Metabolism Characterization and Chemical and Plasma Stability of Casearin B and Caseargrewiin F

Abstract

Oral preparations of Casearia sylvestris (guacatonga) are used as antacid, analgesic, anti-inflammatory, and antiulcerogenic medicines. The clerodane diterpenes casearin B and caseargrewiin F are major active compounds in vitro and in vivo. The oral bioavailability and metabolism of casearin B and caseargrewiin F were not previously investigated. We aimed to assess the stability of casearin B and caseargrewiin F in physiological conditions and their metabolism in human liver microsomes. The compounds were identified by UHPLC-QTOF-MS/MS and quantified by validated LC-MS methods. The stability of casearin B and caseargrewiin F in physiological conditions was assessed in vitro. Both diterpenes showed a fast degradation (p < 0.05) in simulated gastric fluid. Their metabolism was not mediated by cytochrome P-450 enzymes, but the depletion was inhibited by the esterase inhibitor NaF. Both diterpenes and their dialdehydes showed a octanol/water partition coefficient in the range of 3.6 to 4.0, suggesting high permeability. Metabolism kinetic data were fitted to the Michaelis-Menten profile with K_M values of 61.4 and 66.4 µM and V_max values of 327 and 648 nmol/min/mg of protein for casearin B and caseargrewiin F, respectively. Metabolism parameters in human liver microsomes were extrapolated to predict human hepatic clearance, and suggest that caseargrewiin F and casearin B have a high hepatic extraction ratio. In conclusion, our data suggest that caseargrewiin F and casearin B present low oral bioavailability due to extensive gastric degradation and high hepatic extraction.

Key words

Casearia sylvestris - Salicaceae - clerodane diterpene - hepatic metabolism - IVIVE

• References

• 1 Marquete R. Reserva Ecológica do IBGE (Brasília – DF): Flacourtiaceae. Rodriguésia 2001; 52: 5-16
• 2 Ferreira PMP, Costa-Lotufo LV, Moraes MO, Barros FWA, Martins AMA, Cavalheiro AJ, Bolzani VS, Santos AG, Pessoa C. Folk uses and pharmacological properties of Casearia sylvestris: A medicinal review. An Acad Bras Ciênc 2011; 83: 1373-1384
• 3 Xia L, Guo Q, Tu P, Chai X. The genus Casearia: A phytochemical and pharmacological overview. Phytochem Rev 2015; 14: 99-135
• 4 Danuello A, Castro RC, Pilon AC, Bueno PCP, Pivatto M, Vieira Júnior GM, Carvalho FA, Oda FB, Perez CJ, Lopes NP, Dos Santos AG, Ifa DR, Cavalheiro AJ. Fragmentation study of clerodane diterpenes from Casearia species by tandem mass spectrometry (quadrupole time‐of‐flight and ion trap). Rapid Commun Mass Spectrom 2020; 34: e8781
• 5 Ferreira PMP, Bezerra DP, Silva JDN, da Costa MP, Ferreira JRO, Alencar NMN, Figueiredo IST, Cavalheiro AJ, Machado CML, Chammas R, Alves APNN, Moraes MO, Pessoa C. Preclinical anticancer effectiveness of a fraction from Casearia sylvestris and its component Casearin X: In vivo and ex vivo methods and microscopy examinations. J Ethnopharmacol 2016; 186: 270-279
• 6 Spósito L, Oda FB, Vieira JH, Carvalho FA, dos Santos Ramos MA, de Castro RC, Crevelin EJ, Crotti AEM, Santos AG, da Silva PB, Chorilli M, Bauab TM. In vitro and in vivo anti-Helicobacter pylori activity of Casearia sylvestris leaf derivatives. J Ethnopharmacol 2019; 233: 1-12
• 7 Pierri EG, Castro RC, Vizioli EO, Ferreira CMR, Cavalheiro AJ, Tininis AG, Chin CM, Santos AG. Anti-inflammatory action of ethanolic extract and clerodane diterpenes from Casearia sylvestris . Rev Bras Farmacogn 2017; 27: 495-501
• 8 De Ford C, Heidersdorf B, Haun F, Murillo R, Friedrich T, Borner C, Merfort I. The clerodane diterpene casearin J induces apoptosis of T-ALL cells through SERCA inhibition, oxidative stress, and interference with Notch1 signaling. Cell Death Dis 2016; 7: e2070
• 9 Felipe KB, Kviecinski MR, da Silva FO, Bücker NF, Farias MS, Castro LSEPW, de Souza Grinevicius VMA, Motta NS, Correia JFG, Rossi MH, Pedrosa RC. Inhibition of tumor proliferation associated with cell cycle arrest caused by extract and fraction from Casearia sylvestris (Salicaceae). J Ethnopharmacol 2014; 155: 1492-1499
• 10 Sertié JAA, Woisky RG, Cavalheiro AJ, Bolzani V da S, dos Santos AG, Tininis AG. Processo de obtenção de extratos e de frações ativas de Casearia sylvestris e seus usos. Instituto Nacional de Propriedade Industrial (INPI) PI 0306167-1; 2018.
• 11 Itokawa H, Totsuka N, Morita H, Takeya K, Iitaka Y, Schenkel EP, Motidome M. New antitumor principles, casearins A–F, for Casearia sylvestris Sw. (Flacourtiaceae). Chem Pharm Bull (Tokyo) 1990; 38: 3384-3388
• 12 Ferreira PM, Militão GC, Lima DJB, Costa ND, Machado K da C, Santos AGD, Cavalheiro AJ, Bolzani V da S, Silva DHS, Pessoa C. Morphological and biochemical alterations activated by antitumor clerodane diterpenes. Chem Biol Interact 2014; 222: 112-125
• 13 Prieto AM, dos Santos AG, Oliveira APS, Cavalheiro AJ, Silva DHS, Bolzani VS, Varanda EA, Soares CP. Assessment of the chemopreventive effect of casearin B, a clerodane diterpene extracted from Casearia sylvestris (Salicaceae). Food Chem Toxicol 2013; 53: 153-159
• 14 Morita H, Nakayama M, Kojima H, Takeya K, Itokawa H, Schenkel EP, Motidome M. Structures and cytotoxic activity relationship of casearins, new clerodane diterpenes from Casearia sylvestris Sw. Chem Pharm Bull (Tokyo) 1991; 39: 693-697
• 15 dos Santos AG, Ferreira PM, Vieira Júnior GM, Perez CC, Gomes Tininis A, Silva GH, Bolzani Vda S, Costa-Lotufo LV, Pessoa Cdo O, Cavalheiro AJ. Casearin X, its degradation product and other clerodane diterpenes from leaves of Casearia sylvestris: evaluation of cytotoxicity against normal and tumor human cells. Chem Biodivers 2010; 7: 205-215
• 16 Ferreira PMP, Santos AG, Tininis AG, Costa PM, Cavalheiro AJ, Bolzani VS, Moraes MO, Costa-Lotufo LV, Montenegro RC, Pessoa C. Casearin X exhibits cytotoxic effects in leukemia cells triggered by apoptosis. Chem Biol Interact 2010; 188: 497-504
• 17 Basile AC, Sertié JAA, Panizza S, Oshiro TT, Azzolini CA. Pharmacological assay of Casearia sylvestris. I: Preventive anti-ulcer activity and toxicity of the leaf crude extract. J Ethnopharmacol 1990; 30: 185-197
• 18 Oda FB, Crevelin EJ, Crotti AEM, Orlando AB, de Medeiros AI, Nogueira FAR, dos Santos AG. Acidic and hepatic derivatives of bioactive clerodane diterpenes casearins J and O. Fitoterapia 2019; 137: 104197
• 19 Lipinski CA. Lead- and drug-like compounds: The rule-of-five revolution. Drug Discov Today Technol 2004; 1: 337-341
• 20 Kerns EH, Di L. Pharmacokinetics. In: Kerns EH, Di L. editors Drug-like Properties: Concepts, Structure Design and Methods. Amsterdam: Elsevier/AP; 2016: 267-281
• 21 Takács-Novák K. Physicochemical Profiling in Drug Research and Development. In: Mandić Z. editor Physico-Chemical Methods in Drug Discovery and Development. Zagreb: IAPC Publishing; 2012: 1-59
• 22 Moreira da Silva R, Verjee S, de Gaitani CM, Moraes de Oliveira AR, Pires Bueno PC, Cavalheiro AJ, Peporine Lopes N, Butterweck V. Evaluation of the intestinal absorption mechanism of casearin X in Caco-2 cells with modified carboxylesterase activity. J Nat Prod 2016; 79: 1084-1090
• 23 Smith DA, Allerton C, Kalgutkar AS, Van de Waterbeemd H, Walker DK. Pharmacokinetics and Metabolism in Drug Design, 3rd, Revised and Updated Edition Ed. Weinheim: Wiley-VCH; 2012: 1-18
• 24 Koitka M, Höchel J, Gieschen H, Borchert HH. Improving the ex vivo stability of drug ester compounds in rat and dog serum: Inhibition of the specific esterases and implications on their identity. J Pharm Biomed Anal 2010; 51: 664-678
• 25 Moreira da Silva R, de Gaitani CM, Marques LMM, Fraige Baraco K, Cavalheiro AJ, de Moraes LAB, Lopes NP, de Oliveira ARM. Characterization of Casearin X metabolism by rat and human liver microsomes. Planta Med 2019; 85: 282-291
• 26 Nishimuta H, Houston JB, Galetin A. Hepatic, intestinal, renal, and plasma hydrolysis of prodrugs in human, cynomolgus monkey, dog, and rat: Implications for in vitro–in vivo extrapolation of clearance of prodrugs. Drug Metab Dispos 2014; 42: 1522-1531
• 27 Howard ML, Hill JJ, Galluppi GR, McLean MA. Plasma protein binding in drug discovery and development. Comb Chem High Throughput Screen 2010; 13: 170-187
• 28 Obach RS, Baxter JG, Liston TE, Silber BM, Jones BC, MacIntyre F, Rance DJ, Wastall P. The prediction of human pharmacokinetic parameters from preclinical and in vitro metabolism data. J Pharmacol Exp Ther 1997; 283: 46-58
• 29 Zoghbi SS, Anderson KB, Jenko KJ, Luckenbaugh DA, Innis RB, Pike VW. On quantitative relationships between drug-like compound lipophilicity and plasma free fraction in monkey and human. J Pharm Sci 2012; 101: 1028-1039
• 30 Gertz M, Kilford PJ, Houston JB, Galetin A. Drug lipophilicity and microsomal protein concentration as determinants in the prediction of the fraction unbound in microsomal incubations. Drug Metab Dispos 2008; 36: 535-542
• 31 Emoto C, Murayama N, Rostami-Hodjegan A, Yamazaki H. Methodologies for investigating drug metabolism at the early drug discovery stage: Prediction of hepatic drug clearance and P450 contribution. Curr Drug Metab 2010; 11: 678-685
• 32 Zvereva I, Semenistaya E, Krotov G, Rodchenkov G. Comparison of various in vitro model systems of the metabolism of synthetic doping peptides: Proteolytic enzymes, human blood serum, liver and kidney microsomes and liver S9 fraction. J Proteomics 2016; 149: 85-97
• 33 Houston JB, Kenworthy KE. In vitro-in vivo scaling of CYP kinetic data not consistent with the classical Michaelis-Menten model. Drug Metab Dispos 2000; 28: 246-254
• 34 Moreira Fde L, de Souza GHB, Rodrigues IV, Lopes NP, de Oliveira ARM. A non-michaelian behavior of the in vitro metabolism of the pentacyclic triterpene alfa and beta amyrins by employing rat liver microsomes. J Pharm Biomed Anal 2013; 84: 14-19
• 35 Subramanian M, Tracy TS. Methods for Determination of Enzyme Kinetics and Metabolic Rates. In: Encyclopedia of Drug Metabolism and Interactions. Hoboken: John Wiley & Sons, Ltd.; 2012: 1-27
• 36 Houston JB. Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. Biochem Pharmacol 1994; 47: 1469-1479
• 37 Obach RS. Predicting clearance in humans from in vitro data. Curr Top Med Chem 2011; 11: 334-339
• 38 Carvalho FA, Uchina HS, Borges FA, Oyafuso MH, Herculano RD, Gremião MPD, Santos AG. Natural membranes of Hevea brasiliensis latex as delivery system for Casearia sylvestris leaf components. Rev Bras Farmacogn 2018; 28: 102-110
• 39 Bueno PCP, Pereira FMV, Torres RB, Cavalheiro AJ. Development of a comprehensive method for analyzing clerodane-type diterpenes and phenolic compounds from Casearia sylvestris Swartz (Salicaceae) based on ultra high performance liquid chromatography combined with chemometric tools: Liquid Chromatography. J Sep Sci 2015; 38: 1649-1656
• 40 OECD. Test No. 117: Partition Coefficient (n-octanol/water), HPLC Method. OECD Guidelines for the Testing of Chemicals. Paris: OECD Publishing; 2022
• 41 Di L, Kerns EH, Hong Y, Chen H. Development and application of high throughput plasma stability assay for drug discovery. Int J Pharm 2005; 297: 110-119
• 42 Bjornsson TD, Callaghan JT, Einolf HJ, Fischer V, Gan L, Grimm S, Kao J, King SP, Miwa G, Ni L, Kumar G, McLeod J, Obach RS, Roberts S, Roe A, Shah A, Snikeris F, Sullivan JT, Tweedie D, Vega JM, Walsh J, Wrighton SA. The conduct of in vitro and in vivo drug-drug interaction studies: A Pharmaceutical Research and Manufacturers of America (PhRMA) perspective. Drug Metab Dispos 2003; 31: 815-832
• 43 European Medicine Agency. Guideline on bioanalytical method validation. Accessed May 21, 2018 at: https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdf
• 44 Agência Nacional de Vigilância Sanitária. Resolução da Diretoria Colegiada (RDC) n°166, de 24 de julho de 2017. Dispõe sobre a validação de métodos analíticos e dá outras providências. Accessed May 21, 2018 at: http://antigo.anvisa.gov.br/documents/10181/2721567/RDC_166_2017_COMP.pdf/d5fb92b3-6c6b-4130-8670-4e3263763401
• 45 Kanokmedhakul S, Kanokmedhakul K, Buayairaksa M. Cytotoxic clerodane diterpenoids from fruits of Casearia grewiifolia . J Nat Prod 2007; 70: 1122-1126