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Pharmacokinetic disposition of erythraline in rats after intravenous administration

ABSTRACT

Erythraline is the major alkaloid produced by Erythrina verna Vell., Fabaceae, a plant used in folk medicine and phytotherapeutic products to treat anxiety and sleep disorders. This study aimed to investigate the pharmacokinetic parameters of erythraline after intravenous administration in rats. For the analysis of erythraline in rat plasma, a method was developed and validated using UHPLC-MS/MS. Pharmacokinetic parameters were estimated by non-compartmental analysis. The metabolite 8-oxo-erythraline observed in previous biomimetic model studies was monitored during in vivo experiments. The quantification limit was 5 ng/ml within a linear range of 5-2000 ng/ml. After an intravenous dose of 1 mg/kg, the following pharmacokinetic parameters were observed: elimination half-life of 44.2 min; total clearance of 42.1 ml/min/kg and volume of distribution of 2085 ml/kg. In summary, a precise, accurate and selective UHPLC-MS/MS method was developed and successfully applied to investigate the pharmacokinetics of erythraline in rats. The metabolite 8-oxo-erythraline was observed in rat plasma after 20 min of erythraline administration.

Keywords:
Pharmacokinetics; Natural products; Herbal medicine

Introduction

Erythrina genus comprises around 120 species widely distributed in tropical and subtropical regions (Lorenzi and Matos, 2008Lorenzi, H., Matos, F.J.A., 2008. Plantas Medicinais No Brasil: Nativas E Exóticas Cultivadas, 2nd. ed. Plantarum, pp. 576.; da Silva et al., 2013da Silva, M.M.B., Santana, A.S.C.O., Pimentel, R.M.M., Silva, F.C.L., Randau, K.P., Soares, L.A.L., 2013. Anatomy of leaf and stem of Erythrina velutina. Rev. Bras. Farmacogn. 23, 200-206.). In Brazil, about twelve different species from this genus are known, including is Erythrina verna Vell., Fabaceae, which is popularly known as "mulungu" (De Albuquerque et al., 2007De Albuquerque, U.P., De Medeiros, P.M., De Almeida, A.L.S., Monteiro, J.M., Neto, E.M.D.F.L., De Melo, J.G., Dos Santos, J.P., 2007. Medicinal plants of the caatinga (semi-arid) vegetation of NE Brazil: a quantitative approach. J. Ethnopharmacol. 114, 325-354.). Erythrina verna is important in medicine, which extracts from its bark are used in folk medicine as a sedative and to treat anxiety (Decker et al., 1995Decker, M.W., Anderson, D.J., Brioni, J.D., Donnellyroberts, D.L., Kang, C.H., Oneill, A.B., Piattonikaplan, M., Swanson, S., Sullivan, J.P., 1995. Erysodine, a competitive antagonist at neuronal nicotinic acetylcholine-receptors. Eur. J. Pharmacol. 280, 79-89.; Daly, 2005Daly, J.W., 2005. Nicotinic agonists, antagonists, and modulators from natural sources. Cell. Mol. Neurobiol. 25, 513-552.; Flausino et al., 2007aFlausino, O., Santos, L.D., Verli, H., Pereira, A.M., Bolzani, V.D., Nunes-de-Souza, R.L., 2007. Anxiolytic effects of erythrinian alkaloids from Erythrina mulungu. J. Nat. Prod. 70, 48-53., 2007bFlausino, O.A., Pereira, A.M., Bolzani, V.D.S., Nunes-De-Souza, R.L., 2007. Effects of erythrinian alkaloids isolated from Erythrina mulungu (Papilionaceae) in mice submitted to animal models of anxiety. Biol. Pharm. Bull. 30, 375-378.; Dias et al., 2013Dias, S.A., Neves, A.E.O., Ferraz, A.D.F., Picada, J.N., Pereira, P., 2013. Neuropharmacological and genotoxic evaluation of ethanol extract from Erythrina falcata leaves, a plant used in Brazilian folk medicine. Rev. Bras. Farmacogn. 23, 335-341.). Commercial herbal medicines containing E. verna to treat anxiety are available internationally.

The ethnopharmacological use of E. verna is justified by biologically active alkaloids produced by this genus (Serrano et al., 2011Serrano, M.A.R., Batista, A.N.D., Bolzani, V.D., Santos, L.D., Nogueira, P.J.D., Nunes-de-Souza, R.L., Latif, A., Arfan, M., 2011. Anxiolytic-Like effects of erythrinian alkaloids from Erythrina suberosa. Quim. Nova 34, 808-811.; Rosa et al., 2012Rosa, D.S., Faggion, S.A., Gavin, A.S., de Souza, M.A., Fachim, H.A., dos Santos, W.F., Pereira, A.M.S., Cunha, A.O.S., Beleboni, R.O., 2012. Erysothrine, an alkaloid extracted from flowers of Erythrina mulungu Mart. Ex Benth: evaluating its anticonvulsant and anxiolytic potential. Epilepsy Behav. 23, 205-212.; Setti-Perdigao et al., 2013Setti-Perdigao, P., Serrano, M.A.R., Flausino, O.A., Bolzani, V.S., Guimaraes, M.Z.P., Castro, N.G., 2013. Erythrina mulungu alkaloids are potent inhibitors of neuronal nicotinic receptor currents in mammalian cells. PLoS One 8, e82726, http://dx.doi.org/10.1371/journal.pone.0082726.
http://dx.doi.org/10.1371/journal.pone.0...
; Zarev et al., 2017Zarev, Y., Foubert, K., Lucia de Almeida, V., Anthonissen, R., Elgorashi, E., Apers, S., Ionkova, I., Verschaeve, L., Pieters, L., 2017. Antigenotoxic prenylated flavonoids from stem bark of Erythrina latissima. Phytochemistry 141, 140-146.). In fact, the alkaloids isolated from Erythrina sp. can induce effects on the central nervous system (CNS), promoting antifeedant (Cornelius et al., 2009Cornelius, W.W., Akeng'a, T., Obiero, G.O., Lutta, K.P., 2009. Antifeedant Activities of the erythrinaline alkaloids from Erythrina latissima against Spodoptera littoralis (Lepidoptera noctuidae). Rec. Nat. Prod. 3, 96-103.), anticonvulsant (Faggion et al., 2011Faggion, S.A., Cunha, A.O.S., Fachim, H.A., Gavin, A.S., dos Santos, W.F., Pereira, A.M.S., Beleboni, R.O., 2011. Anticonvulsant profile of the alkaloids (+)-erythravine and (+)-11-alpha-hydroxy-erythravine isolated from the flowers of Erythrina mulungu Mart ex Benth (Leguminosae-Papilionaceae). Epilepsy Behav. 20, 441-446.), and other neuronal properties depending on nicotinic receptors modulation (Decker et al., 1995Decker, M.W., Anderson, D.J., Brioni, J.D., Donnellyroberts, D.L., Kang, C.H., Oneill, A.B., Piattonikaplan, M., Swanson, S., Sullivan, J.P., 1995. Erysodine, a competitive antagonist at neuronal nicotinic acetylcholine-receptors. Eur. J. Pharmacol. 280, 79-89.; Dwoskin and Crooks, 2001Dwoskin, L.P., Crooks, P.A., 2001. Competitive neuronal nicotinic receptor antagonists: a new direction for drug discovery. J. Pharmacol. Exp. Ther. 298, 395-402.). Garin-Aguilar et al. (2000)Garin-Aguilar, M.E., Luna, J.E.R., Soto-Hernandez, M., del Toro, G.V., Vazquez, M.M., 2000. Effect of crude extracts of Erythrina americana mill. On aggressive behavior in rats. J. Ethnopharmacol. 69, 189-196. verified tranquilizer and sedative effects of alkaloids fractions from E. americana comparable to diazepam. E. verna, which is composed mostly by erythraline spicocyclic alkaloid, has presented sedative effects in studies performed on animals (Onusic et al., 2002Onusic, G.M., Nogueira, R.L., Pereira, A.M.S., Viana, M.B., 2002. Effect of acute treatment with a water-alcohol extract of Erythrina mulungu on anxiety-related responses in rats. Braz. J. Med. Biol. Res. 35, 473-477., 2003Onusic, G.M., Nogueira, R.L., Pereira, A.M.S., Flausino, O.A., Viana, M.D., 2003. Effects of chronic treatment with a water-alcohol extract from Erythrina mulungu on anxiety-related responses in rats. Biol. Pharm. Bull. 26, 1538-1542.; Vasconcelos et al., 2003Vasconcelos, S.M.M., Oliveira, G.R., de Carvalho, M.M., Rodrigues, A.C.P., Silveira, E.R., Fonteles, M.M.F., Sousa, F.C.F., Viana, G.S.B., 2003. Antinociceptive activities of the hydroalcoholic extracts from Erythrina velutina and Erythrina mulungu in mice. Biol. Pharm. Bull. 26, 946-949., 2004Vasconcelos, S.M.M., Macedo, D.S., de Melo, C.T.V., Monteiro, A.P., Rodrigues, A.C.P., Silveira, E.R., Cunha, G.M.A., Sousa, F.C.F., Viana, G.S.B., 2004. Central activity of hydroalcoholic extracts from Erythrina velutina and Erythrina mulungu in mice. J. Pharm. Pharmacol. 56, 389-393.; Viana et al., 2005Viana, M., Ribeiro, M.D., Onusic, G.M., 2005. Effects of Erythrina velutina and Erythrina mulungu in rats submitted to animal models of anxiety and depression. Eur. Neuropsychopharmacol. 15, S527-S528.; Ribeiro et al., 2006Ribeiro, M.D., Onusic, G.M., Poltronieri, S.C., Viana, M.B., 2006. Effect of Erythrina velutina and Erythrina mulungu in rats submitted to animal models of anxiety and depression. Braz. J. Med. Biol. Res. 39, 263-270.; Flausino et al., 2007aFlausino, O., Santos, L.D., Verli, H., Pereira, A.M., Bolzani, V.D., Nunes-de-Souza, R.L., 2007. Anxiolytic effects of erythrinian alkaloids from Erythrina mulungu. J. Nat. Prod. 70, 48-53., 2007bFlausino, O.A., Pereira, A.M., Bolzani, V.D.S., Nunes-De-Souza, R.L., 2007. Effects of erythrinian alkaloids isolated from Erythrina mulungu (Papilionaceae) in mice submitted to animal models of anxiety. Biol. Pharm. Bull. 30, 375-378.; Faggion et al., 2011Faggion, S.A., Cunha, A.O.S., Fachim, H.A., Gavin, A.S., dos Santos, W.F., Pereira, A.M.S., Beleboni, R.O., 2011. Anticonvulsant profile of the alkaloids (+)-erythravine and (+)-11-alpha-hydroxy-erythravine isolated from the flowers of Erythrina mulungu Mart ex Benth (Leguminosae-Papilionaceae). Epilepsy Behav. 20, 441-446.; Silveira-Souto et al., 2014Silveira-Souto, M.L., Sao-Mateus, C.R., de Almeida-Souza, L.M., Groppo, F.C., 2014. Effect of Erythrina mulungu on anxiety during extraction of third molars. Med. Oral Patol. Oral 19, E518-E524.).

Safety and effectiveness of natural bioactive compounds are crucial to new drug development. Although previous reports have demonstrated that E. verna extract and erythrinian alkaloid show relevant CNS effects, few studies have evaluated the metabolic or pharmacokinetic parameters of these alkaloids or extracts. In a previous study, our group demonstrated that in vitro biomimetic models with erythraline alkaloid produce 8-oxo-erythraline, a potential phase I metabolites that induces the CNS effects (Guaratini et al., 2014Guaratini, T., Silva, D.B., Bizaro, A.C., Sartori, L.R., Humpf, H.U., Lopes, N.P., Costa-Lotufo, L.V., Lopes, J.L.C., 2014. In vitro metabolism studies of erythraline, the major spiroalkaloid from Erythrina verna. BMC Complem. Altern. Med. 14, http://dx.doi.org/10.1186/1472-6882-14-61.
http://dx.doi.org/10.1186/1472-6882-14-6...
). In this context, this work aimed to develop and validate a chromatographic method to analyze erythraline in rat plasma using UHPLC-MS/MS in order to assess the pharmacokinetics in rats. The presence of the previously observed metabolite 8-oxo-erythraline was evaluated to correlate the in vitro metabolism with in vivo pharmacokinetics.

Material and methods

Animals

Five male Wistar rats (220-250 g) were housed in a temperature-controlled room (27 ± 2 ºC) with a 12-h light/dark cycle and free access to water and food. The study was conducted in compliance with the ethical committee of the University of São Paulo Animal Care and Use Committee (Protocol number 14.1.765.53.3). All rats fasted for 8-12 h before the experiment. The immobilization was achieved by wrapping the animal in a felt cloth. After going through the experimental protocol, the animals were euthanized by anesthesia overdose (150 mg/kg thiopental, intravenous).

Chemicals

All chemicals used in UPLC were spectroscopy grade (J.T. Baker) and the water was obtained from Milli-Q system (de-ionized, 18 mΩ, Direct-Q, Millipore). Erythraline was isolated from Erythrina verna Vell., Fabaceae, as previously described by Callejon et al. (2014)Callejon, D.R., Riul, T.B., Feitosa, L.G.P., Guaratini, T., Silva, D.B., Adhikari, A., Shrestha, R.L., Marques, L.M.M., Baruffi, M.D., Lopes, J.L.C., Lopes, N.P., 2014. Leishmanicidal evaluation of tetrahydroprotoberberine and spirocyclic erythrina-alkaloids. Molecules 19, 5692-5703..

Intravenous administration of erythraline

Erythraline dissolved in saline containing no more than 5% DMSO was administered to rats (n = 5) as intravenous (i.v.) bolus dose of 1 mg/kg through the lateral tail vein. The total volume of drug administration varied from 100 to 120 µl. Blood samples (200-250 µl) were collected from lateral tail vein into heparinized tubes at 5; 10; 20; 40; 60; 90; 120; 180 and 240 min. Plasma was obtained by centrifuging blood samples at 2000 × g, at 4 ºC for 10 min and stored at −80 ºC until analysis.

UHPLC-MS/MS conditions

The analytical methodology was developed in a UHPLC-DAD-MS/MS, from Acquity UPLC Waters Corporation, Milford, USA. The stationary phase was a reversed phase column Acquity BEH (Ethylene Bridged Hybrid) (C18, 2.1 mm × 50 mm, 1.7 µm particle size) and pre-column with the same material (Vanguard 2.1 mm × 5 mm). The software used for data acquisition and analysis was MassLynx v.4.1., Waters (Micromass, Manchester, UK). Acetonitrile (B) and water (A), both containing with 0.1% formic acid (v/v), were used as mobile phase. The samples were kept at 10 ºC in the auto-sampler and the column temperature was set at 35 ºC. The flow rate was 0.3 ml/min and the volume of injection was 5 µl. The gradient elution was: 0-3 min: 5-40% B; 3-3.5 m in. 40-80% and 2.5 min for wash and column equilibration.

The electrospray mass spectrometry detection was set at positive ionization mode. For MRM transitions the following parameters were performed: capillary energy 1.0 kV, source and desolvation temperature were 150 ºC and 350 ºC, respectively. The nitrogen was used as desolvation gas (600 l/h) and argon as collision gas (0.16 l/h, pressure in the collision cell: 3.02 × 10−3 mBar). For erythraline detection the MRM transition monitored was 298 > 266, with 15 V of cone energy, for 8-oxo-erythraline was 312 > 280 and 15 V of cone energy and for caffeine (internal standard) was 195 > 138, with 40 V of cone energy. For all transitions the collision energy was 20 eV.

Sample preparation

Protein precipitation of plasma samples was tested using methanol, acetonitrile or equal mixture of these solvents and monitoring the standard signal. Aliquots of 100 µl acetonitrile, which was the solvent used to dilute the internal standard caffeine, were added to 100 µl of plasma samples. The samples were centrifuged (10 ºC, 15 min, 21,000 × g). The supernatants were separated, filtered (0.22 µm, Millex, Millipore) and analyzed in UPLC-MS/MS.

Method validation

ICH guidelines (ICH, 1996ICH, 1996. Validation of Analytical Procedures: Definitions and Terminology. Geneva.) were followed to validate selectivity, linearity, precision, accuracy, and for quantification of limit parameters. The calibration curve was performed using 100 µl of blank plasma, which was added to 50 µl of internal standard caffeine (500 ng/ml) and 50 µl of erythraline solution in different concentrations, both in acetonitrile. Selectivity was evaluated using six blank plasma samples from different animals and monitoring interfering peaks in the MRM transitions for erythraline and internal standard. Linearity was tested analyzing the calibration standards in the range of 0.625-2000 ng/ml, in triplicates. The final concentrations used in analytical curve were: 2000; 1500; 1000; 750; 500; 250; 50; 15; 10; 5 ng/ml. Quality control samples at five different levels were prepared: lower limit of quantification (LLOQ, 5 ng/ml), lower quality control (LQC, 15 ng/ml), mid-quality control (MQC, 750 ng/ml), high quality control (HQC, 1750 ng/ml) and upper limit of quantification (HLOQ, 2000 ng/ml). Precision and accuracy were evaluated by the coefficient of variation (CV) and the relative error (RE) of quality control samples prepared in quintuplicates, respectively, at the same run (intra-assay precision and accuracy) and in three different runs (inter-assay precision and accuracy).

Pharmacokinetic analysis

Non-compartmental pharmacokinetic analysis was performed using the freely available Microsoft Excel add-in program PKSolver (Zhang et al., 2010Zhang, Y., Huo, M., Zhou, J., Xie, S., 2010. PKSolver: an add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput. Methods Programs Biomed. 99, 306-314.). The pharmacokinetic parameters area under the curve plasma concentration vs time (AUC0-t) extrapolated to infinity (AUC0-∞), half-life of elimination (t½), mean residence time (MRT), volume of distribution (Vd) and total drug clearance (CL) were estimated using the trapezoidal method with intravenous bolus input. Descriptive statistics of pharmacokinetic data were presented as medians and percentiles since they do not follow normal distribution.

Results and discussion

Erythrina verna extract is a raw material of several herbal medicines sold around the world, and species of the Erythrina genus are used in folk medicine to treat anxiety, insomnia and pain (Rodrigues and Carvalho, 2001Rodrigues, V.E.G., Carvalho, D.A., 2001. Plantas Medicinais No Domínio Dos Cerrados. UFLA, Lavras, pp. 180.). The scientific evidence of its effectiveness can be verified by an increasing number of studies that demonstrate the activity of alkaloids from these species acting on the CNS (Ribeiro et al., 2006Ribeiro, M.D., Onusic, G.M., Poltronieri, S.C., Viana, M.B., 2006. Effect of Erythrina velutina and Erythrina mulungu in rats submitted to animal models of anxiety and depression. Braz. J. Med. Biol. Res. 39, 263-270.; Serrano et al., 2011Serrano, M.A.R., Batista, A.N.D., Bolzani, V.D., Santos, L.D., Nogueira, P.J.D., Nunes-de-Souza, R.L., Latif, A., Arfan, M., 2011. Anxiolytic-Like effects of erythrinian alkaloids from Erythrina suberosa. Quim. Nova 34, 808-811.). In fact, previous reports demonstrate that hydroalcoholic extracts of the bark, flowers and leaves of E. verna reduced agitation, anxiety and depression in animal models. The effects of these extracts seen in these studies were very similar to diazepam, which acts on the GABAergic receptors and is used as anxiolytic, anticonvulsant and sedatives drugs. In addition, erythrine alkaloids have been shown to induce effects on the CNS by modulation of nicotinic receptors, suggesting that the antagonist effect may be responsible for anticonvulsant and anxiolytic effects reported in previous studies (Decker et al., 1995Decker, M.W., Anderson, D.J., Brioni, J.D., Donnellyroberts, D.L., Kang, C.H., Oneill, A.B., Piattonikaplan, M., Swanson, S., Sullivan, J.P., 1995. Erysodine, a competitive antagonist at neuronal nicotinic acetylcholine-receptors. Eur. J. Pharmacol. 280, 79-89.; Iturriaga-Vasquez et al., 2010Iturriaga-Vasquez, P., Carbone, A., Garcia-Beltran, O., Livingstone, P.D., Biggin, P.C., Cassels, B.K., Wonnacott, S., Zapata-Torres, G., Bermudez, I., 2010. Molecular determinants for competitive inhibition of alpha 4 beta 2 nicotinic acetylcholine receptors. Mol. Pharmacol. 78, 366-375.; Setti-Perdigao et al., 2013Setti-Perdigao, P., Serrano, M.A.R., Flausino, O.A., Bolzani, V.S., Guimaraes, M.Z.P., Castro, N.G., 2013. Erythrina mulungu alkaloids are potent inhibitors of neuronal nicotinic receptor currents in mammalian cells. PLoS One 8, e82726, http://dx.doi.org/10.1371/journal.pone.0082726.
http://dx.doi.org/10.1371/journal.pone.0...
).

Chromatographic methods to analyze erythraline and other alkaloids of Erythrina in plant materials have been developed previously (Garín-Aguilar et al., 2005Garín-Aguilar, M.E.M.E., Valencia del Toro, G., Soto-Hernández, M., Kite, G., 2005. High-performance liquid chromatography-massSspectrometric analysis of alkaloids extracted from seeds of Erythrina herbacea. Phytochem. Anal. 16, 302-306.). For the first time, we report here a chromatographic method for analysis of erythraline in a biological matrix - plasma. The use of UHPLC presents many advantages when compared to conventional HPLC systems such as detectability, speed, and resolution. Tandem mass spectrometry offers several advantages in terms of detectability and selectivity when compared to other detection systems. These characteristics are mainly desirable for methods applied to pharmacokinetic studies of natural products. After administered to experimental animals, the analytes might be present at very low concentration and the interference of components from biological matrices should be avoided (Xu et al., 2017Xu, Y., Li, Y., Zhang, P., Yang, B., Wu, H., Guo, X., Li, Y., Zhang, Y., 2017. Sensitive UHPLC-MS/MS quantitation and pharmacokinetic comparisons of multiple alkaloids from Fuzi-Beimu and single herb aqueous extracts following oral delivery in rats. J. Chromatogr. B Analyt. Technol Biomed Life Sci. 1058, 24-31.).

Although not quantified due to unavailability of certified standard, the metabolite 8-oxo-erythraline was monitored in the MRM transition with m/z 312 > 280. The total conversion of erythraline parent ion (m/z 298) into its fragment (m/z 266) was optimized using 20 eV of collision energy. The fragmentation pathway followed by erythraline and 8-oxo-erythraline involves the loss of methoxyl group (loss of 32 Da), through a remote hydrogen rearrangement reaction (Demarque et al., 2016Demarque, D.P., Crotti, A.E.M., Vessecchi, R., Lopes, J.L.C., Lopes, N.P., 2016. Fragmentation reactions using electrospray ionization mass spectrometry: an important tool for the structural elucidation and characterization of synthetic and natural products. Nat. Prod. Rep. 33, 432-455.). This reaction is favorable once it takes place with groups in axial position and creates a fragment with three conjugated double bounds (Scheme 1). Erythraline and the IS caffeine were resolved within the run time of 4 min. The metabolite 8-oxo-erythraline did not coelute with the analytes (Fig. 1).

Scheme 1
Fragmentation pathway proposed for erythraline.

Fig. 1
Chromatograms of 8-oxo-erythraline (3.29 min), erythraline (2.28 min) and caffeine (internal standard, 1.48 min) in rat plasma samples collected after 40 min of drug administration. Column: Acquity BEH (Ethylene Bridged Hybrid) (C18, 2.1 mm × 50 mm, 1.7 µm particle size), kept at 35 ºC. Mobile phase: acetonitrile (B) and water (A), both containing with 0.1% formic acid (v/v), in the gradient elution mode: 0-3 min: 5-40% B; 3-3.5 m in. 40-80% and 2.5 min of 5% B for wash and column equilibration. The flow rate was 0.3 ml/min.

The analytical method for erythraline analysis in rat plasma was successfully validated considering the figures of merit selectivity, linearity, precision and accuracy. The method was selective since no co-eluting peaks were observed in the retention times of erythraline and the IS. The response was linear within the range of 5-2000 ng/ml with back calculated concentrations within ±15% of nominal values and linear coefficient regression of r2 = 0.99. Precision and accuracy showed coefficients of variation and relative standard errors below 15% for concentration levels (Table 1).

Table 1
Figures of merit of the validation of the method for erythraline analysis in rat plasma samples using UHPLC-MS/MS.

The method developed here was suitable for pharmacokinetic studies of erythraline, where the detection and quantification limits allowed the analysis of erythraline up to 240 min after intravenous drug administration. Erythraline plasma concentrations versus time curves were used to calculate the pharmacokinetic parameters using non-compartmental analysis (Table 2, Fig. 2).

Table 2
Non-compartmental pharmacokinetic analysis for erythraline in rats after intravenous administration of 1 mg/kg (n = 5).

Fig. 2
Plasma concentration of erythraline in rats after intravenous administration of 1 mg/kg (n = 5). Data presented as median (solid line), 5th-95th percentiles (dashed lines). Observed data presented as solid circles.

The AUC0-t/AUC0-inf ratios were used to confirm the suitability of the experimental design. Ratios higher than 80% are observed when the experimental blood sampling protocol are suitable to estimate the pharmacokinetic parameters. Here we observed a median AUC0-t/AUC0-inf ratio of 97.6 ± 1.8%, thus confirming that we accurately estimated the pharmacokinetic parameters (Fig. 2).

After intravenous administration of 1 mg/kg in bolus, the AUC0-inf of erythraline was 23.73 µg.ml/min. The plasma levels of erythraline rapidly decreased (median half-life of the 44.2 min). The fast conversion of erythraline in 8-oxo-erytraline can be the reason for the high total Cl (median: 42.1 ml/min/kg) and short MRT (median: 49.6 min). The metabolite 8-oxo-erythraline was monitored in this pharmacokinetic study and its presence was confirmed by UHPLC-MS/MS, as illustrated in Fig. 3. The 8-oxo-erythraline was verified after 20 min of erythraline administration. The m/z intensities of 8-oxo-erythraline increased over time while the erythraline concentrations were decreasing (Fig. 3).

Fig. 3
Comparison between the intensity erythraline signal and its metabolite 8-oxo-erythraline before (20 min) and after (40 min) i.v. injection.

Evidence herein demonstrates the importance of pharmacokinetic evaluation for future studies focused on metabolites of erythrinian alkaloids, such as 8-oxo-erythraline. Evaluating the metabolism of erythrinian alkaloids is essential to understand the long-term effects on the CNS, plasma concentration, and to predict rational dosing regimens for patients who will use medicines containing the erythrinian alkaloids as active constituent.

Despite some limitations, several studies have been using metalloporphyrins as biomimetic reactions to show that the metabolites formed in these reactions also occur in vivo (Schaab et al., 2010Schaab, E.H., Crotti, A.E.M., Iamamoto, Y., Kato, M.J., Lotufo, L.V.C., Lopes, N.P., 2010. Biomimetic oxidation of piperine and piplartine catalyzed by iron(III) and manganese(III) porphyrins. Biol. Pharm. Bull. 33, 912-916.; Niehues et al., 2012Niehues, M., Barros, V.P., Emery, F.D., Dias-Baruffi, M., Assis, M.D., Lopes, N.P., 2012. Biomimetic in vitro oxidation of lapachol: a model to predict and analyse the in vivo phase I metabolism of bioactive compounds. Eur. J. Med. Chem. 54, 804-812.). This fact is especially important for natural products, since it allows for an assessment of a likely metabolite profile with a reduced number of isolated components. Previous reports from our research group have used this strategy to demonstrate the correlation of metabolites obtained by biomimetic reactions with the metabolites produced in vivo assays. Among these studies we highlight that the main metabolites generated in vitro assays of chlorogenic acid, di-caffeoylquinic acid, piperine, lapachol, and grandisin were also observed in in vivo tests (Santos et al., 2008Santos, M.D., Lopes, N.P., Iamamoto, Y., 2008. HPLC-ESI-MS/MS analysis of oxidized di-caffeoylquinic acids generated by metalloporphyrin-catalyzed reactions. Quim. Nova 31, 767-770.; Schaab et al., 2010Schaab, E.H., Crotti, A.E.M., Iamamoto, Y., Kato, M.J., Lotufo, L.V.C., Lopes, N.P., 2010. Biomimetic oxidation of piperine and piplartine catalyzed by iron(III) and manganese(III) porphyrins. Biol. Pharm. Bull. 33, 912-916.; Ferreira et al., 2012Ferreira, L.D., Callejon, D.R., Engemann, A., Cramer, B., Humpf, H.U., de Barros, V.P., Assis, M.D., da Silva, D.B., de Albuquerque, S., Okano, L.T., Kato, M.J., Lopes, N.P., 2012. In vitro metabolism of grandisin, a lignan with anti-chagasic activity. Planta Med. 78, 1939-1941.; Niehues et al., 2012Niehues, M., Barros, V.P., Emery, F.D., Dias-Baruffi, M., Assis, M.D., Lopes, N.P., 2012. Biomimetic in vitro oxidation of lapachol: a model to predict and analyse the in vivo phase I metabolism of bioactive compounds. Eur. J. Med. Chem. 54, 804-812.). In agreement with other studies, our data confirm the correlation between metalloporphyrins and in vivo experiments.

In summary, the validated UHPLC-MS/MS for analysis of erythraline in rat plasma was successfully applied to estimate its pharmacokinetic parameters in rats after intravenous administration. Previous biomimetic models for in vitro metabolism of erythraline showed to effectively correlate with the in vivo kinetic disposition in rats. Further studies involving erythrian extract or alkaloids must consider short half-life of the alkaloid erythraline and 8-oxoerythraline as a metabolite action, as, on the CNS.

Acknowledgement

Daniel P. Demarque would like to thank the São Paulo Research Foundation (FAPESP) for the doctoral scholarship (2014/18052-0) and ArboControlBrasil Project, Process TED74/2016 (FNS/UnB). The authors thank the Brazilian foundations FAPESP (2014/50265-3), CNPq, and CAPES.

Ethical disclosures

  • Protection of human and animal subjects
    The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).
  • Confidentiality of data
    The authors declare that no patient data appear in this article.
  • Right to privacy and informed consent
    The authors declare that no patient data appear in this article.

Acknowledgement

Daniel P. Demarque would like to thank the FAPESP for the doctoral scholarship (2014/18052-0) and ArboControlBrasil Project, Process TED74/2016 (FNS/UnB). The authors thank the Brazilian foundations FAPESP (2014/50265-3), CNPq, and CAPES.

Appendix A Supplementary data

Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.bjp.2019.07.002.

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Publication Dates

  • Publication in this collection
    3 Feb 2020
  • Date of issue
    Nov-Dec 2019

History

  • Received
    11 May 2019
  • Accepted
    12 July 2019
  • Published
    5 Sept 2019
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