: water (90:10, v/v) containing 5 mM ammonium formate at a flow rate of 0.5 mL/min using an isocratic mode. The mass spectrometry detection was performed on negative ion mode. The deprotonated molecular ions [M-H]- with the mass-to-charge ratio (m/z) 303 and 301 were used for monitoring of CA and KA, respectively. The injection volume was 20 μL and the chromatographic run time was 14 min. The method was developed and validated according to current international guidelines.
2. Material and methods 2.1. Chemicals and reagents Copalic acid (CA) and kaurenoic acid (KA) were isolated from copaiba oleoresin obtained from a cooperative center (Rio Branco, AC, Brazil) as previously described (Nakamura et al., 2017). In 1951, the American botanist John Duncan Dwyer described Copaifera langsdorffii var. krukovii Dwyer in materials from the State of Acre (Brazil), including the large densities of these species in Tarauacá municipality. This data is used by the community for a long term commercialization process. After isolation and purification procedures, samples were 184
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2.5. Plasma sample preparation
Relative error (RE , %) Experimental average concentration = Nominal value
Stock solutions of CA (2000 µM) and KA (2500 µM) were prepared separately in DMSO. Working solutions were prepared by diluting the stock solutions in acetonitrile to reach the concentrations 0.25; 0.5; 1; 2, 5; 16 and 20 μM for KA and 0.2; 0.4; 0.8; 1.6; 4.0; 12.85 and 16 μM for CA. The internal standard (IS) fluvastatin was prepared at the concentration of 1 μM in acetonitrile. All solutions were stored at −20 °C until analysis. Aliquots of 100 μL of plasma were spiked with 20 μL of each working solution, 20 μL of the IS solution (1 μM fluvastatin), 20 μL of 1 M HCl and 300 μL of methyl tert-butyl ether as extractor solvent. The samples were agitated for 20 min using a horizontal reciprocating shaker (130 ± 10 cycles/min) and centrifuged for 10 min (15,000×g, 5 °C). The organic phase (270 μL) of each sample was transferred to 1.5 mL microtubes and evaporated to dryness at 40 °C, using a vacuum sample concentrator (Genevac ™ Centrifugal Duo, Ipswich UK). Residues were reconstituted in 90 μL of mobile phase, vortexed for 1 min and 20 μL were injected into the chromatographic system.
× 100
Stability of the analytes was assessed in the following conditions: a) short-term stability at room temperature (23 ± 2 °C) for 1 h; b) postprocessed stability for 7 h at 18 °C; c) after 3 cycles of freeze (−20 °C) and thaw. 2.7. Cell viability The Caco-2 human colon adenocarcinoma cell line (BCRJ, n° 0059) was purchased from Rio de Janeiro Cell Bank. Caco-2 cells were cultured in DMEM supplemented with 4.5 g/L D-glucose, 20% of FBS, 1% antibiotic mixture containing 5 mg of penicillin, 5 mg of streptomycin and 10 mg of neomycin per mL at 37 °C in an atmosphere of 5% CO2 saturation. The resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide sodium) assay was performed to evaluate the in vitro cell viability (de Grandis et al., 2017). In summary, cells were seeded at a density of 7.5 × 104 cells mL−1 in a 96-well plate and incubated for 24 h (37 °C under 5% CO2). On the next day, CA and KA solutions containing no more than 1% DMSO in DMEM were added to the wells. Dilutions were prepared to obtain CA and KA concentrations ranging from 3.91 to 500 µM (3.91; 7.81; 15.62; 31.25; 62.5; 125; 250 and 500 µM) in DMEM and six replicates of each concentration were evaluated. A solvent control containing 1% DMSO in DMEM was used to assess the viability of cells using only this solvent. DMEM samples were used as negative controls and 15% DMSO (v/v) in DMEM was used as a positive control. After 24 h of incubation (37 °C under 5% CO2), 50 μL of 0.01% resazurin solution was added to each well and plates were incubated for 2 h. Absorbance was evaluated in a microplate reader Synergy H1 (BioTek®) using excitation and emission filters at wavelengths of 530 and 590 nm, respectively. Cell viability was calculated by the following equation:
2.6. Bioanalytical method validation Selectivity was performed to check if unrelated compounds from the biological matrix could interfere with the ability to measure the analytes of interest. Blank plasma samples from six different sources, being four normal, one with in vitro hemolysis (hemolyzed plasma) and one highly concentrated in lipids (lipemic plasma) were evaluated. The absence of interfering components was defined when responses close to the analyte retention time were less than 20% of the lower limit of quantification (LLOQ) and less than 5% for IS. The carry-over effect was evaluated by placing blank plasma samples, one before and two after the calibration standard at the upper quantification limit. Carry-over in a blank sample after a high concentration standard should not be higher than 20% of the LLOQ response and 5% of the IS. The matrix effect was evaluated by comparing peak areas of the analytes and IS in solution with peak areas resulting from plasma samples spiked after extraction. To evaluate the matrix effect, eight different sources of blank plasma were used, being six normal samples, one lipemic and one haemolysed. The coefficients of variation of the ISnormalized matrix factor (NMF) were calculated for both low and high concentration quality controls, by the equation:
Cell viability (%) =
Fluorescence intensity of sample × 100 Fluorescence intensity of solventcontrol
2.8. Permeability across Caco-2 cells Drug permeability was evaluated in Caco-2 cells in line with Hubatsch et al. (2007). To evaluate if CA and KA are P-gp substrates, the transport across Caco-2 monolayer was investigated using verapamil as a P-gp inhibitor (Jin et al., 2013). Cells were cultured at a seeding density of 5 × 104 cells cm−2 in translucent membrane inserts for 12-well plates (ThinCert™, 0.4 µm pore size, Greiner Bio-One, Kremsmünster, Áustria) for 21 days to reach confluence and differentiation. Plates were incubated in an atmosphere of 5% CO2, 95% relative humidity and 37 °C before cell permeability experiments. The integrity of the monolayer was examined by measuring the transepithelial electrical resistance (TEER) with an epithelial voltammeter Millicell-ERS (Millipore Corporation, Bedford, MA) and by using verapamil (44 µM) and fluorescein (45 µM) as high and low permeability controls, respectively (Kratz et al., 2011; Fernandes et al., 2014). Only cell monolayers with a TEER above 200 Ω × cm2 were used for the transport assays (Fernandes et al., 2014; de Grandis et al., 2017). Drug solutions containing 3.9 µM for CA and KA were prepared in Hank's buffer at pH 7.4 containing 200 mM of HEPES. Both apical to basolateral and basolateral to apical transport across the monolayer was evaluated by adding drug solutions in apical and basolateral compartments, respectively. Drug permeability was assessed with and without the P-gp inhibitor verapamil (Jin et al., 2013). Cells were incubated with 50 µM verapamil for 30 min before the incubation with substrates (Lin et al., 2007, 2011). The plates were placed at 37 °C in an orbital shaker (25 rpm) (Ethik Technology model 430 – RDB T5, São Paulo, Brazil) and aliquots of 200 μL were sampled at times 0, 15, 30, 60, 120
NMF =
Nominal value
response of analyte inmatrix / response of internal standardin matrix response of analyte insolution/ response of internal standardin solution
The absence of matrix effect was defined by the relative standard of deviation (RSD, %) of the NMF not greater than 15%. Calibration curves were prepared by spiking the blank matrix (100 μL) with working solutions (20 μL) to reach the range of final plasma concentrations of 0.05–4 μM for KA and 0.04–3.2 μM for CA. The criteria for acceptance of the calibration curve was that recommended by the current guidelines on bioanalytical method validation set by the European Medicines Agency (EMA, 2012) (EMA) and the USA Food and Drug Administration (US FDA, 2018). Precision and accuracy were determined in the same run (withinrun) and in three different runs (between-run) using four quality control levels: LLOQ, low-quality control (LQC), medium-quality control (MQC), and high-quality control (HQC). The method can be considered precise when the RSD does not exceed 15%, except for the LLOQ for which RSD up to 20% is acceptable. Accuracy was evaluated by the relative error (RE), which should be less than ± 15% in terms of the nominal value, except for the LLOQ, for which values less than ± 20% are allowed (EMA, 2012; US FDA, 2018). The RE was calculated according to the equation: 185
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and 180 min. After each sampling, the same volume of a fresh permeability buffer at 37 °C was replaced, in order to keep the volume in the compartment. The apparent permeability (Papp) was calculated according to the following equation.
Papp =
Table 1 Validation parameters of copalic acid (CA) and kaurenoic acid (KA) in human plasma by HPLC-MS.
Inter-assay precision (RSD%, n = 3) LOQ LQC MQC HQC Intra-assay precision (RSD%, n = 5) LOQ LQC MQC HQC Inter-assay accuracy (RE%, n = 3) LOQ LQC MQC HQC Intra-assay accuracy (RE%, n = 5) LOQ LQC MQC HQC Stability (RE%, n = 3) Short duration stability (1 h à 23 °C) LQC HQC Post processing stability (7 h à 16 °C) LQC HQC Freeze/thaw stability (1 cycle) LQC HQC
VR dC × (A × C0) dT
where VR is the volume of the receiver compartment, A is the monolayer surface area (in cm2), C0 is the initial drug concentration in the donor compartment, and dC/dt represents the change of concentration over time. The efflux ratio (ER) was defined as Papp(B-A)/Papp(A-B). The Papp (A-B) of verapamil > 12.2 × 10−6 cm/s and of fluorescein < 3.7 × 10−6 cm/s were used as indicators of a functional Caco-2 monolayer (Park et al., 2015; Hoffmann et al., 2018). The transporter substrate index (TSIA-B) was calculated for P-glycoprotein as in the equation:
TSIapp (A
B)
=
P iapp (A
B)
Papp (A
Papp (A
B)
B)
× 100
Piapp
where (A-B) is measured in the presence of the specific P-gp inhibitor verapamil (Lin et al., 2011). 3. Results and discussion The oral administration of drugs is preferred route in pharmacological therapy due to the convenience, safety and cost for patients, health professionals and the public health system (Skolnik et al., 2010; Eek et al., 2016; Benjamin et al., 2012). In vitro assays to evaluate the oral absorption and bioavailability of drug candidates have shown to minimize the costs of both drug discovery and drug development. By increasing the use of these in vitro ADME assays, the number of withdrawals in drug development reduced from 40% in 1991 to 14% in 2000 (Wang et al., 2007). Thus, in order to evaluate CA and KA as oral drug candidates, stability in the acidic stomach and basic intestinal pH, stability in plasma as well as intestinal permeability using Caco-2 were evaluated. The bioanalytical method was fully validated using human plasma as matrix and considering the parameters selectivity, residual effect, matrix effect, linearity, precision, accuracy and stability (Table 1). Selectivity was demonstrated by the lack of interfering peaks in the retention time of analytes and IS. The absence of matrix effect was shown by the coefficient of variation of IS-normalized matrix factor, which was below 15% at both low and high concentration levels (Supplementary material). The method was linear over the concentration range of 0.05–4 µM for KA and 0.04–3.2 µM for CA, using the 1/x2 weight. Precision and accuracy did not exceed 15% of relative standard deviation and relative error, respectively, except for the LLOQ for which values up to 20% are acceptable. No carry-over effect was observed for CA, KA or the IS. The validated method was slightly modified for the analysis of samples obtained in permeability assays, considering calibration curve in the range of concentrations of 0.0125–5 μM for KA and CA. Methods reporting the analysis of CA and KA in plant material or biological matrix have been reported in the literature by using HPLC with UV detection (Costa et al., 2015; de Matos et al., 2018) or coupled to mass spectrometry (MS) (Gasparetto et al., 2015; Bardají et al., 2016; Jiang et al., 2019), gas chromatography (GC) with flame ionization detection (Tappin et al., 2004) or GC-MS (Gelmini et al., 2013; Miyazaki et al., 2015; Xavier-junior et al., 2017). The bioanalytical methods using HPLC with UV detection (de Matos et al., 2018) have reported LLOQ of 2.48 µM for KA using 100 μL of rat plasma. In the present study, the LLOQ of 0.05 and 0.04 µM for KA and CA, respectively, were observed using 100 μL of human plasma. Other methods using UPLC-MS/MS – which is a technique with better sensitivity than HPLC-MS - have reported lower LLOQ (0.016 µM), using either 100 μL
CA
KA
18.0 6.6 14.0 6.7
13.3 9.9 9.9 8.6
6.6 2.6 2.3 2.9
9.9 5.8 7.9 3.8
−8.5 −3.0 −7.5 −13.2
−7.2 14.9 9.1 −4.8
0.4 1.9 3.9 −7.4
9.9 5.8 7.9 1.9
−12.3 −14.9
−11.8 −14.4
−10.3 −13.1
−12.1 −13.7
−5.8 −13.6
9.7 −12.1
RSD: relative standard deviation or coefficient of variation = [(standard deviation/mean) × 100]; RE: relative error (%) = [(observed concentration – nominal concentration)/nominal concentration] × 100; LOQ: limit of quantification (CA: 0,04 µM; KA: 0.05 µM); LQC: Low concentration quality control (CA: 0.08 µM; KA: 0.10 µM); MQC: Medium concentration quality control (CA: 0.32 µM; KA: 0.40 µM); HQC: High concentration quality control (CA: 2.57 µM; KA: 3.2 µM).
(Jiang et al., 2019) or 200 μL of plasma (Gasparetto et al., 2015). Up to date, this is the first bioanalytical method reporting the determination of both CA and KA in the same chromatographic run and the first reporting CA analysis in plasma. Considering that CA and KA are the main bioactive diterpenes of copaiba oleoresin, this method can be applied in future ADME studies to reduce expenses with solvents and other consumables and to be time-saving when evaluating these two diterpenes. Stability evaluations have shown that CA and KA were rapidly degraded at pH 1.2 with statistical disappearance (p < 0.05) after 0.5 h. After 1 h, concentrations were below the LLOQ of the analytical method, demonstrating that both compounds are unstable at simulated stomach pH. At pH 7.4, CA was stable for up to 24 h and KA for up to 6 h, with statistically significant disappearance (p < 0.05) only after 12 h (Fig. 2). These results are in agreement with the recent findings of pharmacokinetics of KA in rats after oral administration. Non-detectable plasma levels were reported after oral administration of 50 mg/kg of KA (de Matos et al., 2018). Maximum plasma concentrations (Cmax) of only 12.6, 44.7 and 48.0 ng/mL were observed after single oral doses of 10, 20 or 40 mg/kg in rats (Jiang et al., 2019) and these low plasma concentrations can be at least partially explained by the instability of KA in acidic stomach pH. However, it is required to understand if the compounds are converted in the stomach to bioactive compounds. It is already known that oral administration of KA or the oleoresin in rats showed anti-inflammatory and antilipoperoxidative activities (Paiva et al., 2003. Lima-Silva et al., 2009). By the present data, it is suggested that KA in vivo activities could be achieved using lower doses and enteric-coated dosage forms. The cytotoxicity of CA and KA towards Caco-2 cells was evaluated 186
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concentration that caused 50% inhibition (IC50) for CA and KA in Caco2 cells were 237.4 and 154.0 μM, respectively, and the concentration of 3.9 µM was selected for permeability assays to ensure the viability of Caco-2 cells (Fig. 3). The transepithelial electrical resistance (TEER) was measured at 10, 15 and 21 days of cell cultivation on the insert filters as an indicator of the membrane integrity formed by Caco-2 cells (Fernandes et al., 2014; Srinivasan et al., 2015) (Supplementary material). Only cell monolayers with a TEER above 200 Ω × cm2 were used for the transport assays. The membrane integrity was also assessed by determining the permeability of high and low permeability standards, such as verapamil and fluorescein, respectively (Kratz et al., 2011; Bonetti et al., 2018). The Papp (A-B) for verapamil was 115 × 10−6 cm/s. For fluorescein, concentrations in the basolateral compartment at all times sampled were below the limit of quantification of the method. These results are in agreement with the literature in terms of the very low permeability of fluorescein and the high permeability of verapamil (Engman et al., 2003; Koljonen et al., 2006; Chen et al., 2016), and indicates the integrity and viability of Caco-2 cell monolayers during the permeability experiments. Apparent permeability values (Papp) were determined from the amount permeated through the Caco-2 cell membranes at both apicalbasolateral (A-B) and basolateral-apical (B-A) direction. CA and KA exhibited Papp (A-B) values of 4.67 ( ± 0.08) × 10−6 cm/s and 4.66 ( ± 0.04) × 10−6 cm/s, respectively. Compounds with Papp value < 2 × 10−6 cm/s are considered as poorly absorbed; moderately absorbed when the Papp value is between 2 and 10 × 10−6 cm/s; and Papp values higher than 10 × 10−6 cm/s defines compounds with high in vivo intestinal permeability (Flynn and Vohra, 2018). Thus, although requiring enteric coating polymers to protect against gastric fluids, CA and KA shows moderate intestinal permeability and are promising oral drug candidates. In terms of basolateral-apical permeability, CA and KA shows Papp (B-A) of 1.68 ( ± 0.50) × 10−6 cm/s and 2.82 ( ± 0.20) × 10−6 cm/s, respectively. The assessment of transport in both directions can show whether the compounds undergo active efflux. The efflux ratio (ER) is used to evaluate the impact of the efflux on oral permeability. ER values ≥3 indicate predominant efflux activity and ER ˂2 characterize passive transport. When ER is between 2 and 3, oral permeability shows moderate efflux (Skolnik et al., 2010). For CA and KA, ER was < 2, suggesting that passive transport is the main mechanism involved in their oral absorption. The permeability through the Caco-2 cells membranes at the apical-basolateral direction in the presence of the Pglycoprotein inhibitor verapamil (Table 2) shows that this efflux drug transporter does not influence KA and CA oral absorption. Both CA and KA can be considered non-substrates of P-glycoprotein, since TSIPapp of both were < 25% (Lin et al., 2011). Using the four-zone graphical model proposed by Lin et al. (2011), it can be concluded that passive permeability drives both KA and CA in vitro permeability and in vivo oral absorption (Lin et al., 2011). CA and KA can be considered stable in human plasma for 24 h and 12 h at 37 °C, respectively (Fig. 2). For CA, no statistical differences were observed after incubation for up to 24 h. The remaining KA showed a statistical difference in comparison to the basal
Fig. 2. Stability of copalic acid (о) and kaurenoic acid (■) at 37 °C in pH 1.2, pH 7.4 and in plasma. *means statistically significant compared to time 0 by repeated measures ANOVA followed by the Tukey test (p < 0.05). *means statistically significant compared to time 0 by repeated measures ANOVA followed by the Tukey test (p < 0.05).
before the permeability assays to design a protocol using nontoxic concentrations (de Grandis et al., 2017; McMillian et al., 2002). DMSO control, containing no more than 1% of DMSO in DMEM, demonstrated cell viability of approximately 90% in relation to DMEM only. According to the international guide ISO 10993-5, the cytotoxic effect is defined by cell viability lower than 70% (ISO/EN10993-5, 2009). The
Table 2 Apparent drug permeability in Caco-2 cells in the presence and absence of 50 μM verapamil as P-glycoprotein inhibitor. Compound
Copalic acid Kaurenoic acid Verapamil Fluorescein
Papp (A-B) (× 10−6 cm/s)
4.67 ± 0.08 4.66 ± 0.04 115 N.D.
Papp (B-A) (× 10−6 cm/s)
1.68 ± 0.50 2.82 ± 0.20
Efflux ratio (ER) 0.36 0.60
Papp (B Papp (A
A) B)
Piapp(A-B)a
4.48 ± 0.26 5.37 ± 0.72
TSIPapp(A-B)
i (A Papp
B)
Papp (A
Papp (A
B)
B)
×100
−4.07 15.24
Values presented as average ± standard deviation of triplicates; N.D.: not determined (concentrations in the basolateral compartment were bellow the LLOQ). i indicates that experiments were conducted in the presence of 50 μM verapamil. 187
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Fig. 3. Viability of kaurenoic acid (A) and copalic acid (B) in Caco-2 cells after incubation for 24 h at 37 °C, using resazurin assay. The solvent control containing no more than 1% of DMSO in DMEM only was used to determine the relative cell viability.
concentrations only after 24 h of incubation. In rats, a slow elimination has been reported for KA after oral administration (Jiang et al., 2019). However, further studies on KA and CA metabolism and renal excretion are required to identify the main elimination pathways. In conclusion, CA and KA showed moderate intestinal permeability, and passive diffusion is the main mechanism driving its oral absorption, supporting the popular use. Although P-glycoprotein does not impair their oral bioavailability, other intestinal efflux drug transporters were not investigated and this is a limitation of the present findings. Further in vitro studies focusing on the hepatic and intestinal metabolism of both diterpenes should be conducted to better understand their oral bioavailability. As oral drug candidates, CA and KA require entericcoated dosage forms to avoid chemical instability in stomach pH.
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Acknowledgments The authors are grateful to São Paulo Research Foundation (FAPESP, Brazil), Grant no. 14/50265-3. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) – Finance code 001. Author contributions NVM and NPL conceived and planned the study. AB and NPL isolated the compounds. MM carried out all stability and permeability experiments. RGP, FRP and RAG contributed to the permeability experiments. MLC contributed to the stability experiment. MM, NPL and NVM wrote the manuscript. All authors approved and gave critical contributions to the manuscript. Declaration of interest All authors declare no conflict of interest. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jep.2019.02.017. References Albuquerque, K.C.O., da Veiga, A.S.S., Silva, J.V.S., Brigido, H.P.C., Ferreira, E.P.R., Costa, E.V.S., Marinho, A.M.R., Percário, S., Dolabela, M.F., 2017. Brazilian Amazon traditional medicine and the treatment of difficult to heal leishmaniasis wounds with
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